Download Molecular Methods in Milk Quality

Molecular Methods
in Milk Quality
Proceedings of a Symposium
to celebrate the opening
of the new Ithaca facilities of
Quality Milk Production Services
Edited by R. N. Zadoks
Ithaca, NY
September 30, October 1 – 2004
QMPS is a program within the Animal Health Diagnostic Center,
a partnership between the NYS Department of Agriculture and Markets
and the College of Veterinary Medicine at Cornell University
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TABLE OF CONTENTS
Welcome
Y. H. Schukken, R. N. González, R. N. Zadoks
5
Symposium Sponsors
6
Symposium Program
7
Molecular Methods in Food Safety
K. J. Boor
9
Molecular Methods And Mastitis Research With Particular Reference
To Streptococcus uberis
S. P. Oliver and B. E. Gillespie
13
Monitoring Antimicrobial Resistance in USA Agriculture
P. F. Cray
19
Molecular Methods in Antimicrobial Resistance of Mastitis Pathogens
L. L. Tikofsky, R. N. Zadoks, I. Loch
20
Molecular Methods on Dairy Farms: Case Studies
R. N. Zadoks
31
Molecular Medicine, A Reality Coming Through
V. Kapur
40
Decoding The MAP Genome
V. Kapur
41
MLST And Antimicrobial Resistance Of Salmonella
S. Alcaine, S. Sukhnanand, L. D. Warnick, W.-L. Su, P. McDonough, M.
Wiedmann
42
Listeria monocytogenes Contains Two Species-Like Evolutionary Lineages And
Subtypes With Reduced Invasiveness.
K. K. Nightingale, K. Windham, and M. Wiedmann
46
MLST of Streptococcus uberis
R. N. Zadoks, Y. H. Schukken and M. Wiedmann.
49
Real-time PCR in milk – Food Safety in Times of War and Peace
J. Karns
52
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TABLE OF CONTENTS, ctd.
Past, Present And Future Applications Of Bulk Tank Milk Analysis To Assess
Milk Quality And Herd Health Status
B. Jayarao
55
PCR Applications In Food Safety Research
S. P. Oliver and B. E. Gillespie
74
Polymerase Chain Reaction For Detection Of Mycoplasma Bovis
In Clinical Samples
S. Klaessig
84
Bovine Infection Of Coxiella Burnetii (Q Fever) In U.S. Dairy Herds: Use Of
Conventional And Real-Time PCR For Detection Of Coxiella Burnetii In Milk
S. Kim
86
Diagnostic Strategies For Bovine Viral Diarrhea Virus
E. Dubovi
88
Speaker Biographies and Contact Information
91
Acknowledgements
109
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WELCOME
On behalf of Quality Milk Production Services, we would like to welcome you to the two day
symposium on "Molecular Methods for Milk Quality." We hope you will share our enthusiasm
for this program of outstanding speakers and are pleased to have you in our audience. Thank you
for your interest, participation and support.
From the beginning of the "Genomics Initiative" at Cornell University and other universities
around the world, there has been a promise of new opportunities. The publication of the human
genome was a major step in this process. Subsequently, genomes of many organisms that are
important to animal and public health have been deciphered and published. The genomes of
Mycobacterium avium spp. paratuberculosis, Escherichia coli, Staphylococcus aureus, Streptococcus
agalactiae, Listeria monocytogenes and several other species have been fully sequenced. We are now
actively pursuing the opportunities provided by these genomic developments and are putting
them to work towards better understanding of the biology of foodborne, zoonotic and bovine
diseases, particularly mammary gland diseases. With the opening of our “”Molecular
Laboratory” we hope to greatly improve the service we can provide to the dairy industry in these
areas, to the benefit of cows, producers, processors and consumers alike.
This two day program brings a variety of opportunities and applications that will highlight the
new directions in which research and the diagnostic process are moving. You will hear of
developments in Milk Quality and Food Safety research at Cornell University, the Agricultural
Research Service of the USDA and several other prominent research institutes in the U.S.A. The
significance of genome sequencing in diagnostics and disease prevention as well as the use of
molecular methods in the fields of monitoring and improvement of animal health, milk quality,
food safety and antimicrobial resistance of mastitis pathogens will be discussed along with their
practical application to real life problem solving on dairy farms. We hope this symposium will
provide you with information, inspiration and the opportunity to connect with colleagues and
that the impact of this event will last well beyond the two days that you will be our guest.
Again, thank you for you attendance and please enjoy this symposium.
Ynte H. Schukken
Rubén N. González
Ithaca, NY
September 2004
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Ruth N. Zadoks
WE THANKS OUR SPONSORS FOR THEIR FINANCIAL SUPPORT OF THIS
SYMPOSIUM
In alphabetical order:
Fort Dodge
A division of
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SYMPOSIUM PROGRAM
Thursday, September 29, 2004 – Ramada Inn, Ithaca, NY
09:30 Registration and Coffee
10:00
10:15
11:00
11:30
Introduction and Welcome - Dr. Rubén Gozález
Molecular Methods in Food Safety – Dr. Kathryn Boor
Molecular Methods in Streptococcus uberis mastitis – Dr. Stephen Oliver
Monitoring Antimicrobial Resistance in USA Agriculture – Dr. Paula Cray
12:15 Lunch
01:30 Molecular Methods in Antimicrobial Resistance of Mastitis Pathogens
Dr. Linda Tikofsky
02:00 Molecular Methods on Dairy Farms: Case Studies – Dr. Ruth Zadoks
2:30 Break
03:00 Molecular Medicine – A Reality Coming Through – Dr. Vivek Kapur
04:00 Conclusion - Dr. Rubén Gozález
04:30 Official Opening of QMPS Laboratory – 22 Thornwood Drive, Ithaca
Friday, October 1 –College of Veterinary Medicine, Cornell University
09:00 Decoding the MAP genome– Dr. Vivek Kapur
10:00 MLST and antimicrobial resistance of Salmonella – Dr. Martin Wiedmann
10:30 Break
11:00 MLST of Listeria monocytogenes: a tale of two lineages – Ms. Kendra Nightingale
11:30 MLST of Streptococcus uberis: a tale of no lineages – Dr. Ruth Zadoks
12:00 Lunch
01:00 Real-time PCR in milk: Food safety in times of war and peace – Dr. Jeffrey Karns
01:45 Past, present and future applications of bulk tank milk analysis
to assess milk quality and herd health status – Dr. Bhushan Jayarao
02:15 PCR applications in food safety research – Dr. Stephen Oliver
2:45 Break
3:15
3:30
4:00
4:30
PCR for Detection of Mycoplasma bovis – Ms. Suzanne Klaessig
Bovine Infection of Coxiella burnetii (Q fever) in U.S. Dairy Herds – Dr. Sung Kim
Bovine viral diarrhoea virus detecion in milk – Dr. Ed Dubovi
Discussion – Dr. Ynte Schukken
5:00 Refreshments
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NOTES
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MOLECULAR METHODS IN FOOD SAFETY
Kathryn J. Boor
Department of Food Science
Cornell University, Ithaca, NY
ABSTRACT
DNA-based detection and subtyping methods offer improvements in detection and
characterization of foodborne pathogens beyond classical plating and phenotypic methods. The
past 5 years have yielded advancements in development of sensitive, rapid, automated, and
increasingly easy-to-use molecular detection and subtyping methods for a variety of different
foodborne pathogens. This summary highlights key aspects of different DNA-based detection
and subtyping methods and their applications for foodborne pathogens.
DNA-BASED DETECTION METHODS FOR FOODBORNE PATHOGENS
Detection methods currently applied for foodborne pathogens are predominantly based
on nucleic-acid hybridization or polymerase chain reaction. These methods can be designed to
detect either DNA or mRNA. While detection of DNA is often technically more straightforward
than that of mRNA, the stability of DNA leads to the possibility that DNA-based detection
methods may yield positive results from non-viable and/or inactivated pathogens. Detection of
non-viable and inactivated pathogens thus represents a major concern in the direct application of
PCR methods, especially those not designed to include an enrichment step. mRNA, on the other
hand, is less stable than DNA, and has thus has potential as a target for more specific detection of
viable pathogen populations.
Nucleic-acid hybridization-based methods. Nucleic acid hybridization methods have been used
as relatively rapid screening strategies for identification of foodborne pathogens. These methods
also can be used to detect target pathogens in enrichment media. Many commercial nucleic-acid
hybridization-based detection methods use a dip-stick format, e.g., the GenTrack assay. rRNA is
often used as the target for these detection methods, since it provides a high level of sensitivity
due to the presence of a high number of target copies (>1,000) in a single bacterial cell. A
disadvantage of rRNA-based detection methods lies in the limited specificity of these assays due
to the fact that closely related species (e.g., the pathogen L. monocytogenes and the closely related
non-pathogenic species L. innocua) can share highly similar rRNA sequences, which do not allow
their differentiation.
Polymerase chain reaction (PCR). PCR has been used as a research tool for more than 15 years,
and also has been developed into a tool for rapid and specific detection of foodborne pathogens.
While early PCR assays for foodborne pathogen detection were generally developed by research
laboratories and were not suitably developed for routine detection, over the last 3-5 years,
multiple companies have introduced commercial PCR systems for detection of foodborne
pathogens such as Listeria monocytogenes, E. coli O157:H7, and Salmonella. These methods often
allow superior specificity to traditional biochemical identification methods. To date, all
commercial PCR assays still require pre-enrichment steps to achieve appropriate sensitivity.
These pre-enrichment protocols also greatly reduce the risk of false positive results due to
detection of killed organisms, mainly due to the sample dilution step inherent to these protocols.
Another critical component of these commercial assays is the inclusion of an internal positive
control that indicates PCR failures, e.g., through carry over of PCR inhibitors. While most early
PCR commercial assays used detection of PCR products by agarose gel electrophoresis as output,
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at least some commercial assays currently available use a ”real-time format” with either a
5’nuclease probe or a SYBR Green-based detection of PCR products. These formats negate the
need for agarose gel electrophoresis and further speed up completion of PCR-based assays.
DNA-BASED SUBTYPING METHODS FOR FOODBORNE PATHOGENS
The use of subtyping methods to differentiate strains (or subtypes) of bacterial, viral, and
parasitic pathogens has important applications for more rapid, precise, and efficient foodborne
disease surveillance, outbreak detection, and source tracking throughout the food chain.
Differentiation of bacterial foodborne pathogens beyond the species level also provides exciting
opportunities to better understand the biology of bacterial strains and subtypes, including
differences in their ability to cause human foodborne disease.
In the context of subtyping, the terms “subtyping”, “strain typing”, and “fingerprinting”
are often used interchangeably. All of these terms describe the process of differentiating bacterial
isolates beyond the species or subspecies level. The term “fingerprinting” can be somewhat
misleading when used in this context, however, since bacterial subtyping differs significantly from
fingerprinting of humans. Importantly, asexual reproduction in bacteria allows for the parallel
existence of virtually identical organisms. Bacterial subtyping is used to characterize two or more
distinct isolates with the goal of determining their (ancestral) relationship. For example, in
outbreak investigations, the goal of subtyping bacterial isolates is to probe the likelihood that two
or more isolates share a very recent (days to weeks, perhaps months) common ancestor.
Fingerprinting of humans, on the other hand, is used to characterize and track a single specific
individual (Wiedmann, 2002b).
The choice of an appropriate subtyping method (or methods) depends on the intended
application and the goal of the exercise. Commonly used criteria for evaluating subtyping
methods include (i) discriminatory ability; (ii) cost; (iii) standardization and reproducibility; (iv)
automation and ease of use; (v) speed; and (vi) applicability of a given subtyping method to
different bacterial species (Wiedmann, 2002a; de Boer and Beumer, 1999). The discriminatory
ability of a subtyping method can be characterized using Simpson’s Index of Discrimination,
which quantifies the probability that two unrelated strains will be characterized as different
subtypes. No single subtyping method will perform optimally with regard to all of these criteria.
The intended application of subtyping will determine the relative importance of each criterion.
For example, a food testing laboratory that subtypes a limited number of isolates representing a
variety of different foodborne pathogens (e.g., Escherichia coli O157:H7; Listeria monocytogenes,
Salmonella) will have different requirements for a subtyping method than a national or
international subtyping network or reference laboratory that needs to subtype a large number
(>1,000) of isolates of one specific pathogen.
In general, bacterial subtyping methods can be divided into (i) phenotype-based, and (ii)
molecular, genetic or DNA-based methods (Olive and Bean, 1999; Wiedmann, 2002b).
Commonly used phenotype-based strain typing methods for bacterial pathogens include
serotyping, biotyping, phage typing, and multilocus enzyme electrophoresis. While a variety of
shortcomings and concerns may be associated with different phenotype-based strain typing
methods, these methods are still regularly used and have some utility for characterization of
bacterial foodborne pathogens. Phenotype-based methods may lack discriminatory power and
reproducibility. Furthermore, a considerable proportion of bacterial isolates may be untypable
with some of these methods. To overcome these issues and to provide improved strain
differentiation, molecular subtyping methods, which are based on the microbial genotypes, have
been developed (Wiedmann, 2002b).
The widespread development of multiple DNA-based subtyping methods has
dramatically improved our ability to differentiate subtypes of bacterial foodborne pathogens.
Commonly used DNA-based subtyping approaches for bacterial pathogens include plasmid
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profiling, Pulsed-Field Gel Electrophoresis (PFGE), ribotyping, Amplified Fragment Length
Polymorphism (AFLP), random amplification of polymorphic DNA (RAPD) as well as other
PCR-based subtyping methods (Olive and Bean, 1999; Wiedmann, 2002a). Increasingly, DNAsequencing based methods, such as multilocus sequence typing (MLST) are also being developed.
Many DNA-based methods are superior to classical methods (e.g., serotyping) in several respects.
For example, DNA-based subtyping methods often provide more sensitive strain discrimination
and a higher level of standardization and reproducibility as compared to phenotype-based
methods. The use of multiple subtyping methods often improves subtype discrimination and may
thus be appropriate for certain applications and specifically for epidemiological outbreak
investigations. Key aspects of selected and commonly used molecular subtyping methods are
summarized below. For a more comprehensive review the reader is referred to one of the many
review articles on bacterial subtyping methods (Olive and Bean, 1999; de Boer and Beumer,
1999; van Belkum et al., 2001).
Pulsed-Field Gel Electrophoresis (PFGE). PFGE characterizes bacteria into subtypes
(sometimes referred to as “pulsotypes”) by generating DNA banding patterns after restriction
digestion of the bacterial genomic DNA. Specifically, complete bacterial DNA is purified and
subsequently cut into diagnostic DNA fragments using restriction enzymes, which cut DNA
where a specific short DNA sequence is present. Restriction enzymes are chosen such that they
cut DNA only rarely to yield between approximately 8 and 25 large DNA bands ranging from 40
– 600 kb (Wiedmann, 2002a). Since DNA fragments this large cannot be separated by standard
gel electrophoresis techniques, a specific electrophoresis technique using alternating electric fields
needs to be used for size separation of the resulting DNA fragments (i.e., pulsed-field gel
electrophoresis), which will subsequently be visualized as DNA banding patterns. DNA banding
patterns for different bacterial isolates are compared to differentiate distinct bacterial subtypes
from those that share identical (or very similar) DNA fragment patterns.
The CDC and state health departments in the US have developed a national network
(PulseNet) to rapidly exchange standardized PFGE subtype data for isolates of foodborne
pathogens (Swaminathan et al., 2001). PFGE subtyping shows a high level of discrimination for
many foodborne bacterial pathogens and thus is often considered the current gold standard for
discriminatory ability. It is important to realize, however, that PFGE (as well as other subtyping
methods) may also sometimes detect small genetic differences (e.g., 2-3 different bands) that may
not be epidemiologically significant (Tenover et al., 1995). On the other hand, the detection of
an identical PFGE type (or a subtype determined by another method) in two samples (e.g., a food
sample and a sample from a clinically affected human) does not necessarily imply a causal
relationship or a link between these two isolates. Rather, in outbreak investigations, molecular
subtyping information needs to be analyzed in conjunction with epidemiological data to
determine causal relationships between two or more isolates.
Ribotyping. Ribotyping is another DNA based subtyping method in which bacterial DNA is
initially cut into fragments using restriction enzymes. While PFGE uses restriction enzymes that
cut the bacterial DNA in very few large pieces, the initial DNA digestion for ribotyping cuts
DNA into many (>300-500) smaller pieces (approximately 1 to 30 kb). These DNA fragments
are separated by size through agarose gel electrophoresis and a subsequent Southern blot step
uses DNA probes to specifically label and detect the DNA fragments that contain the bacterial
genes encoding the ribosomal RNA (rRNA). The resulting DNA banding patterns are thus based
on only those DNA fragments that contain the rRNA genes. A completely automated,
standardized system for ribotyping (the RiboPrinter® Microbial Characterization system) has
been developed by Qualicon-DuPont (Wilmington, DE) and is commercially available
(Wiedmann, 2002a; Bruce, 1996).
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DNA sequencing-based subtyping. DNA sequencing of one or more selected bacterial genes
represents another genetic subtyping method. Multilocus sequence typing (MLST) refers to a
molecular subtyping approach that uses DNA sequencing of multiple genes or gene fragments to
differentiate bacterial subtypes and to determine the genetic relatedness of isolates. MLST often
refers to sequencing of multiple housekeeping genes (Spratt, 1999), but sequencing of multiple
virulence genes can also be used as a subtyping method (Wiedmann, 2002a). A major advantage
of this approach is that sequence data are considerably less ambiguous (Spratt, 1999) and easier to
interpret than banding pattern-based subtypes obtained through the other DNA-based subtyping
approaches described above. The development of internet accessible databases for MLST
information (such as the MLST database at http://www.mlst.net/) will also facilitate global,
large scale surveillance and tracking of bacterial foodborne pathogens (Spratt, 1999). DNA
sequencing data also provide an opportunity to reconstruct ancestral and evolutionary
relationships among bacterial isolates, allowing scientists to further probe the evolutionary
biology and the ecology of foodborne pathogens. MLST-based approaches for subtyping of
bacterial foodborne pathogens are still in the early developmental stages and optimal target genes
are still being defined for the different bacteria of interest.
REFERENCES
Bruce J. Automated system rapidly identifies and characterizes microorganisms in food. Food
Technol 1996;50:77-81.
De Boer E, Beumer R. Methodology for detection and typing of foodborne microorganisms. Int J
Food Microbiol 1999;50:119-30.
Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial
organisms. J Clin Microbiol 1999;37:1661-9.
Spratt BG. Multilocus sequence typing: molecular typing of bacterial pathogens in an era of
rapid DNA sequencing and the internet. Curr Opin Microbiol 1999;2:312-6.
Swaminathan B, Barrett T, Hunter S, et al. PulseNet: the molecular subtyping network for
foodborne bacterial disease surveillance, United States. Emerg Infect Dis 2001;7:382-9.
Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns
produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol
1995;33:2233-9.
Van Belkum A, Struelens M, de Visser A, et al. Role of genomic typing in taxonomy,
evolutionary genetics, and microbial epidemiology. Clin Microbiol Rev 2001;14:547-60.
Wiedmann M. Molecular subtyping methods for Listeria monocytogenes. J AOAC Int
2002a;85:524-31.
Wiedmann M. Subtyping technologies for bacterial foodborne pathogens. Nutr Rev
2002b;60:201-8.
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MOLECULAR METHODS AND MASTITIS RESEARCH WITH PARTICULAR
REFERENCE TO STREPTOCOCCUS UBERIS
S. P. Oliver and B. E. Gillespie
Food Safety Center of Excellence and the Department of Animal Science
The University of Tennessee, Knoxville, TN
Introduction
Advances in molecular biology in the last decade or so have brought exciting new technology
that can be used to solve complex problems. Utilization of molecular techniques such as the
polymerase chain reaction (PCR), restriction fragment length polymorphism, real-time PCR,
multiplex PCR, pulsed-field gel electrophoresis, ribotyping, single nucleotide polymorphisms,
genomics, proteomics, DNA sequencing, and cloning are used more and more frequently in
many research laboratories in the United States and throughout the world. Use of these
techniques may facilitate the discovery of more effective methods for the prevention, control and
detection of diseases affecting food producing animals. The purpose of this communication is to
describe how the Mastitis/Food Safety Research Program at The University of Tennessee utilizes
molecular-based techniques in our research approach with particular reference to our research on
Streptococcus uberis.
Identification of Disease Susceptible and Resistant Dairy Cows
Novel approaches are currently being developed and utilized to determine what genetic factors
are involved in disease resistance. Identification of such factors will be critical for developing
strategies for eradicating or reducing the incidence of disease. Selection of dairy cows for
enhanced disease resistance without compromising production traits is a very appealing concept
that until the last decade was primarily a theoretical fantasy. However, excellent molecular
techniques have been developed resulting in the identification of new genetic markers that have
been used to identify and characterize genes responsible for production traits and host immunity.
Major histocompatibility complex (MHC) genes, also called bovine lymphocyte antigens or
BoLA, have received much recent attention because of their involvement in host immunity.
Significant associations have been made with some infectious diseases of cattle and BoLA genes.
There is strong evidence indicating that BoLA genes are important in resistance or susceptibility
to diseases such as mastitis, retained placenta and cystic ovarian disease in dairy cattle. For
example, one BoLA-DRB gene pattern in a study of 106 Holstein cows was associated with
resistance to Staphylococcus aureus mastitis.
Results of our research on BoLA-DRB3.2 gene fingerprinting of Jersey cows at The University of
Tennessee Dairy Experiment Station were published by Gillespie et al. (1999a). Jersey cows
(n=172) were genotyped for the BoLA-DRB3.2 allele using polymerase chain reaction and
restriction fragment length polymorphism analysis. Bovine DNA was isolated from aliquots of
whole blood. A two step polymerase chain reaction followed by digestion with restriction
endonucleases RsaI, BstyI, and HaeIII was conducted on the DNA from Jersey cattle. Twenty-four
BoLA-DRB3.2 alleles were identified with frequencies ranging from 0.3 to 22.9%. Thirteen allele
types were similar to those reported previously; eleven were new allele types that have not been
reported previously. Allele types reported previously include: BoLA-DRB3.2*2, *8, *10, *15, *17,
*20, *21, *22, *23, *25, *28, *36, and *37. Their frequencies were 0.3%, 11.3%, 22.9%, 13.6%,
5.5%, 3.7%, 10.7%, 3.5%, 0.9%, 0.3%, 4.7%, 9.3%, and 0.9%, respectively. Of the new allele types
detected, *ibe occurred at the highest frequency (6.1%) in Jersey cows from this herd. The six most
frequently isolated alleles (BoLA-DRB3. *8, *10, *15, *21, *36 and *ibe) accounted for about 74 %
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of the alleles in the population of this herd. Results of our study demonstrated that the BoLADRB3.2 locus is highly polymorphic in Jersey cattle. Thus, the BoLA gene may not be the best
candidate for determining a relationship between genotype and mastitis susceptibility or resistance in
Jersey cows.
A genetic marker associated with inflammatory responses is also being evaluated. One potential
marker is CXCR2, a chemokine receptor required for neutrophil migration to infection sites,
which contains single nucleotide polymorphisms (SNP) within the gene. In a study by
Youngerman et al. (2004a), single nucleotide polymorphisms (SNPs) and resulting haplotypes in
the bovine CXCR2 gene were identified as a potential target for a genetic marker for mastitis
susceptibility. A 311-bp segment of the bovine CXCR2 gene was amplified and sequenced. Five
SNPs at positions 612, 684, 777, 858, and 861 were expressed in both Holstein and Jersey dairy
cattle. Four SNPs resulted in synonymous substitutions, while a nonsynonymous switch at
position 777 (G to C) resulted in a glutamine to histidine substitution at amino acid residue 245.
The five polymorphisms generated ten distinct haplotypes. Six haplotypes were common between
the two breeds, while Holsteins and Jerseys each uniquely expressed two haplotypes. Of the six
common haplotypes, two represented 83% of the Jersey population; whereas four of these
haplotypes represented 95% of the Holstein population.
The association of CXCR2 SNP genotypes with subclinical and clinical mastitis was evaluated by
Youngerman et al. (2004b). Thirty-seven Holstein and 42 Jersey cows that completed at least 2
full lactations were used. A significant association was detected between CXCR2 SNP +777
genotype and percentages of subclinical mastitis cases in Holsteins. Holsteins expressing
genotype GG had decreased percentages of subclinical mastitis, but genotype CC cows had
increased percentages of subclinical mastitis. Significant differences in clinical mastitis incidence
were not detected between genotypes for either breed. This approach of genetically identifying
mastitis resistant cows may represent an effective means of marker-assisted selection for mastitis
and other inflammatory diseases involving neutrophils. The initial work is encouraging and
several studies are ongoing in this exciting research area.
Identifying host mechanisms that contribute to mastitis resistance is difficult due to variability
observed with an outbred population. Progress towards identifying these mechanisms could be
made more quickly with cows that are genetically similar. New techniques such as cloning now
offer a similar opportunity to mastitis researchers. A team at UT scientists led by Drs. Lannett
Edwards and Neal Schrick have successfully cloned Jersey dairy cows from mastitis susceptible
cows and mastitis resistant cows (Pighetti et al., 2003). The mastitis susceptible cow has been
chronically infected with Strep. uberis for about 7 lactations in spite of numerous attempts to
eliminate the infection. Some of these heifers are currently of breeding age, some have been breed
and some have calved and are now in early lactation (http://web.utk.edu/
~taescomm/utcloneproject). By having a unique set of genetically identical animals, it is possible
to develop our understanding of what contributes to mastitis resistance or susceptibility under
different management schemes, vaccination protocols, or stress-situations, without the added
complication of genetic variation. Our first step towards identifying these mechanisms is to
determine if differences in blood leukocyte profiles exist in comparison to age-matched herdmates. Future research will be conducted to determine if immune responsiveness of clones from
mastitis susceptible animals are less than those of herdmates, thus contributing to susceptibility.
Once identified, more basic research altering conditions of the entire animal can begin to dissect
the mechanisms that contribute to susceptibility or resistance to mastitis or other diseases of diary
cattle. Identification of such factors could lead to improved selection strategies and/or novel
approaches for eradicating or reducing incidence of mastitis and other diseases impacting dairy
cows.
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The Streptococcus uberis Story
Streptococcus uberis is an important cause of mastitis in dairy cows -- particularly during the dry
period, the period around calving, and during early lactation -- that is not controlled effectively by
current mastitis control practices. Many Strep. uberis intramammary infections (IMI) that originate
during the nonlactating period and near calving result in clinical and subclinical mastitis during early
lactation. Control programs for reducing Strep. uberis IMI should focus on periods adjacent to the
nonlactating period where opportunities exist to develop strategies to reduce the impact of Strep.
uberis infections in the dairy herd.
We began our Strep. uberis research journey in the early 1990’s. Earlier research in England
demonstrated the presence of two Strep. uberis genotypes designated types I and II. Subsequent
research from England determined the nucleotide sequences of 16S ribosomal RNA of Strep.
uberis genotypes I and II and showed that the two genotypes were phylogenetically distinct and
proposed that Strep. uberis genotype II be designated Streptococcus parauberis. However,
differentiation of Strep. uberis from Strep. parauberis was only possible by DNA hybridization or
16S rRNA sequencing, since cultural, morphological, biochemical and serological characteristics
of the two closely related species are indistinguishable. A technique was developed by Jayarao et
al. (1991) for differentiating Strep. uberis from Strep. parauberis based on DNA fingerprinting.
Results of those studies demonstrated that the predominant organism isolated from infected
mammary glands was Strep. uberis and that Strep. parauberis occurred infrequently. This method
was also used for species identification and differentiation of bacteria of bovine origin. Using the
PCR reaction, oligonucleotide primers complementary to 16S rRNA genes have been used to
amplify the 16S ribosomal gene fragment from bacterial genomic DNA. Characteristic 16S
rDNA fingerprint patterns have been used to correctly identify 11 different Enterococcus and
Streptococcus species (Jayarao et al., 1992).
Research from our laboratory has focused extensively on development of in vivo and in vitro
models to study host-pathogen interactions, and on identification and characterization of
virulence factors associated with the pathogenesis of Strep. uberis mastitis and other environmental
streptococci (Oliver et al., 1998a). We have shown that Strep. uberis was able to adhere to
epithelial cells and that was followed by internalization into the host cell via exploitation of host
cell machinery. Our lab demonstrated that Strep. uberis used host elements like extracellular
matrix proteins to achieve increased adherence, probably utilizing these as a molecular bridge to
attach to host cell membranes. Another of these host cell factors appears to be lactoferrin (LF), a
whey protein found in milk. Use of molecular biology tools such as proteomics, genomics and
bioinformatics has led to the discovery of a novel protein produced by Strep. uberis referred to as
Streptococcus uberis Adhesion Molecule or SUAM.
We have conducted numerous studies on SUAM and a brief summary of these studies follows.
