Clinical manifestations and diagnostic approaches in cases of canine leishmaniasis in Bulgaria
DOI:
https://doi.org/10.12834/VetIt.3128.23106.2Keywords:
Canine leishmaniasis, clinical case, molecular methods, serologyAbstract
Leishmaniasis, a parasitic disease found in many parts of southern Europe, is transmitted in both humans and canines through the bite of phlebotomine sandflies, and can present in a variety of ways, such as cutaneous, mucocutaneous, diffuse, and visceral. In Bulgaria there are endemic areas of canine leishmaniasis, with sporadic cases in humans. However, no detailed studies of the animal population and vectors have been performed. Here we describe a few clinical cases of canine visceral leishmaniasis in two districts in western Bulgaria: one endemic and one without previously detected cases in humans or dogs. Diagnosis was confirmed serologically and molecularly using both real time and conventional PCR. Specific anti-leishmanial antibodies were confirmed in three of the cases via ELISA, with 50% of them returning extremely high values. In the majority of the cases DNA fragments were detected in the skin or lymph node aspirate but not in the blood. This paper highlights the need for further studies updating the current knowledge on the epidemiology, diagnosis, and control of visceral leishmaniasis in the reservoir host population.
Introduction
Canine leishmaniasis (CanL) is a parasitic zoonotic infection of dogs caused by Leishmania spp. parasites and is transmitted by the bite of infected female Phlebotominae (Diptera, Psychodidae) sandflies (Akhoundi et al., 2016). Although cases of CanL due to Leishmania major and Leishmania tropica infection are documented (Baneth et al., 2017), CanL caused by Leishmania infantum is considered the most important vector-borne parasitic diseases of dogs in Europe (Alvar et al., 2004).
In humans, more than 20 Leishmania species have been described as agents of leishmaniasis. All of them are morphologically indistinguishable, but they can be differentiated through molecular methods or isoenzyme analysis. Even though leishmaniasis is classified as a neglected tropical disease (NTD) by the World Health Organization (WHO), between 1,500,000 and 2,000,000 new cases occur each year, causing more than 70,000 human deaths worldwide (Torres-Guerrero et al., 2017).
The first field study on CanL in Bulgaria was performed by Drenovski in 1941 on 100 dogs from Petrich municipality. Thereafter, Matov and Filipov conducted two serological studies in 1958 and 1997, without observing clinical presentation of the disease (Filipov et al., 1997; Matov, 1958). The first autochthonous cases of CanL in Bulgaria were reported in 2006 in two dogs from Petrich showing typical cutaneous lesions. After this report 6 additional dogs from the same municipality tested positive for antibodies presence (Tsachev, 2009; Tsachev et al., 2010). Petrich is situated in south-west Bulgaria close to the borders with North Macedonia and Greece, both of which are known to be leishmaniasis endemic countries. A recent sandfly field investigation performed in this region of Bulgaria, confirmed the presence of competent vectors of CanL (Dvorak et al., 2020). Up to date, Leishmania parasites have not been isolated from sandflies collected in the country. Although sporadic cases in humans are reported annually in southern Bulgaria, canine leishmaniasis in this endemic part of the country remains unmonitored.
The aim of this article is to report the ongoing presence of Leishmania infantum in Bulgaria and to describe the performance of a laboratory algorithm including multiple tests for detection of CanL cases among reservoir hosts, highlighting the importance of this approach for successful detection, control, and prevention of the infection.
Materials and Methods
The study group comprised of four animals: two 6- and 7-year-old mixed breed dogs (cases 1 and 2) living together in Mendovo village, Petrich municipality; a 10-year-old husky (case 3) from Samuilovo village, Petrich municipality; and a 2-year-old mixed breed dog (case 4) located in a shelter in Kostinbrod municipality.
Clinical examination
Dogs were examined for presentation of clinical signs associated with canine visceral leishmaniasis such as enlarged lymph nodes, alopecia, keratoconjunctivitis, blepharitis, onychogryphosis, lesions of the skin, ears, or muzzle, bristle condition, depigmentation, weight loss, decreased appetite, fever, vomiting and diarrhea.
Samples
Blood samples were collected in vacutainer tubes with spray-coated silica and with EDTA for whole blood (BD Vacutainer® Plus Plastic Serum Tubes, Becton Dickinson, Franklin Lakes), from the cephalic vein with a 21G needle. Blood designated for serology was allowed to clot for 30 min at room temperature before centrifugation at 2000 g for 10 min to separate the serum, after which it was stored at -20°C until analyzed. EDTA blood samples, lymph node aspirate (case 4) and skin lesions (cases 1 and 2) were kept at +4°C. The skin samples were homogenised with antibiotic media containing Eagle's Minimum Essential Medium (EMEM), 10 U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 mg/ml gentamicin and 2.5 µg/ml amphotericin B. After 24 h incubation at +4°C, the suspensions were centrifugated at 2000 g for 15 min and the supernatants were used for extraction. EDTA blood samples and lymph node aspirate were directly subjected to analysis.
