Download Comparing Differences in Bacteria Growth in Apartments and

Comparing Differences in Bacteria Growth
in Apartments and Houses with and without
Felines and/or Canines
Vanessa Alcala
May 9, 2007
Bio 401
0
Table of Contents
Page number
Abstract……………………………………………………………………..2
Intro…………………………………………………………………………3
Methods……………………………………………………………………..10
Subject Requirements
10
Preparation of Broth and Sterilized Tubes
10
Obtaining Samples
10
Statistical Analysis
13
Results……………………………………………………………………….14
Discussion……………………………………………………………………16
Acknowledgements…………………………………………………………..19
Appendix 1………………………………………………………………….. 20
Literature Cited………………………………………………………………21
1
Abstract
This study examined if sharing living space with animals exposed the
homeowners to a higher level of bacteria compared to homeowners without animals.
Participants of this study consisted of 60 residences, 30 houses and 30 apartments. The
categories included the following: 10 houses with no animals, 10 houses with 1 cat or dog
and 10 houses with multiple cats and/or dogs, 10 apartments with no animals, 10
apartments with 1 cat or dog and 10 apartments with multiple cats and/or dogs. All
participants reside in the Thurston County area. Every home was swabbed in three
different locations: kitchen, living room and most trafficked area of each home in the
same manner. The method used for swabbing each room of the home was done with a
sterile cotton swab in the motion of a three cornered zig-zag of a 30 cm x 15 cm area.
After 24 hours of incubating the sample in nutrient broth a spectrophotometer reading
was taken to record bacterial levels. A two factor Analysis of Variance (ANOVA) at
p=0.05 was used to compare the 3 treatments across the two factors to determine whether
there was a statistical difference in the levels of bacteria. No differences in bacterial
levels were found among the houses and apartments.
2
Introduction
Zoonotic diseases are diseases caused by infectious agents that can be transmitted
between (or are shared by) animals and humans (Skerget et al., 2003). The popularity of
domesticated animals, pets, in households is greater in America than in any other
country, the most common being the cat and dog. Regardless of where one lives, in a
city or in a rural area, one's pets are bound to be subjected to irritating bug bites and
diseases whilst outside of the home. Pets not only transport fleas and ticks back into the
home, but can also play host to bacteria and microorganisms that attribute to zoonotic
diseases(Kuiken et al., 2004).. The invasion of these outside bacteria and
microorganisms can take over carpets, bedding and furniture. Taking into consideration
the impact this invasion has on human health, I am ultimately looking to investigate
the difference between bacteria levels in apartments and houses, with and without pets.
In exploration of what cats and dogs are capable of hosting, the study of Dubey
(1995), is applicable to my study; he investigated the relationship of felines to
Toxoplasma gondii (T. gonddii), a parasitic protozoan, that can live in any warm blooded
animal. Oocysts are sporozoan zygotes with a thick walled structure that later transfers
them to new hosts. In general, once a cat has excreted oocysts, it develops immunity
toexcretion (Dubey, 1995). The aim of this study was to determine if T. gondiiinfected cats repeated oocyst shedding, or if cats are immune to re-shedding of oocysts.
To study the length of the cat’s immunity to the re-shedding of oocyst, experimental cats
were investigated for 39 days to 77 months. Two other research observances were
undertaken concerning the transportation of oocysts from one cat to another, and whether
oocysts could be found on the hair of cats shedding oocyts (Dubey, 1995). Two groups of
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cats were used: one group as a control and another as the experimental group, which
was given T. gondii antibiotics orally. All cats began to shed oocysts between 4 and 13
days after oral consumption of the parasite. Two of the three cats that became ill between
the 11th and 13th day actually died on the 13th day. The third cat had to be euthanized
because of full infection of T. gondii. In the 7 days after the experimental infection, the
hair of the ten cats had actually shed millions of oocysts. On both the 39th day after
primary infection and 77 months after the primary infection, the remaining cats were
compared to the control cats that had been housed with the infected cats for 6 years.
Those cats that were previously undiagnosed with the parasitic protozoan remained free
from T. gondii. Factors that contributed to the variability of cats contracting or rejecting
the infection had to do with their age at the time of infection and the stage and route T.
gondii used for the infection during the very first contact. For a family to bring a cat into
their home, they are potentially inviting in the prime T. gondii carrier (Dubey, 1995).