Collectively, experiments from our laboratory have provided evidence that: (1) Strep. uberis
produces SUAM (Fang and Oliver, 1999), (2) SUAM bound to LF in milk (Fang et al., 2000), (3)
binding of LF through SUAM enhanced adherence of Strep. uberis to bovine mammary epithelial
cells (Fang et al., 2000). Lactoferrin may function as a bridging molecule between Strep. uberis
and bovine mammary epithelial cells facilitating adherence of this important mastitis pathogen to
host cells, (4) SUAM in the absence of LF influenced adherence to and internalization of Strep.
uberis into bovine mammary epithelial cells, (5) SUAM was isolated, purified and sequenced, (6)
a SUAM-like protein was identified in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus
agalactiae (Park et al., 2002a), (7) SUAM-like proteins produced by Strep. dysgalactiae subsp.
dysgalactiae bound to bovine LF similar to what we observed with Strep. uberis (Park et al., 2002b),
(8) antibodies against SUAM (whole protein) and to a synthetic peptide (pepSUAM)
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encompassing 15 amino acids of the N-terminus of SUAM cross-reacted with homologous
proteins present in other strains of Strep. uberis demonstrating the ubiquity of SUAM across all
strains of Strep. uberis evaluated, (9) pepSUAM and SUAM antibodies cross-reacted with Strep.
agalactiae, Strep. dysgalactiae subsp. dysgalactiae, and Streptococcus pyogenes, (10) antibodies directed
against pepSUAM inhibited adherence to and internalization of Strep. uberis into bovine
mammary epithelial cells suggesting that pepSUAM is biologically active. In addition, we have
determined the theoretical DNA sequence of SUAM and confirmed this by PCR and restriction
digests. Further confirmation of the theoretical SUAM sequence was obtained when the SUAM
gene from the mastitis pathogen Strep. uberis UT888 was amplified, cloned and sequenced.
Sequence analysis demonstrated that UT888 SUAM has 99% sequence identity to the theoretical
SUAM identified in the Sanger Strep. uberis genomic database by homology to the reverse
translated peptide sequence. When the SUAM DNA sequence was compared to GeneBank
(NCBI nr GeneBank), no homologies as an entire gene were found demonstrating that SUAM is
a unique Strep. uberis protein. We hypothesize that SUAM plays a critical role in the pathogenesis of
streptococcal mastitis by facilitating bacterial adherence to bovine mammary epithelial cells. Our
hypothesis is that Strep. uberis expresses SUAM and uses LF in milk and/or on the epithelial cell
surface to adhere to mammary epithelial cells.
Nucleic Acid-Based Methods for Mastitis Pathogen Detection
Detection and subtyping of bacteria for epidemiological evaluation has been made possible by
randomly amplified polymorphic DNA (RAPD) fingerprinting. We have used this technique to
identify Streptococcus species (Gillespie et al., 1997; Gillespie et al., 2004) and other mastitis
pathogens (Jayarao et al., 1996); detect new and persistent Strep. uberis and Streptococcus
dysgalactiae subsp. dysgalactiae IMI in dairy cows (Oliver et al., 1998b). Using phenotypic methods
of streptococcal identification, these new IMI would not have been detected. RAPD
fingerprinting has also been used for confirmation of Strep. uberis after intramammary challenge
with Strep. uberis and identified new Strep. uberis infections in challenged quarters. Subtyping of
Strep. uberis and Strep. dysgalactiae by RAPD fingerprinting demonstrated isolates from New
Zealand were distinct from isolates from the USA Gillespie et al., 1998). RAPD fingerprinting
has been used to study the possibility of Staphylococcus aureus transmission by horn flies to heifers
(Owens et al., 1998; Gillespie et al., 1999b). This technique is also useful in antibiotic efficacy
studies in indicating new IMI or persistent IMI following antibiotic therapy.
Molecular techniques described herein can aide mastitis researchers in identification of bacteria
and subtyping of bacteria isolates for epidemiological applications, identification of genetic
markers associated with disease susceptibility or resistance, and could aid in selection of dairy
cattle that are more or less susceptible to mastitis. Application of these molecular techniques will
allow dairy researchers greater flexibility to explore their area of scientific interest at the
molecular level and may expedite discoveries leading to more effective methods for the control of
mastitis and other diseases affecting dairy cows.
References
Fang, W. and S. P. Oliver. 1999. Identification of lactoferrin-binding proteins in bovine mastitiscausing Streptococcus uberis. FEMS Microbiol. Lett. 176:91-96.
Fang, W., R. A. Almeida and S. P. Oliver. 2000. Effects of lactoferrin and milk on adherence of
Streptococcus uberis to bovine mammary epithelial cells. Am. J. Vet. Res. 61:275-279.
- 16 -
Gillespie, B. E., B. M. Jayarao, and S. P. Oliver. 1997. Identification of Streptococcus species by
randomly amplified polymorphic DNA fingerprinting. J. Dairy Sci. 80:471-476.
Gillespie, B. E., B. M. Jayarao, J. W. Pankey, and S. P. Oliver. 1998. Subtyping of Streptococcus
uberis and Streptococcus dysgalactiae isolated from bovine mammary glands by DNA fingerprinting. J.
Vet. Med. B 45:585-593.
Gillespie, B. E., B. M. Jayarao, H. H. Dowlen, and S. P. Oliver. 1999a. Analysis and frequency
of bovine lymphocyte antigen DRB3.2 alleles in Jersey cows. J. Dairy Sci. 82:2049-2053.
Gillespie, B. E., W. E. Owens, S. C. Nickerson, and S. P. Oliver. 1999b. Deoxyribonucleic acid
fingerprinting of Staphylococcus aureus from heifer mammary secretions and from horn flies. J.
Dairy Sci. 82:1581-1585.
Gillespie, B. E., and S. P. Oliver. 2004. Comparison of an automated ribotyping system, pulsedfield gel electrophoresis and randomly amplified DNA fingerprinting for differentiation of
Streptococcus uberis strains. Biotechnology 3:165-172.
Jayarao, B. M., J. J. E. Dore, Jr., G. A. Baumbach, K. R. Matthews, and S. P. Oliver. 1991.
Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerase chain reaction and
restriction fragment length polymorphism analysis of 16S ribosomal DNA. J. Clin. Micro. 29:27742778.
Jayarao, B. M., J. J. E. Dore, Jr., and S. P. Oliver. 1992. Restriction fragment length
polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine
origin. J. Clin Microbiol. 30: 2235-2240.
Jayarao, B. M., B. E. Gillespie, and S. P. Oliver. 1996. Application of randomly amplified
polymorphic DNA fingerprinting for species identification of bacteria isolated from bovine milk. J.
Food Prot. 59: 615-620.
Oliver, S. P., R. A. Almeida, and L. F. Calvinho. 1998a. Virulence factors of Streptococcus uberis
isolated from cows with mastitis. J. Vet. Med. B 45:461-471.
Oliver, S. P., B. E. Gillespie and B. M. Jayarao. 1998b. Detection of new and persistent
Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain
reaction-based DNA fingerprinting. FEMS Microbiol. Lett. 160:69-73.
Owens, W. E., S. P. Oliver, B. E. Gillespie, C. H. Ray, and S. C. Nickerson. 1998. The role of
horn flies (Haemetobia irritans) in Staphylococcus aureus-induced mastitis in dairy heifers. Am. J. Vet.
Res. 59:1122-1124.
Park, H. M., R. A. Almeida, and S. P. Oliver. 2002a. Identification of lactoferrin-binding
proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from
cows with mastitis. FEMS Microbiol. Lett. 207:87-90.
Park, H. M., R. A. Almeida, D. A. Luther, and S. P. Oliver. 2002b. Binding of bovine lactoferrin
to Streptococcus dysgalactiae subsp. dysgalactiae isolated from cows with mastitis. FEMS Microbiol.
Lett. 208:35-39.
- 17 -
Pighetti, G. M., J. L. Edwards, F. N. Schrick, A. M. Saxton, C. J. Davies, and S. P. Oliver. 2003.
Cloning adult dairy cows: A viable new tool in the fight against mastitis. In: Proc. National Mastitis
Council, pp.360-361.
Youngerman, S.M., A.M. Saxton, and G.M. Pighetti. 2004a. Identification of single nucleotide
polymorphisms, haplotypes and their frequencies within the bovine IL-8 receptor locus in Jersey
and Holstein cattle. Immunogenetics. 56: 355-359.
Youngerman, S. M., A. M. Saxton, S. P. Oliver, and G. M. Pighetti. 2004b. Analysis of bovine
CXCR2 polymorphisms with subclinical and clinical mastitis incidence in Holstein and Jersey
cattle. J. Dairy Sci. 87: 2442-2448.
- 18 -
MONITORING ANTIMICROBIAL RESISTANCE IN USA AGRICULTURE
Dr. Paula J. Fedorka-Cray
Antimicrobial Resistance Research Unit
USDA-ARS, Athens, GA
National Antimicrobial Resistance Monitoring System
CD with NARMS update 2003 here
- 19 -
MOLECULAR METHODS IN ANTIMICROBIAL RESISTANCE OF MASTITIS
PATHOGENS
Linda L. Tikofsky, Ruth N. Zadoks, and Irene Loch
Department of Food Science and Quality Milk Production Services
Cornell University, Ithaca, NY
Introduction
Bovine mastitis is the single most common cause for antibiotic use on dairy farms. Antibiotics
are used for treatment of clinical and subclinical mastitis during lactation and at dry-off. In the
United States, treatment of all quarters of all cows at dry off with a broad-spectrum antibiotic,
regardless of infection status is recommended.
Although the FDA has restricted the use of many drugs previously used in dairy cattle, many
others are still available from veterinarians, feed stores, dairy suppliers, and the internet. Data
from a survey performed by the Animal Health Institute in 1998 found that approximately 17 of
the 50 million pounds of antibiotics produced in the US each year are used in animals2.
Administration of these drugs is often performed by people unfamiliar with basic principles of
pharmacology and therapeutic regimens and may be guided by the concept “If some is good,
more is probably better” rather than by evidence based recommendations. In practice, this can
result in undesirable treamtent choices, such as “If drug A isn’t having an effect after a day,
maybe I should switch to drug B, and if that doesn’t work within a day, what about drug C, even
though I’m really not supposed to use it in cows? Or maybe a combination of drugs?”.
Since it is known that selective pressure from antibiotics can influence the development and
transfer of antimicrobial resistance, there is concern that antibiotic use in animals may create a
reservoir of resistant bacteria or resistance genes that can be transferred to humans via food
products, direct human-animal contact or farm effluents. Current scientific evidence does not
support a widespread, emerging resistance to antibiotics among mastitis pathogens12. However,
information on the relationship between antibiotic use patterns and their influence on
antimicrobial resistance on dairy farms is limited and continued monitoring is needed.
Here at Quality Milk Production Services (QMPS) we have been exploring the question of
antimicrobial susceptibility and resistance in mastitis bacteria for years. Questions we have
attempted to answer through various studies include the following:
•
•
•
•
Does antimicrobial resistance exist in the mastitis bacteria isolated from New York dairy
herds and is it changing over time?
What happens to antimicrobial susceptibility in herds that don’t use antibiotics?
How does antimicrobial use influence the development or acquisition of antimicrobial
resistance by bacteria on the dairy farm?
What resistance mechanisms exist in streptococci and staphylococci isolated from bovine
milk samples?
In this contribution, answers to the above questions will be discussed, and new questions will be
raised, many of which we hope to address with techniques available to us in the QMPS’ new
Molecular Laboratory.
- 20 -
Does antimicrobial resistance exist in the mastitis bacteria isolated from New York dairy herds and is it
changing over time?
QMPS exists to address issues of udder health and milk quality for the dairy farmers of New
York and surrounding states. Over 200,000 milk samples are submitted to the laboratories of
QMPS for bacterial culture each year. Figure 1 depicts the changing prevalence of various
mastitis causing bacteria cultured from milk samples taken at herd surveys over the past ten years
at QMPS.
Culture results for cow and quarter milk samples - central QMPS laboratory
20.0%
S. agalactiae
18.0%
Strep. spp.
S. aureus
16.0%
E. coli
Klebsiella
% of samples
14.0%
12.0%
10.0%
8.0%
6.0%
4.0%
2.0%
er
ag
e
Av
er
ag
e
20
02
Av
Av
er
ag
e
20
01
Av
er
ag
e
20
00
19
99
Av
er
ag
e
er
ag
e
19
98
Av
19
97
Av
er
ag
e
er
ag
e
19
96
Av
Av
er
ag
e
19
95
Av
er
ag
e
19
94
19
93
19
92
Av
er
ag
e
0.0%
Figure 1: Culture results from quarter and composite milk samples taken at QMPS herd surveys over
the past decade.
Results show a decrease in prevalence of Streptococcus agalactiae, relatively stable prevalence of
Staph. aureus, especially in the last six years, and an increasing prevalence of cultures positive for
Streptococcus spp. The number of coliform-positive samples in this graph is low, because coliforms
are mostly found in clinical mastitis and Figure 1 represents milk samples taken at herd surveys,
most of which are not from cows with clinical mastitis.
In addition to data on prevalence of bacteria, data on susceptibility patterns of bacterial isolates
are available. In contrast to the prevalence data that were summarized in Figure 1, the
susceptibility data come from samples that were submitted by veterinarians throughout New
York State. This population represents mostly clinical mastitis samples. The data base covers
approximately 3,400 isolates tested by QMPS between 1985 and 2000 by means of the KirbyBauer agar disk diffusion method. These tests are performed and interpreted according to
NCCLS standards11. Zones of inhibition (lack of growth) of the bacteria are measured around
various antibiotic impregnated disks. Based on the diameters of the inhibition zones, bacteria are
classified as resistant, intermediately resistant or susceptible. For statistical analysis of these
records, two categories were used: susceptible and intermediate/resistant. Statistical analysis was
done using chi-square analysis, and logistic regression to evaluate changes in antibiotic
susceptibility through the years.
Between 1985 and 2000, there was a significant decrease in the susceptibility of Streptococcus spp.
to ampicillin, cloxacillin, penicillin, erythromycin, pirlimycin, and tetracycline. Erythromycin
- 21 -
and pirlimycin are available as lactating cow preparations. Tetracycline is used as an intravenous
antibiotic, as prophylactic in milk replacers and may also (but rarely and off-label) be used as an
intramammary infusion for dry cows. Susceptibility of Streptococcus spp. to both amoxicillin and
cephalothin remained stable at 89% and 98%, respectively.
Susceptibility of Staph aureus
Susceptibility of Strep spp.
100
% susceptible
% susceptible
100
80
60
ampcillin
40
penicillin
20
80
60
amp
40
pen
20
0
0
1
2
6
7
8
1
9 10 11 12 13 14
2
6
7
8
9
10 11 12 13 14
Years since 1985
Years since 1985
Figure 2. Examples of changes in antimicrobial susceptibility results to ampicillin (amp) and penicillin
(pen) observed for Streptococcus spp. and Staphylococcus aureus.
The trend for Staph. aureus is very different than the trend for Streptococcus spp. Where
Streptococcus spp. showed a decrease in susceptibility, Staph. aureus showed significant increases
in susceptibility to both ampicillin and penicillin (Figure 2). Susceptibility to amoxicillin (94%)
and cephalothin (98%) remained stable. Susceptibility to cloxacillin appeared to decrease but the
trend was not significant. Staph. aureus susceptibility to erythromycin, pirlimycin, and
tetracycline did not change significantly over time either.
These analyses were performed for all major mastitis bacteria (Staph aureus, Streptococcus
spp., Staphylococcus spp., E.coli, and Klebsiella spp.) and results are summarized in Table 1.
Similar results have been found in other mastitis research laboratories5,9.
Table 1: Changes in antimicrobial susceptibility over time (proportion susceptible) for
selected milk isolates cultured at QMPS, 1985 to 1999.
Antibiotic
Ampicillin
Amoxicillin
Cephalosporin
(Cl)oxacillin
Erythromycin
Gentamycin
Penicillin
Pencillin-novobiocin
Pirlimycin
Spectinomycin
Streptomycin
Sulfathiazole
TMP- sulfa
Staph. aureus
Strep spp.
E.coli
Klebsiella
I*
NC
NC
NC
NC
I*
NC
NC
NC
-
D*
NC
NC
D*
D*
D*
D*
D*
NC
-
D*
D*
NC
D*
D*
D*
NC
NC
NC
NC
D*
NC
D*
NC
Tetracycline
NC
D*
NC
D*
*p < 0.05
I = increased
D = decreased
- 22 -
NC = no change
In 1975, Davidson performed a similar analysis that covered 10 years of QMPS data on
antimicrobial resistance3. The contrast between the 1975 and 1999 data, a 25 year span, is
striking. Davidson found that 95% of Streptococcus spp. test were susceptible to ampicillin in 1975
while our data indicates as little as 26% susceptibility of Streptococcus spp. in 1999. By contrast,
only 49% of Staph. aureus isolates tested in 1975 were susceptible to ampicillin; in 1999, the
overall susceptibility to ampicillin was 79%. These changes in susceptibility may reflect changes
in mastitis treatments used over the years. Whatever the underlying cause for the observed
changes may be, the data suggest that long-term trends in antimicrobial resistance may be
different from trends measured over a limited number of years.
The apparent decline in susceptibility of Streptococcus spp. to commonly used antibiotics deserves
further study. The term “Streptococcus spp.” refers to a group of species, and the actual species
within this group may differ from each other in prevalence over the years and in antimicrobial
susceptibility. For example, Strep. uberis has been found to be less susceptible to erythromycin,
tetracycline, and streptomycin than Streptococcus dysgalactiae and Strep. uberis is currently the most
common Streptococcus found in NYS bulk tank milk17.
As a whole, QMPS data that were collected over the years show that the development of bacterial
resistance to antibiotics is of concern and warrants careful monitoring, and that results that are
averaged across years or bacterial species may not convey sufficient information on changes in
susceptibility patterns.
What happens to antimicrobial susceptibility in herds that don’t use antibiotics?
Organic livestock production practices are distinguished by the limited use of synthetic
medications including antibiotics. In 2002, the USDA established national standards on organic
agriculture production and handling. Use of non-therapeutic antibiotics and growth promoters is
prohibited under USDA organic livestock production standards, unless animal welfare is
compromised15. As a result antibiotic use on organic dairy farms is rare or infrequent, in contrast
to the situation on most conventional farms.
In 2001, a cross-sectional study of antibiotic susceptibility patterns for Staph. aureus isolated from
bovine milk samples from organic and conventional dairy herds in New York and Vermont was
performed. The antimicrobial susceptibility patterns for 144 isolates from 22 organic herds and
117 isolates from 16 conventional farms were compared.
Antibiotic susceptibility testing was performed as described for isolates from clinical samples.
Antibiotics were chosen based on their activity against Gram positive cocci and included
ampicillin, cephalothin, erythromycin, novobiocin, oxacillin, penicillin, penicillin-novobiocin,
pirlimycin, tetracycline and vancomycin. Many of these antibiotics are routinely employed in
dairy herd health practices. Further details on methods and statistical analyses have been
described previously14.
Using a categorical comparison (susceptible vs. resistant), percent susceptible for cephalothin,
oxacillin, novobiocin, penicillin-novobiocin, and pirlimycin approached 100% and no differences
were found between organic and conventional isolates. For the remaining antibiotics,
conventional herds had fewer isolates in the susceptible range than organic herds: ampicillin
(61.5% vs. 80.5%), penicillin (65.8% vs. 79.9%) and tetracycline (87.2% vs. 99.3%). Percent
susceptible for erythromycin did not differ significantly and was low for conventional as well as
organic herds (49.5% vs. 55.5%). Results are summarized in Table 2.
- 23 -
Table 2. Comparison of susceptible and resistant isolates by category for organic and
conventional herds.
Conventional
n = 117
Susceptible
Resistant
Antibiotic
Ampicillin
Cephalothin
Oxacillin
45
116
28
p = 0.0007
117
0
144
0
No difference
117
0
144
0
No difference
58
59
80
64
No difference
116
1
144
0
No difference
77
40
115
29
p = 0.0106
117
0
144
0
No difference
117
0
144
0
No difference
102
15
143
1
p = .00003
Penicillin
Penicillinnovobiocin
Pirlimycin
Tetracycline
Significance
72
Erythromycin
Novobiocin
Organic
n = 144
Susceptible
Resistant
When results were compared on a continuous scale, that is on the basis of zone of growth
inhibition in millimeters, significant differences between organic and conventional herds were
observed for ampicillin, cephalothin, oxacillin, penicillin, penicillin-novobiocin, pirlimycin, and
tetracycline. Most conventional and organic isolates fell within the ‘susceptible’ category, but
within that category the distribution of zone diameters for isolates from the conventional herds is
shifted to the left (smaller zone diameters) compared to that of the organic herds (Figures 3 and
4), implying that Staph. aureus isolates from conventional herds exhibit decreased antibiotic
susceptibility when compared to those from organic herds.
60
50
N
40
Conventional
30
Organic
20
10
27
25
23
21
19
17
15
13
11
9
0
Zone diameter (mm)
Figure 3. Kirby-Bauer zone diameters (mm) for pirlimycin in Staphylococcus aureus from organic and
conventional herds. Arrow indicates breakpoint for susceptibility.
- 24 -
35
30
25
conventional
15
organic
N
20
10
5
33
31
29
27
25
23
21
19
17
15
13
10
0
Zone diameter (mm)
Figure 4. Kirby-Bauer zone diameters (mm) for oxacillin in Staphylococcus aureus from organic and
conventional herds. Arrow indicates breakpoint for susceptibility.
In this study, most isolates tested expressed antibiotic susceptibilities well within the ‘sensitive’
category. Overall this is good news since the antibiotics currently being used do not appear to be
putting overwhelming pressure on mastitis bacteria and creating resistance. However, the subtle
differences in ‘degree of susceptibility’ warrant further study which can be accomplished through
the implementation of molecular diagnostics. Several hypotheses can be investigated:
•
•
•
Are different strains of Staph. aureus populating organic and conventional dairy herds?
Are the resistance genes present in Staph aureus from organic and conventional herds but
does antibiotic use ‘turn them on’?
Are the strains of Staph aureus similar on both organic and conventional herds but does
antibiotic use encourage the acquisition of resistance genes from other farm bacteria?
QMPS is prepared to answer these questions. All Staph. aureus isolates from this study have been
preserved on Microbeads and frozen at -80 0C for molecular typing in the future. Strain
differences within and between herds and routes of pathogen transmission (cow-to-cow
transmission or infection from multiple environmental sources) can be evaluated. This will
contribute to a better understanding of the impact of antimicrobial usage on antimicrobial
sensitivity, and can lead to changes in management to prevent infection in the future.
How does antimicrobial use influence the development or acquisition of antimicrobial resistance by
bacteria on the dairy farm?
Cross-sectional comparisons of bacterial isolates between herds with no antibiotic usage and
herds with routine antibiotic usages showed subtle but significant differences. To gain more
insight into mechanisms that could underlie such differences, a longitudinal study was performed
in which isolates were compared before (“no antibiotic usage”) and after (“routine antibiotic
usage”) dry cow therapy with intramammary antibiotics. Details of the field study, that was
designed as a Staph. aureus treatment trial, have been published4. In this section, we will focus on
analysis of the antimicrobial susceptibility of the Staph. aureus isolates.
Briefly, approximately 300 different isolates of Staph.aureus from seventy-five dairy herds in
Canada were derived from a year-long study of the efficacy of tilmicosin as a dry cow treatment.
Cows infected with Staph. aureus in at least one quarter were randomly assigned to be treated
- 25 -
with either a beta-lactam antibiotic, 500 mg benzathine cloxacillin (DryClox, Ayerst Laboratory,
Guelph, Ontario, Canada) or with 1500 mg of tilmicosin phosphate, a broad spectrum
macrolide antibiotic (supplied by Provel, Division of Eli Lilly Canada, Inc, Guelph, Ontario,
Canada). For all Staph. aureus isolates, minimum inhibitory concentrations (MIC) for multiple
antibiotics were determined by broth microdilution (Sensititre, Westlake, Ohio). Data were
analyzed in a manner similar to the comparison of isolates from organic and conventional farms.
Sixty-four pairs of isolates (before and after treatment) from the same quarter of the same cow
were available for additional study. Twenty-six of the pairs showed a shift in MIC values for one
of the Macrolide-Lincosamide-Streptogramin (MLS) antibiotics before and after treatment.
Twelve of the pairs exhibiting the shift, representing eight cows from five herds were selected for
molecular typing. Strain typing was performed with restriction enzyme EcoRI at Cornell
University’s Laboratory for Molecular Typing using the RiboPrinter® Microbial
Characterization (Qualicon, Wilmington, DE USA).
Isolates from cows receiving treatment with tilmicosin exhibited statistically significant changes
in resistance patterns for the MLS antibiotics (tilmicosin, erythromycin, pirlimycin) and
tetracycline (Figure 5). No differences were observed for penicillin, oxacillin, ceftiofur,
sulfadimethoxine, penicillin-novobiocin, and cephalothin. Significant increases in the proportion
of resistant isolates after the dry period were likewise observed for tilmicosin, erythromycin, and
pirlimycin. No significant differences were observed in either MIC patterns or proportion
resistant for Staph. aureus isolates from cows receiving the cloxacillin treatment.
Cephalothin
Tetracycline
After Calving
Penicillin/Novobiocin
Before Dry Off
Pirlimycin
Erythromycin
Ceftiofur
Tilmicosin
Oxacillin
Penicillin
0
2
4
6
8
10
12
Percent of Isolates Resistant
Figure 5: Percent of Staphylococcus aureus isolates resistant to specified antibiotics before (cross-hatched
bars) and after (black bars) treatment with tilmicosin at dry-off
From the twelve pairs of isolates submitted for molecular characterization, five ribotypes (169-5,
1104-1, 1106-3, 1106-1, 1106-7) were identified (Figure 6). Ribotype 169-5 was present in seven
cows from five herds. Each of the other ribotypes was present in one cow per herd in four
separate herds. In herd 52, there were two ribotypes present; herd 53 had one cow with the same
strain in two quarters, and herd 62 had one strain infecting three separate cows. In all cases,
- 26 -
isolates before and after treatment were the same ribotype and were thus likely to represent
persistent infections rather than acquisition of a new infection.
Molecular Typing
12-25-RH-2
12-25-RH-5
26-45-RH-3
26-45-RH-4
55-45-RH-3
55-45-RH-5
85-45-RH-3
85-45-RH-6
1106-1
1106-3
169-5
1106-7
Figure 6. Examples of riboprint patterns for four pairs of Staph. aureus isolates collected before and
after treatment with Tilmicoson at dry off. Isolate identification is show on the right hand side and
strains (1106-1, 1106-3, 169-5, 1106-7) on the left hand side.
Members of the MLS family of antibiotics are widely used in both animal and human medicine.
The primary resistance mechanism, coded for by various erm genes, is ribosomal methylation,
which decreases binding of the macrolide. Resistance in staphylococci is the result of expression
of primarily the ermA or ermC genes genes6,8. The majority of erm genes identified in bovine
Staph aureus are ermA1. Coagulase negative staphylococci are a large reservoir of both ermA and
ermC, which are present on mobile genetic elements (transposons and plasmids, respectively).
Expression of erm genes generally results in high levels of resistance to multiple members of the
macrolide family.
Efflux pump activity is a another macrolide resistance mechanism that has been identified in
streptococci and staphylococci and that results from the expression of mefA/E and msrA,
respectively. Efflux pumps may confer a low, medium, or high resistance to macrolide antibiotics
and resistance levels may differ between different members of the MLS group and can also be
induced by exposure to antibiotics16.
In this study, strains from the same quarter of the same cow were indistinguishable before and
after treatment. The change in MIC values after exposure to tilmicosin but not after exposure to
cloxacillin suggests induction by the antibiotic even after a single exposure. This increase in
resistance may be due to acquisition of resistance genes from the environment, e.g. coagulase
negative staphylococci, or up-regulation of an existing mechanism, such as an efflux pump.
Further molecular investigation into the presence resistance genes in these isolates is planned.
What resistance mechanisms exist in streptococci isolated from bovine milk samples?
In a recent study, we explored which genes encode macrolide resistance in streptococcal isolates
from bovine milk. Strep. uberis and Strep. dysgalactiae isolates from composite milk and bulk tank
samples from herds in the Southwestern USA were tested for the presence of macrolide resistance
genes belonging to the classses of ribosomal methylaseas (ermA/TR, ermB, ermC) and efflux
- 27 -
pumps (mefA/E). The isolates in this study originated from herds that used macrolide antibiotics
for mastitis control. All streptococcal isolates tested positive for the presence of the ermB gene by
means of PCR, while ermA/TR, ermC, and mefA/E were not detected. PCR amplicons were
subsequently sequenced and the streptococcal isolates were further characterized by automated
ribotyping with PvuII. Results are summarized in Figure 7, which shows a phylogenetic tree of
ermB alleles (a, b and c) and includes information on herd of origin (numbers 1-6), ribotype
(letters A-N), species (SUB = Strep. uberis; SDY = Strep. dysgalactiae), and isolate identification
(starting with Z3). Alleles “a” and “b” were both associated with very high phenotypic resistance
to erythromycin and pirlimycin (MIC ≥ 64 for both macrolides), while allele “c” was associated
with slightly lower resistance (MIC ≥ 64 for erythromycin and ≥ 16 for pirlimycin.)
1 - A - SUB - z3070
1 - C - SUB - z3075
4 - K - SUB - z3069
4 - K - SUB - z3093
4 - K - SUB - z3095
Alelle ‘a’
4 - K - SUB - z3097
Very high resistance
5 - M - SDY - z3088
5 - M - SDY - z3099
5 - M - SDY - z3100
5 - M - SDY - z3101
2 - F - SUB - z3074
2 - F - SUB - z3076
6 - N - SUB - z3096
Allele ‘b’
Very high resistance
6 - N - SUB - z3102
2 - E - SUB - z3072
Allele ‘c’
4 - L - SUB - z3094
High resistance
Figure 7. Relatedness of ermB alleles from Streptococcus uberis (SUB) and Streptococcus dysgalactiae (SDY)
from bovine milk. Numbers (1 through 6) indicate herds. Letters (A through N) indicate strains based
on ribotyping. Isolates are identified by numbers starting with “z3”. Isolates with allele “a” or “b”
had MIC ≥ 64 for erythromycin and pirlimycin. Isolates with allele “c” had MIC ≥ 64 for erythromycin
and ≥ 16 for pirlimycin.
