Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

A survey on canine leishmaniasis and phlebotomine sand flies in central Italy

Zoonotic visceral leishmaniasis (ZVL) is a vector-transmitted zoonosis caused by the parasitic protozoan Leishmania infantum. Bloodsucking sand flies of the subfamily Phlebotominae are the obligatory insect hosts, and the dog is the only domestic reservoir.This study reports data from a survey of canine infection and sand fly phlebotomine monitoring in the province of Perugia in central Italy. The overall seroprevalence in a total of 100 dogs tested was 8% (95% confidence interval: 3.8-15.6%). Data analysis revealed that serological positivity was statistically associated with age (p-value = 0.03) and the area where dogs lived. Standard blacklight traps employed for sampling Culicoides midges in bluetongue disease surveillance were used in phlebotomine monitoring. A total of 5698 sand flies were collected and the two species, Leishmania competent vectors, were identified, Phlebotomus perfiliewi (50%) and Phlebotomus perniciosus (30%).     

Methods for diagnosis of canine leishmaniasis and immune response to infection

Canine leishmaniasis (CanL) caused by Leishmania infantum (syn. L. chagasi, in Latin America), which is transmitted by the bite of phlebotomine sand flies, is endemic and affects millions of dogs in Europe, Asia, North Africa and South America. It is an emergent disease in North America. Early detection and treatment of infected animals may be critical in controlling the spread of the disease and is an essential part of human zoonotic visceral leishmaniasis control. The laboratory diagnosis of CanL still poses a challenge, despite progress made in the development of several direct and indirect methods. An effective diagnosis test, apart of being able to confirm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high sensitivity, specificity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or adaptable for field conditions. Ideally, it should detect all Leishmania-infected dogs, preferentially using non-invasive collection of biological samples. In this paper we review the advantages and shortcomings of the available procedures for CanL diagnosis in the different phases, e.g. pre-patent and patent period of the infection and methods to determine the related immune response.     

A review of parasitic zoonoses in a changing Southeast Asia

Parasitic zoonoses are common and widely distributed in the Southeast Asian region. However, the interactions between parasites, hosts and vectors are influenced by environmental, socio-cultural and livestock production changes that impact on the distribution, prevalence and severity of disease. In this review we provide an update on new knowledge in the context of ongoing changes for the food-borne pig associated zoonoses Taenia solium and Trichinella spp., the food-borne trematodes Opisthorchis viverrini and Clonorchis sinensis, the water-borne trematodes Schistosoma spp., the vector-borne zoonotic protozoa Plasmodium knowlesi and Leishmania spp. and the soil-borne zoonotic hookworm Ancylostoma ceylanicum. These various changes need to be considered when assessing or developing regional control programs or devising new research initiatives in a changing SE Asia.     

Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (≥1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR–RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.     

Clinical, Serologic, and Parasitologic Follow-Up after Long-Term Allopurinol Therapy of Dogs Naturally Infected with Leishmania infantum

Canine leishmaniasis usually is treated with antimony compounds, but frequent relapses, adverse effects, high costs, and development of resistance to long-term antimonial therapy emphasize the importance of searching for alternative antileishmanial drugs. Allopurinol was used at a dosage of 10 mg/kg/day PO to treat 10 dogs naturally infected with Leishmania infantum for a period of 2-24 months. Nine dogs recovered within 2-6 months of chemotherapy, and no relapses were observed during the treatment of up to 20 months. However, 3 of 4 dogs relapsed after treatment was discontinued. These dogs again recovered clinically when therapy was resumed. Parasite-specific immunoglobulin concentrations (IgG2) were high in all dogs before therapy and remained high even in clinically cured dogs during or after therapy. On the other hand, specific IgG1 reactions, which have been shown to be detectable in symptomatic animals, persisted in 7 dogs for long periods after clinical recovery. Three of these dogs relapsed within 2-4 weeks after interrupting therapy. However, 1 dog with no detectable specific IgG1 reaction at the end of therapy did not relapse in the following 4 months. Parasites could be detected in 8 of 9 dogs after clinical improvement by in vitro cultivation or polymerase chain reaction (PCR) testing of lymph node aspirates. In 4 of these dogs, parasites also were detected in blood samples by PCR. Hence, these clinically cured dogs must be regarded as reservoirs of Leishmania and allopurinol cannot be recommended in endemic areas.     

