|Year : 2018 | Volume
| Issue : 3 | Page : 230-234
Low susceptibility of domestic cats to experimental Leishmania infantum infection
Baharak Akhtardanesh1, Reza Kheirandish2, Iraj Sharifi3, Ali Mohammadi4, Ali Mostafavi5, Tohid Mahmoodi6, Mohadesse Ebrahimi6
1 Department of Clinical Sciences; Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
2 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
3 Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran
4 Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, Iran
5 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran
6 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
|Date of Submission||13-May-2018|
|Date of Acceptance||01-Aug-2018|
|Date of Web Publication||4-Jan-2019|
Faculty of Veterinary Medicine, Shahid Bahonar University, P.O. Box 76169133, Kerman
Source of Support: None, Conflict of Interest: None
Background and objectives : The dogs are considered the main reservoir of visceral leishmaniasis (VL), but lately the disease incidence has been reported in cats also. In this study, the susceptibility of domestic cats to experimental Leishmania infantum infection was assessed by different diagnostic methods.
Methods : A total of 12 healthy adult male cats were captured by double door live trap cages containing baits. Of them eight cats were intraperitoneally inoculated with 107 L. infantum promastigotes (stationary phase), and four cats were used as controls. Whole blood and serum samples were collected at weekly intervals for 16 wk after inoculation for testing by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods. Aspirates of prescapular lymph nodes and bone marrow were obtained at monthly intervals. Clinical examination was performed twice weekly and histopathological evaluation was done on necropsy samples at the termination of the study. Results: One week after inoculation, blood nested PCR was able to detect the L. infantum infection and it remained positive until 16 wk. ELISA test remained negative during the study. Amastigote phase of parasite was not observed in bone marrow aspiration and necropsy samples.
Interpretation and conclusion : The feline model described in this work would be useful in further understanding of L. infantum immunopathogenensis in cats. The results of this preliminary study suggest that cats might be resistant to VL as the inoculation dose which induces pathognomonic clinical features in dogs, just creates asymptomatic parasitaemia in cats. Though, due to long-lasting parasitaemia, cats may act as appropriate reservoir for transmission of VL to human population. Further studies are needed to describe the possible role of cats in the epidemiology of VL in endemic areas.
Keywords: Experimental infection; feline; histopathology; Leishmania infantum; PCR; serology; visceral leishmaniasis
|How to cite this article:|
Akhtardanesh B, Kheirandish R, Sharifi I, Mohammadi A, Mostafavi A, Mahmoodi T, Ebrahimi M. Low susceptibility of domestic cats to experimental Leishmania infantum infection. J Vector Borne Dis 2018;55:230-4
|How to cite this URL:|
Akhtardanesh B, Kheirandish R, Sharifi I, Mohammadi A, Mostafavi A, Mahmoodi T, Ebrahimi M. Low susceptibility of domestic cats to experimental Leishmania infantum infection. J Vector Borne Dis [serial online] 2018 [cited 2019 Jan 23];55:230-4. Available from: http://www.jvbd.org/text.asp?2018/55/3/230/249481
| Introduction|| |
Visceral leishmaniasis (VL) or kala-azar is a systemic infection caused by L. donovani and L. infantum, and nearly 500,000 new cases of the disease are reported annually all over the world. In Iran, kala-azar in the most areas is sporadic and in some provinces, including Ardabil (Meshkinshar and Garmi), Fars (Jahrom and Firuz Abad), East Azarbaijan (Kalibr and Ahar), Qom, Khorasan, Chaharmahal-Bakhtiari, Khuzestan, Bushehr and Kerman (Baft and Orzoeih) is endemic. Based on the life-long persistence of parasites in the skin and reticuloendothelial organs of infected dogs, they are considered as major domestic reservoirs of VL playing a key role in transmission of infection to humans. Cats have been suggested as a secondary reservoir in areas where L. infantum is endemic but their actual role in the life cycle of the organism is not well-established. While subclinical feline infections are common in endemic areas for canine leishmaniasis, clinical illness due to L. infantum in cats is rare and commonly associated with coinfections by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV),. The prevalence rate of feline infection with L. infantum in serological or molecular-based surveys has been reported, to range from 0 to >60%. The ulcerative or nodular cutaneous and mucocutaneous lesions, lymphadenomegaly and ocular lesions are the most prevalent lesions reported in infected cats. Chronic gingivostomatitis is also a common clinical finding and has been reported in about one fourth of L. infantum infected cats. Another important point is that large numbers of cats do not show any obvious clinical signs and could be asymptomatic reservoir,. Experimental studies in the field of feline visceral leishmaniasis (FVL) are very scarce but they are highly valuable because clinical and histopathological pattern of VL and validity of different diagnostic methods are still unknown in cats,. In a study conducted in endemic regions of Iran, 10% of stray cats were found to be infected with L. infantum which suggest the role of felines in the epidemiology of kala-azar in endemic provinces,. The present study was aimed to evaluate the susceptibility of domestic cats to VL after experimental intraperitoneal inoculation of L. infantum promastigotes.