Figure 7 shows that: (i) phenotypic resistance profiles are associated with specific genotypic
profiles (allele “c” encodes lower resistance than allele “a” or “b”; (ii) S. uberis isolates from
different herds and belonging to different strains may carry the same ermB allele (e.g. strains F
and N from herds 2 and 6); and (iii) within a herd, multiple alleles of the ermB gene may occur
(herd 2, strains E and F, alleles “b” and “c”; herd 4, strains L and K, alleles “a” and “c”).
Furthermore, it can be seen that the same ermB allele was found in Strep. uberis and in Strep.
dysgalactiae. The fact that the same allele for a resistance gene can be found in multiple
streptococcal strains and species suggests that horizontal transfer of the resistance gene may have
taken place. To assess whether other bacterial species might carry the same resistance alleles, data
from our study were compared to DNA-sequence data that are available via de World Wide Web
(http://www.ncbi.nlm.nih.gov/) (Figure 8).
- 28 -
Enterococcus hirae - source unknown
Arcanobacterium.pyogenes - cow
Bacillus cereus – soil
Allele ‘b’
Clostridium perfingens – source unknown
Strep. gallolyticus –pigeon, human
Strep. uberis - z3102 - cow
Lactobacillus reuteri – source unknown
Allele ‘a’
Strep. uberis - z3097 - cow
Enterococcus faecium – source unknown
Strep. uberis –z3072 - cow
Allele ‘c’
Staph. intermedius - dog
Strep. pneumoniae - human
Strep. agalactiae – cow
Strep. agalactiae – cow
Figure 8. Genetic relatedness of ermB alleles from Streptococcus uberis and other bacteria, originating
from bovine milk and from a variety of host species and sources, respectively.
Comparison of ermB sequence data from Strep. uberis with ermB sequence data from other
bacterial species showed that the same alleles can be found in streptococci that infect other host
species, and in other genera of bacteria, including bacteria isolated from soil. This reinforces the
idea that horizontal gene transfer may occur, and adds the possibility of interspecies gene
transfer7 which could potentially occur between different bacteria carried by the same cow or in
the dairy farm environment.
Summary and Conclusion
Data collected by QMPS over the past decades show that the population of mastitis bacteria
changes over time. This is true for the occurrence of bacterial species and for the occurrence of
antimicrobial resistance within a bacterial species. For some bacterial species and antimicrobials,
susceptibility increased over the years, while it decreased for others. Staph. aureus isolates from
milk samples from organic dairy farms show higher levels of susceptibility to antibiotics than
Staph. aureus isolates from conventional farms, although most isolates from both types of farms
would be considered susceptible under current guidelines. While dry-cow treatment with betalactam antibiotics did not cause a shift in antimicrobial susceptibility among Staph. aureus isolates,
treatment with tilmicosin, a member of the MLS group of antibiotics, causes a significant loss of
susceptibility. Resistance to MLS antibiotics also occurs in streptococci from bovine milk, where
it is encoded by alleles of the ermB gene. The same alleles are also found in other bacterial
species, including bacteria from humans and soil. This suggests that horizontal transfer of
resistance genes has occurred, and implies that such transfer may continue to happen, especially
if use of antimicrobials on dairy farms creates an environment that favors the survival of resistant
bacteria. Such transfer may lead to uptake of resistance genes by mastitis pathogens so that
treatment of cows becomes unsuccessful, or it may lead to transfer of resistance genes by mastitis
pathogens to other bacteria, including those that may reach and/or affect humans. Currently,
antimicrobial resistance is not a major problem in mastitis pathogens or the dairy industry, but
antimicrobial use is associated with differences and changes in antimicrobial susceptibility,
- 29 -
specifically susceptibility of mastitis-causing streptococci and staphylococci. Hence, monitoring
of antimicrobial resistance and use of management and treatment strategies that minimize the
risk of development of antimicrobial resistance continue to be necessary.
References
1. Aarestrup FM, Agerso Y, Ahrens P, Jørgensen JC, Madsen M, Jensen LB. 2000.
Antimicrobial susceptibility and presence of resistance genes in staphylococci from poultry.
Vet Microbiol. 74: 353-364.
2. Animal Health Institute. Survey on Antibiotic Use in Humans and Animals in 1998. Feb.
2000. (www. AHI.com)
3. Davidson JN. Antibiotic Resistance Patterns of Bovine Mastitis Pathogens. Proc. 19th
National Mastitis Council Meeting. 1980. 181-185.
4. Dingwell RM, Leslie KE, Duffield TF, Schukken YH, DesCoteaux, Keefe GP, Kelton DF,
Lissemore KD, Shewfelt W, Dick P, Bagg R. 2003. Efficacy of intramammary tilmicosin and
risk factors for cure of Staphylococcus aureus infection in the dry period. J Dairy Sci. 86:159-68.
5. Erskine RJ, Walker RD, Bolin CA., Bartlett PC, White DG. 2002. Trends in Antibacterial
Susceptibility of Mastitis Pathogens During a Seven-Year Period. J Dairy Sci. 85:1111-1118.
6. Khan SA, Nawasz MS, Khan AA, Cerniglia CE. 2000. Transfer of erythromycin resistance
from poultry to human clinical strains of Staphylococcus aureus. J. Clin Microbiol. 38:1832
7. Kimpe A, Descostere A, Martel A, Devriese A, Haesebrouck F. 2003. Phenotypic and
genetic characterization of resistance against macrolides and lincosamides in Streptococcus
gallolyticus strains isolated from pigeons and humans. Microb Drug Resist. 9 Suppl1:S35-S38.
8. Lina G, Quaglia A, Reverdy M, LeClercq R, VanDenNesch F, Etienne J. 1999. Distribution
of genes encoding resistance to macrolides, lincosamides and streptogramins among
staphylococci. Antimicrob. Agents Chemother. 43:1062-1066.
9. Makovec JA and Ruegg PL. 2003. Results of milk samples submitted for microbiological
examination in Wisconsin from 1994 to 2001. J Dairy Sci. 86:3466-3472.
10. Martel A, Meulenaere V, Devriese LS, Decostere A, and Haesebrouck F. 2003. Macrolide
and lincosamide resistance in the gram-positive nasal and tonsillar flora of pigs. Microb. Drug
Resist. 9:293-297.
11. NCCLS Performance Standards of Antimicrobial Disk Susceptibility Tests. 6th edition. Vol.
17. Jan. 1997.
12. National Mastitis Council Research Committee Report. Bovine Mastitis Pathogens and
Trends in Resistance to Antibacterial Drugs. http://www.nmconline.org/docs/ResPaper.pdf
13. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. 1996. Detection of erythromycinresistant determinants by PCR. Antimicrob.Agents Chemother. 40:2562-2566.
14. Tikofsky LL, Barlow JW, Santisteban. C, Schukken YH. 2003. A Comparison of
Antimicrobial Susceptibility Patterns for Staphylococcus aureus from Conventional and Organic
Farms. Microb. Drug Resist. 9 Suppl. 1:S39-45.
15. USDA. 2002. USDA National Standards on Organic Agricultural Production and handling.
(http://www.ams.usda.gov/nop/nop2000/nop2/finalrulepages/finalrulemap.htm;
last
accessed October 7, 2002)
16. Van Bambeke F, Balzi E, Tulkens PM. 2000. Antibiotic efflux pumps. Biochem Pharm.
60:457-470.
17. Zadoks RN, González RN, Boor KJ, Schukken YH. 2004. Mastitis-Causing Streptococci
are Important Contributors to Bacterial Counts in Raw Bulk Tank Milk. Journal of Food
Protection. In press.
- 30 -
MOLECULAR METHODS ON DAIRY FARMS: CASE STUDIES
Ruth N. Zadoks
Department of Food Science and Quality Milk Production Services
Cornell University, Ithaca, NY
Introduction
How does use of molecular methods contribute to improvement of udder health and milk quality
on dairy farms? Why would a farmer or a veterinarian consider their use? Why pay for it?
Because it provides information that regular bacterial culture can’t provide. Molecular typing,
also called strain typing or DNA-fingerprinting, gives a level of detail and insight that is not
available with traditional culture methods. It provides answers to riddles that can’t be solved
otherwise. It provides the data to convince people of things they are reluctant to believe because it
goes against the common knowledge of the day. By “molecular methods” we mean methods to
identify and characterize bacteria based on detection of their DNA. The fact that molecular
methods are DNA-based sets them apart from bacterial culture methods, where it is the growth of
bacteria that is observed. Molecular methods can be used to detect the presence of bacterial
species (e.g. E. coli, Klebsiella, Staphylococcus aureus and other staphylococci, Streptococcus agalactiae
and other streptococci, etc.), bacterial strains (groups of bacteria within a species that have a
specific characteristic in common, compare to breeds within animal species), or specific genes
(e.g. genes for resistance to antibiotics). In this contribution, examples of on-farm applications of
molecular methods will be given. All examples are from work that has been done with real-world
dairy farms to address their mastitis or milk quality concerns. Questions that we answered with
molecular methods include:
•
•
•
•
I have had a closed and Streptococcus agalactiae-free herd for years and now the lab found
Strep. agalactiae in my milk. How can that be?
What is the cause of the high bacteria count in my bulk tank milk?
Can I get rid of environmental streptococci?
My milking hygiene is perfect, and yet my bulk tank somatic cell count is too high. What
else can I do to lower the SCC so that I can get a quality premium?
For questions about any of these examples, to discuss whether your milk quality questions could
be answered with molecular methods, or to submit samples, please contact Ruth Zadoks at
QMPS, phone (607) 255-8202, or by e-mail: [email protected].
How can milk from a Streptococcus agalactiae-free herd be positive for Strep. ag.?
Streptococcus agalactiae is a contagious pathogen of dairy cows. It spreads very easily between
cows, mostly during milking, and causes high somatic cell counts (SCC). Because the mastitis
that is caused by Strep. agalactiae is often subclinical (invisible), it may go unnoticed until there are
so many cows with high SCC that the bulk tank SCC rises and even exceeds PMO limits
(750,000 cells/ml). In a Strep. agalactiae eradication program, it is extremely important to detect
all infected cows. To show how dangerous and contagious Strep. agalactiae can be, here is an
example: QMPS worked with a herd of approximately 700 cows and bulk tank SCC around
300,000 cells/m. The herd was expanded with 140 purchased animals, and within two months
the bulk tank SCC shot up to 700,000 cells/ml. Whole herd milk culture revealed that 68 cows
- 31 -
were infected with Strep. agalactiae, which had probably been brought into the herd by the new
cows. It took 18 months to control the disease, or rather, to get to the point where the disease
appeared to be under control. With only 3 cases left, control measures started to lapse. Within
five months, the number of infected cows was back up to 26 and bulk tank SCC was on the rise
again.
Strep. agalactiae is very common in people, although it is mostly known by a different
name: group B streptococcus or GBS. A large proportion of people carry GBS or Strep. agalactiae
in their gastrointestinal or urogenital tract10. Both men and women can be carriers, usually
without any signs or symptoms. The danger lies mostly in infection of children at birth (for more
information see http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5111a1.htm). GBS is also
considered an emerging pathogen in immunocompromised adults and the elderly. Dogs and cats
can be carriers of Strep. agalactiae14, but this not very common.
Do cows and people have the same Strep. agalactiae? This question has been studied by a
number of research groups3,11, including Cornell University's Food Safety Laboratory (FSL) in
collaboration with QMPS. In 2002, 52 human and 52 bovine Strep. agalactiae isolates were
collected in New York State. For each of these isolates, strain typing was performed by several
methods, including DNA sequencing and ribotyping. Based on these methods, it could be shown
that people and cows each have their own strains of Strep. agalactiae (Figure 1; based on 13).
Although one clone of S. agalactiae that may affect people has arisen from a bovine ancestor at
some point in the past1, there is no evidence that S. agalactiae from bovine milk currently presents
a risk to human health.
FSL S3-027
FSL S3-048
(116-533-S-8)
FSL S3-043
(116-533-S-7)
FSL S3-034
(116-533-S-5)
FSL S3-030
(116-522-S-4)
FSL S3-062
(116-549-S-7)
FSL S3-026
FSL C1-487
(116-522-S-1)
FSL C1-496
(116-522-S-2)
FSL S3-012
(116-522-S-5)
FSL S3-018
(116-522-S-5)
FSL S3-008
(116-522-S-5)
FSL F2-338
(116-522-S-4)
FSL F2-347
(116-549-S-1)
FSL S3-009
(116-522-S-5)
Figure 1. Molecular typing results (DNA-sequencing of hyaluronidase gene) for
Streptococcus agalactiae: Evolutionary tree showing the relatedness of isolates from cows
and humans13. Bovine and human strains of Strep. agalactiae form separate groups.
So what about Strep. agalactiae in closed dairy herds? How can a bulk tank sample test positive for
Strep. agalactiae when all the cows are Strep. agalactiae-free according to the herd-survey? And how
can a cow test positive in a closed herd that has been free for years? And how can it be only one
- 32 -
cow? Does that imply it is the very start of a major mastitis outbreak? Based on reports that
milkers may infect their animals with Staph. aureus2, we postulated that people might infect their
cows with Strep. agalactiae. After all, many healthy people are carriers of Strep. agalactiae. If a
human were the source of Strep. agalactiae that could also explain how a bulk tank can test
positive while none of the cows does. Ribotyping confirmed our suspicions: in a herd where only
the bulk tank tested positive, the Strep. agalactiae that was identified belonged to a human type. In
three herds where only one cow tested positive, each Strep. agalactiae case was caused by a human
type. The human types of Strep. agalactiae are not specifically adapted to survival in the cow. They
may cause clinical mastitis, which is unusual for Strep. agalactiae in cows, and the infections may
not last long, again unusual for Strep. agalactiae in cows8. Additional work in our laboratories has
shown that human and bovine Strep. agalactiae also differ in their resistance to antimicrobials,
with higher resistance levels in human than in bovine isolates4. Dogs and cats, by the way, also
carry the human rather than the bovine strains14.
In summary: there are exceptions to the Strep. agalactiae rules. If you find Strep. agalactiae
in a closed herd or in a herd with bulk tank SCC below 200,000 cells/ml, with only one cow
testing positive, possibly manifesting as a clinical mastitis, and resistant to antibiotics (mostly
tetracycline and macrolides), or maybe only in the bulk tank milk and not in any cows at all, that
Strep. agalactiae may belong to a human type. To be on the safe side, treatment of cows infected
with human strains is recommended. That will most likely be the end of it, and an outbreak of
mastitis is unlikely to occur.
What is the cause of the high bacteria count in my bulk tank milk?
Bacteria counts of bulk tank milk are monitored routinely. The PMO limit is 100,000 cfu/ml (cfu
= colony forming units), but ideally bacteria counts do not exceed 10,000. If they do, there may
be a problem with milking time hygiene, equipment cleaning, milk cooling or cow health5,7,12.
Sometimes, people look for the explanation in the field they are most familiar with. A
veterinarian may be more likely to focus on cows, while an equipment expert may be more likely
to focus on cleaning and cooling. Alternatively, people may try to “shift the blame” to the field
that is not their area of expertise, which contributes as little to a solution as focusing on the wrong
cause.
Collaborative work between FSL and QMPS has shown that high bulk tank counts are
most likely to be caused by Streptococci, specifically Strep. uberis, and coliform bacteria,
predominantly E. coli6. Both Strep. uberis and E. coli are considered to be environmental
pathogens, i.e. bacteria that are common in the environment of the cow. Because the bacteria are
common in the environment, their presence in bulk tank milk is often attributed to poor milking
time hygiene or poor equipment cleaning5,12. If environmental contamination was the source of a
high bulk tank bacterial count, the population in the bulk tank milk would be expected to reflect
the high heterogeneity of the environmental bacterial population. In other words: we would
expect a large variety of bacterial strains in the tank if the bacteria came from the environment. If
a cow has mastitis, she is usually infected with one strain of bacteria. Hence, if a cow with
mastitis were the source of a high bacteria count in bulk tank milk, one would expect to find the
strain from the cow to be the dominant strain in the tank.
To determine whether cows could be the source of high counts of streptococci or E. coli in
bulk tank milk, we used molecular methods 1) to explore the diversity of the bacterial population
in the bulk tank milk; and 2) to compare the strains from cows with mastitis to the strains found
in the bulk tank. Table 1 shows two examples of results for Streptococcus uberis, the most common
non-agalactiae streptococcus.
- 33 -
Table 1. Molecular typing results (ribotyping) for Streptococcus uberis from bulk tank
milk isolates and from all Strep. uberis infected cows in each herd.
Farm Source
A
E
Ribotype
Bulk Tank
116-795-5
Bulk Tank
116-795-5
Bulk Tank
116-795-5
Cow 1
116-795-5
Cow 2
116-793-3
Bulk Tank
116-788-6
Bulk Tank
116-788-6
Bulk Tank
116-788-6
Cow
116-788-6
RiboPrinterTM pattern
For five dairy herds, multiple Strep. uberis isolates were available from the bulk tank milk so that
the strain diversity in the bulk tank could be assessed. For all five herds, a dominant strain was
identified. For herd A, this was strain 116-795-5 and for herd E, this was strain 116-788-6. The
presence of a dominant strains shows that a point source was more likely to be the cause of high
bulk tank count than environmental contamination. The bulk tank milk sample had been taken
on the day that a whole herd survey was performed so that individual results were available for
all cows that had contributed to the bulk tank milk. All cows were tested for environmental
streptococci, and the species and strain of the mastitis-causing bacteria was determined. In each
of the five herds, a cow was shown to shed the strain that was found in the bulk tank milk. Thus,
cows with mastitis were the most likely source of high bacteria counts in those herds.
After seeing the results shown above for S. uberis, one of the QMPS veterinarians brought
in some client samples. A whole herd survey for the client’s herd had just been completed.
Among the results was a high bacteria count in the bulk tank milk, caused by E. coli, and one cow
that was infected with E. coli. The veterinarian and the farmer discussed the possibility of the cow
being the source of the bulk tank count. Although that seemed plausible, it went against
everything the farmer had always been told about high coliform counts in milk. Therefore, we
decided to use strain typing to show whether the bacteria in the bulk tank belonged to one or
multiple strains, and also whether the strain from the infected cow was found in the bulk tank.
Again, a dominant strain was found in the bulk tank, and the same strain was indeed found in the
cow (Figure 2). With those results, veterinarian and farmer were
now convinced that the cow had been the cause, and the bulk tank
bacterial count problem could be resolved by dealing with the cow,
without adjustments to milking procedures, equipment cleaning or
cooling installations.
BTM
Figure 2. Molecular typing results (RAPD-PCR) for three bulk
tank milk (BTM) isolates and one cow isolate (C) of E. coli
from a herd with high coliform counts. Left hand lane shows
molecular size marker. No other cows were infected with E.
coli in this herd.
- 34 -
C
Can I get rid of environmental streptococci?
So cows may be the source of bacteria in bulk tank milk. But where do the cows get their
bacteria? If the source of environmental bacteria could be identified, targeted intervention
measures might be possible, such as use of different bedding material. Some studies have
suggested that specific animals in the herd may be carriers of Strep. uberis in their gastro-intestinal
tract9. Fecal dispersal of the bacteria in the environment by such carrier animals may put the rest
of the herd at risk of mastitis. If such animals could be identified, environmental contamination
and subsequent environmental mastitis could potentially be prevented.
To find out whether specific cows or specific environmental sites could be pinned down
as the source of Strep. uberis on a dairy farm, we did a 10-month study in central New York. Every
month, we collected samples from feed (hay, haylage, grass/pasture greens), water (trough,
ponds, streams), lying areas (bedding from stalls or body imprints in pasture), and
gathering/traffic areas (doorway, around watering trough, under stand of shade providing trees).
In addition, fecal and milk samples were collected from cows at dry-off and at calving. Molecular
methods were used to determine whether Strep. uberis was present in environmental, fecal and
milk samples, and also to explore which strains were
present in the different sources. Strep. uberis was found
in 63% of environmental samples, 25% of fecal samples
and 4% of milk samples. Many environmental sites and
fecal samples harbored multiple different strains of
Strep. uberis (example shown in Figure 3).
Figure 3. Molecular typing results (ribotyping)
for Strep. uberis from a soil sample. Among
eight isolates from one sample, five strains
(indicated by RiboGroups on the left hand
side) were identified.
Close to 90% of soil samples, two-thirds of grazing matter, and 40% of water samples tested
positive for the presence of Strep. uberis. The strains of Strep. uberis that were found in
environmental samples did not differ from those in fecal samples or milk samples. Thus, any
environmental site could potentially be a source of Strep. uberis infection and there is no particular
site that could be targeted for preventive efforts. Targeting fecal shedders did not seem to be an
option either: many animals tested positive for Strep. uberis at some point in time, but very few
animals tested positive repeatedly, and persistent fecal shedders were not identified. For this herd,
promoting cow health and mastitis resistance through measures such as adequate nutrition and
selective breeding is a more promising road to mastitis prevention than elimination of potential
sources of infection.
What can I do to lower my bulk tank SCC?
A farmer in Vermont struggled to lower his bulk tank somatic cell count (BMSCC). The current
BMSCC level was around 300,000 cells/ml and the farmer felt he did everything he possibly
could to prevent transmission of mastitis pathogens during milking. Still, the target of lowering
BMSCC to 200,000 cells/ml and the associated quality premium seemed elusive. To add insult to
injury, one of his advisors insisted that something must be wrong with his milking time hygiene
for the BMSCC to remain that high.
- 35 -
Through a different advisor, the farmer heard about the use of molecular methods for milk
quality improvement. As shown in Figure 4, molecular methods can be used to determine
whether mastitis cases are caused by one strain that is spread from cow to cow as contagious
pathogen (contagious, dotted black circle), or by a variety of strains that come from the cow’s
environment (environmental, thin black ellipse)15.
18
16
Contagious
14
12
number of
infected quarters
10
8
6
Environmental4
2
17
15
19
21
23
25
27
farm visit
13
0
11
B A C
D F
G I
J L
M P
S. uberis strain
Q 1
9
7
5
3
Figure 4. Molecular typing results (RAPD) for Streptococcus uberis isolates from a dairy
herd. Samples were collected from all quarters of all cows at 3-weekly farm visit. Molecular
typing was performed for isolates from all udder quarters (strains identified by letters). For
each visit, the number of quarters infected with a specific strain was determined (vertical
axis). Adapted from 15.
To help the farmer solve his BMSCC problem, a combination of approaches was used: a herd
visit, inspection of DHIA data, culture of cow milk samples, and molecular methods. During the
herd visit, milking time hygiene seemed impeccable. Inspection of DHIA data on somatic cell
counts of individual cows showed that most cows were healthy, new infections were rare and
mostly occurred at calving, most cows with high SCC had chronic mastitis, and few cows cured
(Figure 5).
Routine culture results showed that environmental streptococci were the most common mastitis
pathogens. They were found in eight milk samples. Molecular methods (species-specific PCR)
showed that two isolates belonged to the species Strep. dysgalactiae and six isolates belonged to the
species Strep. uberis. Because two species were involved, at least two sources of infection had
played a role and not all infections were the result of transmission in the milking parlor.
- 36 -
Figure 5. DHIA data for
linear score of individual
cows. Horizontal and vertical
axis show previous and
current score (PLS and LS),
respectively. Dividing lines
indicate LS=4 and PLS = 4.
Values above 4 are considered
too high. Based on the
combination of PLS and LS,
cows can be categorized as
healthy (PLS and LS low),
new cases (PLS low, LS
high), chronic infections (PLS
and LS high) or cures (PLS
high, LS low).
New cases
Chronics
Healthy
Cures
Subsequently, molecular methods were used to differentiate Strep. uberis at the strain level.
Among six isolates, originating from six quarters of four cows, four strains were identified, with
one strain per cow (Figure 6). Clearly, with each cow having her own species or her own strain of
streptococcus, the infections were not due to cow-to-cow transmission during milking, but rather
to independent infections of each cow with bacteria from a variety of sources. Thus, herd
inspection, SCC data and molecular results all confirmed the herd owner’s notion that his
milking time hygiene was excellent. In this herd, room for improvement was to be found in
prevention of new infections around calving and in detection and treatment or culling of
chronically infected cows.
M 1 2
3 4
5 7 + + -
A B B C C D E F
1 2
3 4
5 7
+ + -
M
A B B C C D E F
I
II
III
IV
RF LF LF LH
Figure 6. Molecular typing (RAPD) of six Streptococcus uberis isolates (identified by numbers)
from four cows (indentified by roman numerals) with strain designation (letters A-F). Typing
performed in duplicate. Molecular markers (M), positive (+) and negative (-) controls are
included. RF = right front quarter, LF = left front quater, LH = left hind quarter.
- 37 -
Conclusion and future prospects
This paper covered a few examples of herd health and milk quality issues that were addressed
with the help of molecular methods. Many more applications are conceivable, including testing
of raw milk for presence of food borne pathogens before consumption, detection of potential fecal
carriage of Klebsiella by dairy cows resulting in contamination of bedding material that was
originally Klebsiella free, monitoring for presence of antimicrobial resistance genes, and
evaluation of the success of treatment with differentiation between non-cures and re-infections of
cured quarters. Molecular methods have been used with incredible success in research
laboratories for the past 15 years. Now that techniques are becoming increasingly user-friendly
and affordable, they are slowly starting to make their way into the diagnostic laboratory.
Although their routine implementation still faces challenges, for example in terms of cost
recovery and turn-around times, it is inevitable that in another few years, molecular methods will
be among the routine tools used in diagnostic laboratories, mastitis management, and the
promotion of udder health and milk quality.
References
1. Bisharat, N., D. W. Crook, J. Leigh, R. M. Harding, P. N. Ward, T. J. Coffey, M. C.
Maiden, T. Peto, and N. Jones. 2004. Hyperinvasive neonatal group B streptococcus has
arisen from a bovine ancestor. J. Clin. Microbiol. 42:2161-2167.
2. Devriese, L. A. and J. Hommez. 1975. Epidemiology of methicillin-resistant Staphylococcus
aureus in dairy herds. Res. Vet. Sci. 19:23-27.
3. Dmitriev, A., E. Shakleina, L. Tkacikova, I. Mikula, and A. Totolian. 2002. Genetic
heterogeneity of the pathogenic potentials of human and bovine group B streptococci. Folia
Microbiol. (Praha) 47:291-295.
4. Dogan, B., Y. H. Schukken, C. Santisteban, and K. J. Boor. 2004. Serotype distributions and
antimicrobial resistances of Streptococcus agalactiae isolates from bovine or human origin. In
preparation.
5. Farnsworth, R. J. 1993. Microbiologic examination of bulk tank milk. Vet. Clin. North Am.
Food Anim Pract. 9:469-474.
6. Hayes, M. C., R. D. Ralyea, S. C. Murphy, N. R. Carey, J. M. Scarlett, and K. J. Boor. 2001.
Identification and characterization of elevated microbial counts in bulk tank raw milk. J.
Dairy Sci. 84:292-298.
7. Jayarao, B. M. and D. R. Wolfgang. 2003. Bulk-tank milk analysis. A useful tool for
improving milk quality and herd udder health. Vet. Clin. North Am. Food Anim Pract.
19:75-92, vi.
8. Jensen, N. E. 1982. Experimental bovine group-B streptococcal mastitis induced by strains of
human and bovine origin. Nord. Vet. Med. 34:441-450.
9. Kruze, J. and A. J. Bramley. 1982. Sources of Streptococcus uberis in the dairy herd. II.
Evidence of colonization of the bovine intestine by Str. uberis. J. Dairy Res. 49:375-379.
- 38 -
10. Manning, S. D., P. Tallman, C. J. Baker, B. Gillespie, C. F. Marrs, and B. Foxman. 2002.
Determinants of co-colonization with group B streptococcus among heterosexual college
couples. Epidemiology 13:533-539.
11. Martinez, G., J. Harel, R. Higgins, S. Lacouture, D. Daignault, and M. Gottschalk. 2000.
Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly
amplified polymorphic DNA analysis. J. Clin. Microbiol. 38:71-78.
12. Saran, A. 1995. Disinfection in the dairy parlour. Rev. Sci. Tech. 14:207-224.
13. Sukhnanand, S., B. Dogan, M. O. Ayodele, R. N. Zadoks, M. P. J. Craver, N. B. Dumas, Y.
H. Schukken, K. J. Boor, and M. Wiedmann. 2004. Molecular subtyping and population
genetics of bovine and human Streptococcus agalactiae isolates. J. Clin. Microbiol. Manuscript
JCM00977-04.
14. Yildirim, A. O., C. Lammler, R. Weiss, and P. Kopp. 2002. Pheno- and genotypic properties
of streptococci of serological group B of canine and feline origin. FEMS Microbiol. Lett.
212:187-192.
15. Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H.
Schukken. 2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis
infections in dairy herds. Epidemiol. Infect. 130:335-349.
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MICROBIAL PATHOGENOMICS, A REALITY COMING THROUGH
Vivek Kapur
Department of Microbiology
University of Minnesota, St. Paul, MN
Microbial pathogenomics is an emerging discipline that is concerned with the application of
genomics based approaches to the study of biological processes associated with infectious agents
and the disease they cause.
