Download Feline visceral leishmaniasis in Kerman, southeast of Iran

J Vector Borne Dis 54, March 2017, pp. 96–102
Feline visceral leishmaniasis in Kerman, southeast of Iran: Serological and
molecular study
Baharak Akhtardanesh1–2, Iraj Sharifi2, Ali Mohammadi2-3, Mahshid Mostafavi2, Mojdeh
Hakimmipour1 & Neda Ghasemi Pourafshar1
1
Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman; 2Leishmaniasis
Research Center, Kerman; 3Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, Iran
ABSTRACT
Background & objectives: Visceral leishmaniasis (VL) is a fatal zoonotic disease in tropical and sub-tropical countries
including Iran. Dogs constitute the main domestic reservoir for VL (kala-azar) in Iran but incidence of the disease
in cats from Fars and East Azerbaijan provinces has led to propose them as secondary reservoirs, and possible
expansion of the feline role in the transmission of disease. The aim of this study was to evaluate the prevalence of
Leishmania infantum infection in stray cats in Kerman City by ELISA and PCR methods.
Methods: In this cross-sectional descriptive study, 60 stray cats were randomly live trapped from different parts
of Kerman City during a six month period between March and September 2014. About 3 ml blood samples were
drawn from jugular vein of captured cats and a detailed questionnaire about demographic characteristics and clinical status of each cat was recorded by attending veterinarian. The complete blood counts and biochemistry analysis
were performed for all cats. Finally collected sera samples were tested by an indirect enzyme-linked immunosorbent
assay (ELISA) kit and PCR amplification method.
Results: Prevalence of Leishmania infantum infection was 6.7 and 16.7% by ELISA and PCR assays, respectively.
Infection rate was significantly higher in leukopenic cats, which were older than 3 yr.
Interpretation & conclusion: The results of the study indicate that stray cats are at risk of L. infantum infection in
Kerman City. Further, studies are required to elucidate the role of cats as potential reservoir host in the epidemiology of VL in endemic regions.
Key words Cat; Iran; PCR; serology; visceral leishmaniasis
INTRODUCTION
Leishmaniasis represents a spectrum of disease condition with significant health impacts, caused by different
species of Leishmania genus. This disease is currently
endemic in 98 countries of the world. Overall, annual
prevalence is 12 million and the population at risk is approximately 350 million1. Cutaneous leishmaniasis (CL)
and visceral leishmaniasis (VL) are present in 14 of the 22
countries of the Eastern Mediterranean Region2. Iran is an
endemic country for both CL and VL3.
Leishmania infantum is transmitted by various species of female phlebotomine sandflies4. Domestic dogs
(Canis familiaris) are major reservoir for L. infantum
which play a key role in transmission of infection to humans, whereas cats have been suggested as a secondary
reservoir in endemic areas5–6. Unfortunately, there are
still several obscure points about host factors, clinical
outcome and transmission of VL to other vertebrates7–8;
hence it is necessary to evaluate their infection status and
role in the epidemiology of zoonotic VL. The main foci
of kala-azar in southern Iran are Kazeroun, Nourabad,
Firouzabad and Darab districts in Fars province, where
dogs are the principle domestic reservoir9–12. Based on a
hospital record, 260 cases of VL have been recorded during 2001–2009 from these regions12. Reports of feline VL
from the aforementioned areas indicate that cats might
also be involved as potential hosts in the epidemiology of
the disease13–14.
Detection of antibodies to Leishmania by different
diagnostic tests consisting of enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody (IFA),
and the direct agglutination test (DAT), proved suitable
for screening canine and human population in different
studies15–18.
In the last decade, the use of polymerase chain reaction (PCR) for detection of Leishmania DNA was shown
to be highly sensitive and specific. A variety of canine tis-
Akhtardanesh et al: Feline visceral leishmaniasis in Iran
sues, including bone marrow, spleen, lymph nodes, skin,
and conjunctival biopsy specimens and peripheral blood
have been used for the identification purpose19.
Cats might be implicated as a secondary reservoir in
Fars and Azerbaijan provinces in Iran where kala-azar is
endemic, and hence evaluation of their infection status
and role in the epidemiology of zoonotic leishmaniasis
is very important8. The aim of this study was to assess the
prevalence of L. infantum infection within the population
of stray cats in Kerman City using ELISA and PCR methods. The results of the present study identified another
potential animal reservoir host of L. infantum (other than
dog) and this data could be used for planning an effective
future control strategy for VL in Iran.