Hematology and blood biochemical analysis
All samples were tested for complete blood count (CBC) and blood biochemical parameters using the hematology analyzer BC-2800 and the bio-chemistry analyzer BA-88A (Mindray, Shenzen, China).
Serology
For antibodies detection two methods were used: An in clinic rapid Leishmania Ab Test kit (BioNote, Big Lake, USA) and a commercially available enzyme-linked immunosorbent assay (ELISA) (ID Screen® Leishmaniasis Indirect, IDVet, Grabels, France) following the manufacturer’s protocols.
Molecular methods
Extraction was performed with IndiSpin® Pathogen Kit (Indical Bioscience, Leipzig, Germany) and specific DNA fragments were detected with real time and conventional polymerase chain reaction (PCR). For the real time PCR, a multiplex assay including primers and probe targeting the parasite specific kinetoplast DNA (kDNA) minicircle and primers and probe detecting an exogenous internal DNA control was applied (Applied Biosystems, Waltham, USA) (Lombardo et al., 2012). Conventional PCR was carried out by using 18 µl of Virotype Mix +IC-DNA (Indical Bioscience), 1 µM of each primer, amplifying a 145 base-pair (bp) size fragment from the conserved region of the kDNA minicircles, and 5 µl of DNA (le Fichoux et al., 1999). The sequences of the specific primers/probe and the temperature regimes of the reactions are shown in Table I.
Differential diagnosis
SNAP 4Dx Plus Test (IDEXX Laboratories Inc., Westbrook, USA) rapid test was used for additional testing for Dirofilaria immitis, Еhrlichia canis, Еhrlichia ewingii, Borrelia burgdorferi, Anaplasma platys and Aanaplasma phagocytophilum.
Results
Clinical examination revealed alopecia and necrosis on the ears, exfoliative dermatitis, nasal and auricular hyperkeratosis, onychogryphosis (cases 1 and 2), conjunctivitis and keratitis (case 1) (Figure 1). Furthermore, all dogs showed progressive weight loss and decreased appetite. One of the animals (case 3) exhibited nasal lesions, chronic diarrhea, and epistaxis. Clinical manifestation in case 4 included periorbital skin lesions. In none of the animals fever and solitary or generalized lymphadenomegaly was observed.
Blood biochemical profiling (Table II) and CBC (Table III) were performed on all dogs. The most important biochemical changes found in the plasma were mild to severe hyperproteinemia, hypoalbuminemia with a decrease in the albumin/globulin (A/G) ratio and in cases 2 and 3, increased plasma creatinine and urea. In all cases, leukocytosis and mild to moderate anemia were detected.
Due to the present clinical symptoms consistent with CanL, dogs were screened with a rapid test for antibody detection to confirm the clinical observations. All four dogs yielded positive results. High amounts of specific anti-leishmanial antibodies were confirmed in cases 3 and 4 when serum samples were subsequentially tested with ELISA, while IgG levels were marginally above the assay’s positive cut-off limit (S/P% ≥50%) in case 2, and in the gray zone in case 1 (S/P%: >40% – <50%). The results obtained from the laboratory investigations are presented in Table IV.
CanL infection was confirmed in all animals through the real time PCR method. Leishmania infantum specific DNA fragments were detected only in one of the examined EDTA samples (case 3), while the presence of the parasite in the rest of the affected animals was discovered in the skin (cases 1 and 2) and the lymph node aspirate (case 4). Afterwards, results were verified with the conventional PCR and a 145 bp fragment was detected in all samples which were positive on the real time PCR (Figure 2). No co-infection with heartworm or antibodies against Lyme disease and anaplasmosis were identified in any of the four dogs.
Discussion
Clinical manifestation of CanL is often variable, from subclinical to severe progressive disease, depending on the immune response of the host (Solano-Gallego et al., 2009). All four dogs in the present study showed clinical signs associated with leishmaniasis. During physical examination exfoliative dermatitis, nasal and auricular hyperkeratosis, conjunctivitis, keratitis, weight loss and chronic diarrhea were observed. The main blood biochemical abnormalities detected in all animals were related to changes in the protein levels, which are common findings, especially in the acute phase of the disease (Paltrinieri et al., 2010). Renal disease, due to deposition of immune complexes at the glomerular level (Grauer, 2005) can be present in approximately 50% of the cases (Cortadellas et al., 2006). Kidney function loss, associated with proteinuria (Almeida et al., 2005) can cause further decrease in the A/G ratio. Deviations in the creatinine level were present in two of the examined cases.