The rapid progress in genomics technologies has provided an important tool for the study of
infectious diseases during the twenty-first century. These microbial pathogenomics investigations
have provided important insights on pathogen and host biology as well as on host-pathogen
interactions. For instance, the availability of the human genome sequence along with the
complete genomic sequences of nearly all of the major pathogens associated with disease in
humans has provided key missing insights on the basic mechanisms of infectious disease
pathogenesis (5, 7). In addition, the development of methods for detection of gene expression
profiles using microarays – tools for studying how large numbers of genes interact with each
other and how a cell’s regulatory networks control vast batteries of genes simultaneously, or for
high-throughput genotyping (such as SNP analysis), have added considerably to the repertoire in
the toolkit of the modern day infectious disease researcher. Finally, the rapidly emerging fields of
computational molecular biology and bioinformatics that together deal with the development and
application of computational tools and approaches for expanding the use of biological, medical,
and health-related data, including tools to acquire, store, organize, archive, analyze, or visualize
such data, have proved to be essential for the extraction of biological insights and knowledge
from the vast quantities of information generated during a “typical” microbial pathogenomics
experiment.
Recent research activities in my laboratory have focused on functional genomics and proteomics
applications relating to microbe-host interactions. Our group has participated in the complete
genome sequencing of various microbial pathogens (for e.g. 1, 6, 8, 16) as well as functional
genomics investigations for these agents including
Microbial Pathogen
Size (Mbp)
genome-scale studies of host-pathogen interactions
Brucella abortus
2.1
Cryptosporidium parvum
9.1
using custom and Affymterix-based microarrays
Lawsonia intracellularis
1.8
Mycobacterium paratuberculosis
4.8
and other sophisticated tools for pathogen and
Pasteurella multocida
2.2
host gene expression analysis (1-4,6,8-19). A list
Staphylococcus aureus RF122
2.4
Staphylococcus aureus TSS
2.5
of microbial pathogen genomes that have been
Streptococcus pyogenes
1.9
completely sequenced in our Laboratory at the
University of Minnesota along with the size of the genome is provided in the adjacent table.
Taken together, these sequencing and functional genomics studies have led to the appreciation of
the commonalities and particularities amongst microbial pathogens and the strategies they adopt
to successfully infect, colonize, infect, and cause disease in their hosts.
What are these commonalities? What, if anything, of practical utility has resulted from these
microbial pathogenomics investigations? My presentation at the symposium will highlight the
results of recent investigations carried out in our laboratory in an attempt to answer these and
related questions.
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40 - -
Literature Cited.
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Abrahamsen, M. S., T. J. Templeton, S. Enomoto, J. E. Abrahante, G. Zhu, C. A. Lancto, M. Deng,
C. Liu, G. Widmer, S. Tzipori, G. A. Buck, P. Xu, A. T. Bankier, P. H. Dear, B. A. Konfortov, H. F.
Spriggs, L. Iyer, V. Anantharaman, L. Aravind, and V. Kapur. 2004. Complete genome sequence of
the apicomplexan, Cryptosporidium parvum. Science 304:441-5.
Baechler, E. C., F. M. Batliwalla, G. Karypis, P. M. Gaffney, W. A. Ortmann, K. J. Espe, K. B.
Shark, W. J. Grande, K. M. Hughes, V. Kapur, P. K. Gregersen, and T. W. Behrens. 2003. Interferoninducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl
Acad Sci U S A 100:2610-5.
Boyce, J. D., I. Wilkie, M. Harper, M. L. Paustian, V. Kapur, and B. Adler. 2002. Genomic scale
analysis of Pasteurella multocida gene expression during growth within the natural chicken host. Infect
Immun 70:6871-9.
Boyce, J. D., I. Wilkie, M. Harper, M. L. Paustian, V. Kapur, and B. Adler. 2004. Genomic-scale
analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl
cholera. Microbes Infect 6:290-8.
Cummings, C. A., and D. A. Relman. 2000. Using DNA microarrays to study host-microbe
interactions. Emerg Infect Dis 6:513-25.
Herron, L. L., R. Chakravarty, C. Dwan, J. R. Fitzgerald, J. M. Musser, E. Retzel, and V. Kapur.
2002. Genome sequence survey identifies unique sequences and key virulence genes with unusual rates
of amino Acid substitution in bovine Staphylococcus aureus. Infect Immun 70:3978-81.
Manger, I. D., and D. A. Relman. 2000. How the host 'sees' pathogens: global gene expression
responses to infection. Curr Opin Immunol 12:215-8.
May, B. J., Q. Zhang, L. L. Li, M. L. Paustian, T. S. Whittam, and V. Kapur. 2001. Complete
genomic sequence of Pasteurella multocida, Pm70. Proc Natl Acad Sci U S A 98:3460-5.
Munir, S., and V. Kapur. 2003. Regulation of host cell transcriptional physiology by the avian
pneumovirus provides key insights into host-pathogen interactions. J Virol 77:4899-910.
Munir, S., and V. Kapur. 2003. Transcriptional analysis of the response of poultry species to
respiratory pathogens. Poult Sci 82:885-92.
Munir, S., S. Singh, K. Kaur, and V. Kapur. 2004. Suppression subtractive hybridization coupled with
microarray analysis to examine differential expression of genes in virus infected cells. Biol Proced
Online 6:94-104.
Paustian, M. L., B. J. May, D. Cao, D. Boley, and V. Kapur. 2002. Transcriptional response of
Pasteurella multocida to defined iron sources. J Bacteriol 184:6714-20.
Paustian, M. L., B. J. May, and V. Kapur. 2001. Pasteurella multocida gene expression in response to
iron limitation. Infect Immun 69:4109-15.
Paustian, M. L., B. J. May, and V. Kapur. 2002. Transcriptional response of Pasteurella multocida to
nutrient limitation. J Bacteriol 184:3734-9.
Scamurra, R. W., D. J. Miller, L. Dahl, M. Abrahamsen, V. Kapur, S. M. Wahl, E. C. Milner, and E.
N. Janoff. 2000. Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells. J Immunol
164:5482-91.
Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee, G. L. Sylva, D. E.
Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins, S. B. Beres, D. S. Campbell, T. M. Smith, Q.
Zhang, V. Kapur, J. A. Daly, L. G. Veasy, and J. M. Musser. 2002. Genome sequence and
comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute
rheumatic fever outbreaks. Proc Natl Acad Sci U S A 99:4668-73.
Stratmann, J., B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q.
Zhang, V. Kapur, and T. J. Bull. 2004. A 38-kilobase pathogenicity island specific for Mycobacterium
avium subsp. paratuberculosis encodes cell surface proteins expressed in the host. Infect Immun 72:126574.
Yarwood, J. M., J. K. McCormick, M. L. Paustian, V. Kapur, and P. M. Schlievert. 2002. Repression
of the Staphylococcus aureus accessory gene regulator in serum and in vivo. J Bacteriol 184:1095-101.
Yarwood, J. M., J. K. McCormick, M. L. Paustian, P. M. Orwin, V. Kapur, and P. M. Schlievert.
2002. Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3.
Implications for the evolution of staphylococcal pathogenicity islands. J Biol Chem 277:13138-47.
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DECODING THE MAP GENOME
Vivek Kapur
Department of Microbiology
University of Minnesota, St. Paul, MN
Selected Recent Publications
•
Motiwala AS, Amonsin A, Strother M, Manning EJ, Kapur V, Sreevatsan S. Molecular
epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild
animal species. J Clin Microbiol. 2004 Apr;42(4):1703-12.
•
Amonsin A, Li LL, Zhang Q, Bannantine JP, Motiwala AS, Sreevatsan S, Kapur V.
Multilocus short sequence repeat sequencing approach for differentiating among
Mycobacterium avium subsp. paratuberculosis strains. J Clin Microbiol. 2004 Apr;42(4):1694-702.
•
Stratmann J, Strommenger B, Goethe R, Dohmann K, Gerlach GF, Stevenson K, Li, LL,
Zhang Q, Kapur V, Bull TJ. A 38-kilobase pathogenicity island specific for Mycobacterium
avium subsp. paratuberculosis encodes cell surface proteins expressed in the host. Infect Immun.
2004 Mar;72(3):1265-74.
•
Bannantine JP, Hansen JK, Paustian ML, Amonsin A, Li LL, Stabel JR, Kapur V.
Expression and immunogenicity of proteins encoded by sequences specific to
Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol. 2004 Jan;42(1):106-14.
•
Dohmann K, Strommenger B, Stevenson K, de Juan L, Stratmann J, Kapur V, Bull TJ,
Gerlach GF. Characterization of genetic differences between Mycobacterium avium subsp.
paratuberculosis type I and type II isolates. J Clin Microbiol. 2003 Nov;41(11):5215-23.
•
Bannantine JP, Zhang Q, Li LL, Kapur V. Genomic homogeneity between Mycobacterium
avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent
growth rates. BMC Microbiol. 2003 May 9;3(1):10.
•
Motiwala AS, Strother M, Amonsin A, Byrum B, Naser SA, Stabel JR, Shulaw WP,
Bannantine JP, Kapur V, Sreevatsan S. Molecular epidemiology of Mycobacterium avium
subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification
of unique targets for diagnosis. J Clin Microbiol. 2003 May;41(5):2015-26.
- 41 -
MLST AND ANTIMICROBIAL RESISTANCE OF SALMONELLA
Sam Alcaine1, Sharinne Sukhnanand1, Lorin D. Warnick2, Wan-Lin Su1,
Patrick McDonough2, and Martin Wiedmann1
2
1
Department of Food Science, Department of Population Medicine and Diagnostic
Sciences, Cornell University, Ithaca, NY
INTRODUCTION
Salmonella is an important zoonotic pathogen. According to the Centers for Disease
Control and Prevention (CDC), an estimated 1.4 million cases of human disease due to nontyphoidal salmonellosis occur annually in the U.S. (Mead et al., 1999). Within the genus
Salmonella, almost 2,500 serotypes can be differentiated using the standard Kauffman-White
scheme. Serotypes within subspecies Ι (enterica) are responsible for the vast majority of
salmonellosis infections in warm-blooded animals. These serotypes differ widely in a variety of
features, most notably, their host range and the severity and type of disease typically caused by
them. For example, Salmonella serotype Typhimurium causes gastroenteritis in a multitude of
hosts, whereas Salmonella serotype Dublin causes enteric fever and induces abortion primarily in
cattle. While the overall number of salmonellosis cases has been decreasing, there has been a rise
in the number of antibiotic resistant isolates encountered. For example, while the incidence of
the two most common serotypes found in humans, Typhimurium and Enteritidis, has fallen 23%
and 35%, respectively, from 1997 to 2002, the incidence of Salmonella Newport, a serotype
recently associated multi-drug resistance, has risen 165% in the same period (CDC, 2002). These
resistant strains pose a serious risk to human and animal populations. It has been speculated that
the rise of these strains may be linked to improper administration of antibiotics in hospitals
and/or use of antibiotics in livestock.
Children under the age of 5 account for a quarter of all salmonellosis cases (CDC, 2002),
and represent a vulnerable portion of the population. Of particular concern is the appearance of
Salmonella strains resistant to ceftriaxone, the drug of choice for treatment of invasive
salmonellosis cases in children. Ceftriaxone is closely related to ceftiofur, a 3rd generation
cephalosporin with widespread use in cattle herds. Beef and dairy products account for 10% of
reported food borne Salmonella outbreaks (CDC, 2002), and concerns have been raised on
possible transmission of antibiotic resistant Salmonella, including ones resistant to ceftiofur, from
cattle to people (Fey et al., 2004).
While serotyping has been widely used to differentiate Salmonella subtypes, this method
has limited discriminatory power and does not reveal the genetic relationships of strains within
the same or different serotypes. More discriminatory methods for subtyping of Salmonella isolates
include phage typing as well as pulsed field gel electrophoresis (PFGE). Multi-Locus Enzyme
Electrophoresis (MLEE) has also been used successfully to subtype Salmonella isolates and to
study the evolution and population genetics of various Salmonella serotypes (e.g., Selander et al.,
1990). However, MLEE is technically difficult and hard to standardize between laboratories and
thus does not represent a subtyping method suitable for routine surveillance. The advent of
automated DNA sequencing technology has led to the development and implementation of DNA
sequence-based subtyping techniques, such as Multi-Locus Sequence Typing (MLST). MLST is
based on the concepts of MLEE except that allelic types are determined from nucleotide
sequences of housekeeping genes rather than by the electrophoretic mobilities of the enzymes they
encode (Maiden et al., 1998). One key advantage of MLST over MLEE and other banding
pattern-based subtyping techniques is that the sequence data generated is non-ambiguous and can
- 42 -
be readily compared between laboratories, thus facilitating global, large-scale surveillance
(Maiden et al., 1998).
The changing epidemiology of Salmonella infections and the emergence of new Salmonella
strains (e.g., multi-drug resistant Salmonella Typhimurium DT 104 and multi-drug resistant
Salmonella Newport) make it imperative to develop new Salmonella subtyping methods that not
only allow for sensitive subtype discrimination, but also provide data that can be used for
evolutionary analyses of Salmonella. In addition, molecular subtyping methods for Salmonella
should also allow for serotype prediction, thus obviating the need for maintenance of specialized
serotype reagents for Salmonella. Thus, our goal was to develop an MLST scheme for Salmonella
enterica serotypes that (i) provides sensitive subtype discrimination, (ii) reliably predicts Salmonella
serotypes and (iii) provides data that can be used for evolutionary analyses. We subsequently
applied the MLST scheme we developed to probe the emergence and transmission of ceftiofur
resistant Salmonella on dairy farms.
MLST DEVELOPMENT
The results of our efforts to develop a Salmonella MLST scheme have been submitted for
publication (Sukhnanand et al., 2004). A set of 25 Salmonella enterica isolates, representing five
clinically relevant serotypes (Agona, Heidelberg, Schwarzengrund, Typhimurium, and
Typhimurium var. Copenhagen) was initially used to develop a multi-locus sequence typing
(MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panB, fimA,
aceK, mdh, icdA, manB, and spaN). A total of 8 MLST types were found among the 25 isolates
sequenced. A good correlation between MLST types and Salmonella serotypes was observed;
only one Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for
Typhimurium isolates. Since manB, fimA, and mdh allowed for highest subtype discrimination
among the initial 25 isolates, we choose these three genes to perform DNA sequencing of an
additional 41 Salmonella isolates representing a larger diversity of serotypes. This “three-gene
sequence typing scheme” allowed discrimination of 25 sequence types (STs) among a total of 66
isolates; STs correlated well with serotypes and allowed within serotype differentiation for 9 of
the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and
Newport could each be separated into two distinct, statistically well supported, evolutionary
lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype
prediction and for limited subtype discrimination among clinically relevant serotypes of
Salmonella. Three-gene sequence typing also supports that Salmonella serotypes represent both
monophyletic and polyphyletic lineages.
EVOLUTION AND POPULATION GENETICS OF CEFTIOFUR RESISTANT
SALMONELLA
Our studies on the application of the three-gene sequence typing scheme described above
to study the evolution and transmission of ceftiofur resistant Salmonella are currently being
prepared for submission (Alcaine et al., 2004). Both ceftiofur resistant and sensitive isolates from
eight farms, which had previously been found to be characterized by the presence of ceftiofur
resistant Salmonella strains (Warnick et al., 2003), were analyzed for genetic relatedness (using the
three-gene sequence typing scheme described above) as well as for the presence of class I
integrons and CMY-2. Ceftiofur susceptibility was determined using an automated broth dilution
method (Sensititre, Trek Diagnostics). It should be noted that cut-offs for classifying Salmonella
isolates as sensitive or resistant to ceftiofur have not been validated. For this study, the
breakpoints were those typically used for National Antimicrobial Resistance Monitoring System
- 43 -
reports. CMY-2 is a beta-lactamase gene associated with broad resistance to cephalosporins,
which has been shown to confer resistance to ceftiofur. Class I integrons are mobile genetic
elements, that tend to carry and transmit antibiotic resistance genes. The isolates grouped into
six sequence types (STs), three of which contained isolates with ceftiofur resistance. Each ST
contained isolates from multiple farms, and included both resistant and sensitive isolates. CMY2 was found in all except three isolates that had ceftiofur resistance, and DNA sequencing
showed that the CMY-2 genes carried in all isolates were 100% identical, suggesting horizontal
gene transfer of CMY-2. Furthermore, on the farm level, each farm contained only one
Salmonella ST that had resistance, though not all isolates of the same ST type on the farm had
resistance, suggesting recent acquisition or loss of resistance. Presence of class I integrons and
CMY-2 were not correlated, suggesting that CMY-2 is not carried in an integron, consistent with
other studies that have shown CMY-2 to be associated with a plasmid. Overall, our data are
consistent with a model that both horizontal and vertical transfer contribute to spread of ceftiofur
resistant Salmonella among cattle.
SUMMARY AND CONCLUSIONS
Using an initial collection of 25 Salmonella isolates, we developed a seven-gene MLST scheme
targeting a combination of housekeeping and virulence genes. Based on the initial data obtained
with this seven-gene MLST scheme, we chose three genes with the highest discriminatory ability
to develop and apply a more economical three-gene sequence typing scheme using 66 Salmonella
isolates, representing 12 serotypes. Our results show that (i) a three-gene sequence typing scheme
allows for serotype prediction and for limited subtype discrimination within serotypes, and (ii)
Salmonella serotypes represent both monophyletic and polyphyletic lineages. Application of the
three-gene sequence typing scheme furthermore allowed us to initially probe the evolution and
transmission of ceftiofur resistant Salmonella. Our preliminary data are consistent with a model
that both horizontal and vertical transfer contribute to spread of ceftiofur resistant Salmonella
among cattle. On-going studies on larger sets of human and animal Salmonella isolates are
designed to provide further insight into the evolution and transmission of Salmonella (with a
particular focus on antibiotic resistant strains) among and between humans and farm animals.
REFERENCES
Alcaine, S., L. D. Warnick, P. McDonough, K. J. Boor, and M. Wiedmann. 2004. Ceftiofurresistant Salmonella represent multiple widely distributed subtypes that evolved by independent
horizontal acquisition of CMY-2. J. Clin. Microbiol. (in preparation)
Centers for Disease Control and Prevention. 2002. Salmonella Annual Summary 2002.
Fey P.D., T. J. Safranek, M. E. Rupp, E. F. Dunne, E. Ribot, P. C. Iwen, P. A. Bradford, F. J.
Angulo, and S. H. Hinrichs. 2000. Ceftriaxone-resistant Salmonella infection acquired by a child
from cattle. N. Engl. J. Med. 342:1242-1249.
Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J.
Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus
sequence typing: A portable approach to the identification of clones within populations of
pathogenic microorganisms. Proc. Nat. Acad. Sci. USA 95:3140-3145.
- 44 -
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R.
V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607625.
Selander, R. K., P. Beltran, N. H. Smith, R. Helmuth, F. A. Rubin, D. J. Kopecko, K. Ferris, B.
D. Tall, A. Cravioto, and J. M. Musser. 1990. Evolutionary genetic relationships of clones of
Salmonella serovars that cause human typhoid and other enteric fevers. Infect. Immun. 58:22622275.
Sukhnanand, S., S. Alcaine, W.-L. Su, J. Hof, M. P. J. Craver, L. D. Warnick, P. McDonough,
K. J. Boor, and M. Wiedmann. 2004. DNA sequence-based subtyping and evolutionary analysis
of selected Salmonella enterica serotypes. J. Clin. Microbiol. (submitted 9/16/04; JCM01716-04).
Warnick, L. D., K. Kanistanon, P. L. McDonough, and L. Power. 2003. Effect of previous
antimicrobial treatment on fecal shedding of Salmonella enterica subsp. enterica serogroup B in
New York dairy herds with recent clinical salmonellosis. Prev. Vet. Med. 56:285-297.
- 45 -
LISTERIA MONOCYTOGENES CONTAINS TWO SPECIES-LIKE EVOLUTIONARY
LINEAGES AND SUBTYPES WITH REDUCED INVASIVENESS
Kendra K. Nightingale, Katy Windham, and Martin Wiedmann
Department of Food Science
Cornell University, Ithaca, NY
Background and significance.
Listeria monocytogenes is a facultative intracellular human foodborne and animal pathogen
that may cause severe invasive disease in immunocompromised individuals (Schlech, 2000).
Clinical manifestations of invasive human listeriosis include meningitis, encephalitis, late-term
spontaneous abortion, and septicemia. While invasive listeriosis is a rare disease with an
estimated frequency of 2 to 15 cases/million population, the case mortality for this disease is very
high (13-34%) (Roberts and Wiedmann, 2003). Apparently healthy individuals infected by L.
monocytogenes may experience a mild noninvasive form of listeriosis characterized by
gastrointestinal illness (Roberts and Wiedmann, 2003). The vast majority of human listeriosis
infections (99%) are thought to be foodborne (Mead et al., 1999).
Listeriosis is a systemic bacterial infection characterized by diffusion of L. monocytogenes
from the intestinal lumen to the central nervous system and the placenta (Lecuit et al., 2001). A
systemic infection requires that bacteria be internalized both by professional phagocytes such as
macrophages and induce their own uptake in non-professional phagocytes including epithelial,
endothelial and hepatic cells (Braun et al., 2000). Internalin (InlA) is a surface protein encoded
by inlA that facilitates the entry of L. monocytogenes into epithelial cells that express specific Ecadherin alleles, the receptor for InlA. Because E-cadherin is expressed in various intestinal cells,
InlA may be critical for L. monocytogenes to cross the intestinal barrier as well as later on in
infection (Lecuit et al., 2004). Researchers have shown that InlA is sufficient to promote
internalization L. monocytogenes into several cell lines expressing human E-cadherin (Lecuit et al.,
1997).
Traditionally, L. monocytogenes has been differentiated into 13 serotypes; however, only
four of these serotypes (1/2a, 1/2b, 1/2c and 4b), however, have been reported to cause the
majority (98%) of human listeriosis cases (Doumith et al., 2004). Molecular subtyping methods
(e.g., automated ribotyping and pulsed field gel electrophoresis) allow for more sensitive
discrimination of L. monocytogenes subtypes as compared to phenotypic methods (e.g., serotyping)
and have provided an initial understanding of the population structure of this pathogen
(Wiedmann, 2002). Data generated with most molecular subtyping methods have shown that L.
monocytogenes isolates can be grouped into two major genetic divisions or lineages, termed lineage
I and lineage II (Wiedmann, 2002). Interestingly, previous reports have shown a statistically
significant epidemiological associated between the isolation lineage I strains from human clinical
listeriosis cases as compared to their isolation from contaminated foods (Gray et al., 2004). On
the other hand, lineage II appears to contain two distinct subpopulations one of which has been
associated with isolation from contaminated food samples while to other appears to be associated
with isolation from human listeriosis cases (Gray et al., 2004).
Most epidemiological and population genetics studies on L. monocytogenes have used
DNA banding pattern-based subtyping methods (e.g., pulsed-field gel electrophoresis and
ribotyping). However, these methods are difficult to standardize and data are not easily
compared between laboratories. A reliable high-throughput universal subtyping method such as
multilocus sequence typing (MLST) is needed to study the molecular epidemiology and evolution
of L. monocytogenes. Similar to multilocus enzyme electrophoresis, MLST surveys several loci
- 46 -
expected to operate under neutral genetic variation (Dingle et al., 2001; Maiden et al., 1998).
MLST typically targets conserved loci such as housekeeping genes that diversify slowly and are
not heavily influenced by evolutionary forces other than point mutation (i.e., positive selection
and recombination). However, including virulence genes in a MLST scheme may provide an
enhanced ability to differentiate isolates and allow researchers to make inferences on the
evolution of virulence in L. monocytogenes clonal groups (Cai et al., 2002).
L. monocytogenes contains two species-like evolutionary lineages.
We assembled a representative geographically matched set of 120 L. monocytogenes isolates
from humans and animals with clinical listeriosis as well as from foods to analyze the genetic
diversity, population genetics, and evolution of this L. monocytogenes with a specific focus on those
molecular subtypes associated with foodborne disease transmission. Partial sequencing of four
housekeeping (gap, prs, purM and ribC), one stress-response (sigB), and two virulence (actA and
inlA) genes revealed between 11 (gap) and 33 (inlA) allelic types (unique combination of
polymorphisms). actA, ribC, and purM demonstrated the highest levels of nucleotide diversity (π
> 0.05). Based on a concatenated data set, 52 unique sequence types (unique combination of
allelic types) were differentiated among the 120 L. monocytogenes isolates characterized.
Further analyses showed that actA and inlA may be affected by positive selection and that
these virulence genes along with hypervariable housekeeping loci ribC and purM may have a
history of intragenic recombination. Molecular phylogenies of all seven genes, inferred by
maximum likelihood methods, indicated that L. monocytogenes contains two deeply-separated
evolutionary lineages. Lineage I appears to be highly clonal which may be explained by this
lineage experiencing a population bottleneck, while lineage II shows greater diversity and
evidence of ancient horizontal gene transfer events. Nucleotide distance within evolutionary
lineage was much lower than that observed between lineages suggesting a barrier for genetic
exchange between lineages. Additionally, alleles were not shared between lineage I and II with
respect to all genes except gap for which a single lineage II isolate showed a lineage I allele. Our
data show that (i) L. monocytogenes is a highly diverse species with at least two deeply-separated
species-like evolutionary lineages, which differ in their population structure, and (ii) horizontal
gene transfer as well as positive selection contributed to the evolution of L. monocytogenes
currently present in the food system.
L. monocytogenes contains molecular subtypes with reduced invasiveness.
DNA sequencing of the C-terminal region of InlA (ca. 270 codons) for L. monocytogenes
revealed three unique nonsense mutations upstream of the membrane anchor resulting in a
truncated form of InlA. These truncated InlA alleles were observed in 6 L. monocytogenes strains
(assigned by automated EcoRI ribotyping) including DUP-1052A and DUP-16635 (mutation type
1); DUP-1025A and DUP-1031A (mutation type 2); and DUP-1046B and DUP-1062A (mutation
type 3). Searches of the PathogenTracker (www.pathogentracker.net) database which contains
epidemiological information for more than 5000 L. monocytogenes isolates from human and
animal clinical listeriosis cases, food, and various environments were performed to probe the
distribution of truncated InlA strains. Results indicated that truncated InlA strains are more
commonly isolated from food as compared to their isolation from human listeriosis cases. A
PCR-RFLP assay and inlA sequencing were used to determine prevalence of these three observed
unique nonsense mutations in human clinical and food isolates representing L. monocytogenes
strains shown to harbor a truncated form of InlA. Results showed that nonsense mutation were
present in ca. 40% L. monocytogenes isolates representing truncated InlA strains resulting from
mutation types 1 (DUP-1052A and DUP-16635A) and 2 (DUP-1025A and DUP-1031A).
Further, mutation type 3 was detected in 100% of DUP-1046B and DUP-1062A isolates.
Nonsense mutations were observed more frequently in L. monocytogenes isolates from food as
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compared to human clinical cases. A Caco-2 cell invasion assay showed that truncated InlA
strains have significantly (P<0.0001) reduced invasiveness as compared to full-length InlA strains.
Our data indicate that specific L. monocytogenes subpopulations commonly isolated from
contaminated food carry a truncated InlA and thus show reduced virulence in human intestinal
epithelial cells.
References
Braun, L., B. Ghebrehiwet, and P. Cossart. 2000. gC1q/p32, a C1q-binding protein, is a receptor
for the InlB invasion protein of Listeria monocytogenes. EMBO. 19:1458-1466.
Doumith, M., C. Cazalet, N. Simones, L. Frangeul, C. Jacquet, F. Kunst, P. Martin, P. Cossart,
P. Glaser and C. Buchrieser. 2004. New aspects regarding evolution and virulence of Listeria
monocytogenes revealed by comparative genomics and DNA arrays. Infect. Immun. 72:1072-1083.
Gray, M. J., R. N. Zadoks, E. D. Fortes, B. Dogan, S. Cai, Y. Chen, V. N. Scott, D. E. Gombas,
K. J. Boor, and M. Wiedmann. 2004. Food and human isolates of Listeria monocytogenes form
distinct but overlapping populations. Appl. Environ. Microbiol. (accepted 6/21/04; AEM
00611-04).
Jonquieres, R., H. Biern, J. Mengaud and P. Cossart. 1998. The inlA gene of Listeria
monocytogenes LO28 harbors a nonsense mutation resulting in release of Internalin. Infect.
Immun. 66:3420-3422.
Lecuit, M., S. Vandormael-Pournin, J. Lefort, M. Huerre, P. Gounon, C. Dupuy, C. Babinet,
and P. Cossart. 2001. A transgenic model for listeriosis: role of Internalin in crossing the
intestinal barrier. Science. 292:1722-1724.
Lecuit, M., H. Ohayon, L. Braun, J. Mengaud, and P. Cossart. 1997. Internalin of Listeria
monocytogenes with an intact leucine-rich repeat is sufficient to promote internalization. Infect.
Immun. 65:5309-5319.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCraig, J. S. Bresee, C. Shapiro, P. M. Griffin and R.
V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607625.
Roberts, A. J. and M. Wiedmann. 2003. Pathogen, host and environmental factors contributing
to the pathogenesis of listeriosis. Cell Mol. Life Sci. 60:904-918.
Wiedmann, M. 2002. Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int.
85:524-531.
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MULTI-LOCUS SEQUENCE TYPING OF STREPTOCOCCUS UBERIS SHOWS
RETICULATE EVOLUTION BETWEEN HOUSEKEEPING GENES AND POSITIVE
SELECTION IN A PUTATIVE VACCINE TARGET
1
Ruth N. Zadoks1, 2, Ynte H. Schukken2 and Martin Wiedmann1.
Department of Food Science, 2 Quality Milk Production Services
Cornell University, Ithaca, NY.