| Material & Methods|| |
This study was conducted in Kerman province which is located in the southeast of Iran with an area of 180,726 km2. The population of the province is about three million. The stray cats were gathered during April to September 2015, in a Trap-Neuter-Return program which is managed by the Corporation of Kerman Veterinary Faculty and Municipality. Cats were captured by double door live trap cages containing baits, by a volunteer cat rescue team. Before contraception surgery, all animals were clinically monitored for three consecutive days and a detailed questionnaire was completed for each animal, recording the age, sex and observations of clinical status.
Haematological and biochemical evaluation
From each cat 3 ml blood sample was taken from jugular vein for haematological and biochemical evaluations. Complete blood counts were performed manually for all cats and the presence of haematological disorders were recorded. Serum samples were separated by centrifugation at 3000 rpm for 3–5 min and stored at –20°C for serological and biochemical examinations. Total protein, globulin, blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by an autoanalyser (AMS SABA-18, Autolab, China).
In the next phase, rapid immunochromatography test (BVT Company, France) was used for exclusion of FELV and FIV infected cats.
DNA extraction and nested PCR assay
DNA extraction from blood samples was carried out by commercial Viral DNA/RNA extraction kit (Bioneer Company, South Korea) and two steps of nested PCR were used on extracted DNA to amplify the variable fragments of kinetoplast DNA (kDNA) of the Leishmania species for ruling out the in captured cats.
For the study, 12 clinically normal adult male cats aged between 12 and 18 months with normal haematology and biochemistry findings were selected. The cats were placed in separate cages in the animal house of Kerman Veterinary School, Kerman. The animals were fed daily with cooked chicken and had unlimited access to water. During an acclimatization period, they received treatment against intestinal parasite (Cestal, Hungry) and were vaccinated against feline parvovirus, herpesvirus, calcivirus (Biofel PCH, Czech Republic) and rabies virus (Biocan R, Czech Republic). The insecticide-repellent collar (Royalist, Spain) used for all selected cats and the animal house was monthly sprayed by cypermethrin 10% (Meli Agro Chemical Company, Iran) for prevention of mosquitoes' access to cats.
The study underwent ethical review by research council of the Shahid Bahonar University of Kerman, Kerman and was given approval on 10 April 2015 by an Institutional Animal Care Committee (Approval No.: C/95.10.10). All the procedures were performed by the appropriately qualified scientific colleagues.
Leishmania infantum promastigotes were prepared in the Leishmaniasis Research Center (School of Medicine, Kerman Medical University, Kerman). Before experimental infection, eight cats were anesthetized with an intramuscular injection of 2% xylazine (1 mg/kg) and 10% ketamine (15 mg/kg) (Netherlands, Alfasan Co). Venipuncture from jugular vein was done and the cats were inoculated with 107 live stationary-phases of L. infantum promastigotes in 1 ml RPMI 1640 (Roswel Park Memorial Institute, Sigma-Aldrich, Germany) intraperitoneally. The other four cats were selected as control group and received distilled water.