Cats are the starting place in the transmission of T. gondii to humans. My study does not
specifically search for active T. gondii cultures, but will measure the amount of bacteria
found in homes with cats.
Due to our attempts to keep our houses germ-free and sanitary, bacteria can
become resistant to our cleaning efforts (Cole et al.,2003). The relationship between
resistance in
bacteria found in homes is described in the study of Cole
etople who used antibacterial products
and people who cleaned without antibacterial products were tested, and both were
sampled environmentally and clinically. Testing from three geographical locations was
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performed to isolate target bacteria and test their resistance to cleaners. Cole et al., (2003)
focused on organisms that are recognized as potential viruses or cause diseases for
humans. Their method for collection from randomized home samples began with the
completion of questionnaires to make sure the homes fulfilled all the necessary
requirements so that different variables did not affect the study. The participants were
screened over the phone. For qualification requirement information, they were asked
questions such as: the types of products used, recent antibiotic therapies, their occupation,
their type of dwelling, recent water damages to their home, etc. The team collected
samples from three different geological locations: North Carolina, New Jersey, and
England. Prospective participants were excluded from the participation if they had been
on antibiotic therapy in the last 30 days or long-term antibiotic therapy at all in the last
year, worked in a health care occupation, had preschool children under the age of 5 in a
day care more than 20 hours per week, or had reported water damage. Once the results of
the questionnaire were gathered, 10 randomly-selected homes from each of the 3 sites
were selected from the 15 that met the criteria. From the 30 antibacterial users and 30
nonusers, more virus and disease causing bacteria were found in the nonusers' home than
the users of antibacterial products. These results show that there was no correlation
between antibiotic resistance in gram-positive and gram-negative potential human
pathogens and their ability to resist four of the most popular antibacterial products on the
market. Incorporating a survey method, as Cole et al. (2003) did for their experiment,
allowed me to choose home samples randomly from a pool of qualified environments.
Test subjects excluded those who had experienced fungal outbreaks in the last year,
because this would cause differences in bacteria levels compared to other samples.
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Additionally, bladder problems of the animals were recorded because higher levels of
ammonia in the carpet could have been recognized as an outside variable. Further, Cole
et al’s. (2003) research inspired my study to compare rooms amongst homes, because of
his antibacterial results. I explored the difference in bacteria levels of floor surfaces that
can be cleaned with liquid antibacterial agents (kitchen floors) and surfaces that are
vacuumed cleaned (carpet floors).
At one point, domestic cats were categorized as resistant to diseases, such as
Influenza A virus infection according to Kuiken et al. (2004). In this experiment, 3 cats
had the disease implanted into their tissue. Of those 3 cats, one died unexpectedly after
the sixth day of infection. All 3 cats, including the one that died, had damage that spread
to the respiratory track, resulting in a death and two clinically infected cats. Infection
through another cat was also tested, as was infection by feeding on infected birds. All
three methods of infection resulted in the excretion of the virus and pulmonary damage.
This study changed the thought that domestic cats were not susceptible to viruses. Kuiken
et al. (2004) found that cats are able to catch the Influenza A virus by being in the
presence of an infected cat and by eating infected chicks. Because Influenza A is
transferable through contact with infected cats, humans are at risk of acquiring the
influenza virus from their pets. The findings of this study show that cats are making this
avian virus possible for mammals, increasing the risk of a human influenza plague. The
results of this study are related to my hypothesis that bringing animals into one's home
increases the risk of ill health. This virus, in particular, indicates that danger may exist.
A fact that may bring relief to pet owners is that animals have cells that kill
foreign bacteria and the breeding of insects on their body. But, according to Cavassani et
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al., (2004), mosquitoes, flies, sand flies and ticks, have evolved the ability to adapt to
their host's immune response. These insects have saliva that prevents and stops the
animal's natural defense responses. To test this, a study was done on 20 dogs by planting
60 adult, lab-grown ticks on their backs. The saliva that the ticks produced was
micropipetted and stored. A protein produced in the saliva is a direct indicator of the
amount of cytokine production by T- cells that help the body fight off what it does not
recognize. Animals' bodies, like humans', have functioning cells that protect against
harmful bacteria detected in the body, yet they are still susceptible to infections. The
extent of dangerous diseases that cats and dogs can harbor on their body is immense
Approximately 152 species of mammals were reported to have been infected with C.