MATERIAL & METHODS
Study area
This epidemiological study was conducted in the City
of Kerman, the center of Kerman province. Kerman is located in the southeast of Iran, the second largest province
of the country with an area of 180,726 km2 and population
of over 800,000 people (2015 census).
Sampling
In total 60 stray cats were captured by a double door
live trap cages containing baits by a volunteer cat rescue
group in a Trap-Neuter-Return program. The cages were
placed at five different locations of Kerman City near
garbage dumpsters. These cats were randomly selected
with no limitation for age, sex, and clinical status between
September and March 2014. Before neutering surgery, all
animals were clinically monitored for three consecutive
days and a detailed questionnaire consisting of age, sex
and clinical status of each animal was recorded by attending veterinarian. From each cat 3 ml blood samples were
collected from jugular vein and transferred into falcon
tubes containing anticoagulant, ethylenediaminetetraacetic acid (EDTA).
All clinical procedures were performed by
appropriately qualified scientific colleagues and the
project underwent ethical review, and was given approval by an Institutional Animal Care (Approval number:
C/93.6.14).
Haematology, biochemistry and serology
Complete blood counts were performed manually for
all cats and the presence of haematological disorders such
as anaemia (haematocrit <20), leucopenia or leukocytosis
(<5500 to >19500 leukocyte/µl of blood) and changes in
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differential leukocyte count were recorded20. Serum samples were separated by centrifugation at 3000 rpm for 3–5
min and stored at –20 °C for serological and biochemical
examination. Total protein, globulin, blood urea nitrogen
(BUN), creatinine, alkaline phosphatase (ALP), alanine
aminotransferase (ALT) and aspartate aminotransferase
(AST) level were measured by an autoanalyser (Autolab,
AMS -18A, China)21. All the serum the samples were
tested by an indirect ELISA kit (ID Screen Canine
Leishmaniasis, ID-Vet Company, France) following the
manual’s instruction of the manufacturing company, and
the samples were read at 450 nm by an ELISA reader
(ELX800 BioTek, USA).
The ELISA test was validated if the mean value of
the positive control optical density (ODPC) was > 0.350
(ODPC > 0.350), and the ratio of the mean values of the
positive and negative controls (ODPC and ODNC, respectively) was > 3(ODPC/ODNC > 3).
The proportion rate of each sample over positive (S/P)
control was calculated by the following formula:
S/P =
OD (Sample) – OD (NC)
× 100
OD (PC) – OD (NC)
The sample was interpreted as positive if the rate was
≥50%. The ratio >40 and <50% was considered doubtful
and ≤40% was recorded as negative.
Molecular identification
DNA extraction: DNA extraction from blood samples of
cat was carried out by Viral Gene-spin™ Viral DNA/RNA
Extraction kit (VeTeK™, South Korea) according to manufacturer’s instructions. Template DNA was measured
using a NanoDrop–2000 spectrophotometer (Thermo
Fisher Scientific) and extracted DNA was stored at –20°C.
Nested PCR assay: The two step of nested PCR were
used on extracted DNA to amplify variable fragments
of kinetoplast DNA (kDNA) of the Leishmania species.
The chosen primers were designed specifically for this
PCR analysis as previously described by Noyes et al22. In
the first-step of the PCR, external primers CSB2XF (5'CGAGTAGCAGAAACTCCCGTTCA-3') and CSB1XR
(5'- ATTTTTCGCGATTTT CGCAGAACG-3') and in the
second-step internal primers 13Z (ACTGGGGGT TGGTGTAAAATAG) and LiR (TCGCAGAACGCCCCT)
were used. In the first round of each 25 μl reaction mixture
of PCR 5 μl template DNA, 12.5 μl Taq DNA Polymerase
Master Mix Red (Ampliqon, Denmark) and 30 picoM of
each CSB2XF/ CSB1XR primers were used. The ther-
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J Vector Borne Dis 54, March 2017
mocycler program consisted of an initial step at 95°C for
5 min, followed by 35 cycles, 30 sec each at 94°C, 1 min
at 55°C and 1 min at 72°C, and then a final extension for
5 min at 72°C. For the second round of PCR, 1 μl of diluted (1 : 9) PCR product of the first round was used as
template and this step was performed with the same conditions and reaction mixture as in the first one, but with different specific primers LiR and 13Z. The amplified DNA
was subjected to electrophoresis in a 2% agarose gel, prestained with ethidium-bromide and viewed under ultraviolet light. The presence of specific bands was recorded
in positive samples.