The main abnormalities detected in the CBC were leukocytosis and anemia. The observed leukocytosis may be a consequence of a systemic inflammatory response and often is prominent when ulcerative cutaneous lesions, with secondary bacterial infection are present. The most consistent hematological change in dogs naturally infected with Leishmania infantum is anemia (Kiral et al., 2004). The pathogenesis of the anemia in affected dogs has different mechanisms (Day, 2010; Smith et al., 2004; Tvedten, 2010; Weiss, 2010) and may appear six months after exposure to infection (Foglia Manzillo et al., 2013). Based on the blood biochemical results obtained in our study, pathogenesis can be attributed to depression of the metabolic activity of the bone marrow and the renal failure in cases 2 and 3, leading to decreased iron availability for erythropoiesis and reduced erythropoietin synthesis.
Immunochromatographic tests (ICT) and ELISA methods are extensively used for serological analysis of CanL. Although ICTs have been established as a screening assay performed routinely in the clinics, they have several limitations, including low diagnostic sensitivity (30-70%) and qualitative presentation of the results (EFSA Panel on Animal Health and Welfare, 2015; Maia and Campino, 2008). Due to these limitations, it is advisable to confirm the presence of antibodies with methods with better diagnostic performance, such as immunofluorescent antibody test (IFAT) or ELISA, which are designed to measure their level (Solano-Gallego et al., 2011).
Laboratory algorithms with high diagnostic sensitivity are essential for the detection and management of CanL. The most successful approach to identifyLeishmania infected dogs is the combination of serology and molecular methods (Miró et al., 2008). This methodology could be further improved by testing various types of samples for DNA detection. In the present study, three of the animals were confirmed as clinical cases based on the positive results from both ELISA and real time PCR tests. In these patients, parasite DNA fragments were detected in the skin or lymph node aspirate but not in the EDTA blood samples. Skin and lymph node aspirate/tissue are among the recommended types of samples to be tested in order to identify symptomatic and asymptomatic dogs with CanL (Morales-Yuste et al., 2022; Reis et al., 2006). Although PCR on whole blood is considered as less sensitive method, which was confirmed in this investigation also, PCR testing on EDTA samples is accepted as eligible approach in certain scenarios. In addition, all results obtained through the real time PCR were confirmed with a highly sensitive conventional PCR targeting kDNA, which enhanced the reliability of the performedlaboratory diagnosis.
Conclusion
In Bulgaria CanL remains unreported and infected dogs are usually diagnosed at the late stages of the disease, when the cost of the treatment is high and the prognosis for the outcome is poor. Delayed diagnosis affects not only the quality of life of the CanL cases but also favors the spread of the infection among other susceptible animals. Reliable diagnostic tests are crucial for the successful management of this infection and multiple analytical modalities can overcome limitations of singular analytical techniques for confirming the presence or absence of the disease. The combination of different methods increases the effectiveness of the laboratory algorithm and should be implemented in field investigations, particularly in endemic regions, where the transmission of the infection is high. The detection of subclinical cases is an additional challenge and should be considered when the strategy for disease control is framed.
Acknowledgment
The authors would like to thank Dr John Bianco for critical reading of the manuscript. This article was made with the financial support of Project № NIS-B-1326 “Canine Leishmaniasis in endemic regions of Bulgaria. Etiological, Epidemiological and clinical studies”, NIS, University of Forestry.
Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of this article.
Animal Rights Statement
None required.
References
Akhoundi M., Kuhls K., Cannet A., Votýpka J., Marty P., Delaunay P., et al. 2016. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies. PLOS Neglected Tropical Diseases, 10, e0004349.
Almeida M.A.O., Jesus E.E.V., Sousa-Atta M.L.B., Alves L.C., Berne M.E.A. & Atta A.M. 2005. Clinical and serological aspects of visceral leishmaniasis in Northeast Brazilian dogs naturally infected with Leishmania chagasi. Veterinary Parasitology, 127, 227–232.
Alvar J., Cañavate C., Molina R., Moreno J. & Nieto J. 2004. Canine Leishmaniasis. 1–88.
Baneth G., Yasur-Landau D., Gilad M. & Nachum-Biala Y. 2017. Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology. Parasites & Vectors, 10, 113.
Cortadellas O., del Palacio M.J.F., Bayón A., Albert A. & Talavera J. 2006. Systemic hypertension in dogs with leishmaniasis: prevalence and clinical consequences. Journal of veterinary internal medicine, 20, 941–947.
Day M.J. 2010. Immune-mediated anemias in the dog. Schalm’s Veterinary Hematology, 216–222.