parvum parasites are the source of danger in isolation of the genotypes (Kuiken et
al.,2004). Without genetic analysis, it is impossible to accurately predict host specificity
of infectious potential of a specific isolate of the cryptosporidium, Cryptosporidiurn canis
has been found in domestic dogs (Kuiken et al., 2004). There is a genetic difference
between dog and human reaction to the exposure of infected ticks and the mouse
reaction when compared in the lab. Deoxyribonucleic acid (DNA) was taken from both
dogs and humans and exposed to the polymerase chain reaction, and then sequences were
compared with the sequences from other humans and animals that had species. Had there
been the time and equipment, I would have genetically analyzed the cryptosporidiunz
dogs and cats participating in my study to determine if they had been potential carriers of
cryptosporidiunz and compared the bacteria found in the home in observance of a correlation
between the two factors. Instead, I compared the amounts of bacteria in apartments and
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houses with animals, versus those with no animals without knowledge as to what the
animals potentially carried.
Skerget et al. (2005) performed a study with a pool of 202 people with either
domestic dogs, cats, or both and compared it with 174 people who had no domestic pet
contact for at least one year, and had not lived with a pet in their life. Volunteers who
had gotten a tick bite within the past year were eliminated from the study. Each
participant submitted the following information: age, sex, area of residence, and medical
history. Serum samples were tested by several methods that were run through a software
program called Sigma Stat Statistical Software, version 2.0 for a biostatistical analysis.
This analysis gave the chi-square test results and a two tailed p value. The chi-square
tests were used to determine the differences between the presence of antibodies to the test
organism and demographics that came from the various methods that the samples were
tested. Antibodies against the cat-scratch disease Bartonella henselae (B. henselae)
existed in 88 people of the 376, and through the software program it is evident that there
was not a difference caused by variables such as age, sex, urban or rural residence, or
affiliated diseases. When comparing the differences in people with and without domestic
pets, a distinction could not be made.
The same results were seen for the antibodies against Q fever, Coxiella burnetii
(C. burnetii). Both B. henselae and C. burnetii had no difference or relationship
correlating sex, age, urban or rural residence, concomitant diseases, or if they lived with
or without domesticated pets. It is the veterinarian's job to educate pet owners on the risk
of contracting B. henselae and C. burnetii. Although the Skerget et al.,(2003) study
suggests that cat or dog ownership was not related to the increased incidence of
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antibodies in the study, clients should be informed that this is a threat. They do, however,
mention that Ixodes ricinu, tick should be considered a hazard to dogs and cats although it
is most associated with livestock and deer. I. ricinus is recognized as the principal vector
in Europe and since it is considered an organism that has transferable abilities without
detection, Skerget et al., (2003) suggests infections be specifically checked for I. ricinus .
The population considered most at risk includes the very people who cannot be subjected
to study: pregnant women, the elderly, immune suppressed patients, HIV-positive
persons and infants (Skerget et al., 2003). While my study did not attempt to examine
correlation of cat or dog ownership to incidences of humans contracting B. henselae and
C. burnetii, bacteria concentration was taken into consideration in studying and
comparing levels of those without dogs or cats in the home.
Although cat-scratch disease (CSD) is identified as a disease mostly contracted by
children and young adults, all age groups were included in the population of this study
(Ben-Ami et al., 2005). Patients with CSD ranged from infants to elderly individuals.
The results were separated into two age groups, >60 years (elderly group) and <60 (non
elderly), and compared to see which age group was most receptive; 846 infected patients
volunteered for the study and were surveyed on their demographic, epidemiologic, and
clinical data. In order to qualify for the study, they had to be positive in one of the
following testing categories: PCR, EIA for anti-B. Henselae antibodies (IgM and/ or IgG),
or culture for B. henselae. There were 52 individuals who qualified for the study and
were categorized as elderly. Non-elderly volunteers were most commonly found to have
lymphadenopathy, yet general malaise was most commonly found in elderly patients.
9
This study brings attention to the fact that cats and dogs can serve as means for bacterial
diseases and a potential source of infection for humans.