PCR assay for sequencing and phylogenic analysis:
Specific amplification was used on extracted DNA to
amplify partial 7SL RNA gene of the Leishmania species
as described previously in a study by Zelazny et al23. The
PCR amplification with primers TRY7SL (5'-TGCTCTGTAACCTTCGGGGGCT-3') and TRY7SL (5'- GGCTGCTCCGTYNCCGGCCTGACCC-3') generated a
product of 137–139 bp (excluding the primers). The
PCR assay was performed in 25 μl reaction mixture
containing 5 μl template DNA, 12.5 μl Taq DNA Polymerase Master Mix Red (Ampliqon, Denmark) and 10
picoM of each primer. The PCR program was set for
initial step at 95°C for 5 min, followed by 34 cycles at
95°C for 30 sec, 65°C for 30 sec, and 72°C for 45 sec
and a final incubation at 72°C for 5 min. All amplicons
were sequenced in both directions by an ABI-3730XL
capillary machine (Macrogen Inc., South Korea). Nucleotide sequence analysis was performed by the basic
local alignment search tool (BLAST) from the National
Center for Biotechnology (http://www.ncbi.nlm.nih.gov)
and Bioedit software (ver.7.2)24. All individual sequences from partial 7SL RNA region of Leishmania species
(KMC3, KMC4, and KMC7 and KMC8) were identified and submitted to GenBank under accession numbers
KU870696 to KU870699.
Evolutionary analyses for partial 7SL RNA region
were conducted in MEGA 625. The analyses of multiple
sequence alignments for evolutionary history were inferred employing the maximum-likelihood (ML) method
based on the Kimura 2-parameter model. Initial tree(s)
for the heuristic search were obtained automatically by
applying neighbour-join (NJ) and bio-NJ algorithms to
a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting
the topology with a superior log likelihood value. The tree
was drawn to scale, with branch lengths measured in the
number of substitutions per site.
Data analysis
The PCR method was considered as a gold standard
for determining the sensitivity, specificity, positive and
negative predictive value of ELISA. The degree of agreement between ELISA, and kDNA PCR was determined by
calculating Kappa. Finally positive PCR test was set as an
outcome variable while sex, age, health status, biochemical and haematological alterations were considered independent variables. Analysis of prevalence values relative
to independent variables was conducted using the statistical package for the social sciences (SPSS) software version 15 (SPSS Inc., Chicago, IL) considering a probability
(p) value of <0.05 as statistically significant.
RESULTS
By using ELISA, anti-Leishmania antibody was detected in 4 (6.7%) of the studied cats, whereas by using
PCR method, 10 cats (16.7%) were found infected by
L. infantum and one (1.7%) by L. tropica. The specific
kDNA amplicon lengths of 650 bp for L. infantum were
amplified from nine samples but the suspected mixed
750 bp band for L. tropica was amplified from one of
the blood samples. Amplification was not detected in 2
(2%) cases and in the negative controls (Fig. 1). All infected cats were apparently healthy at clinical examination. The factors associated with positive PCR test were
age (p = 0.01), elevated creatinine (p = 0.01) and globulin
N
1
2
3
4
L. tropica L. infantum
M
Fig. 1: Agarose gel electrophoresis. Lanes 2–4: Positive samples
with size specific 650 bp band for L. infantum; Lane 1:
Suspected positive samples with non-specific bands; Lane
M: Size of marker 100 bp (Thermo Scientific, USA); Lane
N: Negative control (Non-template control); Lanes L.
tropica MHOM/Sudan/58/OD strain) with 750 bp band and
L. infantum (MHOM/TN/82/IPT1 strain) with 650bp are
shown as positive control. Cats with negative samples are not
shown.