Background. Streptococcus uberis is an important udder pathogen of dairy cows. For years, a
vaccine has been sought after as a means to protect cows from S. uberis mastitis, with GapC and
PauA as the main vaccine targets9,12, but no field-trials demonstrating vaccine efficacy have been
published yet. Improvement of S. uberis control programs has been hampered by insufficient
knowledge of the epidemiology and pathophysiology of infections. Knowledge of strain
characteristics associated with patterns of transmission, infection or cure could contribute to
improvement of herd and cow specific recommendations with respect to mastitis treatment and
control, potentially reducing the current reliance on use of antimicrobials as primary control
strategy. While DNA banding pattern based typing methods have contributed to insights in the
epidemiology of S. uberis mastitis10,11,13, they have limitations in terms of typeability,
discriminatory power, and reproducibility. For several streptococcal species, including S.
agalactiae, S. pneumoniae, S. pyogenes, and S. suis, banding pattern based typing methods have been
superseded by multilocus sequence typing (MLST), which provides more standardized and
informative strain typing data than banding pattern based methods1,2,4,8. We developed a multilocus sequence typing scheme for S. uberis, and used phylogenetic analyses to explore the
evolutionary mechanisms behind S. uberis diversity.
MLST is more discriminatory than DNA banding pattern based strain typing and provides
taxonomic information. Among 50 S. uberis isolates from the USA (n=30) and The Netherlands
(n=20), 35 ribotypes (index of discrimination D = 0.973), and 12 (cpn60), 10 (gap), 14 (oppF), 11
(pauA), 9 (sod) and 11 (tuf) alleles were identified, with a total of 39 sequence types (D = 0.989).
One strain of S. parauberis was included in the analysis. S. parauberis did not yield amplicons of
oppF or pauA, and had unique alleles for the remaining four housekeeping genes. In addition to
being more discriminatory than banding pattern based methods, MLST differentiated within
ribogroups or RAPD-patterns in accordance with epidemiological data (herd of origin, time of
collection). Comparison of DNA sequences for four housekeeping genes (cpn60, gap, sod, tuf)
showed that S. uberis isolate FSL Z1-015 was only distantly related to the remaining S. uberis
isolates and showed higher homology with S. parauberis then with S. uberis. Based on sequencing
of the rnp and rpoB genes, isolate FSL Z1-015 was subsequently confirmed to belong to the
species S. uberis. Isolates with the same MLST type as FSL Z1-015 have been recovered from
environmental samples in farm environments and this group appears to constitute a subtaxon
within S. uberis with as yet unexplored epidemiological and pathophysiological characteristics.
Reticulate evolution occurs within and between genes of S. uberis. Evolution of three
housekeeping genes (gap, sod, tuf) could be represented by a bifurcating tree, while reticulate
evolution was detected in two housekeeping genes (cpn60, oppF) and a virulence gene (pauA). In
addition, there was reticulate evolution between genes so that a core phylogeny for S. uberis
evolution could not be constructed. Reticulate evolution between genes also plays an important
role in overall sequence diversity for other streptococcus species, such as S. pyogenes, group C
streptococci (GCS) and group G streptococci (GGS), while only a limited number of
housekeeping genes show evidence of within-gene reticulate evolution in these species6. Despite
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widespread occurrence of reticulate evolution between genes, clonal groups may be preserved in
some streptococcal species. These clonal complexes may be niche specific, as has been shown for
S. pyogenes5 and S. suis 8. To cover the full spectrum of genetic diversity within S. uberis, isolates
from a variety of niches, including the mammary gland and environmental sources, should be
included in additional population genetic studies.
Evolution of pauA differs from the evolution of housekeeping genes. Unlike the other genes
included in our MLST scheme, pauA is a virulence gene and it is not uncommon for virulence
genes to be present in a proportion of strains only. Absence of a pauA amplicon was observed in 2
of 50 isolates in our study (4.0%) and in 4 of 130 S. uberis isolates (3.1%) in a study from
Germany7. Average G+C content was 0.39 for housekeeping genes and 0.34 for pauA, suggesting
acquisition of pauA through horizontal transfer. The average dN/dS ratio was 0.07 for
housekeeping genes and 1.2 for pauA, indicating positive selection in pauA, which encodes a
streptokinase (plasminogen activator A) that is a putative vaccine target9. Some of the observed
non-synonymous mutations in pauA are likely to be inconsequential, while other nonsynonymous substitutions may affect the conformation and/or functionality of the protein.
Differences between S. uberis isolates in plasminogen activity have been reported3, but it remains
to be elucidated whether any of the amino acid polymorphisms in PauA have an impact on its
functionality.
Conclusion and Outlook. MLST-based typing of S. uberis was superior to banding pattern-based
methods in terms of discriminatory ability, concordance with epidemiological data, and
quantitative information regarding relatedness of isolates and taxonomic groups. Reticulate
evolution contributes to a limited extent to genetic variability within the genes covered by our
typing scheme, but plays a major role in overall sequence variability of S. uberis. Reticulate
evolution may provide S. uberis with the ability to diversify in response to control measures taken
to reduce the incidence of S. uberis infections in dairy cattle. The evolution of the virulence gene
pauA, which encodes a putative vaccine target, differed from the evolution of housekeeping genes
and was subject to positive selection. In the development and evaluation of vaccines, creation of
sub-unit vaccines targeting conserved epitopes and continued monitoring of evolution of vaccine
targets should be considered.
References
1. Enright, M. C. and B. G. Spratt. 1998. A multilocus sequence typing scheme for Streptococcus
pneumoniae: identification of clones associated with serious invasive disease. Microbiology
144 ( Pt 11):3049-3060.
2. Enright, M. C., B. G. Spratt, A. Kalia, J. H. Cross, and D. E. Bessen. 2001. Multilocus
sequence typing of Streptococcus pyogenes and the relationships between emm type and clone.
Infect. Immun. 69:2416-2427.
3. Johnsen, L. B., K. Poulsen, M. Kilian, and T. E. Petersen. 1999. Purification and cloning of a
streptokinase from Streptococcus uberis. Infect. Immun. 67:1072-1078.
4. Jones, N., J. F. Bohnsack, S. Takahashi, K. A. Oliver, M. S. Chan, F. Kunst, P. Glaser, C.
Rusniok, D. W. Crook, R. M. Harding, N. Bisharat, and B. G. Spratt. 2003. Multilocus
sequence typing system for group B streptococcus. J. Clin. Microbiol. 41:2530-2536.
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5. Kalia, A. and D. E. Bessen. 2004. Natural selection and evolution of streptococcal virulence
genes involved in tissue-specific adaptations. J. Bacteriol. 186:110-121.
6. Kalia, A., M. C. Enright, B. G. Spratt, and D. E. Bessen. 2001. Directional gene movement
from human-pathogenic to commensal-like streptococci. Infect. Immun. 69:4858-4869.
7. Khan, I. U., A. A. Hassan, A. Abdulmawjood, C. Lammler, W. Wolter, and M. Zschock.
2003. Identification and epidemiological characterization of Streptococcus uberis isolated from
bovine mastitis using conventional and molecular methods. J. Vet. Sci. 4:213-224.
8. King, S. J., J. A. Leigh, P. J. Heath, I. Luque, C. Tarradas, C. G. Dowson, and A. M.
Whatmore. 2002. Development of a Multilocus Sequence Typing Scheme for the Pig
Pathogen Streptococcus suis: Identification of Virulent Clones and Potential Capsular Serotype
Exchange. J. Clin. Microbiol. 40:3671-3680.
9. Leigh, J. A., J. M. Finch, T. R. Field, N. C. Real, A. Winter, A. W. Walton, and S. M.
Hodgkinson. 1999. Vaccination with the plasminogen activator from Streptococcus uberis
induces an inhibitory response and protects against experimental infection in the dairy cow.
Vaccine 17:851-857.
10. McDougall, S., T. J. Parkinson, M. Leyland, F. M. Anniss, and S. G. Fenwick. 2004.
Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk. J.
Dairy Sci. 87:2062-2072.
11. Oliver, S. P., B. E. Gillespie, and B. M. Jayarao. 1998. Detection of new and persistent
Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain
reaction-based DNA fingerprinting. FEMS Microbiol. Lett. 160:69-73.
12. Perez-Casal, J., T. Prysliak, and A. A. Potter. 2004. A GapC chimera retains the properties of
the Streptococcus uberis wild-type GapC protein. Protein Expr. Purif. 33:288-296.
13. Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H.
Schukken. 2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis
infections in dairy herds. Epidemiol. Infect. 130:335-349.
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REAL-TIME PCR IN MILK – FOOD SAFETY IN TIMES OF WAR AND PEACE
Jeffrey S. Karns
Environmental Microbial Safety Lab
USDA/ARS, Beltsville, MD
In October 2001 the USA was rocked by a terrorist attack in which spores of Bacillus anthracis
were distributed through the U. S. Postal system, killing five people due to inhalation anthrax,
causing cutaneous anthrax in several others, and causing a fair degree of economic disruption. It
instantly became clear that, as a nation, we were unprepared to deal with such an unconscionable
act, lacking (at least in the civilian arena) the necessary methods for quick, reliable detection of
agents that might be used for biological terror in a large number of samples. Our laboratory
responded to the needs of the USDA and several other Federal Agencies in the Washington,
D.C., area by setting up a mobile laboratory in which real-time PCR assays were used to screen
over 4,500 environmental samples for the presence of B. anthracis. None of the PCR reactions
run on DNA extracted directly from the air or surface swabs were positive. Tests with spiked
samples indicated that several thousand spores would have to be present in the samples to be
detected by PCR, partly due to the small volume of sample added to the PCR reaction and partly
because of the dirty condition of the samples. In this case, the real value of the real time PCR
assay was rapid and positive identification of suspect colonies as B. anthracis, saving days of
confirmatory culture and serological testing.
Analysis of possible methods that bioterrorists might use indicated that the nation’s food supply
might be a means for the delivery of agents to a susceptible public. Milk might be a target for B.
anthracis contamination because there are several stages in the production process at which the
spores might be added and because the spores would survive the pasteurization process.
Although gastrointestinal anthrax is less deadly than inhalation anthrax, such an attack would
likely shatter public confidence in the milk supply. We were able to use a commercial kit for realtime PCR detection of B. anthracis to detect 8 spores per reaction (2,500 spores/ml milk). The
spores tended to concentrate in the cream fraction when the milk was subjected to low-speed
centrifugation, an observation that may be exploited in future experiments to lower the limits of
detection of the organism.
In other studies we have shown that real-time PCR combined with enrichment culture can be
used to detect low levels of Salmonella and pathogenic forms of Escherichia coli in bulk-tank milk.
When a commercial kit was used to analyze samples taken as part of the NAHMS 2002 dairy
survey, Salmonella contamination was indicated in 101 of 854 samples tested (12%).
Conventional enrichment culture followed by plating on selective agar yielded Salmonella in only
26 (3%) of the samples (and 6 of those only upon re-culture after obtaining the PCR results).
When the samples were examined for the presence of E. coli using a real-time PCR assay specific
for the lacZ gene, 826 out of 859 samples were positive (96%). A real-time PCR assay to detect
the eaeA gene (encoding intimin) indicated that this virulence factor was present in 199 samples
(23%). These 199 samples were then tested for the presence of the allele of the tir gene (encoding
the translocated intimin receptor) associated with E. coli O157:H7 and 61 were found to be
positive (7% of total milk samples). Further examination of these 61 samples with a real-time
PCR assay for the detection of rfbO157 (associated with the production of the O157 antigen)
showed that 5 samples potentially contained pathogenic E. coli O157. Culture of the preserved
enrichments from these 5 samples resulted in the isolation of a strain of O157 from one sample.
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Real-time PCR assays of the E. coli enrichments also showed that shiga toxin genes (stx1 and stx2)
were present in 69 samples (8% of total milk samples).
In summary, real-time PCR offers a quick and sensitive alternative to conventional culture
techniques for the analysis of the loads of pathogenic bacteria in milk and dairy products. One
advantage of real-time PCR over conventional PCR is the time savings gained by eliminating the
need to run gels to visualize PCR products. A second advantage is that real-time PCR can be
used in a quantitative fashion to obtain information on the actual number of target bacteria in a
sample. As the genomes of more pathogenic bacteria are sequenced, providing the information
needed to design real-time PCR assays to detect those organisms, we will be able to design the
assays needed to assure a safe food supply.
Related Publications
Gagliardi, J. G. and Karns, J. S. 2000. Leaching of Escherichia coli O157:H7 in diverse soils
under various agricultural management practices. Appl. Environ. Microbiol. 66: 3512-3517.
Shelton, D. R. and Karns, J. S. 2001. Quantitative detection of Escherichia coli O157 in surface
waters using immunomagnetic-electrochemiluminescence (IM-ECL). Applied and
Environmental Microbiology 67:2908-2915.
Higgins, J.A., M.C. Jenkins, D.R. Shelton, R. Fayer and J.S. Karns. 2001. Rapid Extraction of
DNA from Escherichia coli and Cryptosporidium parvum for Use in PCR. Applied and
Environmental Microbiology 67:5321-5324.
Gagliardi, J. V. and J.S. Karns. 2002. Persistence of E. coli O157:H7 in soil and on plant roots.
Environmental Microbiology 4:89-96.
Higgins, J.A., S. Nasarabadi, J.S. Karns, D.R. Shelton, M. Cooper, A. Gbakima, and R.P.
Koopman. 2003. A handheld real-time thermal cycler for bacterial pathogen detection. Biosen.
Bioelect. 18:1115-1123.
Higgins, J.A., Cooper, M., Schroeder-Tucker, L., Black, S., Miller, D., Karns, J.S., Manthey, E.,
Breeze, R., and Perdue, M.L. 2003. A Field Investigation of Bacillus anthracis Contamination of
U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax
Attack of October 2001. Appl. Environ. Microbiol. 69:593-599.
Van Kessel, J. S., Karns, J. S. and Perdue M. L. 2003. Using portable real-time polymerase
chain reaction (RT-PCR) assays to detect Salmonella spp. in raw milk. Journal of Food
Protection 66:1762-1767.
Perdue, M. L., Karns, J. S., Higgins, J. and Van Kessel, J. S. 2003. Detection and fate of Bacillus
anthracis (Sterne) vegetative cells and spores added to bulk tank milk. Journal of Food Protection
66:2349-2354.
Shelton, D.R., Van Kessel, J. S., Wachtel, M. R., Belt, K. T. and Karns, J. S. 2003. Evaluation
of parameters affecting detection of Escherichia coli O157 in enriched water samples using
immunomagnetic electrochemiluminescence. Journal of Microbiological Methods 55:717-725.
- 53 -
Shelton, D. R., Pachepsky, Y. A., Sadeghi, A. M., Stoudt, W. L., Karns, J. S. and Gburek, W. J.
2003. Release Rates of Manure-borne Coliform Bacteria from Data on Leaching through Stony
Soil. Vadose Zone Journal 2:34-39.
Van Kessel, J.A.S., Karns, J.S. Gorski, L. McCluskey, B.J. and Perdue M.L. 2004. Prevalence
of Salmonellae, Listeria monocytogenes and Fecal Coliforms in Bulk Tank Milk on U.S. Dairies.
Journal of Dairy Science (accepted with revision April 2003).
Shelton, D.R., Higgins, J.A., Van Kessel, J.A.S. ,Pachepsky, Y., Belt, K. and Karns, J.S. 2004.
Estimation of Viable Escherichia coli O157 In Surface Waters Using Enrichment in Conjunction
with Immunological Detection. Journal of Microbiological Methods (Accepted April 2004)
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PAST, PRESENT AND FUTURE APPLICATIONS OF BULK TANK MILK ANALYSIS
TO ASSESS MILK QUALITY AND HERD HEALTH STATUS
Bhushan Jayarao
Department of Veterinary Science
The Pennsylvania State University, University Park, PA
Introduction
In the United States by late 1950s, collection and storage of farm raw milk gradually shifted from
cans to refrigerated bulk tanks (Thomas et al., 1971). With this change the concept of using raw
milk in the bulk tank to assess milk quality and mastitis pathogens emerged. Over time, our
knowledge on the bacteriology of raw milk, mastitis, and farm management have greatly
improved. This coupled with rapid advancement in the areas of biochemical, microbiological and
molecular methods has allowed us to formulate strategies to improve milk quality and reduce the
incidence of mastitis on dairy herds. Bulk tank is an ideal site to sample, when sampled correctly
serves as a unique representative sample of all the cows that were milked prior to sample
collection (Jayarao et al., 2004). This paper attempts to provide an overview of different
microbiological and molecular assays that have been developed around using BTM, and potential
diagnostic tests that could be developed to assess milk quality and herd health status using BTM.
Historical perspective on BTM analysis
In the early 1960s several studies were conducted to determine the number of somatic cells and
the type mastitis pathogens present in BTM. There was a general agreement that analysis of BTM
samples provided a fairly accurate picture of mastitis at the herd level (Gray and Schalm, 1960;
Spencer and Simon, 1960; and Frank and Pounden, 1963). Around the same time, several
researchers utilized BTM samples to establish the relationship between farm management
practices and milk quality. Jackson and Clegg (1966) showed that when the bacterial count was
less than 20,000 cfu/ml, micrococcus was the predominant organism. With the increase in the
bacterial count, gram negative rods and streptococcal organisms usually increased in BTM. The
microflora varied considerably between BTM samples collected from different farms. A multi
provincial collaborative project was conducted in Canada by Morse et al. (1968a). They reported
that BTM bacterial counts ranged from 690 to15,500 cfu/ml. They were of the opinion that
improperly washed and dried udders and milk house water with high bacterial counts could lead
to high bacterial counts in BTM. In a subsequent study, Morse et al (1968b) observed that high
preliminary incubation counts in BTM samples was as result of poor sanitizing procedures and
unsatisfactory bulk tank conditions.
The use of BTM to identify mastitis pathogens began in California in the late 1970s.
Jasper et al. (1979) conducted an extensive study to determine the prevalence of Mycoplasma
bovine mastitis in California. Nearly 4% of all dairy herds surveyed had Mycoplasma of potential
animal health significance in BTM. The findings of the study showed that BTM could be used for
surveying herds with Mycoplasma. Meek and Barnum (1982) investigated the possibility of using
BTM somatic cell counts to monitor the level of subclinical mastitis in dairy herds. The findings
of the study showed that BTM somatic cell counts did not accurately depict the level of
subclinical mastitis in dairy herds. The first comprehensive guide to interpret somatic cells,
bacterial counts and mastitis pathogens in BTM was proposed Guterbock and Blackmer (1984).
This report was followed by work done by Farnsworth (1993) in Minnesota with emphasis of
using BTM culture results for interpreting mastitis and milk quality issues on dairy herds.
Subsequent studies conducted over the last decade have shown that examination of BTM is
useful for diagnosing multiple problems (current and potential) that might exist in a dairy herd
- 55 -
related to milk quality and mastitis pathogens (Godkin and Leslie, 1993; Bray and Shearer, 1996;
Jayarao and Wolfgang, 2003; Jayarao et al., 2004).
Current outlook on use of BTM for managing milk quality and udder health status
The use of BTM analysis has received a lot of attention, especially from veterinarians and dairy
health consultants who view milk quality and mastitis as an important part of their consulting
service for their clients (Britten and Emerson, 1996; Keeter, 1997; Mickelson et al., 1998). Our
knowledge on the bacteriology of raw milk, mastitis, and farm management practices related to
milking and milk hygiene has increased considerably, making it possible to formulate strategies to
improve milk quality and reduce the incidence of mastitis in dairy herds (Houghtby et al., 1992;
Fenlon et al., 1995; Murphy, 1997). More recently, Jayarao et al. (2004) have developed
microbiological guidelines for interpreting BTM analysis based on herd size and farm
management practices.
Review of BTM diagnostic tests
Like other diagnostic applications, BTM analysis has its benefits and limitations. The likely
circumstances that would augment and or impede BTM analysis and interpretation have been
listed in Table 1. To date diagnostic tests reported in literature have focused on enumeration and
or detection of; 1) somatic cells, 2) bacterial pathogens (foodborne, animal and zoonotic), 3)
bacteria associated with lowering milk quality, 4) viruses of dairy cattle, 5) antibodies in milk
against bacterial, viral and gastrointestinal parasites, and 6) substances such as antibiotics,
metabolites, drugs, toxins and trace minerals. Diagnostic test that have been developed and used
for BTM analysis are listed in Tables 2-5.
Bacteria
A large majority of the diagnostic tests used currently for detection of bacterial pathogens
(foodborne, animal, zoonotic and mastitis) rely quite extensively on bacteriological methods. Use
of selective media for enrichment and cultivation followed by rapid identification kits have now
become a standard practice in diagnostic laboratories (Table 2). ELISA-based assays that detect
antibodies against specific pathogens such as Salmonella, Mycobacterium avium subsp.
paratuberculosis, and Coxiella brunetti have been developed. PCR-based assays are available for vast
array of pathogens including foodborne, animal and of public health importance. Many of the
initial PCR assays relied on pure bacterial cultures and as better DNA isolation techniques and
purification matrices were made available, DNA isolation techniques were directly coupled to the
PCR assay eliminating the need of culturing the organism. Among mastitis causing pathogens,
PCR-based diagnostic test for Mycoplasma was first developed and tested and is now widely used
by many diagnostic laboratories.
Diagnostic tests based on BTM must be critically evaluated for its reliability, sensitivity,
specificity and predictive values. A validated diagnostic test would permit proper interpretation of
BTM milk analysis results before implementing changes on the farm. A constant criticism of
many of the PCR assays is that information on their sensitivity and specificity are not made
available. For example a test that can detect the organism consistently at 100 cfu/ml would be
better than an assay that is able to detect the organism at10, 000 cfu/ml. In general, current
PCR-based assays have a detection threshold of 100-10,000 cfu/ml. However under
circumstances when the number of the organisms is low (example <10 cfu/ml) the PCR assay
would fail to detect the target DNA. To overcome this hurdle two unique approaches have been
developed. The first approach is based on the use of short selective enrichment (4-12 h) coupled
with DNA isolation and PCR assay. Kits that rely on this technique are now available
commercially for Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7. The second
approach employs selective concentration of the organism present in milk using
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immunomagnetic beads. Organisms bound to the immunomagnetic beads are then subjected to
culture and or PCR analysis. Immunomagnetic bead coupled PCR assays have been developed
for many foodborne and animal pathogens such as Mycobacterium avium subsp. paratuberculosis.
The concept of detecting multiple pathogens or multiple gene sequences for a given
pathogen in a single PCR assay has received a lot of attention. To date there are several multiplex
PCR assays have been developed for foodborne pathogens (Tables 2 and 3). As with uniplex PCR
assays, multiplex assays have also to contend with optimization of primer concentration, PCR
mixture, multiple DNA templates, and stringency of PCR reactions. However standardization of
multiplex assay is much more time consuming and generally are less precise as compared to their
respective uniplex assays. Recently, in our laboratory we have successfully developed a multiplex
PCR assay to detect three contagious mastitis pathogens directly from milk. The sensitivity and
specificity of the multiplex PCR for detection of M. bovis was 89%, 97%; S. aureus-67%, 94%; S.
agalactiae- 83%, 84%, respectively (Tmanova, 2003). The Gastroenteric Disease Center at Penn
State has developed a multiplex PCR assay for simultaneous detection of Salmonella, Listeria
monocytogenes and Escherichia coli directly from raw milk. The sensitivity and specificity of the
assay ranges from 89-98% and 95-99%, respectively (DebRoy and Jayarao, 2003, unpublished
data).
The foot and mouth disease in Britain in 2000 and the 9/11 terrorist act has accelerated
the development of real time detection methods for potential agents of bio- and agro-terrorism.
The Agricultural Research Service at Beltsville has developed real time PCR assays for Bacillus
anthracis (Perdue et al., 2003) and Salmonella (Van Kessel et al., 2003) in milk. Khare et al. (2004)
have developed a real time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis.
A real time PCR assay with high sensitivity and specificity was developed by Sreevatsan et al.
(2000) to detect Mycobacterium bovis and Brucella abortus simultaneously.
Viruses
BTM has been used for detection of antibodies to viruses including bovine viral diarrhea virus
(BVDV), bovine leucosis virus, food and mouth disease virus, bovine herpes virus, bovine
respiratory syncytial virus, and bovine corona virus using ELISA-based tests (Table 2). Use of
BTM to detect BVDV has received considerable attention (Table 2). RT-PCR assay using BTM
for detection of BVDV could become a standard test for screening herds for BVDV or prior to
purchase of animals.
Considerations for developing a diagnostic test using milk from bulk tank
Three specific aspects have to be considered before developing a diagnostic test using BTM. They
include; 1) the biology and epidemiology of the organism, 2) characteristics of the food matrix
that hosts the organism, 3) the technology that will be used for detection, and 4) validation of the
test under laboratory and field conditions.
Biology and epidemiology of the organism
The following factors need to be considered with regard to the biology and epidemiology of the
organism: 1) likely pathways through which organisms can gain access into BTM, 2) frequency,
type, number and distribution of the organisms in the milk phase, 3) viability of the organism
under refrigeration temperatures, 4) ability of the target organism to compete for milk-based
nutrients with other organisms and survive for extended period of time in milk, and 5) unique
target molecule that can be used to detect the organism (number of targets, location, and factors
associated with its expression).
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Characteristics of the food matrix
As compared to other food, milk may appear as a simple mixture of nutrients that is easy to
collect, store, handle, process and analyze, in fact it is a complex matrix of nutrients. Milk favors
growth and survival of many organisms and also protects the organisms by allowing them to
attach to milk proteins and fat globules and evade isolation and detection. Further macrophages
in milk have the ability to internalize bacteria such as Staphylococcus aureus, Streptococcus uberis,
Salmonella spp., and Listeria monocytogenes. Once within the macrophage, some bacterial
pathogens have developed cellular mechanisms to evade their destruction and even multiply
within the macrophage.
Detection system
DNA- and biosensor-based methods are by far the most rapidly growing segment of molecular
diagnostics. A detection system needs to take into account the biology of the organism and the
characteristics of the food matrix with special reference to factors that would hinder the process
of selecting the target molecule. Bickley et al. (1996) showed that calcium in milk could reduce
the efficiency of DNA isolation and inhibitory substances in the DNA lysates prepared directly
from milk could interfere with the PCR reaction. DNA-based test needs to be rigorously tested
and optimized using experimental conditions that would mimic field samples. In other words, a
PCR-based assay that is developed using experimentally inoculated milk sample would be more
realistic that using pure cultures in broth samples to standardize and optimize the assay.
Validation of a diagnostic test
The true value of a diagnostic test can be determined after it has been tested using farm BTM or
quarter milk samples. A diagnostic test that has been meticulously evaluated for its sensitivity,
specificity and predictive values will give the diagnostician a better appreciation of the test
(Martin and Meek, 1986).
Future prospects of BTM-based diagnostics tests
The use of BTM for monitoring herd health has continued to gain popularity and it is expected
that many more tests, particularly PCR-based real time assays will be developed. Existing PCR
assays will be adapted to real time assays. It in the near future, real time assays kits may become
commercially available. Multiplex PCR assays may not gain popularity unless it has a very high
level of sensitivity and specificity. There is a widespread notion that microbiological assays are
the “gold standard” to which PCR-based assays need to be compared. This may prove to be one
of the constraints for implementing PCR-based assays, as several studies have repeatedly shown
that PCR-based assays detect a higher number of positive samples as compared to established
culture techniques. Use of concordant analysis or other statistical methods can be used to address
this discrepancy (Johnson et al., 2004; Puppe et al., 2004).
It may soon be possible to tailor a set of BTM diagnostic assays based on the type
investigation that needs to be done. For example, a module consisting of contagious mastitis
pathogens, Mycobacterium paratuberculosis and BVDV may be used for screening animals on a herd
or animals that are being considered for purchase. BTM analysis is ideally suited for herds that
collect milk in a bulk tank. However on many large farms (> 500 cows in milk) one would expect
to see more sophisticated milking operations in which cooled milk is directly pumped into a tank
of a milk hauler truck. Sampling milk under such situations may be challenging. To overcome
some of these issues, milk-line sampling devices have been developed and validated (Godden et
al., 2002). One would expect more milk-line devices being employed for sampling milk for
analysis. Milk-line sampling device could permit sequential analysis of milk samples collected
over time during a milking session and may be valuable for troubleshooting problems related to
contagious mastitis, particularly Mycoplasma mastitis. A diagnostic test is not of much value,
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unless the result is interpreted correctly in context with the farm’s management practices which would
allow making decisions on improving milk quality and herd udder health. Decision matrices or
guidelines need be developed for efficient implementation of results derived from BTM-based
diagnostic tests (Jayarao et al., 2004). It is envisioned that biosensor-based techniques for
detection of bacteria, toxins, drugs and metabolites in milk will complement and in some
instances replace many of the existing technologies (Yang et al., 2004; Rasooly, 2001; Gillis et
al., 2002 and Gustavsson and Sternesjo, 2004).
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Table 1. Benefits and limitations of BTM analysis
Benefits
Limitations
Provides a logical approach for troubleshooting herds
with multiple milk quality and mastitis related
problems.
Does not provide information about milk quality and
mastitis at individual cow level.
Less expensive than quarter milk sampling the whole
herd.
Understanding milk quality and mastitis problems in a
herd cannot be done effectively using a single BTM
sample.
BTM analysis can be done in about 96 hours.
Information on herd management practices on milking
cows, mastitis prevention, milk sanitation and general
farm hygiene are required to interpret BTM analysis
results.
A reliable tool for veterinarians to troubleshoot milk
quality and herd level mastitis.
Proper interpretation of BTM milk analysis results is
critical before implementing changes on the farm.
An important component of total herd health
management or veterinary practice consultancy
services.
Samples have to be analyzed within 36 hours of
collection. Samples collected for somatic cell and milk
quality cannot be frozen. Samples have to be held at 4 oC
till analyzed.