My study did not consist of checking for any of the mentioned diseases or
infections. My experiment examined whether cats and dogs as house pets increased the
amount of bacteria in the home. I tested the homes of residents in the Thurston County
area. Three different locations were swabbed within each home. My research experiment
was an extensive study on the comparison of bacteria amounts found in households with
and without animals. Apartments with dogs and/or cats and houses with dogs and/or cats
both went through the same sampling procedure and were compared to apartments and
houses without either of the two pets. A requirement of Skerget et al. 's, (2005) volunteers
without pets was that they had never had a dog or cat live with them, my study only
required that they not have a pet living with them for the last six months. My hypothesis
was that houses and apartments with one or more pets (dog and/or cat) will have a higher
level of bacteria than those homes without animals.
Methods
Subject Requirements:
My participants consisted of 60 residents, 30 houses and 30 apartments. The
samples included the following: 10 houses without cats or dogs, 10 houses with 1 cat or
dog, and 10 houses with multiple cats and dogs; 10 apartments without cats and dogs, 10
apartments with 1 cat or dog and 10 apartments with cats and dogs. All participants
resided in the Thurston County area. The names of the participants, who allowed me into
their homes, were kept confidential. Each participant in the study had signed a
10
confidentiality agreement and filled out a survey (Appendix 1). Each home was swabbed
in three different locations: kitchen, living room and the most trafficked area, which was
the entry/door way.
Preparation of Broth and Sterilized Tubes:
In preparation, all test tubes were sterilized using a Tuttnauer™ 3870E autoclave.
Empty test tubes were loosely capped and put into the autoclave. After all the tubes were
removed from the autoclave, the caps were tightened to prevent bacterial contamination.
Until time of sampling the sterilized test tubes were stored in the refrigerator, inhibiting
bacterial growth. The swabs were taken from the various households and the following
samples were brought back to the lab. Fisher® nutrient broth was prepared. Two
separate batches of 14.4g (14.4g x 2 = 28.8g, the total amount needed to distribute 15 mL
to each of the 240 test tube) of dry nutrient broth was mixed into two separate beakers.
Each beaker contained 1800 ml distilled water. The nutrient broth was complete when all
dry mixture was entirely dissolved it was then autoclaved for sterilization.
Obtaining Samples:
The location for sampling was taken by swabbing 1.2 m from the entrance of the
various rooms. A sterile swab and sterile test tube were used to obtain the bacterial
sample from each floor surface location. The bacterial sample was obtained by one
swiping motion of the swab from left to right over a 30 x 15 cm sample area three times,
as shown in Figure 1.
11
Figure 1: The method of swabbing each room of a home with a sterile cotton swab. The
area is 30 cm x 15 cm.
The measurement of 30 cm x 15 cm was chosen because it is a large enough area
to get a sample of the bacteria in the room. The wooden end of each swab was then
broken to fit into each test tube and the tube was closed tightly for transport. A total of
240 capped test tubes were brought to the Saint Martin’s University biology laboratory
where the bacteria levels were compared. The control of this experiment was the houses
and apartments without dogs and or cats. Each tube was color coded to indicate house,
apartment, and presence of pets and then numbered to signify which room it had come
from. Fifteen mL of nutrient broth was then pipetted into each of the 240 test tubes.
Each test tube was then placed into the incubator for 24 hours at 37˚ C, the normal
body temperature, a temperature that assists in optimal bacterial growth (Brown, 2005).
After 24 hours of incubation the samples were placed into a spectrophotometer to
measure percent transmission (Benson, 2002). A spectrophotometer is used to measure
12
the turbidity of each sample. While a measure of turbidity does not report the actual
number of cells present in the sample, the light that passed through the sample is
decreased according to an increased number of bacterial cells present (Benson, 2002).
First the wavelength of the spectrophotometer was set to 686 nm (Benson, 2002).
The spectrophotometer was blanked with a disposable cuvette containing sterilized
nutrient broth. Another cuvette was rinsed with distilled water. After mixing each
sample via gentle shaking of the tube, the solution was carefully poured in the cuvette.
After taking the reading, the cuvette was then rinsed with nutrient broth between sample
readings to eliminate the remains of the previous test and then reused.
Next, the percent transmission of each sample was recorded. This process was
repeated for each sample using disposable cuvettes. Each home had three data values
that were recorded: living room, kitchen, and hallway.