Akhtardanesh et al: Feline visceral leishmaniasis in Iran
level (p = 0.03) and leukopenia (p = 0.05). There were no
other significant alterations in haematological and biochemical findings in the infected cats. The sensitivity and
specificity of ELISA method was 44.4 and 100%, respectively while positive and negative predictive values for
ELISA method were 100 and 90.9%, respectively (considering PCR as gold standard). Mild agreement (Kappa=
0.368) was seen between these diagnostic methods.
Multiple sequence alignments compression of the
amplificons of partial 7SL RNA region showed much more
polymorphism in KMC3 sequence than in other Leishmania species (Fig. 2). A consensus tree with the highest
log likelihood (–556.2154) is shown in Fig. 3. The 7SL
RNA phylogeny analysis of three individual sequences
showed high similarity to the other L. infantum whereas
the KMC3 was clustered in a separate branch next to the
other L. infantum and as sister clade of L. donovani23–24.
The numbers on the branches represent the percentage of 1000 bootstrap samples supporting the branch.
Initial tree(s) for the heuristic search were obtained by
applying the NJ method to a matrix of pairwise distances estimated using the maximum composite likelihood
(MCL) approach. All positions containing gaps and missing data were eliminated. There were total 134 phylogenic
positions in the final dataset.
DISCUSSION
In Iran, visceral leishmaniasis is caused by L. infantum and the main reservoirs of the disease are dogs; however, infection in other animals such as cats has also been
99
reported13. The cats (Felis domesticus) are still regarded
as unusual hosts for VL, although the first record of feline
leishmaniasis dates back to 1912 in Algeria, in a bone marrow sample from a 4-month-old house hold kitten living
in the same house with a dog and a child which together
were affected by CL and VL26.
In recent years, asymptomatic or symptomatic infection caused by L. infantum in cats has been reported in several countries where zoonotic VL is prevalent; however,
the actual susceptibility of cats to infection by Leishmania
spp. is still poorly understood14.
The common techniques for detecting VL are indirect
fluorescent antibody test (IFAT), ELISA, direct agglutination test (DAT) and PCR. ELISA has been widely used
as serodiagnostic method, with high sensitivity in dogs,
but its specificity depends upon the antigen. Earlier studies on dogs have reported moderate agreement between
results obtained by serological and molecular methods27.
Serological investigations in endemic VL areas revealed
different seroprevalence in cats ranging from 0.6% in Portugal to as high as 69% in southern Italy26–28 .
In Middle East, in a nonendemic place of Jerusalem,
seroprevalence of 6.7% was reported among cats29. In a
previous study carried out on felines for detection of VL,
28 and 25% of sampled cats revealed antibodies reacting
with L. infantum by DAT and IFAT method, respectively in
the endemic provinces of Fars and East Azerbaijan, Iran,
whereas in another study in the same regions, prevalence
of feline VL was estimated to be 10% when determined
by parasitological and molecular methods using liver and
spleen samples13-14.
Fig. 2:Alignment of partial 7SL RNA sequences from Leishmania spp. Dots indicate identity with the
Leishmania infantum sequence. Since all isolates belonging to the same species had an identical 7SL
RNA sequence, only KMC3 sequence showed highly nucleotide polymorphism in some positions.
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J Vector Borne Dis 54, March 2017
Fig. 3:Phylogenetic tree of 15 reference blood isolates of Leishmania spp. and the L. collosoma 7SL RNA gene as the out group.
Numbers on the branches represent the percentage of 1000
bootstrap samples supporting the branch. Initial tree(s) for
the heuristic search were obtained by applying the neighbourjoining method to a matrix of pairwise distances estimated
using the maximum composite likelihood (MCL) approach.
The tree is drawn to scale, with branch lengths measured in
the number of substitutions per site. The present study is among the first epidemiological
investigations performed on feline Leishmania infection
in south eastern Iran; and the overall seroprevalence of
feline VL by ELISA and PCR was 6.7 and 16.7% respectively. In comparison to earlier studies, frequency of positive samples in the present study was considerably higher
by the PCR method13-14. A similar study was carried out in
Ahar City in northwest of Iran, wherein no any infection
was detected in cats by PCR and culture, but 6% of cats
had a positive titer in DAT test31.
As reported in other studies, the diagnostic sensitivity
of all serological tests (except PCR) could be very low in
cats because the production of antibodies against Leishmania spp. is lower, which can render them seronegative32.