Dvorak V., Kasap O.E., Ivovic V., Mikov O., Stefanovska J., Martinkovic F., et al. 2020. Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey. Parasites & Vectors, 13, 573.
EFSA Panel on Animal Health and Welfare. 2015. Scientific Opinion on canine leishmaniosis. EFSA Journal, 13, 77.
Filipov G., Getcheva G. & Kostova T. 1997. Epidemiological study of clinical cases of visceral leishmaniosis in Bulgaria. Problems of Infectious and Parasitic Diseases, 24, 5–7.
Foglia Manzillo V., Di Muccio T., Cappiello S., Scalone A., Paparcone R., Fiorentino E., et al. 2013. Prospective Study on the Incidence and Progression of Clinical Signs in Naïve Dogs Naturally Infected by Leishmania infantum. PLoS Neglected Tropical Diseases, 7, e2225.
Grauer G.F. 2005. Canine glomerulonephritis: new thoughts on proteinuria and treatment. The Journal of small animal practice, 46, 469–478.
Kiral F.K., Seyrek K., Paşa S., Ertabaklar H. & Ünsal C. 2004. Some haematological, biochemical and electrophoretic findings in dogs with visceral leishmaniasis. Revue De Medecine Veterinaire, 155, 226–229.
le Fichoux Y., Quaranta J.-F., Aufeuvre J.-P., Lelievre A., Marty P., Suffia I., et al. 1999. Occurrence of Leishmania infantum Parasitemia in Asymptomatic Blood Donors Living in an Area of Endemicity in Southern France. Journal of Clinical Microbiology, 37, 1953–1957.
Lombardo G., Pennisi M.G., Lupo T., Migliazzo A., Caprì A. & Solano-Gallego L. 2012. Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Veterinary parasitology, 184, 10–17.
Maia C. & Campino L. 2008. Methods for diagnosis of canine leishmaniasis and immune response to infection. Veterinary Parasitology, 158, 274–287.
Matov K. 1958. Parasitology and arachnoentomology.
Miró G., Cardoso L., Pennisi M.G., Oliva G. & Baneth G. 2008. Canine leishmaniosis – new concepts and insights on an expanding zoonosis: part two. Trends in Parasitology, 24, 371–377.
Morales-Yuste M., Martín-Sánchez J. & Corpas-Lopez V. 2022. Canine Leishmaniasis: Update on Epidemiology, Diagnosis, Treatment, and Prevention. Veterinary Sciences, 9, 387.
Paltrinieri S., Solano-Gallego L., Fondati A., Lubas G., Gradoni L., Castagnaro M., et al. 2010. Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Journal of the American Veterinary Medical Association, 236, 1184–1191.
Reis A.B., Teixeira-Carvalho A., Vale A.M., Marques M.J., Giunchetti R.C., Mayrink W., et al. 2006. Isotype patterns of immunoglobulins: Hallmarks for clinical status and tissue parasite density in brazilian dogs naturally infected by Leishmania (Leishmania) chagasi. Veterinary Immunology and Immunopathology, 112, 102–116.
Smith B.E., Tompkins M.B. & Breitschwerdt E.B. 2004. Antinuclear Antibodies Can Be Detected in Dog Sera Reactive to Bartonella vinsonii subsp. berkhoffii, Ehrlichia canis, or Leishmania infantum Antigens. Journal of Veterinary Internal Medicine, 18, 47.
Solano-Gallego L., Koutinas A., Miró G., Cardoso L., Pennisi M.G., Ferrer L., et al. 2009. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veterinary Parasitology, 165, 1–18.
Solano-Gallego L., Miró G., Koutinas A., Cardoso L., Pennisi M.G., Ferrer L., et al. 2011. LeishVet guidelines for the practical management of canine leishmaniosis. Parasites Vectors, 4, 86.
Torres-Guerrero E., Quintanilla-Cedillo M.R., Ruiz-Esmenjaud J. & Arenas R. 2017. Leishmaniasis: a review. F1000Research, 6, 750.
Tsachev I. 2009. Exotic Zoonoses Among Dogs in Bulgaria (Monocytic Ehrlichiosis, Granulocytic Anaplasmosis, Visceral Leishmaniasis). . DSc Thesis, Trakia University, Stara Zagora Bulgaria. 315p.
Tsachev I., Kyriazis I.D., Boustini S., Karagouni E. & Dotsika E. 2010. First report of canine visceral leishmaniasis in Bulgaria. Turkish Journal of Veterinary & Animal Sciences, 34, 465–469.
Tvedten H. 2010. Laboratory and clinical diagnosis of anemia. Schalm’s Veterinary Hematology, 152–161.
Weiss D.J. 2010. Myelodysplastic syndromes. Schalm’s Veterinary Hematology, 467–474.