Statistical Analysis
The concentrations of the bacteria that were found in the various rooms within
each household were compared. A comparison was drawn against households where pets
reside versus households that do not have cats and/or dogs. I conducted a two-way
Analysis of Variance (ANOVA) test and minitab (2005) was used for the statistical
analysis. ANOVA tests requisite two factors, respectfully number of pets and type of
home, (apartment or house).
13
Results
A two-way analysis of variance (ANOVA) was performed via MINITAB (2005)
to evaluate the difference in bacteria levels among apartments and houses with pets and
without pets by testing three rooms from each residence. Graphs created from this data
exhibit bacterium mean absorbance levels. There was a wide range of Bactria found in
each home, more specifically in Figure 1. ranging from 0.0464 to 0.1038 in residences
with more than one pet. Absorbance shown in residents without a pet in Figure 2.
demonstrates a range from 0.039 to 0.063. All residents with one pet depicted in Figure 3.
appear to have a wide absorbance range from 0.1277 to 0.0599. ANOVA results indicate
there was no significant difference between the bacterial levels of houses and apartments
regardless of whether they have a pet and regardless of the number of pets (F=0.53; d.f. =
10; p= 0.868). Since no statistically significant difference was found between residences,
I compared bacterial levels among the rooms of each residence. The optical densities
from from the living room, kitchen, and dining room show no statistically significant
difference (F=0.53; d.f. = 10; p=0.868).
14
0.25
Absorbance
0.2
0.15
0.1
0.05
0
Living room
kitchen
House with more than 1 pet
hallway
Apartments with more than 1 pet
Figure 1. The graph illustrates the mean absorbance of a culture from swab sample
lactation placed in nutrient broth and incubated for 24hr. at 37ْ C. from 10 apartments
and 10 houses with more than 1 pet after 24 hours of incubation. The error bars represent
one standard error.
0.25
Absorbance
0.2
0.15
0.1
0.05
0
living room
kitchen
Houses without pets
hallway
Apartments without pets
Figure 2. The graph illustrates the mean absorbance of a culture from swab sample
lactation placed in nutrient broth and incubated for 24hr. at 37ْ C. from 10 apartments
and 10 houses after 24 hours of incubation. The error bars represent one standard error.
15
0.25
0.2
0.15
0.1
0.05
0
Living room
kitchen
Houses with only 1 pet
hallway
Apartments with only 1 pet
Figure 3. The graph illustrates the mean absorbance of a culture from swab sample
lactation placed in nutrient broth and incubated for 24hr. at 37ْ C. from 10 apartments
and 10 houses with only 1 pet after 24 hours of incubation. The error bars represent one
standard error. Absorbance appears to range from 0.1277-0.0599.
Discussion
The results obtained in this study suggest no difference in bacteria on the floors
among the tested homes, despite the presence of a cat and/or dog. Owning a pet in an
apartment or in a home did not have a significant increase in bacterial levels compared to
those homes where only humans reside. Little research has been conducted on the actual
amounts of bacteria in homes, studies like that of Dubey, 1995 who studied parasitic
protozoan’s in felines, collected data from shedding cat fur. Fur shedding in large
quarters, would be thinned over floor surface reducing data collection for absorbance. I
structured my hypotheses on the thought that, people with less living space and more
occupants would result in a higher bacterial level. The results of my study failed to
support my hypothesis that a given apartment or house without animals would have a
16
higher bacterial level than an apartment or house without a cat or dog. Contrary to
expectation, the data depicted in Figure 3 indicates that the largest mean difference was
found in houses and apartments with only one pet, more specifically in the kitchen.
Evidently those differences were not significantly increased in the homes with more than
one pet, as shown in Figure 1.
On average, houses with animals appeared to have a larger bacteria growth in the
living room compared to the kitchen and hallway in Figure 2. This may be due to the fact
that if let outside, the animals enter through the front door. This possibility could be
further investigated by proposing that a sample be specifically taken from exactly where
the pets enter the home. There is no evidence of any generalities, or patterns from the
data represented by the Figures.