This study used nested-PCR as a rapid and sensitive
method for detection and characterization of Leishmania
species, as this method has been widely used in many
other studies32–34. In concordance with these studies, all
positive samples displayed similar 650 bp bands for L.
infantum whereas the suspected KMC3 sample showed
nonspecific bands in the range of 600 to 800 bp. Hence, to
confirm the results of the nested PCR, sequencing method
was used. The mini-exon gene act as good genetic marker, but the sequencing process is cumbersome32. Due to
the limitations of sequencing technique for kinetoplast
mini circle amplification and the risk of improper readings for Noyes’ nested PCR methods, the 7SL RNA region
was used for sequencing and analysis of the nucleotide
composition of the samples31-33. The differences between
common Leishmania species in Iran could be clearly dis-
tinguished by sequencing this region34- 35.
Although, nucleotide polymorphism was observed
among samples, phylogenetic analysis did not show a significant difference between positive samples in the present study and other L. infantum records. However, due
to the presence of more nucleotide differences in KMC3
sample, the sample was placed in a separate branch from
the L. infantum cluster.
Eslami et al36 have reported similar results from the
conserved region inside kDNA when compared with
other genomic DNA regions. They had suggested further studies to find more significant molecular markers
to differentiate the Leishmania species. Thus, based on
the results of the two amplification region and phylogenetic analysis, although KM3 was considered as positive
sample, in the absence of a clear species border within suspected samples, it was named as Leishmania spp. according to Auwera et al32 studies. The species characterization
for this suspected sample is essential for further study on
other gene regions.
The specificity and sensitivity of the commercial
ELISA test used in other studies have been reported as
high as 99.1 and 98.5% in the detection of canine VL in
endemic areas37. In this study, the sensitivity and specificity of ELISA method was 44.4 and 100% respectively in
cats. Based on the considerable specificity and predictive
value of ELISA, this method could be effectively used to
rule out disease in suspected feline cases.
Leishmania infantum grows poorly in culture media,
and culture method has low sensitivity; therefore culture
and isolation were not preformed in the present study1.
On the other hand, for ethical consideration, noninvasive
methods were used for primary investigations.
Till date, 28 clinical confirmed cases of feline visceral leishmaniasis (FVL) have been reported across the
world, 11 (39.3%) of them occurred in the New World
(10 cases in South America) and 17 (60.7%) in the Old
World38. The most common reported clinical finding in
infected cats was skin lesions which was not detected in
the current study39. In addition, it is important to emphasize that previous studies have shown that cats are more
attractive and are more preferred as blood meal sources by
phlebotomine sandflies38.
As there are limited studies in the field of feline leishmaniasis, hematological and biochemical alteration have
not been obviously defined, though elevated creatinine
and globulin levels and leucopenia have been reported in
infected VL cases40. Elevated creatinine level might not
be directly related to leishmaniasis as most of elderly cats
are predisposed to chronic renal failure41.
In the present study, there was a significant asso-
Akhtardanesh et al: Feline visceral leishmaniasis in Iran
ciation between age of cats and disease prevalence This
finding is consistent with other reports which noted that
seropositivity is higher in geriatric animals14-42. However,
there was no significant association between prevalence
and sex which is in agreement with the findings of other
studies43-45.
CONCLUSION
In conclusion, the results of the study indicate that domestic cats might serve as potential secondary reservoirs
for Leishmania. The lack of accurate diagnostic method
for feline leishmaniasis in endemic areas might imply that
the animal will continue to be a potential reservoir for L.
infantum. Further, epidemiological studies are necessary
to determine the exact role of cats in the transmission of
leishmaniasis in endemic areas of Iran.
Conflict of interest
None of the authors of this paper have any financial or
personal relationship with other people or organizations
that could inappropriately influence or bias the content of
the paper.
ACKNOWLEDGEMENTS
The support and facilities provided by the Faculty
of Veterinary Medicine, Shahid Bahonar University
Kerman, Iran are gratefully acknowledged.
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Correspondence to: Dr Akhtardanesh Baharak, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University
of Kerman; and/or Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran.
E-mail: [email protected]; akhtardanesh@ uk.ac.ir
Received: 27 September 2016
Accepted in revised form: 25 November 2016