Skin Lesions in Canine Leishmaniasis (Kala-Azar): A Clinical and Histopathological Study on 22 Spontaneous Cases in Greece

Abstract— Skin lesions occurring in 22 dogs with leishmaniasis (Kala-azar) were studied clinically and histopathologically, so as to better characterize the range of abnormalities encountered in this disease. The main clinical dermatological patterns observed were exfoliative dermatitis (90.9 per cent of dogs), ulcerations (63.6 per cent), onychogryposis (54.5 per cent), sterile pustular dermatitis (13.6 per cent), and paronychia (13.6 per cent). Most of the dogs (86.4 per cent) had more than one type of skin lesion. The most commonly seen histopathological patterns were granulomatous perifolliculitis and sebaceous adenitis (68.2 per cent), superficial and deep perivascular dermatitis (54.5 per cent), and interstitial dermatitis (50.0 per cent). Parasites, mostly within macrophages, were detected in the skin biopsies from 50.0 per cent of the dogs. Résumé— Les lesions cutanés de 22 chiens atteints de leishmaniose ont étéétudiées cliniquement et histopathologiquement, afin de mieux caractériser les anomalies observées lors de cette maladie. La principale manifestation dermatologique observée était une dermatitie exfoliative (90,9% des chiens), des ulcérations (63,6%), une onychogriphose (54,5%) une dermite pustuleuse stérile (13,6%) et une paronychie (13,6%). La plupart des chiens (84,6%) avaient plus d'un type de lésion cutanée. Les lesions histopathologiques les plus communes étaient des périfolliculites granulomateuses et des adénites sébacées (68,2%), une dermite superficielle et profonde périvasculire (54,5%), une dermite intersticielle (50%). Les parasites, généraiement dans les macrophages, ont été isolés dans une biopsie sur deux. Zusammenfassung— Hautveränderungen, die bei 22 Hunden mit Leishmaniose (Kala-azar) auftraten, wurden klinisch und histopathologisch untersucht, um die Spannweite der Veränderungen, die bei dieser Krankheit auftreten, besser charakterisieren zu können. Als hauptsächliche klinische dermatologische Symptome wurden exfoliative Dermatitis (90,9% der Hunde), Ulzerationen (63,6%), Onychogrypose (54,5%)), sterile pustulöse Dermatitis (13,6%) und Paronychie (13,6%) beobachtet. Die meisten Hunde (86,4%) wiesen mehr als einen Typ Hautveränderung auf. Die häufigsten histopathologischen Befunde bestanden in granulomatöser Perifollikulitis und Talgdrüsenadenitis (68,2%), oberflächlicher und tiefer perivaskulärer Dermatitis (54,5%) und interstitieller Dermatitis (50,0%). Die Parasiten, die meistens in den Makrophagen liegen, wurden in den Hautbiopsien bei 50,0% der Hunde entdeckt. Resumen Se estudiarion clínica e histopatológicamente las lesiones cutáneas padecidas por 22 perros con leismaniasis (Kala-azar), con el propósito de caracterizar el rango de abnormalidades producidas por la enfermedad. La sprincipales características patológicias observadas fueron: dermatitis exfoliativa (90.9% de los perros), ulceraciones (63.6%), onicogriposis (54.5%), dermatitis postular esteril (13.6%), y paronquia (13.6%). La mayoría de los perros, (86.4%), presentaban más de un tipo de lesion dérmica. Los modelos histopatológicos reconocidos más frequentemente fueron: perifoliculitis granulomatosa y adenitis sebácea (68.2%)), dermatitis perivascular superficial y profunda (54.5%), y dermatitis intersticial (50%). Las biopsias cutáneas detectaron parásitos, la mayoría en los macrófagos, en 50% de los perros.     

Leishmania infantum secreted iron superoxide dismutase purification and its application to the diagnosis of canine Leishmaniasis

Leishmania spp. are digenetic parasites whose infection occurs inside the mononuclear phagocitary system. The iron superoxide dismutase secreted (Fe-SODe) by promastigotes of Leishmania spp. seems to plays an important role in the defense to environmental detoxification and neutralization of oxidative stress damage caused by reactive oxygen species (ROS) produced by macrophages during the infection. Parasites Fe-SODe is involved in establishing the infection and manifestation of Leishmaniasis. Its high immunogenicity makes it a useful molecular marker in diagnosing trypanosomatids infections. The aim of this study is demonstrate that purified Fe-SODe from Leishmania infantum is much more sensitive than un-purified Fe-SODe for diagnosis canine Leishmaniasis. We have purified a Fe-SODe of L. infantum using an ion exchange and a molecular sieve chromatographies and its application in diagnosis of canine Leishmaniasis was tested. One hundred and forty-five dogs’ sera from Andalusia Autonomous Community, Spain were tested by ELISA and Western blot and the antigen Fe-SODe purified is compared with two different antigens: the total parasites soluble lysate and the unpurified Fe-SODe. To validate the results obtained using the Fe-SODe purified we tasted 10 L. infantum infected dogs’ sera from Lombardy, Italy as positive control.