There are many possible explanations as to why there were not significant
differences among the mean household bacterial loads found in homes with animals and
homes without animals. First, the intensity of cleaning for each home is random and
varies greatly. Vacuuming and cleaning the carpet could lead to a lower bacteria
concentration even if there are more animals, than a home with no animals that does not
get vacuumed regularly. It could also be argued that the vacuuming of bacterial infested
carpet could actually aid in spreading the bacteria throughout the home. Another possible
explanation for my results may be the number of people living in the home. A home with
four adults may have more bacteria than a home with only one adult and a dog. Taking
into consideration the amount of people and the size and type of the animal could have
explained the results.
17
It would be interesting to conduct the research again with more than ten
participants in each group to account for variability in the treatments. Also, testing once a
day over several days could affect the results by creating a more accurate mean. Testing
the homes of only those who take off their shoes at the door might also generate different
results, since bacteria from outside can also be brought in by shoes on humans. Finally,
separating the investigation into two studies; one of only homes with dogs, and a separate
study with only homes with cats. Some homes could have had a cat that sheds hair more
than a dog in another home, which would affect the bacterial growth in the nutrient broth
after sampling, and hair thickness could have affected the amount of hair swabbed onto a
sample.
The results of this study are important for homeowners with and without pets.
Since there was no statistical difference between bacterial levels found from residences
with animals and without, it is noted from this study that ultimately pets do not determine
the amount of bacteria found in the home, it is a matter of many variables. The amount of
bacteria is ultimately determined by the cleaning habits of the pet owner, the products
used when cleaning and how often cleaning occurs. Therefore, be it an apartment or
house, with or without pets, it would be beneficial for the homeowner to practice
thorough cleaning habits in order to decrease the amount of bacteria found throughout.
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Aknowledgements
I am grateful for the help I found in Dr. Mary Jo Hartman, for her guidance and
giving me numerous suggestions and crucial advice, and teaching me how to make
graphs on the computer. I am grateful to Dr. Margaret Olney for her guidance and
suggestions. I would also like to thank the participants in my study who shall forever
remain anonyms’ yet the sole vital piece of my research. I would like to thank my
personal assistant and best friend Takara Thale, Thank you very much for always making
time to edit my papers. Finally, thanks to Cheryl Guglielmo who was a tremendous
assistance in the lab, and I am thankful for all the help with the autoclave, obtaining
supplies, and preparing nutrient broth. Lastly, thank you to my senior seminar peers of
the class of 2007.
19
Appendix1.
20
Literature Cited
Ben-Ami, R.Ephros, M., Avidor, B., Katchman, E., Varon, M., Leibowits, C.,
Comanesnter, D., Giladi, M. 2005.Cat-Scratch Disease in Elderly Patients. Clinical
Infectious Diseases.2005; 41:969-74.
Benson, H. 2002. Microbiological applications: Laboratory Manual in General
Microbiology, 8th ed. McGraw Hill, NY, pp. 96-136.
Brown, A. 2005. Benson's Microbiological Applications Short version: Laboratory
Manual in General Microbiology, 9th ed. McGraw Hill, NY, pp. 45-139.
Cavassani, K. A., Aliberti, J.e., Silva, D. J., Ferreira, B. R2004. Tick saliva inhibits
differentiation, maturation and function of murine bone-marrow-derived
dendritic cells. Immunology. 114: 235-245.
Cole, E.e.Addison, RM Rubion, lR Leese, K. E. Dulaney, P.D. Newell, M.S. Wilkins, J.
Gaber, D.J. Wineinger, T. Criger,D. A. 2003. Investigation of antibiotic and
antibacterial agent cross-resistance in target bacteria from homes of antibacterial
product users and nonusers. Journal of Applied Microbiology. 95:664-676.
Dubey, J. P. 1995.Duration of Immunity to Shedding of Toxoplasma Gondii Oocysts By
Cats. The Journal ofParasitology.81:410-415.
Kuiken,T.,Rimmelzwaan,G.,Vanriel,D.,Van AmerongenG., Baars, M., Fouchier, R,
Osterhaus, A., 2004. Avian H5Nl influenza in cats. Science. 306: 241-241.
Minitab ® release 14.20. 2005 Minitab Inc.
Skerget, M. Wenisch, e. Daxboeck, F. Krause, R Haberl, R. Stuenzner, D. 2003. Cat or
Dog Ownership and Seroprevalence of Ehrlichiosis, Q Fever, and Cat Scratch
Disease. Emerging Infectious Diseases. 9: 1337-1339.
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