BTM analysis report becomes documentary evidence of
milk quality assurance protocol practiced on the farm.
Milk is a complex matrix consisting of water, cells,
proteins, fats, minerals, and carbohydrates. This inherent
nature of the matrix could hinder the isolation of some
types of bacteria and viruses that are intracellular in
nature.
Non invasive technique, when collected properly serves
a representative herd sample.
Volume of milk in the bulk tank, the number of organisms
and their relative distribution in the milk phase may
influence the results of the BTM analysis.
Milk held over period of time or subjected to temperature
abuse may lead to overgrowth of certain types of bacteria.
This could result in: 1) false picture on the bacterial flora
in the sample, and 2) hinder in the isolation of organisms
that are very few in number.
A diagnostic test that has not been validated for its
reliability, sensitivity, specificity and predictive value
could be a serious limiting factor. Interpretation of results
based on an un-validated test could lead to erroneous
conclusions.
Interpreting BTM analysis results for large herds may be
difficult, unless multiple samples or string samples are
collected and analyzed.
- 66 -
Table 2. Use of BTM for detection of foodborne pathogens and pathogens of animal and public
health importance.
Pathogens in BTM
Technique
Bacillus anthracis
Real time PCR
Brucella abortus
Fluorescence
polarization assay
Campylobacter jejuni
Campylobacter spp.
Real time PCR
Bacterial Culture
Coxiella burnetii
ELISA
Escherichia coli
Multiplex PCR
E. coli O157:H7
Bacterial culture
Foodborne Pathogens
Bacterial Culture
Foodborne Pathogens
Bacterial Culture
Foodborne Pathogens
Bacterial Culture
Foodborne Pathogens
Bacterial Culture
Comments
Pasteurization of milk inoculated with B.
anthracis spores (2 separate occasions) did
not kill all of the spores.
The assay had a sensitivity and specificity
of 100 and 95.9%, respectively.
Surveillance and eradication of B. abortus
would be more cost-effective using this test.
The whole assay was completed in 60 min
with a detection limit of approximately 1
CFU/ml. 27.3% (82/300) of milk samples
were positive for C. jejuni.
1.6% BTM samples were positive for
Campylobacter.
21% of BTM samples showed serological
evidence of C. burnetii infection.
A multiplex PCR allowed detection of
pathogenic genes of enteropathogenic,
enterotoxigenic and verocytotoxinproducing E. coli.
2 of 268 (0.75%) BTM samples positive for
E. coli O157:H7.
Campylobacter jejuni, Listeria
monocytogenes, Salmonella spp., and
Yersinia enterocolitica were detected in
12.3, 4.1, 8.9, and 15.1 % of BTM samples,
respectively.
Campylobacter jejuni, shiga toxin
producing E. coli Listeria monocytogenes,
and Salmonella spp., were detected in 0.47,
0.87, 2.7, and 0.17 % of BTM samples,
respectively.
Campylobacter jejuni, shiga-toxin
producing Escherichia coli, Listeria
monocytogenes, Salmonella spp., and
Yersinia enterocolitica were detected in 9.2,
3.8, 4.6, 6.1, and 6.1% of bulk tank milk
samples, respectively.
Salmonella (n=9 serotypes) was detected in
2.6% of BTM samples. Listeria
monocytogenes (n=5 serotypes) was
detected in 6.5% of BTM samples.
- 67 -
Reference
Perdue et al. (2003)
Gall et al. (2002)
Yang et al. (2003)
Whyte et al. (2004)
Paiba et al. (1999)
Bottero et al. (2004)
Murinda et al. (2002)
Rohrbach et al.
(1992)
Steele et al. (1997)
Jayarao and Henning
(1999)
Van Kessel et al.
(2004)
Table 2. continued…
Pathogens in BTM
Listeria monocytogenes
Salmonella Dublin
Technique
Bacterial Culture
Indirect ELISA
Salmonella spp.
VIDAS
Salmonella assay
Salmonella spp.
Bacterial Culture
Salmonella spp.
Bacterial Culture
Salmonella spp.
Real Time PCR
Staphylococcus aureus
ELISA
Staphylococcus aureus
Bacterial Culture
Staphylococcus aureus
Bacterial Culture
Staphylococcus aureus
Multiplex PCR
assay
Mycobacterium bovis
Brucella abortus
Multiplex PCR
assay
Salmonella and Listeria
monocytogenes
Immunomagnetic
separation Multiplex PCR
assay
(IMS-mPCR)
Comments
Farm milk contamination with L.
monocytogenes was a sporadic event. Low
numbers of L. monocytogenes in milk could
be due to environmental contamination.
Elisa titer values can be used to predict
seroconversion status in a herd and for a
geographical region
VIDAS Salmonella assay using a modified
sampling method compared favorably to the
conventional culture method.
Six of 268 (2.24%) of BTM positive for
Salmonella.
1.1% of BTM samples positive for
Salmonella.
12.6% milk line filters positive for
Salmonella.
Real-time PCR system allowed detection of
Salmonella in raw milk. The combination of
enrichment and real-time PCR techniques
yielded results in 24 h.
The ELISA test had a sensitivity and
specificity of 90%, 97% respectively.
S. aureus was isolated from 183 of 300 raw
milk samples at a Milk Cooperative in
Kenya. 72 of 97 (74.2%) of the isolates
produced one or more enterotoxins.
The mean counts of S. aureus in BTM
ranged from 5,900 to 12,000 cfu/ml. 45 of
105 (42.9%) isolates produced
Staphylococcal enterotoxin A, B, C, D or a
combination of these toxins.
The sensitivity of the multiplex PCR was
100 CFU/ml of skim milk. The assay
allowed presumptive identification and
differentiation of enterotoxigenic S. aureus
in less than 6 h.
The multiplex assay provides a highly
sensitive, cost effective and economically
viable alternative to serological testing.
IMS-mPCR was completed within 7 h. A
detection level of 10(3) cfu/ml was
achieved in the simultaneous detection for
both pathogens.
- 68 -
Reference
Meyer-Broseta
(2003)
Wedderkopp et al.
(2001)
Walker et al. (2001)
Murinda et al. (2002).
Warnick et al. (2003)
Van Kessel et al.
(2003)
Grove and Jones
(1992)
Ombui et al. (1992)
Adesiyun et al.
(1998)
Tamarapu et al.
(2001)
Sreevatsan et al.
(2000)
Li et al. (2000)
Table 2. continued …
Parasites and
Pathogens
Ostertagia ostertagi
Technique
ELISA
Mycobacterium avium
subsp.
paratuberculosis
IS900 PCR assay
Mycobacterium avium
subsp.
paratuberculosis
IS900 PCR assay
Mycobacterium avium
subsp.
paratuberculosis
Immunomagnetic
separation - PCR
assay (IMS-PCR)
Mycobacterium avium
subsp.
paratuberculosis
Bacterial Culture
PCR assay
Mycobacterium avium
subsp.
paratuberculosis
ELISA
Mycobacterium avium
subsp.
paratuberculosis
Immunomagnetic
separation – real
time PCR assay
(IMS-rPCR)
Comments
Indirect ELISA using an O. ostertagi crude
antigen was a useful technique for
monitoring gastrointestinal parasite burdens
in adult dairy cows and perhaps could be
used as a potential predictor of response to
anthelmintic treatment.
273 (19.7%) of the 1384 BTM samples
were positive by IS900 PCR-assay. The
prevalence of M. paratuberculosis varied
considerably for different regions of
Switzerland.
M. paratuberculosis can be detected
directly from quarter milk and bulk tank
milk by IS900 PCR.
IMS-PCR correctly identified 97. 5% of
milk samples (sensitivity 100%, specificity
95%), including spiked milk samples.
Conventional IS900 PCR correctly
identified only 72.5% of the same 40 milk
samples (sensitivity 23%, specificity
100%).
Milk samples all cultured negative, but
analysis of milk samples by PCR resulted in
68% of herds positive for M.
paratuberculosis DNA including 24 of 31
herds with positive fecal cultures and 11 of
21 herds with negative fecal cultures.
Contamination of bulk tank milk samples
with M. paratuberculosis does occur in
seropositive herds, even in some with
negative fecal cultures.
The technical performance of the ELISA
was not sufficient to provide a tool for
surveillance.
IMS-rPCR allowed detection of M.
paratuberculosis from milk. Ten or fewer
M. paratuberculosis organisms were
consistently detected in milk (2-ml).
- 69 -
Reference
Sanchez et al (2002)
Corti and Stephan
(2002)
Pillai and Jayarao
(2002)
Grant et al (2000)
Stabel et al (2002)
Nielsen et al (2000)
Khare et al (2004)
Table 3. Use of BTM for determination of milk quality and herd udder health
Pathogens in BTM
Technique
S. agalactiae
Bacterial culture
Mycoplasma
Nested PCR assay
Milk Quality and Herd
Udder health
Bacterial Culture
Mycoplasma
Bacterial Culture
Streptococci and
Streptococci like
organisms
Bacterial Culture
Bacterial counts
Bacterial Culture
Trichosporon beigelii
Culture
Staphylococcus aureus
RPLA
ELISA
Culture
Staphylococcus aureus
Culture
ELISA
Comments
BTM milk samples need to be analyzed
frequently to identify herds with S.
agalactiae infection.
100% sensitivity; 99.8% specificity.
Bacterial and somatic cell count (SCC)
estimation of BTM when interpreted within
the context of the farm's management
practices provided a basis for evaluating
current and potential milk quality and
mastitis problems in a herd.
Milk quality components were not strongly
related to the presence of Mycoplasma spp.
in BTM. The presence of other contagious
mastitis pathogens was also not related to
the presence of Mycoplasma.
Edwards modified medium supplemented
with colistin sulfate (5 mg/L) and oxolinic
acid (2.5 mg/L) was evaluated using BTM
samples, the sensitivity and specificity of
this medium was observed to be 100 and
87.5%, respectively.
The bacterial composition of BTM milk (n=
13 farms) was tested over a 2-wk period to
study sudden elevations ("spikes") in the
total bacterial count. Twenty standard plate
count spikes were observed. S. uberis was
the predominant organism in 11 spikes.
T beigelii could be associated with high
counts in BTM.
TSST was detected in 25 of 43(58.1%)
isolates from clinical mastitic cow’s milk,
79 of 103(76.7%) isolates from subclinical
mastistic cow’s milk and 85 of 126(67.4%)
of farm bulk milk.
37.5% of the isolates of mastitic samples
isolates were S. aureus, none of these
isolates encoded for enterotoxins or TSST1. 4 of 13 isolates and 6 of 20 isolates from
Washington State and Korea expressed
enterotoxins, respectively. Isolates from
different geographical locations varied
considerably.
- 70 -
Reference
Andersen et al.
(2003)
Baird et al. (1999)
Jayarao and
Wolfgang (2003)
Fox et al. (2003)
Sawant et al. (2002)
Hayes et al. (2001)
Gonzalez et al.
(2001)
Takeuchi et al.
(1998)
Lee et al. (1998)
Table 3. continued…
Mastitis pathogens
and milk quality
indicators
Technique
Somatic Cells
Antibiotic Residues
Epidemiological
analysis of monthly
official state
regulatory data
Polymorphonuclear
Neutrophils (PMN)
IR analysis
Gram Negative Bacteria
Bacterial Culture
Mycoplasma spp.
Bacterial Culture
Bacterial Counts
Bacterial Culture
Prototheca zopfii
Bacterial Culture
Staphylococcus aureus
Streptococcus agalactiae
Streptococcus uberis
Streptococcus dysgalactiae
Detection of Gram + / Bacteria
Staphylococcus aureus
Streptococcus agalactiae
Mycoplasma
Multiplex PCR
Flow cytometry
Multiplex PCR
Comments
SCC values were significantly higher
for samples with positive antibiotic
residue tests for grade A milk during all
4 yr tested. The SCC values were
significantly higher for samples with
positive antibiotic residue tests for
grade B milk for 3 of 4 yr.
the concentration of PMN may be a
useful indicator of herd status in bulk
tank monitoring schemes.
Examination of BTM for coliforms and
non-coliform bacteria could provide an
indication of current and potential
problems associated with milk quality
Mycoplasma bovis (243/499; 48.6%)
was the most commonly isolated
species. Distribution of Mycoplasma
spp. varied by year, number of colonies
isolated per sample, season, and herd.
Bacteriological counts increased at
each stage as the milk passed through
the milking machine. A strong
correlation (0.98) between total and
streptococcal counts of the bulk milk
was observed.
New selective Prototheca enrichment
method was developed. Prototheca spp.
were recovered from 28 of 787 BTM
samples.
Regular analysis of BTM using the
multiplex PCR assay could be a useful
tool for monitoring S. agalactiae, but
was of less value for monitoring other
organisms.
BTM inoculated with Staphylococcus
aureus and Escherichia coli can be
detected using flow cytometry without
precultivation.
The sensitivity and specificity of the
multiplex PCR for detection of M.
bovis was 89%, 97%; S. aureus-67%,
94%; S. agalactiae- 83%, 84%,
respectively.
- 71 -
Reference
Ruegg and Tabone
(2001).
Kelly et al. (2000)
Jayarao and Wang
(1999)
Kirk et al. (1997)
McKinnon et al.
(1990)
Pore et al. (1987)
Phuektes et al. (2003)
Holm and Jespersen
(2003)
Tmanova Lyubov
(2003). MS Thesis
The Pennsylvania
State University
Table 4. Use of BTM for detection of viruses of dairy cattle
Viruses in BTM
Technique
Bovine Viral Diarrhea Virus
Indirect ELISA
Bovine Leukosis Virus
ELISA
Bovine Leukosis Virus
ELISA
Foot and Mouth Disease Virus
Liquid phase
blocking ELISA
Bovine Viral Diarrhea Virus
RT-PCR assay
Bovine Herpes Virus-1
ELISA
Bovine Viral Diarrhea Virus
Virus isolation
RT-PCR
Bovine Viral Diarrhea Virus
Bovine Herpes Virus 1(BHV-1)
Bovine Respiratory Syncytial
Virus (BRSV)
Bovine Corona Virus (BCV)
ELISA
Bovine Viral Diarrhea Virus
PCR assay
Virus isolation
Comments
Reference
The proportion of positive herds in
Chile ranged from 71.2 to 83%.
BTM samples were tested for BLV
for the purpose of eradication and
monitoring of BLV in Finland.
Examination of pooled milk samples
with the ELISA provided a reliable,
practical, and economic procedure
for identification of BLV-infected
herds.
Significant overall correlation
(R=0.53; n=624) was obtained
between serum titers and milk IgG
(1) results derived from the modified
specific isotype assay (SIA).
RT-PCR had high sensitivity and can
be used as an alternate technique to
current standard methods for
detecting BVDV from BTM.
The BHV-1 blocking ELISA on
BTM allowed detection of
seropositive herds.
RT-PCR was superior to virus
isolation with respect to sensitivity,
specificity, and turnaround time.
Both methods are recommended for
successful detection of the virus.
65 % of the herds had a high level of
BTM antibody suggestive of recent
infection with BVDV 69% of the
herds were BHV-1 antibody-positive
and all the herds tested were antibody
positive to BRSV and BCV.
PCR assay was 14.6 times more
sensitive than virus isolation in
detecting BVDV RNA in purified
milk somatic cells. BVDV RNA was
detected in 33 of 136 BTM samples.
Melendez and
Donovan (2003)
- 72 -
Nuotio et al. (2003)
Gutierrez et al.
(2001)
Armstrong and
Matthew (2001)
Scheibner et al.
(2000)
Nylin et al. (1999)
Renshaw et al. (2000)
Paton et al. (2000)
Radwan et al. (1995)
Table 5. Use of BTM for detection of trace minerals, toxins, off flavors and metabolites.
Analytes and Devices
Selenium
Technique
Atomic Absorption
Spectophotometry
Aflatoxin M (1)
Liquid
Chromatography
Bulk Tank Milk Urea
Nitrogen (BTMUN)
Infra red analysis
Off-flavors
Organoleptic
analysis
Salmonella Typhimurium
Microelectrode
impedance sensor
Beta-lactams
Surface plasmon
resonance (SPR)
biosensor
Progesterone
Surface plasmon
resonance (SPR)
biosensor
Staphylococcal enterotoxin B
Surface plasmon
resonance (SPR)
biosensor
Comments
Selenium concentration in BTM
could be related to levels of SE in
feed.
78% of BTM samples had >5ng/L
aflatoxin M (1). None of the samples
exceeded EU limit of 50ng/L.
The BTMUN had good correlation
with weighted average of the
individual cow MUN levels (CC=
0.91).
Poor air quality in the lactating cows’
barn, baled silage as the main forage,
as well as feeding roughage before
milking was significantly associated
with the incidence of transmitted
flavors in BTM.
The test could detect 4.8 and 5.4 x
105 CFU/ml of Salmonella in 9.3 and
2.2 h, respectively. The technique
allowed detection of 1 bacterial
cell/sample.
The results of the 2 biosensor assays
showed good agreement with those
of the other assays including
Delvotest SP, Penzym S, BetaSTAR, SNAP, and Parallux.
The assay could be used in-line in the
milk parlor and could be an
important tool for reproductive
management for detecting heat and
predict pregnancy in cattle.
The assay showed that SPR
biosensor may be a useful tool for
real-time analysis of toxin in foods.
- 73 -
Reference
Wichtel et al. (2004)
Roussi et al. (2002).
Arunvipas et al.
(2004)
Mounchili et al.
(2004)
Yang et al. (2004)
Gustavasson and
Sternesjo (2004)
Gillis et al. (2002)
Rasooly (2001)
PCR APPLICATIONS IN FOOD SAFETY RESEARCH
S. P. Oliver and B. E. Gillespie
Food Safety Center of Excellence and the Department of Animal Science
The University of Tennessee, Knoxville, TN
Introduction
Molecular techniques such as the polymerase chain reaction (PCR), multiplex PCR, and realtime PCR are very useful tools used frequently in many research laboratories in the United States
and throughout the world. Use of PCR-based techniques has facilitated the discovery of more
effective methods for the detection of foodborne pathogens associated with food-producing
animal environments and foodborne pathogens causing disease in humans. These techniques
have also been quite useful to delineate virulence factors as well as antimicrobial resistance genes
of important foodborne pathogens. The purpose of this communication is to briefly describe some
of the PCR techniques in use today, and to demonstrate how these molecular-based techniques
are used in our research approach on food safety and foodborne pathogens at The University of
Tennessee Food Safety Research Center of Excellence.
PCR
The PCR method is based on in vitro amplification of target DNA sequences and involves the
application of primers (carefully selected oligonucleotides) and heat stable Taq DNA polymerase.
With PCR, it is theoretically possible to detect pathogens in the sample (food or environmental)
directly – but so far this has been done in very few investigations. Major disadvantages of this
technique are the inability to distinguish between live and dead cells, the presence of polymerase
inhibitors in test samples, and the inability of the method to facilitate further identification. Preenrichment of test samples overcomes most of these problems and is presently needed for
detection of specific pathogens in food by PCR. Conventional PCR formats are limited in that
they only provide for detection of a single pathogen. However, many clinical diseases manifest in
a nonspecific or syndromic fashion thereby necessitating the simultaneous assessment of multiple
suspect pathogens.
Multiplex PCR
Multiplex PCR can be used for specific identification and profiling of several gene sequences
from the same pathogen or from a mixture of pathogens simultaneously. The major advantage of
multiplex PCR over conventional PCR methods is its cost-effectiveness. It reduces the amounts
of reagents such as Taq DNA polymerase used in each assay. It is more applicable to routine
diagnostic use and automation and requires less preparation and analysis time than systems in
which several pathogens are analyzed individually. In many cases, more than 4 pairs of primers
can be used.
Real-Time PCR
Several types of fluorogenic probes have been used to quantitate bacteria in real-time such as
TaqMan and molecular beacons (MBs). TaqMan differs from MBs in that it has no “hairpin”
structure and exploits the 5’ - 3’ nucleolytic activity of Taq DNA polymerase to cleave a reporter
- 74 -
dye, such as fluorescein, from the 5’ end of a labeled linear probe that has hybridized downstream
from the forward PCR primer. When this cleavage occurs, the reporter dye is separated from the
quencher and fluorescence occurs. Common reporters (5’ end) for TaqMan are 6carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein, or hexachloro-6-carboxyfluorescein, with the quencher dye 6-carboxytetramethylrhodamine (TAMRA) attached 2 or
more bases downstream from the reporter. The proximity of the reporter and quencher reduces
emission intensity of the reporter until the reporter is cleaved.
Molecular beacons (MBs) are “hair-pin” oligonucleotide probes that fluoresce upon hybridization
with complimentary sequences. MBs have been used to construct probes that are useful for realtime detection of nucleic acids of target pathogens. MB probes are based on single-stranded
nucleic acid molecules that possess a stem-and-loop structure. The loop portion contains a
sequence complimentary to a target gene sequence; the stem is formed by annealing of two
complimentary arm sequences that are not related to the target gene sequence. A fluorescent
moiety is attached to the end of one arm and a non-fluorescent quenching moiety is attached to
the other end. When the two moieties are close together no fluorescence is produced due to the
quenching effect. However, when the probe is in proximity to a single stranded target
oligonucleotide, it hybridizes with the target and undergoes a spontaneous conformational
change that forces the moieties apart resulting in fluorescence.
The interaction of TaqMan probes and MBs with their targets is extraordinarily specific. As target
strands synthesized in PCR accumulate, the fraction probe bound to targets increases causing a
brighter fluorescence. Measurement of fluorescence permits simultaneous quantitation and
monitoring progress of the reaction in real-time in the PCR tube. MBs are more sensitive than
TaqMan since they can distinguish single base differences thus allowing multiple allele
discrimination in the same reaction. TaqMan has been applied to detect food pathogens like
Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7. Recently, several studies have
successfully applied MBs in a variety of PCR reactions for study of pathogens like
enterohemorrhagic E. coli and Salmonella and as few as 2 colony forming units (CFU) can be
detected with MB-based PCR assays.
Analysis of fluorescence data recorded at each annealing stage using real-time PCR gives a clear
profile of the amplification process. The critical cycle (Ct), which is the cycle at which a
significant increase in fluorescence is first recorded, increases as the initial number of template
DNA molecules decreases. Samples containing low concentrations of template DNA would
require more PCR cycles to replicate enough copies to produce a significant fluorescence signal.
The Ct is inversely proportional to the logarithm of the initial number of target molecules. These
data can be used to formulate a standard quantitation curve for detection of a specific pathogen.
Detection of Foodborne Pathogens and Virulence Factor Genes Using Different PCR Formats
The need for rapid, sensitive and reproducible techniques for bacterial strain identification is
evident in many areas of public health, agriculture, and national security. Bacterial detection
methods for differentiating bacterial species and strains are based on both phenotype and
genotype. Techniques based on phenotype, such as metabolic studies, serotyping and
immunological methods, are not specific enough to completely distinguish among different
genera, species and strains of bacteria and are not general enough to apply to a diverse set of
pathogens. Additionally, genes may not be expressed under certain cultural conditions. Methods
based on the genotype examine differences in DNA sequences and are much more successful in
- 75 -
discriminating among different bacterial strains. The most definitive methods in use for bacterial
subtyping are restriction fragment length polymorphism (RFLP), PCR-based methods and
ribotyping. PCR-based methods require only small quantities of DNA, whereas RFLP requires
relatively large amounts of DNA. Unique bacterial DNA-sequences (chromosomal and/or
plasmidal) can be used for detection to the genus and in many cases to the species level.
Current methods used for routine identification and confirmation of foodborne pathogens such as
Campylobacter, Salmonella spp., Listeria monocytogenes and Shiga toxin-producing E. coli (STEC;
O157 and non-O157 STEC) are generally slow, inadequate, laborious and non-existent in the
case of critical detection of pathogens like non-O157 STEC. Many clinical laboratories do not
routinely report non-O157 STEC serotypes since they cannot easily identify these
microorganisms. Conventional diagnostic methods are often too cumbersome and timeconsuming to be useful for timely monitoring of foods, especially those with limited shelf-lives.
Rapid detection methods could be used effectively for quality control in food processing facilities
to rapidly screen incoming ingredients and raw materials. Rapid detection methods allow: (1)
timely monitoring of food processing equipment and the immediate environment, (2) brisk
corrective action (product recall and release of lots/batches of product for distribution), and (3)
faster intervention in the case of threats of disease or potential death, without having to wait
several days for results, as in the case of most current microbiological methods.
Shiga toxin-producing E. coli are of immense economic and public health significance. STEC
O157:H7 are characterized by low infectious doses (1-100 colony-forming units) and are highly
pathogenic in humans where they cause serious acute illness and long-term sequelae.
Manifestations of illnesses caused by STEC that are linked to production of Shiga toxins include,
non-bloody diarrhea, diarrhea-associated hemorrhagic colitis, hemolytic uremic syndrome (HUS)
and thrombotic thrombocytopenic purpura. Intimin and enterohemolysin are among the
prominent ancillary virulence factors elaborated by STEC. There is a general consensus that
ruminants are the main source of human pathogenic STEC. An array of food products that
include, beef, apple cider, salad, fruits and contaminated well water have been implicated in
foodborne disease involving STEC. Sufficient evidence has been presented on the zoonotic nature
of bacterial enteric pathogens and the role companion animals play as reservoirs of some human
pathogenic STEC serotypes.
We used a multiplex PCR format to identify E. coli O157:H7 strains that target common
virulence genes encoding Shiga toxin 1 and 2 (stx1 and stx2), enterohemolysin (hly933), intimin
(eaeA), and flagellar H7 (flicCh7) gene sequences (Murinda et al., 2002; 2004a; 2004b; 2004d). The
objective of this study was to characterize 400 E. coli isolates from dairy cows/feedlots, calves,
mastitis, pigs, dogs, parrot, iguana, human disease and food products for prevalence of STEC
virulence markers. The rationale of the study was that isolates of the same serotype that were
obtained from different sources and possessed the same marker profiles could be cross-species
transmissible. Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and
Stx2) and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle
and food) isolates were stx-positive. Sixty-five percent of O157 STEC produced both Stx1 and
Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had
sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128
and 4 of 10 O26 were stx-positive. Five of 6 stx-positive O26 and O111 produced Stx1, however,
stx-positive O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were
common attributes of O157 STEC, whereas, non-O157 stx-positive strains exhibited 38.5% and
30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and
cattle, whereas, all isolates from mastitis (n=105), and pigs, dogs, parrot and iguanas (n=48) were
- 76 -
stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles
and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease
shared similar toxigenic profiles, whereas isolates from other disease sources had few
characteristics in common with the former isolates. These data suggest interspecies
transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle.
We have also used a multiplex PCR format to confirm and identify Campylobacter jejuni isolated
from the dairy farm environment and from dairy cows (Murinda et al., 2004c). Campylobacter is
a leading cause of bacterial foodborne illness in the USA and in many other industrialized
countries. This organism is widespread in nature and can be isolated from the gastrointestinal
tracts of many animal species, including poultry, freshwater and bulk tank milk. The ubiquity and
low infectious dose of Campylobacter makes its presence in the food supply a significant health
hazard. It is therefore important to have accurate and reliable methods for isolation and detection
of Campylobacter spp. in particular C. jejuni, which is the most common species associated with
acute bacterial enteritis. The major disadvantages of the commonly used phenotype-based typing
schemes, such as biochemical tests, including serology, are that they are time-consuming,
technically demanding and may lead to a high number of untypable strains. Consequently, there
is an increasing need for highly sensitive and reliable DNA-based methods for typing C. jejuni.
Targets that we used for identification of C. jejuni were the hippuricase gene (hip) and a 23S
rRNA gene specific for thermophilic Campylobacter. All 265 bulk tank milk samples analyzed
were negative for C. jejuni, whereas, 5 of 411 (1.2%) fecal samples tested positive. This is the first
report that has used a combination of sequences of the two genes in a multiplex format to identify
C. jejuni to the species level. The method described has potential for routine use in the detection of
thermophilic Campylobacter in farm environmental samples as well as other samples. This
multiplex PCR assay can decrease the time for identification and confirmation of C. jejuni.
PCR-ELISA
Molecular techniques can also be utilized to serogroup bacterial isolates. We used a polymerase
chain reaction-based enzyme linked immunosorbent assay (PCR-ELISA) to identify Salmonella
somatic groups B, C1, C2, D and E1 (Gillespie et al., 2003). Salmonella are important foodborne
pathogens that are responsible for serious cases of foodborne illness. Salmonella may be
transmitted by a wide variety of agricultural products and processed foods. Foods of animal
origin such as beef, pork, chicken, eggs, and milk have been shown to carry these pathogens.
Salmonellosis is commonly diagnosed in dairy cows and calves, and the presence of Salmonella
on dairy farms has been well documented. Several serogroups of this bacterium occur with
varying degrees of relevance to human and animal health. Identification of Salmonella is
important for surveillance, prevention, and control of foodborne diseases. An accurate and rapid
procedure for identification of Salmonella is needed to identify sources, reservoirs, and transfer of
these foodborne pathogens through the food chain. However, there are many problems
associated with differentiating Salmonella species, subspecies and serovars. Current available
screening tests only provide presumptive identification of Salmonella as a group without
identification of serogroups. Negative results are considered definitive, but positive results must
be confirmed by conventional methods and serology.