All experimental animals were checked on every alternate day by clinical examination. Skin lesions (dry exfoliative dermatitis, ulcerative dermatitis, nodular dermatitis, pustular dermatitis, hyperkeratosis), pale mucous membranes, lymphadenopathy, visceromegaly, weight loss, conjunctivitis and/or blepharitis, uveitis and/or keratitis, mouth ulcers or nodules and other related clinical manifestation were evaluated up to 16 wk post-infection.
Haematological and biochemical evaluation
Blood samples (5 ml) were weekly drawn from jugular vein, of which 2 ml were transferred in tubes with EDTA for haematological evaluation and DNA extraction, and the rest were used for serological and biochemical evaluations as described in earlier section,.
Total genomic DNA was extracted from EDTA-anticoagulated whole blood which was collected weekly during the study. Two steps of the nested PCR assay were performed with primers which were designed specifically as described earlier by Noyes et al. 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 (5′-ACTGGGGGTTGGTGTAAAATAG-3′) and LiR (5′-TCGCAGAACGCCCCT-3′) were used. In the first round of PCR containing 25 μ reaction mixture, 5 μ template DNA, 12.5 μ Taq DNA Polymerase Master Mix Red (Ampliqon, Denmark) and 30 picoM of each CSB2XF/CSB1XR primers were used. The thermocycler 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 μ diluted (1 : 9) PCR product of the first round was used as template, and was performed with the same conditions and reaction mixture as in the first one, but with different specific primers LiR and 13Z. 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.
Serum samples which gathered weekly 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 proportion rate of each sample over positive control was calculated by the following formula:
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. 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 optical density (ODPC and ODNC, respectively) was >3 (ODPC/ODNC >3).
Bone marrow and lymph node aspiration
Popliteal lymph node (PLN) aspirate and impression smear slides from bone marrow tissues were methanolfixed, Giemsa-stained and examined by direct microscopy for amastigotes (Leishman-Donovan bodies) at 2 wk intervals after inoculation.
At Week 16, all cats were euthanized by intravenous (IV) injection of 20% pentobarbital solution. Cats were evaluated for enlargement of reticuloendothelial system (RES) organs such as spleen liver and lymph nodes. The RES organs and lymph nodes were transported to the laboratory. Tissue slices were preserved in 10% formalin and embedded in paraffin; and 4 μm thick tissue sections were stained with Haematoxylin and Eosin (H & E) for further histopathological study.
| Results|| |
No any specific clinical signs attributable to infection with L. infantum were observed in the infected cats and they were apparently healthy during the study. Blood PCR was positive at first week and afterwards remained positive throughout the study. Antibody titres were not significantly elevated during 16 wk of evaluation. In comparison to control group, significant decrease in haematocrit (21.35 ± 8.5%) and elevation in serum total protein (10.75 ± 1.4 g/dl) level was seen in the infected cats. All the bone marrow and lymph nodes aspiration were negative during the study. At histopathological evaluation, no parasites were detected in the lymph nodes, bone marrow, spleen, and liver of inoculated cats but some pathological changes were seen [Table 1]. Hence, the present experimental study indicates that cats are apparently less susceptible than dogs to the development of a clinical disease after L. infantum infection. Furthermore, it was concluded that domestic cats show slower serological response in comparison to dogs. PCR is a good diagnostic method and viscerotropic Leishmania species induce a long-lasting infection in domestic cats.
