INTRODUCTION
Leishmaniasis represents a spectrum of disease con- dition 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 ap- proximately 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 spe- cies of female phlebotomine sandflies4. Domestic dogs (Canis familiaris) are major reservoir for L. infantum which play a key role in transmission of infection to hu- mans, 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 dur- ing 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 immunosor- bent 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 reac- tion (PCR) for detection of Leishmania DNA was shown to be highly sensitive and specific. A variety of canine tis-
Feline visceral leishmaniasis in Kerman, southeast of Iran: Serological and molecular study
Baharak Akhtardanesh
1–2, Iraj Sharifi
2, Ali Mohammadi
2-3, Mahshid Mostafavi
2, Mojdeh Hakimmipour
1& Neda Ghasemi Pourafshar
11Department 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 clini- cal 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 epidemiol- ogy of VL in endemic regions.
Key words Cat; Iran; PCR; serology; visceral leishmaniasis
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 meth- ods. 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 lo- cated 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 attend- ing veterinarian. From each cat 3 ml blood samples were collected from jugular vein and transferred into falcon tubes containing anticoagulant, ethylenediaminetetraace- tic acid (EDTA).
All clinical procedures were performed by appropriately qualified scientific colleagues and the project underwent ethical review, and was given approv- al 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
differential leukocyte count were recorded20. Serum sam- ples 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, re- spectively) was > 3(ODPC/ODNC > 3).
The proportion rate of each sample over positive (S/P) control was calculated by the following formula:
OD (Sample) – OD (NC) S/P = OD (PC) – OD (NC) × 100
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 man- ufacturer’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 TG- GTGTAAAATAG) 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-
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 di- luted (1 : 9) PCR product of the first round was used as template and this step was performed with the same condi- tions and reaction mixture as in the first one, but with dif- ferent specific primers LiR and 13Z. The amplified DNA was subjected to electrophoresis in a 2% agarose gel, pre- stained with ethidium-bromide and viewed under ultra- violet 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'-TGCTCT- GTAACCTTCGGGGGCT-3') and TRY7SL (5'- GGCT- GCTCCGTYNCCGGCCTGACCC-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 Poly- merase 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). Nu- cleotide 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 sequenc- es from partial 7SL RNA region of Leishmania species (KMC3, KMC4, and KMC7 and KMC8) were identi- fied 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 in- ferred 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 maxi- mum 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 agree- ment 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, biochemi- cal and haematological alterations were considered inde- pendent variables. Analysis of prevalence values relative to independent variables was conducted using the statisti- cal package for the social sciences (SPSS) software ver- sion 15 (SPSS Inc., Chicago, IL) considering a probability (p) value of <0.05 as statistically significant.
RESULTS
By using ELISA, anti-Leishmania antibody was de- tected 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 in- fected cats were apparently healthy at clinical examina- tion. The factors associated with positive PCR test were age (p = 0.01), elevated creatinine (p = 0.01) and globulin
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.
N 1 2 3 4 L. tropica L. infantum M
level (p = 0.03) and leukopenia (p = 0.05). There were no other significant alterations in haematological and bio- chemical findings in the infected cats. The sensitivity and specificity of ELISA method was 44.4 and 100%, respec- tively while positive and negative predictive values for ELISA method were 100 and 90.9%, respectively (con- sidering 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 Leish- mania 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 percent- age 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 distanc- es estimated using the maximum composite likelihood (MCL) approach. All positions containing gaps and miss- ing data were eliminated. There were total 134 phylogenic positions in the final dataset.
DISCUSSION
In Iran, visceral leishmaniasis is caused by L. infan- tum and the main reservoirs of the disease are dogs; how- ever, infection in other animals such as cats has also been
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 mar- row 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 infec- tion caused by L. infantum in cats has been reported in sev- eral 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 agglutina- tion 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 stud- ies 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 Por- tugal 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.
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% respec- tively. In comparison to earlier studies, frequency of posi- tive 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 Leish- mania 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 mark- er, but the sequencing process is cumbersome32. Due to the limitations of sequencing technique for kinetoplast mini circle amplification and the risk of improper read- ings 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 sig- nificant difference between positive samples in the pres- ent 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 fur- ther studies to find more significant molecular markers to differentiate the Leishmania species.Thus, based on the results of the two amplification region and phyloge- netic analysis, although KM3 was considered as positive sample, in the absence of a clear species border within sus- pected samples, it was named as Leishmania spp. accord- ing 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 specific- ity 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 vis- ceral 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 empha- size 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 leish- maniasis, 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-
Fig. 3: Phylogenetic tree of 15 reference blood isolates of Leishma- nia 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 neighbour- joining 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.
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 do- mestic 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: bakhtardanesh@yahoo.com; akhtardanesh@ uk.ac.ir Received: 27 September 2016 Accepted in revised form: 25 November 2016