The concept of targeting gene sequences that encode for species specificity is promising. In
Salmonella, the rfb gene clusters are responsible for biosynthesis of the O antigens of Salmonella
lipopolysaccharide. Variations among different O antigen structures are manifested in the types of
sugar present or arrangement of sugars. This variability provides the basis for serotyping
Salmonella into serogroups. This highly polymorphic rfb gene cluster has been targeted as a
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molecular marker for the organism for detection of Salmonella serovars. In our study (Gillespie et
al., 2003), a PCR-ELISA procedure was developed to identify Salmonella serogroups A, B, C1,
C2 and D. Primers were selected from the rfb gene cluster, which is responsible for biosynthesis of
O antigens of Salmonella lipopolysaccharide. Previously serogrouped Salmonella isolates (n=169)
were evaluated by the PCR-ELISA procedure. DNA from all isolates was amplified using the
PCR procedure for selected somatic groups and subjected to the ELISA procedure. This
technique correctly identified 93% of Salmonella isolates belonging to somatic groups B, C1, C2,
D and E1. The sensitivity of this procedure to correctly identify Salmonella somatic groups was
96% and the specificity was 98%. Utilization of this procedure circumvents the need to have
Salmonella isolates serogrouped state or regional reference laboratories.
Real-Time PCR
We have dedicated much time and many resources attempting to develop real-time PCR
techniques for detecting pathogens directly from milk. Real-time PCR is a relatively new DNAbased technique that monitors amplification of target DNA in real-time by monitoring
florescence. Real-time PCR can be used to quantify bacteria from various samples including milk,
feces, food and water. Real-time PCR can be used for processing, detecting and confirming
pathogens in multiple samples at one time in a 96-well plate format. Additional post-detection
methods are not utilized, therefore, eliminating potential cross-contamination that may occur
after amplification.
A multiplex real-time PCR method for simultaneous detection of Staphylococcus aureus,
Streptococcus agalactiae and Streptococcus uberis directly from milk has been developed (Gillespie
and Oliver, 2004). These three mastitis pathogens frequently cause mastitis in dairy cows
throughout the world. Targets that we used for the multiplex real-time PCR were a Staph. aureus
specific genetic marker, the cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP)
factor for Strep. agalactiae, and the plasminogen activator gene for Strep. uberis. A total of 192
quarter milk samples were analyzed by the multiplex real-time PCR assay and conventional
microbiological techniques. This technique correctly identified 97.7% of all quarter milk samples
and correctly identified 91% of Staph. aureus, 98% of Strep. agalactiae and 100% of Strep. uberis.
The overall sensitivity of this procedure to correctly identify Staph. aureus, Strep. agalactiae and
Strep. uberis directly from milk was 95.5% and the specificity was 99.6%. Using an enrichment
step, the detection limit was one colony forming unit/ml. No cross-reactivity was detected with
53 American Type Culture Collection reference strains representing common mastitis pathogens.
Results of this study indicate that the multiplex real-time PCR procedure is a rapid and accurate
method for identification of Staph. aureus, Strep. agalactiae and Strep. uberis directly from milk.
Manipulation of this multiplex real-time PCR method could be done to include additional or
other frequently encountered pathogens found in milk including foodborne pathogens.
We have also developed real-time PCR methods for identification of foodborne pathogens in
food, dairy environmental samples and dairy cows. Real-time PCR assays utilizing dual labeled
probes have been developed to identify E. coli O157:H7 and L. monocytogenes from beef products
(Nguyen et al., 2004). Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA,
respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were
specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was
positive for (9/10) E. coli O157:H7 and all L. monocytogenes (7/7) strains evaluated. Detection
sensitivity ranged from 103 to 104 CFU/g of raw ground beef or hotdog without enrichment for
E. coli and L. monocytogenes. Approximately 1.4 - 2.2 CFU/g of E. coli O157:H7 in raw ground
beef were detected following an enrichment step of 4 hours. Approximately 1.2 – 6.0 CFU/g of
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L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 hours. Realtime PCR assays used in this study for detection of E. coli O157:H7 and L. monocytogenes in raw
ground beef and beef hotdogs were specific, sensitive, rapid and appear promising.
We have also used SYBR Green in real-time PCR assays to detect C. jejuni from dairy farm
environmental samples (Nam et al., 2004b). The melting temperature (Tm) obtained for C. jejuni
was 77.5°C. The detection limit for samples spiked with C. jejuni was >103 CFU/ml. However,
after a 24 hour enrichment step, the detection limit for samples spiked with C. jejuni was <10
CFU/ml. Eighty-two dairy farm environmental samples including fecal slurry, feed/silage,
lagoon water, drinking water, bulk tank milk, farm soil, and bedding material were analyzed. The
SYBR Green real-time PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of
these samples being culture positive for C. jejuni. All samples that were positive by standard
culture methods were also positive by the SYBR Green real-time PCR assay.
A real-time PCR method utilizing SYBR Green I dye and a 119-bp fragment of the invA gene
was evaluated for detection of Salmonella spp. in dairy farm environmental samples (Nam et al.,
2004a). A total of 240 bacterial strains were evaluated including 124 Salmonella spp. type strains
and 116 non-Salmonella strains. Only the Salmonella strains tested positive for the invA gene by
analyzing the melting temperature (Tm = 79oC) of the amplicon. The detection limit from spiked
environmental samples was 103 to 104 CFU/ml in broth without enrichment. The detection limit
was reduced to <10 CFU/ml in broth after 18 hours of enrichment.
Detection of Antimicrobial Resistance Genes in Veterinary and Foodborne Pathogens
Antimicrobials are used extensively in food-producing animals to combat disease and to improve
animal performance. On dairy farms, antimicrobials such as tetracyclines, penicillins, and
sulfonamides are used to treat or prevent diarrhea and pneumonia, both of which are important
diseases in dairy calves. Antimicrobials such as penicillins, cephalosporins, erythromycin and
oxytetracyclines are used for treatment and prevention of mastitis, an important disease caused
by a variety of Gram-positive and Gram-negative bacteria. Such drugs are often administrated
routinely to entire herds to prevent mastitis during the nonlactating period. Benefits of antibiotic
use in animal production systems include improved growth and/or feed efficiency, decreased
nitrogen excretion and thus reduced environmental impact, decreased pathogen loads, and a
lower incidence of disease.
In contrast to the above benefits, however, are suggestions that agricultural use of antibiotics may
be partly responsible for the emergence of antimicrobial resistant bacteria, which in turn may
decrease the efficacy of similar antimicrobials used in human medicine. While investigations
have focused on emergence of drug resistant bacteria, persistence of resistant bacteria and effects
on human medicine, little information is available with regard to antimicrobial resistance of
commensal bacteria and veterinary and foodborne pathogens on dairy production facilities, or
management conditions that affect antimicrobial resistance. Information on prevalence of
antimicrobial resistance, effects of stressors on the host animal, and the effect of management and
environment at the farm level are especially lacking. Furthermore, much of the current available
antimicrobial resistance data is derived from evaluating clinical isolates originally obtained from
sick animals. Consequently, this information may be biased by several factors, including housing
or husbandry conditions, age and condition of animals tested, and previous antibiotic therapies.
Because transferable resistance may originate from a variety of bacteria and associated hosts
under a number of conditions, it is important that confounding factors are characterized so that
more definitive conclusions can be derived.
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The objective of our research is to gain insight into antimicrobial resistance gene flow from
commensal bacteria of dairy farms to animal and human pathogenic bacteria. There is only
limited information on rates and extent of gene exchange from commensal bacteria to animal and
human pathogenic bacteria. We hope to define in detail the predominant resistance constructs in
bacterial populations of dairy cows and their environment; and identify reservoirs, how
antimicrobial resistance is transferred, and the relationships of antimicrobial use with
development of antimicrobial resistance. Molecular tools such as DNA probes and PCR-based
detection systems have greatly facilitated the study of the epidemiology of antimicrobial
resistance genes and mobile genetic elements (MGE) and their transfer to other bacteria at the
genetic level. Antimicrobial resistant commensal bacteria of dairy farms may play a pivotal role
in the spread of antimicrobial resistance to pathogens that can cause disease in humans and
animals. Since dairy cows are treated with many antimicrobial compounds for prevention and
control of different diseases, commensal bacteria of cows and bacteria normally found in the
dairy farm environment may acquire antimicrobial resistance.
We have used PCR to detect several different antimicrobial genes in a variety of veterinary and
foodborne pathogens (Murinda et al., 2004e; Srinivasan et al., 2004a; 2004b; 2004c).
Campylobacter jejuni (n=39), Listeria monocytogenes (n=38) and Salmonella spp. (n=12) isolated from
dairy farms were evaluated for antimicrobial resistance gene patterns (Srinivasan et al., 2004a).
All foodborne pathogens were screened for 21 antibiotic resistance genes using PCR.
Campylobacter jejuni (5.1%), L. monocytogenes (31.6%) and Salmonella spp. (100%) contained more
than one antibiotic resistance gene. Tetracycline resistant determinant (tetA) was found in 15.4%,
31.6% and 100% of C. jejuni, L. monocytogenes and Salmonella spp., respectively; tetC was found
only in Salmonella spp.; and tetB, tetC, tetE, and tetG were not detected in any of the foodborne
pathogens evaluated. The only other antimicrobial resistance gene detected in at least one isolate
of each of the foodborne pathogens evaluated was sulI. A high frequency of floR (65.8%), penA
(36.8%), and strA (34.2%) was found in L. monocytogenes. In Salmonella spp., strA (100%), strB
(83.3%), sulI (100%), ermB (58.3%) and penA (50%) were amplified and all Salmonella spp. were
multi-drug resistant. Results of this study indicate that a high prevalence of foodborne pathogens
isolated from the dairy farm environment contain antimicrobial resistance genes. The potential
exists for foodborne pathogens carrying antimicrobial resistance genes to acquire additional
resistance genes as well as to spread this genetic material to commensal and pathogenic bacteria
in the dairy farm environment. In another study (Srinivasan et al., 2004b), antimicrobial
resistance gene patterns of 131 E. coli isolated from dairy cows with clinical mastitis were
evaluated. All E. coli contained more than one antimicrobial resistance gene. Tetracycline
resistance determinants, tetA and tetC, were found in 8.4% and 64.1% of isolates, respectively.
Other tetracycline resistant determinants (tetB, tetD, tetE and tetG) were not observed in any of
the isolates studied. Even though many E. coli carried the tetC gene, they were sensitive to
tetracycline. Thus, tetracycline MIC data were negatively correlated with the presence of the tetC
gene and positively correlated with the presence of tetA genes. Streptomycin resistance genes strA
(7.6%) and strB (9.9%) and streptomycin-spectinomycin adenyltransferase gene (aadA) were
found in 77.9% of test isolates. Ampicillin resistance gene (ampC) was the predominant gene
(94.7%) found in E. coli from cows with mastitis. Over 99% of E. coli were resistant to ampicillin
and this correlated with the presence of the ampC gene. Other resistance genes, penA (49.6%),
sulI (9.9%) and sulII (8.4%) were observed by PCR. Vancomycin resistance gene, vanA, was
found in most E. coli (94.7%) but vanB was not present in any of the E. coli evaluated. Only one of
131 E. coli carried the floR gene. None of the isolates carried cmlA, aac(3)IV, ermB, ereA or ereB.
In conclusion, all E. coli from cows with mastitis were multi-drug resistant and carried more than
one antimicrobial resistance gene. Escherichia coli causing bovine mastitis may be a reservoir for
antimicrobial resistance genes and may play a role in dissemination of antimicrobial resistance
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genes to other pathogenic and commensal bacteria in the dairy farm environment. However,
further research is necessary to substantiate this hypothesis.
Enterobacteriaceae isolated from soil samples using standard isolation protocols were screened
for antimicrobial resistance (Srinivasan et al., 2004c). Among 36 bacteria isolated from different
soils, tetA was found in 2 isolates (5.6%), tetB in one isolate (2.8%), floR in 5 isolates (13.9%), strA
in 27 isolates (75%), strB in one isolate (2.8%), and no isolates carried cmlA. The prevalence of
antimicrobial resistance genes was four-fold higher in bacteria isolated from the soil of dairy
farms than in bacteria isolated from non-agricultural soils. About 50% of the bacteria isolated
from dairy farm soils were multi-drug resistant and carried more than one antimicrobial
resistance gene. These soil bacteria could serve as a reservoir for antibiotic resistance genes in the
environment and spread to other environmental sources through vertical or horizontal gene
transfer mechanisms. PCR amplification of antimicrobial resistance genes in soil metagenomic
DNA revealed that all dairy farm soils carried all antimicrobial resistance genes in different
combinations whereas only one ‘virgin’ non-agricultural soil carried only floR.
Conclusions
PCR-based techniques are very useful tools to study a variety of complex phenomenon and are
used frequently in many research laboratories throughout the world. Use of PCR-based
techniques have facilitated the discovery of more effective methods for the detection of foodborne
pathogens associated with food-producing animal environments and foodborne pathogens
causing disease in humans. These techniques have also been quite useful to delineate virulence
factors and antimicrobial resistance genes of several important foodborne pathogens
The challenges to providing a safe and nutritious food supply are complex because all aspects of
food production – from farm to fork – need to be considered. Given the considerable
national/international demand for food safety and the formidable challenges of producing and
maintaining a safe food supply, food safety research and educational programs has taken on a
new urgency. As the system of food production and distribution changes, the food safety system
needs to change with it. A strong science-based approach that addresses all the complex issues
involved in continuing to improve food safety and public health is necessary to prevent foodborne
illnesses. Not only must research be conducted to solve complex food safety problems, results of
that research must be communicated effectively to producers and consumers. Research and
educational efforts identifying potential on-farm risk factors will better enable dairy producers to
reduce/prevent foodborne pathogen contamination of dairy products leaving the farm.
Identification of on-farm reservoirs could aid with implementation of farm-specific pathogen
reduction programs. Foodborne pathogens, mastitis, milk quality and dairy food safety are
indeed all interrelated. A safe, abundant and nutritious milk and meat supply should be the goal
of every dairy producer in the world.
References
Gillespie, B. E., A. G. Mathew, F. A. Draughon, B. M. Jayarao, and S. P. Oliver. 2003. A PCRELISA technique for detection of somatic group-specific Salmonella spp. J. Food Prot. 66:23672370.
Gillespie, B. E., and S. P. Oliver. 2004. Simultaneous detection of Staphylococcus aureus,
Streptococcus agalactiae and Streptococcus uberis in milk by multiplex real-time polymerase chain
reaction. (In preparation).
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Murinda, S. E., L. T. Nguyen, S. J. Ivey, B. E. Gillespie, R. A. Almeida, and S. P. Oliver. 2002.
Prevalence and molecular characterization of Escherichia coli O157:H7 in bulk tank milk and fecal
samples from cull dairy cows: a 12-month survey of dairy farms in East Tennessee. J. Food Prot.
65:752-759.
Murinda, S. E., L. T. Nguyen, H. M. Nam, R. A. Almeida, S. J. Headrick, and S. P. Oliver.
2004a. Detection of Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter
jejuni, and Salmonella spp. in dairy farm environmental samples. Foodborne Pathogens & Disease
1(2) 97-104.
Murinda, S. E., S. D. Batson, L. T. Nguyen,B. E. Gillespie, and S. P. Oliver. 2004b. Phenotypic
and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26
strains from North America. Foodborne Pathogens & Disease 1(2):125-135.
Murinda, S. E., L. T. Nguyen, and S. P. Oliver. 2004c. Problems in isolation of Campylobacter
jejuni from frozen-stored raw milk and bovine fecal samples: genetic confirmation of isolates by
multiplex PCR. Foodborne Pathogens & Disease 1(3):166-171.
Murinda, S. E., L. T. Nguyen, T. L. Landers, F. A. Draughon, A. G. Mathew, J. S. Hogan, K.
L. Smith, D. D. Hancock, and S. P. Oliver. 2004d. Comparison of Escherichia coli isolates from
humans, food, farm and companion animals for presence of Shiga-toxin producing Escherichia coli
virulence markers. Foodborne Pathogens & Disease 1(3):178-184.
Murinda, S. E., P. D. Ebner, L. T. Nguyen, A. G. Mathew, and S. P. Oliver. 2004e. Class 1
integrons, antimicrobial and acid resistance in pathogenic Escherichia coli isolates from dairy cow
mastitis milk and feces J. Appl. Microbiol. (Submitted).
Nam, H. M., V. Srinivasan, B. E. Gillespie, S. E. Murinda, and S. P. Oliver. 2004a. Application
of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm
environmental samples. Int. J. Food Microbiol. (Accepted).
Nam, H. M., V. Srinivasan, S. E. Murinda and S. P. Oliver. 2004b. Specific detection of
Campylobacter jejuni in dairy farm environmental samples using SYBR Green real time PCR.
Foodborne Pathogens & Disease (Submitted).
Nguyen, L. T., B. E. Gillespie, H. M. Nam, S. E. Murinda, and S. P. Oliver. 2004. Detection of
Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain
reaction. Foodborne Pathogens & Disease (In press).
Srinivasan, V., H. M. Nam, L. T. Nguyen, B. Tamilselvam, S. E. Murinda, and S. P. Oliver.
2004a. Prevalence of antibiotic resistance genes and integrons in foodborne pathogens isolated
from dairy farms. In: Proc. Natl. Mastitis Counc. pp. 369-370.
Srinivasan, V., B. E. Gillespie, L. T. Nguyen, M. J. Lewis, Y. H. Schukken, and S. P. Oliver.
2004b. Phenotypic and genotypic antibiotic resistance patterns of Escherichia coli isolated from
dairy cows with mastitis. In: Proc. Natl. Mastitis Counc. pp. 371-372.
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Srinivasan, V., B. Tamilselvam, H. M. Nam, L. T. Nguyen, and S. P. Oliver. 2004c. Prevalence
of antibiotic resistant bacteria and antibiotic resistance genes in soil from dairy farms. In: Proc.
Natl. Mastitis Counc. pp. 373-374.
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POLYMERASE CHAIN REACTION FOR DETECTION OF MYCOPLASMA BOVIS IN
CLINICAL SAMPLES
Suzanne Klaessig
Department of Population Medicine and Diagnostic Sciences
Cornell University, Ithaca, NY
Mycoplasmas are parasites widely distributed in nature. Hosts include mammals, plants, reptiles,
fish and insects. Over 180 species have been identified thus far (1). Mycoplasmas differ from
other bacteria of the class Mollicutes by their minute size (they are the smallest self-replicating
organisms), and lack of cell wall. They usually grow symbiotically with their host, often coinfecting with other pathogens. The use of molecular techniques, including the comparison of the
highly conserved 16S rRNA gene sequence, has greatly expanded the understanding of these
organisms. Mycoplasmas can exist both extra- and intracellularly, and have multiple, complex
methods of interaction with their host cells. The discovery of variable surface proteins (Vsps) on
the outer surface of the plasma membrane has led to many studies of the interaction of
mycoplasmas with the immune system. These Vsps may stimulate or help evade the host immune
system, and possibly mediate adherence to cells.
Mycoplasma bovis is the most important agent of bovine mycoplasmal mastitis, although at
least eleven other Mycoplasma and Acholeplasma species have been isolated from milk (2). It can
cause both chronic and acute infections, and like most mycoplasmas exhibits organ and tissue
specificity, infecting the mucosal surfaces of the respiratory and urogenital tracts, eye, alimentary
canal, mammary gland and joints. Cows can be infected at any age and lactational stage (2).
Mycoplasma is easily contagious by airborne transmission and direct contact, as well as via
milking machines or milker’s hands. In lactating cows, it can cause severe clinical mastitis that
resists treatment, and also chronic infections with little or no decrease in milk production. As few
as one-hundred colony-forming units (cfu) can cause intramammary infection (IMI) (3), and the
infected animal can shed the organism for prolonged periods of time. Because vaccines and
antibiotics are not available for treatment, current methods for controlling Mycoplasma include
culling, segregation and hygienic measures. Identification of the infected animals at an early stage
is critical to prevention of transmission in the herd.
Diagnosing M. bovis is most commonly done by culture and subsequent serology to
identify individual species. Mycoplasmas are notoriously slow to grow and difficult to culture.
Traditionally, very complex media have been used for culture, based on beef heart infusion,
including peptones, yeast extract and serum. These rich growth media have recently been found
to be inhibitory in some cases. Genomic work has shown that Mycoplasmas have few genes
involved in biosynthetic pathways, for example, both M. genitalium and M. pneumoniae lack all
genes involved in amino acid synthesis. When collecting milk samples for culture they must be
kept cool and plated promptly. Overgrowth by other species of bacteria can occur even when
antibiotic inhibitors are included in the media. Plates must be viewed daily, both to scan for
contaminants and to watch for the distinctive “fried egg” morphology of the Mycoplasma on solid
medium (2). Incubation and observation should continue for 7-10 days before plates are
considered negative, and false-negatives are common due to low numbers of organisms in the
sample, or the fragility of Mycoplasma itself. The use of DNA-based tests promises to be faster,
more sensitive and more specific.
The detection of Mycoplasma by polymerase chain reaction (PCR) is based on the in vitro
amplification of the highly-conserved 16S rRNA gene (3, 4). PCR can amplify the target DNA
sequence by as much as five orders of magnitude, thus potentially solving the two largest
problems dealing with Mycoplasma: early detection, and low numbers of organisms in the clinical
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samples. Detection levels as low as 5 cfu/ml in milk samples have been reported (3). Detection of
M. bovis from broth cultures or DNA extracts can be carried out on a routine basis, but detection
in milk presents a number of problems. The presence of high levels of protein, calcium, and
unidentified proteases has an inhibitory effect on amplification. Numerous approaches have been
investigated to increase the yield of amplifiable bacterial DNA (5).
We report our preliminary results of a PCR procedure for detection of M. bovis DNA
using the Pennsylvania State University Animal Diagnostic Laboratory protocol (6). Further
work will include comparing methods of DNA extraction from milk for PCR, and PCR detection
of other species of Mycoplasma.
References
(1) Razin S, Yoger D, Naot Y. Molecular Biology and Pathogenicity of Mycoplasmas. Microbiol
& Molec Biol. Reviews. Dec 1998:1094-1156.
(2) Gonzales R, Wilson D. Mycoplasmal Mastitis in Dairy Herds. Vet Clin Food Anim. 2003
(19):199-221.
(3) Pinnow C, Butler J, Sachse K, Hotzel H, Timms L, Rosenbusch R. Detection of Mycoplasma
bovis in Preservative-Treated Field Milk Samples. J Dairy Sci 2001 (84):1640-1645.
(4) Hirose K, Kawasaki Y, Kotani K, Tanaka A, Abiko K, Ogawa H. Detection of Mycoplasma
in Mastitic Milk by PCR Analysis and Culture Method. J Vet Med Sci 2001 63(6):691-693.
(5) Hotzel H, Sachse K, Pfutzner H. Rapid Detection of Mycoplasma bovis in Milk Sample and
Nasal Swabs Using the Polymerase Chain Reaction. J Applied Bact 1996 (80): 505-510.
(6) The Pennsylvania State University Animal Diagnostic Laboratory. Polymerase Chain
Reaction for detection of Mycoplasma in Clinical Samples. SOP. 2002.
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BOVINE INFECTION OF COXIELLA BURNETII (Q FEVER) IN THE U.S. DAIRY
HERDS: USE OF CONVENTIONAL AND REAL-TIME PCR FOR THE DETECTION OF
COXIELLA BURNETII (Q FEVER) IN MILK
Sung Kim
Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences
Cornell University, Ithaca, NY
Introduction
Q (Query) fever is a ubiquitous zoonosis caused by Coxiella burnetii, an obligate
intracellular rickettsial organism. Since its first independent report by Australian and American
investigators in 1935, the disease has been reported from all over the world except New Zealand.
C. burnetii infections are reported in humans, farm animals, pet animals, wild animals, and
arthropods. Among farm animals, dairy cattle, sheep and goats are implicated as the major
reservoirs of human C. burnetii infection. Animals are often naturally infected but most animals
usually do not show any typical symptoms of C. burnetii infection. C. burnetii could be isolated
from the blood, lungs, spleen, and liver in the acute phase. The female uterus and mammary
glands are primary sites in the chronic phase of C. burnetii infection. Shedding of C. burnetii into
the environment occurs mainly during parturition by birth products, particularly the heavily
contaminated placenta. Shedding of C. burnetii in milk by infected dairy cows is also well
documented. The clinical signs associated with C. burnetii infection are abortion, significantly in
sheep and goats, and reproductive disorders in cattle.
Though shedding of C. burnetii in their milk by infected dairy cows has been known, only
limited information by some earlier investigators in 1940’s and 1950’s is currently available.
Previous studies on the prevalence of Q fever in dairy cattle were mostly based on serological
tests, including complement fixation (CF), indirect inmmunofluorescene (IF) , and enzymelinked immunosorbent assay (ELISA). Recent seroepidemiological studies for cattle have
indicated that C. burnetii antibody seroprevalence in these animals is higher nowadays than 20 or
30 years ago. However, the real prevalence of C. burnetii infection in such animals is still not
available, partly due to the lack of surveillance. The objects of this presentation are to increase the
awareness of Q fever and to report the prevalence of Q fever in the U.S. dairy herds.
Epidemiology and Transmission
The primary mode of transmission is by inhalation of infected aerosols with
contaminated droplets or dusts from birth products, milk, and excreta of infected animals.
Infectious aerosols containing viable organisms can be spread in distance by the wind. Direct
contact with infected animals or other contaminated materials and ingestion of contaminated raw
milk with the organisms also can be potential routes of transmission. There is relatively little
information on the prevalence of the infection. Farm animals, particularly cattle, goats and sheep
are considered the primary reservoirs. A California study conducted at 20 dairy herds in 17
counties showed 100% (20/20) of herds had seropositive cows and 82% of 1,052 had serum
antibodies to C. burnetii. Of 1,634 whey samples, 51% were positive for antibodies Cows that
were seropositive were usually also whey positive. A total of 23% of 840 cows shed C. burnetii in
their milk. A sharp increase in the prevalence of Q-fever was noted in Ontario dairy cattle herds
between 1964 and 1984 from 2.3% to 66.8%. Wild animals, such as coyotes, foxes, rabbits and
deer and pet animals, cats and dogs, are also known to be seropositive, indicating as possible
reservoirs. Ticks are also known to transmit the disease.
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PCR as a Study Tool
Working with the Q-fever agent based on isolation techniques is not an easy task due to
the agent’s high infectivity and rigorous compliance requirements in handling a selective agent.
Recently polymerase chain reaction (PCR) assays have been used to detect C. burnetii. Tran-PCR
assay was developed to detect C. burnetii in milk targeting a transposon-like sequence only found
in C. burnetii. Trans-PCR assay detects the Q fever agent, C. burnetii, unlike serological assays
which detect antibodies. A real-time PCR was developed in this study to measure numbers of C.
burnetii shed in milk. The objective of our study was to conduct a preliminary nation-wide
prevalence surveillance of Q-fever in the United States dairy herds based on bulk tank milk
testing by PCR.
Bovine Herd Infection of Q fever in the U.S.
We tested bulk tank milk samples from the dairy herds in the United States over a 3-year
period by Trans-PCR. Positive results were confirmed by nested PCR and DNA sequencing. The
sequencing results of the 687-bp PCR product were consistent with the published sequence of
IS1111 with 100% homology. The overall prevalence of C. burnetii in the United States dairy herds
was 94.3% with little variations year to year from 93.2 to 94.7%. When compared the prevalence
in New York State and the other states, there were no significant variations indicating the Q-fever
infection of the dairy herds was persistent or steady with little temporal and regional variations.
Conclusions
Bulk tank milk has been used to diagnose dairy herds for several bovine diseases
including bovine viral diarrhea. We reports that greater than 90% of the United States dairy herds
were infected with Q-fever based on bulk tank milk testing over a 3-year period. This high
infection rate did not show temporal and regional variations, suggesting the Q fever infection in
the dairy herds is prevalent throughout the United States. Our report of the high prevalence of Q
fever in the dairy herds is not surprising to take into a consideration of earlier reports regarding a
great increase of bovine infection in North America. An early investigator concluded that Q fever
was already endemic throughout the United States, and predicted that the high bovine infection
may occur in other parts of the country in a similar fashion as in Southern California where a
98% of the herd infection was reported. Widespread and increasing bovine C. burnetii infection
was reported. Frequent chronic Q fever infection of dairy cows could be the most important
source of human infection. Though the mode and extent of transmission from bovine infection to
human has not been determined, epidemiologic studies indicate that Q fever develops in persons
in contact with domestic animals including farmers, veterinarians, slaughterhouse workers, and
laboratory personnel working with C. burnetii.
- 87 -
DIAGNOSTIC STRATEGIES FOR BOVINE VIRAL DIARRHEA VIRUS
Edward J. Dubovi
Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences
Cornell University, Ithaca, NY
The challenge which is still with us over 50 years after the first clinical descriptions of BVDV
infections in cattle is to develop effect means to control BVDV infections. To do this, the
practitioner must be able to deliver a clear, concise, and consistent message to the producer. This
message must include a simple description of the pathogenesis of BVDV, the role management
plays in this problem, diagnostic testing programs and the limitations of vaccines in the context of
the producer's management philosophy. Control of BVDV will need to include the establishment
of best management practices combined with a validated immunization program supported by an
active surveillance system.
For a good control program for BVDV, there are three essential elements. First, one
should determine whether the virus currently exists on the farm or ranch. With the use of
vaccines, clinical disease is not necessarily a true indication of the presence of BVDV. If the
presence of BVDV is detected, then a strategy for its elimination should be instituted. Once the
virus has been eliminated, then a program to keep it out must be developed. Over the past ten
years, a variety of diagnostic tests have been developed which can accurately detect the presence
of BVDV in an animal or in a herd. The problem is not that good tests are not available. The
tests that are available are not being used properly or consistently. Too often practitioners shop
around the country looking for the perfect test when the issue is the testing program and not the
test. Unfortunately, each farm and ranch presents a set of unique conditions that must be taken
into account when a testing strategy is developed. The worst thing to happen is to initiate an
expensive testing program when the virus does not exist on the premises. Good evidence of
infection is not that the owner or the practitioner “thinks” that all of the problems on the farm are
related to BVDV. From the laboratory perspective, good evidence of infection is the isolation of
the virus from an animal on the farm, detection of antibody in unvaccinated animals, and
detection of viral nucleic acid or antigen in a clinical sample.