|Table 1: Histopathological changes in study cases (experimentally infected by L. infantum) and control cats|
Click here to view
| Discussion|| |
For a long period, it was believed that cats did not play any role in the epidemiology of L. infantum in endemic areas due to low frequency of clinical manifestations compared to dogs, and hence cats have also been considered more resistant to experimental infection. Over the last few decades, many studies have confirmed that FVL may be relatively common in areas where Canine leishmaniasis is endemic. Seroprevalence ranges from 0 to 68.5% and molecular rates of infection range between 0 and 60.7% in endemic regions of the Old World. Sporadic clinical cases of FVL, with confirmed L. infantum parasites, have been reported in cats from European countries such as Italy, France, Spain and Portugal. Although, dogs are the main reservoirs of the parasite in countries of the Mediterranean basins but the role of cats in the epidemiology of L. infantum needs further attention. The low seroprevalence titres, along with the commonly asymptomatic infection in cats, can contribute to the underestimation of FVL occurrence in Iran whereas surveys have shown that the percentage of infected cats is not negligible in some endemic areas and they could be implicated as a secondary reservoir in such areas (Kerman, Fars and Azerbaijan provinces),,.
Therefore, it is necessary to determine their exact role in the maintenance and transmission of L. infantum in experimental studies. In Kirkpatric et al study, two weeks after intravenous inoculation of 8×108 of L. chagasi and L. denovani promastigotes, the liver and spleen impression smear were positive and antibody titre increased up to 30 fold at 24 wk post-infection. Intradermal injection of 5 × 107 promastigote of L. chagasi was not able to improve visceral involvement and bone marrow biopsy was negative till 24 wk after inoculation. The result was completely different from this study, however, the selected species, route of infection and infective dose was not similar. In an another study, stray cats were inoculated intradermally with 107 promastigote of L. braziliensis. Papules emerged at the injection site four wk after infection and skin lesions progressed to ulcerated nodules. Antibody titre appeared 20 wk after infection and lasted for 18 months. The skin lesions spontaneously healed after 32 wk.
Low susceptibility of domestic cats to experimental L. infantum infection was observed in the present study. The infective dose, which has provided serological and histopothological evidences of CVL in other experimental studies, did not cause any symptomatic infection in the studied cats. Feline hepatic lipidosis which was observed in six infected and two control cats might be related to environmental and nutritional stress which is common in stray cats, but sinus histocytosis may be the first sign of splenic pathological changes which was related to FVL. On the other hand, PCR showed that infection in cats may persist for long periods. Cats have been demonstrated to be infectious to sandflies in experimental xenodiagnostic studies both in the Old and New Worlds.
Recently it has been reported that disease progression in cats is proportional to increase in anti L. infantum specific antibody, and it may take longer time. The absence of antibody titre and clinical signs in infected cats in the present study may be related to the short maintenance time after inoculation. Strong association between retroviral, coronavirus, Toxoplasma and L. infantum coinfections has been documented and it is hypothesized that the coinfection may have impaired the cellular immune response allowing active multiplication of the parasite and the visceral dissemination,. Though, in the present experimental study, healthy immunocompetent cats were inoculated and the potency of immune system might have prevented L. infantum visceral dissemination. Feline immunodeficiency virus (FIV), feline leukaemia virus (FeLV) is endemic in Iran and retroviral-associated immunosuppression may increase the risk of L. infantum infection in stray cats and increase the risk of kala-azar transmission to humans.
| Conclusion|| |
The results of this preliminary study suggest that cats might be resistant to VL as the inoculation dose which induced pathognomonic clinical features in dogs, created asymptomatic parasitaemia in the cats. Though, due to longlasting parasitaemia, cats may be appropriate reservoir for transmission of kala-azar to human population. The feline model described in this work will be of use for further understanding of L. infantum immunopathogenensis in cats. The epidemiological role of cats in the maintenance and transmission of L. infantum should be further investigated in future experimental studies by using of different infective doses and other Leishmania species.
Conflict of interest
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
| Acknowledgements|| |
The authors are grateful to the Research Council of Shahid Bahonar University for financial supporting the study. They are thankful to Messrs Mohammadi and Keihani for their technical help and many veterinary students who supplied blood samples during the research period.
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