For years, the “gold” standard for BVDV detection was the isolation of the virus. Most
diagnostic laboratories are now capable of dealing with BVDV once the problems of
contaminated reagents were recognized. However, the existence of the noncytopathic biotype of
BVDV was a diagnostic nightmare because quality antisera used for its detection were not
available. The first technological breakthrough in the diagnosis of BVDV was the development
of monoclonal antibodies in the late 80’s. The antigenic variation of field isolates of BVDV was
determined by the Mabs and selected Mabs were then used to detect BVDV in cell cultures and
tissue samples. For the first time, a consistent reliable reagent was available that could detect
BVDV regardless of its biotype.
The second major development with regard to Mabs was the discovery that one Mab,
namely 15c5, could be used to detect BVDV in formalin-fixed tissue. Monoclonal antibodies to
BVDV had been used for immunohistochemistry detection of viral antigens, but these efforts had
been done on frozen sections. A Mab that could detect antigen in formalin-fixed tissues opened
the door to many retrospective studies on the involvement of BVDV in clinical events that were
not initially linked to BVDV. In particular, animals persistently infected (PI) with BVDV have no
lesions in tissue samples that can be detected by standard histological techniques. The IHC
technique permitted the detection of these animals and extended the link between PI animals and
other clinical presentations. A Mab that works on formalin-fixed tissues also eliminates that need
for rapid transit of samples from the field to the laboratory. In countries without rapid courier
services, the diagnosis of BVDV was now possible on fixed tissue.
- 88 -
The third advance with regard to BVDV and Mabs was the development of an antigencapture ELISA (ACE) test for the detection of persistently infected animals using serum as the
test substance. ACE tests for BVDV had been developed using whole blood as the sample, but
these tests required treatment of the sample prior to testing. A test that could work on serum was
a great benefit because the serum samples could also be used for other herd testing programs such
as Johne’s ELISA tests or BLV. As the ACE test became more widely used, an alternative test
became available which was based on an IHC test on skin biopsies. With PI animals, an
abundance of antigen exits in the epithelial cells of the skin. IHC test became the test of choice
particularly in the beef industry. It soon became evident that the ACE test using a skin biopsy
specimen was equivalent to the IHC test. Currently either test is used as a herd screening tool.
With the advent of molecular techniques, nucleotide sequencing of BVDV isolates
became an important issue with the outbreak of severe clinical disease in Canada in the early
90’s. Along with the realization that there was great genetic diversity among BVDV isolates
came the challenge for designing PCR primers that would detect all possible isolates. There are
numerous papers describing RT-PCR systems for detecting BVDV and with no national
standards, it is up to each lab to establish the validity of their testing system. Standard RT-PCR
protocols shifted to nested PCRs to increase sensitivity, but this also increased the chances of
false positives. With the advent of “real-time PCR systems, the contamination problem became
less of an issue since the reaction tubes never had to be processed for product detection.
PCR tests for BVDV can be used in any situation where virus isolation is used. If the
question is simply is there BVDV in the system, then PCR is a viable alternative to virus
isolation. If, however, the question is whether there is any pathogen in the system, then PCR
becomes less useful. The analytical sensitivity of PCR permits pooling of serum, milk or whole
blood samples when detection of a PI animal in a herd is the goal. However, a positive PCR
reaction can be generated from at least three possible events: vaccination of a herd with modifiedlive vaccine, acute infection or a persistent infection. PCR cannot in of itself distinguish these
three possibilities.
Current Testing Strategies at the AHDL
The consensus of opinion is that BVDV maintains itself in the bovine population through the
generation of persistently infected animals. Therefore, there is an emphasis on being able to
screen herds for the presence of one of these animals in 100-200 normal cows. Cost is the
obvious overriding concern with this testing process. Over the years, the AHDL has modified its
approach to herd testing to take advantage of technology where appropriate. Currently in dairy
herds, we use several protocols that are somewhat dependent upon the management of the farm.
We have championed the use of bulk tank milk testing for detecting PI animals in the lactating
herd. If BVDV is detected by this approach, then individual cow testing is necessary. This can be
achieved either through pooled PCR testing or by use the ACE test directly on all animals in the
herd. This approach has generated considerable cost savings over simply testing all animals
individually from the very beginning.
References
There are numerous references available for all aspects of BVDV. For a good comprehensive
starting point, I would recommend: Bovine Viral Diarrhea Virus: Persistence is the Key. (2004).
Veterinary Clinics of North America: Food Animal Practice. Vol 20 (1).
- 89 -
NOTES
- 90 -
Speaker Biographies
and
Contact Information
- 91 -
NOTES
- 92 -
Kathryn J. Boor, PhD
Associate Professor
Cornell Institute of Food Science
413 Stocking Hall
Cornell University
Ithaca, NY 14853
[email protected]
http://www.foodscience.cornell.edu/faculty/boor.htm
Kathryn Boor earned a BS in Food Science from Cornell University and an M.S. in Food Science
from the University of Wisconsin. She spent two years in Kenya, East Africa, working with
impoverished farmers to enhance their animal-based food production and preservation systems,
then earned her PhD in Microbiology at the University of California, Davis. She established the
Food Safety Laboratory as an Assistant Professor in the Department of Food Science at Cornell
in 1994. Dr. Boor currently also directs Cornell’s Milk Quality Improvement Program. She
serves on the New York State Interagency Task Force on Food Safety and Security, on the
editorial boards for Journal of Food Protection, Applied and Environmental Microbiology, and
Foodborne Pathogens and Disease, and as Scientific Advisor to the New York State Cheese
Manufacturers’ Association. Dr. Boor received a 2000 USDA Honor Award as a member of the
Listeria Outbreak Working Group, the 2000 Foundation Scholar Award of the American Dairy
Science Association, the 2000 Cornell University Constance E. Cook and Alice H. Cook
Recognition Award, and the 2002 Samuel Cate Prescott Award for Research from the Institute of
Food Technologists. She is also Past President (2000) of the New York State Association of Milk
and Food Sanitarians.
- 93 -
Dr. Paula J. Fedorka-Cray, PhD
Microbiologist, Research Leader
Richard B. Russell Agricultural Research Center
USDA-ARS-RRC-ARRU
950 College Station Road
Athens, GA 30605
[email protected]
http://www.arru.saa.ars.usda.gov/
Dr. Fedorka-Cray received her BS from Penn State University (Microbiology), MS from North
Dakota State University (Bacteriology), MAS from Johns Hopkins University (Administration)
and a Ph.D. from the University of Nebraska Medical School (Veterinary Microbiology). She
was the recipient of the Joseph J. Garbarino Achievement Award for Excellence in Agricultural
Research awarded by the Animal Health Institute Foundation. She has been employed by the
USDA - ARS since 1991 and is a Microbiologist, Research Leader for the Antimicrobial
Resistance Research Unit. Her program focuses on antimicrobial resistance in food borne
pathogens with an emphasis on Salmonella and Campylobacter. In particular she is interested in
the host/pathogen relationship as well as the ecological impact of antimicrobial resistance. She
also directs the testing efforts for the veterinary arm of the National Antimicrobial Resistance
Monitoring System - Enteric Bacteria (NARMS).
Antimicrobial Resistance Research Unit
The mission of the Antimicrobial Resistance Research Unit is to study Antimicrobial resistance
in zoonotic food borne pathogens and commensal bacteria. Epidemiology, microbiology, risk
factor analysis, and molecular techniques are used to 1) gain an understanding of the prevalence
of resistance among food borne pathogens and factors which may affect the development and
persistence of resistance in production facilities and in the environment, 2) study the molecular
mechanisms that are associated with the development of resistance, and 3) define the role of
commensal bacteria in the development and transfer of resistance. The veterinary arm of the
National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) is also
located in our Unit. This program is a multi-agency endeavor involving scientists from the USDA
- ARS, FDA - Center for Veterinary Medicine, USDA - FSIS, USDA - APHIS, and the CDC.
The goal of the program is to track the development of antimicrobial resistance in veterinary
isolates as it arises and disseminate the information to all stakeholders in an attempt to arrest the
development and spread of resistance, especially among food borne pathogens. The results
generated by these endeavors will enhance our knowledge of antimicrobial resistance and provide
the scientific data that is critically needed to direct research among the scientific community and
to develop policy in a number of agencies, including the USDA and FDA.
- 94 -
Edward J. Dubovi, PhD
Director – Virology Section
Animal Health Diagnostic Center
Department of Population Medicine and Diagnostic Sciences
College of Veterinary Medicine
PO Box 785
Cornell University
Ithaca, NY 14851-0786
[email protected]
http://www.popmed.vet.cornell.edu/bios/dubovi.asp
Received a Masters degree in Virology from Purdue University in 1968 and a PhD in
microbiology from the School of Medicine, University of Pittsburgh in 1975. Following
postdoctoral training at the University of Virginia and the University of North Carolina, assumed
position as Director of the Virology Section, Diagnostic Laboratory, College of Veterinary
Medicine at Cornell in 1981. Areas of research: Viral diseases of cattle with particular emphasis
on bovine viral diarrhea virus and bovine retroviruses; viral diseases of horses with emphasis on
equine arteritis virus and equine morbilli-like virus. Within the context of the Department of
Population Medicine and Diagnostic Sciences, current efforts also included herd health programs
that emphasize biosecurity and effective use of veterinary biologicals.
- 95 -
Rubén N. González, DVM, PhD
Senior Research Associate and Associate Director
Quality Milk Production Services
Department of Population Medicine and Diagnostic Sciences
College of Veterinary Medicine, Cornell University
22 Thornwood Drive
Park View Technology Center I
Ithaca, New York 14850-1263, USA
Tel:
(607) 255- 202
Fax: (607) 257-8485
[email protected]
http://qmps.vet.cornell.edu/qmps.html
Dr. González graduated as a Doctor of Veterinary Medicine from the Faculty of Veterinary
Sciences, National University of La Plata, Argentina. After working for two years as a veterinary
practitioner in a mixed rural practice, he returned to academia as an Assistant Professor of
Epidemiology and Infectious Diseases at the Faculty of Agronomy and Veterinary Medicine,
National University of Río Cuarto, Argentina. Later, as a Senior Bacteriologist, he joined
Argentina's National Institute for Agricultural Technology and the United Nations/FAO Project
in Animal Health Argentina 75/072 where he helped to set up and supervise a net of regional
veterinary diagnostic laboratories in five provinces in Northwestern Argentina. In 1982, Dr.
González came to the U.S. and while pursuing graduate studies, worked as a Post-Graduate
Research Associate in the Department of Clinical Pathology and the Cooperative Extension
Mastitis Laboratory, School of Veterinary Medicine, University of California at Davis. From this
university, Dr. González received both a Master in Preventive Veterinary Medicine (1984) and a
PhD in Comparative Pathology (1988). He joined Quality Milk Production Services in 1988.
Areas of research are epidemiology, diagnosis and control of bovine mastitis, with emphasis in
Mycoplasma and other non regular agents of the disease.
- 96 -
Bhushan Jayarao, PhD
Extension Veterinarian/Associate Professor
Department of Veterinary Science
111 Henning Bldg
The Pennsylvania State University
University Park, PA 16802
[email protected]
http://www.vetsci.psu.edu/personnel/faculty/jayarao.cfm
Dr. Jayarao received his Bachelor’s degree in Veterinary Medicine from Bombay Veterinary
College in 1980. In 1982, he received his Master’s degree in Veterinary Public Health. During
the time between 1980 and 1986, Dr. Jayarao taught Zoonoses, Epidemiology and Veterinary
Public Health, as well as practiced companion and food animal medicine. In 1985, Dr. Jayarao
attended the University of Veterinary Sciences, Budapest where he received his PhD in
Microbiology and Epidemiology in 1989. One year later, he joined Dr. Stephen Oliver’s mastitis
research group at the University of Tennessee. Over the next five years (1990-1995), Dr. Jayarao
worked on developing diagnostic tests and understanding the epidemiology of S. uberis IMI.
During the same period, Dr. Jayarao earned a degree in Master’s in Public Health with
specialization in communicable diseases. In 1995, Dr. Jayarao joined the Department of Dairy
Science at the South Dakota State University. He was engaged in teaching Microbiology courses
to undergraduate and graduate students. At this time, his research program focused primarily on
pre-harvest food safety. In July of 1998, Dr. Jayarao joined the department of Veterinary Science
at the Pennsylvania State University as an extension Veterinarian. Dr. Jayarao’s primary
responsibility was to develop an extension program that is focused on infectious diseases and
public health. His research appointment allows him to pursue research interests related to
diagnosis and detection of infectious diseases, molecular epidemiology of antimicrobial
resistance, development of plant-based vaccines and food safety. Dr. Jayarao has been successful
in obtaining federal, state and industry funding as principal or co-investigator on 24 peer
reviewed external grants equivalent to about $ 1.5 million dollars to support extension and
research work at Penn State. In 2002, Dr. Jayarao received the 2002 ADSA West Agro award for
his significant contributions in the area of understanding the molecular epidemiology of
streptococcal infections in dairy cattle. The same year Dr. Jayarao received early tenure and was
promoted to Associate Professor. Currently, Dr. Jayarao has 2 post-doctoral research associates,
3 PhD and 3 master’s and 2 honor’s students, and 4 technicians in his laboratory. His research
group conducts research in the following areas: 1) Epidemiology of antimicrobial resistance in
companion and food animals, 2) Plant-based vaccines; development of transgenic plants that
encode for bacterial antigens, and 3) Mastitis and Milk Quality Dr. Jayarao has published more
than 50 peer reviewed journal articles. Dr. Jayarao is an active member of a number of
professional societies, including American Dairy Science Association, American Veterinary
Medical Association, Pennsylvania Veterinary Medical Association, American Association of
Extension Veterinarians, and National Mastitis Council.
- 97 -
Vivek Kapur, PhD
Professor, Department of Microbiology
Dept of Vet Path 300 A Vet Science Bldg
University of Minnesota
1971 Commonwealth Avenue
St. Paul, MN 55108
Education:
• Veterinary Medicine-UAS, Bangalore, India, 1986
• Ph.D. - Pennsylvania State University, University. Park, PA 1991
• Post Doc - Baylor College of Medicine, Houston, TX, 1992-1995
Research Interests:
Microbial Pathogenomics, Host-Pathogen Interactions, Functional Genomics.
Research in the Kapur Laboratory seeks to define the basic mechanisms by which pathogenic
microbes successfully infect, colonize, and cause disease in their hosts. The research effort is
organized along three thematic lines: microbial population genetics; microbial pathogenesis; and
host response to infection. A translational research component seeks to translate the results of the
investigations into improved diagnostic tests and methods for microbial identification, as well as
investigate opportunities for developing new generations of antimicrobial vaccines and
therapeutics.
Synergistic Activities:
Dr. Kapur is also Director of the Advanced Genetic Analysis Center (www.agac.umn.edu) and
co-Director of the Biomedical Genomics Center (www.bmgc.umn.edu) at the University of
Minnesota and is a member of various genomics and computational biology related advisory
committees at the University, national and international levels.
- 98 -
Jeffrey S. Karns, PhD
Environmental Microbial Safety Laboratory, Agricultural Research Service,
U. S. Dept. Of Agriculture, Building 173 BARC-East
10300 Baltimore Ave, Beltsville, MD 20705
Phone: 301-504-6493, FAX: 301-504-6608
[email protected]
http://www.anri.barc.usda.gov/emsl/
Education
1981 Virginia Commonwealth University, Microbiology; PhD
1975 The Pennsylvania State University, Medical Technology, BS
Experience
1981-1983
1983-1984
1984-1994
1994-2000
2000-
Post-Doc, University of Illinois Health Sciences Center, Chicago, IL
Post-Doc, University of Maryland, College Park, MD
Research Microbiologist, Pesticide Degradation Lab, USDA/ARS, Beltsville,
MD
Research Microbiologist, Soil Microbial Systems Lab, USDA/ARS,
Beltsville, MD
Research Microbiologist, Environmental Microbial Safety Laboratory (formerly
Animal Waste Pathogens Lab), USDA/ARS,
Beltsville, MD
Research Accomplishments
Characterized the genes and enzymes responsible for the degradation of numerous
agrochemicals. Developed methods for the use of pesticide degrading enzymes for the benefit of
agriculture.
Developed methods for APHIS to control microbial processes in cattle dipping vats so that the
acaricide in the vat was preserved in order to get maximum usage and so that microbial processes
could be exploited for waste treatment. The methods have become standard operating procedure
for the APHIS-VS Tick Eradication Program.
Developed real-time PCR methods for the detection of E. coli O157:H7 in soil and water.
Investigated the leaching of the organism in soil.
Participated in emergency response to anthrax threat in Washington, DC through preparation
and operation of a mobile laboratory. Developed protocols for processing of environmental
samples and detection of Bacillus anthracis using commercial real-time PCR kits.
Honors and Awards
Certificates of Merit for outstanding performance in 1995, 1997, 2000 and 2001.
Certificates of Merit for superior performance in 1990, 1992, 1998, 1999, 2002 and 2003.
2002
USDA Group Honor Award for Excellence from the Secretary of Agriculture For
emergency activities, communications, initiatives and employee security measures taken
in response to Sept. 11, 2001 and the anthrax threat to the Department of Agriculture.
- 99 -
Sung G. Kim, PhD
Research Associate
Director, Molecular Diagnostics Laboratory
Animal Health Diagnostic Center
College of Veterinary Medicine
Cornell University
P.O. Box 5786
Ithaca, NY 14853-5786
[email protected]
http://www.diaglab.vet.cornell.edu/
Dr. Kim received his Masters and Ph. D. degrees from Cornell University. He did his
postdoctoral program with Dr. Carl Batt in the area of molecular evolution studies of
bacteriophages with an emphasis on DNA sequencing of phage genomes and antisense RNA
technology. Since he joined the Animal Health Diagnostic Laboratory at Cornell’s College of
Veterinary Medicine in 1994, he has been involved in development and application of molecular
diagnostic methods to detect pathogens causing animal diseases including BVD, Johne’s disease,
Lyme disease, Potomac horse fever, E. coli O157/H7, Q-fever, Erhlichiosis, and avian influenza.
His research interest is use of molecular diagnostic tools to assure animal and public health. He is
currently section head of Molecular Diagnostics at Cornell Animal Health Diagnostic
Laboratory.
- 100 -
Suzanne C. Klaessig
Research Support Specialist
Department of Population Medicine and Diagnostic Sciences
College of Veterinary Medicine
S3-118 Schurman Hall
Cornell University
Ithaca, NY 14853
[email protected]
Suzanne Klaessig has been the supervisor of the Cell Biology Laboratory at Cornell for 15 years.
The Cell Biology Laboratory is a fee-for-service lab and a section of Quality Milk Production
Services. Her responsibilities include training of graduate students, faculty and undergraduates.
Suzanne has multi-dimensional experience in research, with extensive experience in mammalian
tissue culture. For 10 years, she has also been a staff participant in the Microinjection Techniques
section of the Cell Biology course taught at the Marine Biological Laboratory at Woods Hole.
- 101 -
Kendra K. Nightingale
Ph.D. candidate
Department of Food Science
412 Stocking Hall
Cornell University
Ithaca, NY 14853
[email protected]
http://www.foodscience.cornell.edu/wiedmann/graduates.htm
Kendra Nightingale is a Ph.D. candidate in the Department of Food Science at Cornell
University, majoring in Food Science with a concentration in Food Microbiology and pursuing
minors in Epidemiology and Microbiology. Kendra is originally from a small farming
community in Western Kansas. Her most recent work includes studying the molecular
epidemiology, ecology, and evolution of the human foodborne and animal pathogen Listeria
monocytogenes. Kendra holds a M.S. degree from Kansas State University in Food Science, where
her research evaluated the use of lactoferrin, a milk-derived protein, to decontaminate and extend
the shelf-life of beef products. She also holds a B.S. degree, cum laude, from Kansas State
University, where she participated in the undergraduate honors program. She is a member of
several agricultural honor societies including Phi Tau Sigma, and has been recognized with
national graduate research fellowships from Institute of Food Technologists, National Milk
Producers Federation, and International Association for Food Industry Suppliers as well as
awards from the Department of Food Science at Cornell.
- 102 -
Stephen P. Oliver, PhD
Professor, Department of Animal Science
Co-Director Food Safety Center of Excellence
University of Tennessee
59 McCord Hall
2640 Morgan Circle Drive
Knoxville, Tennessee 37996
[email protected]
http://www.foodsafe.tennessee.edu/
Principle Duties:
Research on mammary gland physiology, immunology, and microbiology with emphasis on
development of nonantibiotic approaches for the prevention and control of mastitis in dairy cows;
and development and evaluation of strategies to control/reduce foodborne pathogens in food
producing animals.
Educational Background:
• PhD 1980, Dairy Science (Lactation Physiology), The Ohio State University
• MS 1978, Dairy Science (Lactation Physiology), The Ohio State University
• BS 1976, Animal Science, North Carolina State University
Other Organizations, Committees, Awards, Honors, Etc.
EDITOR-IN-CHIEF of Foodborne Pathogens & Disease, a new peer-reviewed quarterly
international journal published both in print and online by Mary Ann Liebert, Inc.
Former Chairman and current member of NMC Research Committee
Recipient of the 2003 United States Food and Drug Administration’s Group Recognition Award as
a member of the Tissue Residues & Strategies for Case Development Organization and Training
Team.
Recipient of several research awards from the American Dairy Science Association (2002 Land
O’Lakes Award, 1998 Merck AgVet Dairy Management Research Award, 1992 West Agro
Award, 1989 Agway Young Scientist Award) and from The University of Tennessee.
- 103 -
Ynte Hein Schukken, DVM, PhD
Director of the Quality Milk Production Services
22 Thornwood Drive
Park View Technology Center I
Ithaca, New York 14850-1263, USA
Tel:
(607) 255- 202
Fax: (607) 257-8485
Professor of Epidemiology and Herd Health
Department of Population Medicine and Diagnostic Sciences,
College of Veterinary Medicine
Cornell University
S3 119 Schurmann Hall
[email protected]
http://qmps.vet.cornell.edu/qmps.html
EDUCATION:
DVM, University of Utrecht
M.Sc., Cornell University
Ph.D., University of Utrecht
SPECIALTY
CERTIFICATION:
Bovine Medicine (Royal College of Dutch Veterinarians)
Epidemiology (Dutch National Science Foundation)
RESEARCH
INTEREST:
1985
1987
1990
Udder health in well managed dairy herds. A research and service
approach based on understanding epidemiology and pathobiology of the
diseases affecting mammary health.
Understanding population dynamics of infectious diseases in animal
populations.
Application of epidemiological, statistical and mathematical methods to
animal disease research.
- 104 -
Linda L. Tikofsky, DVM
Senior Research Associate Quality Milk Production Services
Department of Population Medicine and Diagnostic Sciences
College of Veterinary Medicine, Cornell University
22 Thornwood Drive
Park View Technology Center I
Ithaca, New York 14850-1263, USA
Tel:
(607) 255- 202
Fax: (607) 257-8485
[email protected]
http://qmps.vet.cornell.edu/qmps.html
Linda graduated with a DVM degree from the University of Illinois in 1984, completed an
internship at the University of Missouri and worked in private practice from 1985 through 1997
prior to joining QMPS. Linda began work at QMPS in December of 1997 as a Post Doctoral
Associate and now holds the position of Senior Extension Veterinarian. Linda is a field
veterinarian and coordinator of the Milker Training Program. On top of her clinical
responsibilities, she conducts research into epidemiology of antimicrobial resistance of
Staphylococcus aureus. In recent years, Linda established a network of contacts with the organic
dairy community, resulting in a series of workshops for organic and transitioning dairy farmers in
2003, a homeopathic treatment trial for S. aureus mastitis in 2004 and most recently, a $518,000
USDA grant for a four-year project entitled "The Transitioning Dairy: Identifying and
Addressing Challenges and Opportunities In Milk Quality and Safety".
- 105 -
Martin Wiedmann, DVM, PhD
Assistant Professor
Department of Food Science
412 Stocking Hall
Cornell University
Ithaca, NY 14853
[email protected]
http://www.foodscience.cornell.edu/faculty/wiedmann.htm
Dr. Martin Wiedmann received a DVM from the Ludwig-Maximilians University in Munich in
1992 and a doctorate in Veterinary Medicine from same university in 1994. In 1997, he received
a Ph.D. in Food Science from Cornell University. After a two year postdoc in the Cornell Food
safety Laboratory with Professor Kathryn Boor, he joined the Department of Food Science at
Cornell as an Assistant Professor in 1999. His research interests focus on the molecular biology,
epidemiology, and transmission of foodborne and zoonotic pathogens.
Dr. Wiedmann has published more than 60 peer-reviewed publications, 8 book chapters and
reviews, 7 proceedings articles, more than 80 abstracts, and 2 patents. He has given more than 50
invited presentations. Current research in his laboratory is supported by grants from the National
Institutes of Health, the USDA National Research Initiative, the USDA Food Safety Initiative,
and the International Life Sciences Institute. He was a member of the Listeria Outbreak
Working Group, which was honored by a USDA Secretary’s Award for Superior Service in 2000
for the detection of a multistate listeriosis outbreak. He also received the Young Scholars award
from the American Dairy Science Association in 2002, the Samuel Cate Prescott Award from
Institute of Food Technologists’, and most recently the International Life Science Institute (ILSI)
North America Future Leaders Award.
- 106 -
Ruth N. Zadoks, DVM, PhD
Research Associate
Department of Food Science
401 Stocking Hall
Cornell University, Ithaca, NY 14853
Phone: (607) 254-4967
[email protected]
http://www.foodscience.cornell.edu/boor/fac.html
Ruth Zadoks is a Research Associate in the Laboratory of Food Safety and the Laboratory of
Food Microbiology, Department of Food Science, Cornell University, Ithaca NY, and will be
heading the new Molecular Laboratory at Quality Milk Production Services, College of
Veterinary Medicine, Cornell University.
Ruth obtained her DVM (1995, cum laude), MSc (1998, Veterinary Epidemiology and Herd
Health), and PhD (2002, cum laude) degrees from the Faculty of Veterinary Medicine, Utrecht
University, the Netherlands where she worked as a bovine clinician, and taught bovine herd
health and epidemiology of infectious diseases to undergraduate and graduate students from 1996
to 2002. The focus of her PhD work, which was carried out in collaboration with the Department
of Population Medicine and Diagnostic Sciences at Cornell University, the University of
Tennessee, Knoxville TN, and Washington State University, Pullman, WA, was the
epidemiology of streptococcal and staphylococcal mastitis in dairy herds. Both mathematical and
molecular approaches were used in those studies of mastitis epidemiology. Her current research
at Cornell University deals with the ecology, pathogenesis and consequences of contagious and
environmental mastitis in dairy herds. In particular, Dr. Zadoks explores the usefulness of
bacterial strain typing methods as diagnostic tools in mastitis control, and studies the
epidemiology of streptococcal mastitis and antimicrobial resistance in bovine streptococci.
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NOTES
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ACKNOWLEDGEMENTS
Without the contributions of many people, organization of the symposium on “Molecular
Methods for Milk Quality”, the remodeling of QMPS facilities in Ithaca, and the opening of the
Molecular Laboratory would not have been possible. We would like to thank everybody for their
contributions, small or large, anonymous or named, without which none of this could have taken
place.
We would like to thank all presenters at the symposium for their wonderful papers and
presentations. It has been a joy to compile these symposium proceedings and to realize how far
we have come with the application of molecular methods in dairy medicine, milk quality, and
food safety. In addition, we would like to thank all symposium attendees for participating. Some
of you have traveled far to be here. We have guests from many places in the U.S.A. and abroad,
including Canada, Italy and India.
The staff at QMPS has gone above and beyond to organize the symposium. In particular, we
would like to acknowledge the work behind the scenes done by Belinda Gross, Tollie Stuprich,
and Victoria Thomas. In addition, we would like to thank office, field and laboratory personnel
for the effort they put into preparations for the official opening and open house.
Thanks to Roberta Militello for the design of the logo for the new Molecular Laboratory.
We would also like to extend our thanks to our colleagues, clients and students. Without farmers,
veterinarians, research collaborators, extension personnel, summer scholars, and many others
much of our work could not be as successful and rewarding as it has been over the years. Dr.
Martin Wiedmann and Dr. Kathryn Boor from Cornell University’s Department of Food Science
have been particularly active as research collaborators, and supportive of our initiatives in the
field of molecular epidemiology. We hope that the addition of the Molecular Laboratory will
further enhance the width and depth of service and knowledge that QMPS can provide to the
dairy and research communities.
Our corporate sponsors are also our partners in research, service and development. We thank
them for their input in this symposium and, in general, for their stimulating collaborations.
Sponsors that provided funds for this symposium are presented next to contents page of these
proceedings.
Finally, we would like to express our appreciation to our two ‘parents’: The State of New York
and Cornell’s College of Veterinary Medicine. Form the State of New York we have enjoyed so
much support from both the legislators (Senate and Assembly alike) and the Department of
Agriculture and Markets. The Animal Health Diagnostic Center and the College of Veterinary
Medicine have always been helpful and supportive in efforts to develop this new laboratory. It is
with the greatest possible pleasure that we learned that representatives of all these organizations
have agreed to perform the official opening of our new Laboratory.
Thank you all so much!
Ruth Zadoks
Ynte Schukken
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