Canine Leishmaniosis Research Articles

Use of the nitroblue tetrazolium reduction test for the evaluation of Domperidone effects on the neutrophilic function of healthy dogs

The nitroblue tetrazolium reduction test (NBT) is an assay based on the activation percentage of neutrophils in peripheral blood, that has been proposed for the follow up of canine leishmaniosis owing to the narrow relationship between the molecules involved in the oxidative burst and the leishmanicidal activity of phagocytes. Domperidone is a drug used for the treatment of canine leishmaniosis having been claimed to stimulate the dogs’ cell-mediated immune response. The aim of this study was to evaluate the degree and the lasting of phagocytic activation induced by a 30-day course treatment with Domperidone (0.5mg/kg/day) in healthy dogs, by using the NBT. A statistically significant increase in the percentages of activated phagocytes was observed in the treated group during treatment, thereafter remaining high for up to one month after the end of treatment. In contrast, untreated dogs maintained the baseline percentage of activated neutrophils all along the study. It is concluded that the NBT is a useful tool for the follow up of the stimulating effects of Domperidone on the neutrophilic response of healthy dogs and that these effects persist for up to one month after treatment with this molecule.

Monitoring the reverse to normal of clinico-pathological findings and the disease free interval time using four different treatment protocols for canine leishmaniosis in an endemic area

Twenty-four dogs naturally infected by Leishmania spp. were treated with four different protocols using meglumine antimoniate (aNm) and allopurinol in combination or in monotherapy. Aiming to compare the efficacy of the different treatments the reverse to normal of clinico-pathological findings and the disease free interval time (DFIT) were evaluated. Treated dogs were monitored for 1year and, in absence of relapses, the DFIT was postponed to the last available follow-up. Seven dogs treated with aNm alone showed relapses during the year of observation. In the group of dogs treated with the combination of aNm (50mg/kg/SC 12 hourly up to clinico-pathological recovery) and allopurinol (15mg/kg/PO 12 hourly administered for 6months) no relapses were registered in the year of monitoring and the DFIT reached up to 65months. Our results showed that this combination represents the best choice to treat canine leishmaniosis compared to other protocols.

Canine leishmaniosis: in vitro efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum

Despite the availability of different therapeutic options, canine visceral leishmaniosis (CVL) remains a challenging disease to treat. Recently miltefosine has been registered for use in dogs, and different studies have demonstrated its leishmanicidal effect. Moreover, it has been suggested that fluoroquinolones, compared to standard chemotherapeutic agents, could be an effective and pragmatic alternative to treat CVL. The efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum was assessed in vitro by incubating increasing concentrations of the drugs with a standard parasite inoculum. Miltefosine was significantly more efficacious than marbofloxacin (P < 0.05) against the two strains of L. infantum either alone or in combination with allopurinol. Both drugs were significantly (P < 0.05) more efficacious when associated with allopurinol than alone.

Down-regulation OF Anti-inflammatory Cytokines in the Brain During Canine Visceral Leishmaniosis

Down-regulation OF Anti-inflammatory Cytokines in the Brain During Canine Visceral Leishmaniosis

One-year clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis

The purpose of this international, multicentric, and non-comparative field trial was to obtain complementary data on long-term clinical and parasitological follow-up of dogs treated with marbofloxacin for canine leishmaniosis (CanL). Seventy-four dogs with clinical signs of CanL and without severe renal failure were recruited in France, Spain and Italy, and 61 of them were part of the analysis. Each dog was treated with palatable tablets of marbofloxacin at 2mg/kg once a day for 28 days. A clinical and parasitological follow-up was performed regularly up to 12 months. Efficacy was demonstrated in 42 dogs (68.9%), within 51 days (mean value), 10 of them (23.8%) being clinically cured after 3 months. A decrease of 61% in the sum of clinical scores was observed after 3 months. Haemato-biochemical parameters improved in general, supporting the observed clinical efficacy. Relapse was observed in 20/38 dogs (52.6%) approximately 5.5 months after treatment completion. The blood parasite load generally developed in conformity with the clinical outcome, even if exceptions were not rare. Lymph nodes remained positive by culture or PCR for a long time, even in dogs for which a good clinical response was observed. Despite the incomplete parasite clearance, as is also the case with other anti-leishmanial drugs, these results nevertheless confirm the relevance of marbofloxacin as a CanL treatment.

In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these...

Detection of Leishmania amastigotes in peripheral blood from four dogs — Short communication

The authors carried out microscopic examination of blood smears of 1438 dogs infected with Leishmania infantum. Unusual findings of leishmaniosis associated with circulating parasitised cells are described in four dogs. Most of the dogs presented severe illness, with lethargy, dysorexia, emaciation and alterations of the haematological pattern (anaemia, thrombocytopenia, neutrophilia and monocytosis). In three cases, leishmaniosis was associated with ehrlichiosis. On examination of peripheral blood smears, Leishmania sp. amastigotes were observed both in various circulating leukocytes (neutrophil, monocyte, macrophage) and free. In conclusion, parasites can rarely be detected in blood smears (in 0.28% of the animals examined); thus, the time-consuming microscopic search for amastigotes can make only a weak contribution to the conventional diagnosis of canine leishmaniosis.

LeishVet guidelines for the practical management of canine leishmaniosis

The LeishVet group has formed recommendations designed primarily to help the veterinary clinician in the management of canine leishmaniosis. The complexity of this zoonotic infection and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of canine leishmaniosis challenging. The recommendations were constructed by combining a comprehensive review of evidence-based studies, extensive clinical experience and critical consensus opinion discussions. The guidelines presented here in a short version with graphical topic displays suggest standardized and rational approaches to the diagnosis, treatment, follow-up, control and prevention of canine leishmaniosis. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient.

English

&nbsp; An epidemiological survey of visceral leishmaniosis among domestic and wild canines as well people was carried out in the new endemic focus of Azarshahr (northwest of Iran) to assess the distribution of the disease and the possible association between infection in dogs and people during 2008-2009. At first using direct agglutination test (DAT), an examination of visceral leishmaniosis was carried out among 1500 Azarshahr district residents children in the age from 6 month to 10 years old. Then, one hundred and twenty domestic dogs, ten jackals, ten foxes and ten wolves from the district were examined for clinical and serological signs of canine leishmaniosis. The sera were evaluated by DAT then parasitological study was performed for all seropositive animals. Human sera samples, the overall seropositive rate of disease was 1.3% (19/1500) (95% CI, 0.7 to 1.8) by DAT (1:3200 and above). Data analysis revealed that serological positivity was statistically associated with age groups (&chi;2&nbsp;=7.46, P =0.023). Of the 120 serum sampled collected from 120 domestic and stray dogs, 19.2% (23/120) (95% CI, 12.1-26.2) were positive by DAT (1:320 and above). No statistically significant difference was found between male (17.9%) and female (37.5%) seroprevalence (P=0.172). Also, no statistically significant difference was found between serological positivity and age groups (P=0.107). Serology and parasitology tests that were performed in 30 wild canines showed 10% of these animals were infected by&nbsp;Leishmania infantum. Amastigotes of Leishmania were observed in 14 seropositive dogs after dissection and parasitological examinations. A polymerase chain reaction (PCR) genotyping tool was developed and used to identification of species of the seropositive dog isolates. This isolates were identified as&nbsp;L. infantum. The present study provides, for the first time, information on the distribution of the visceral leishmaniosis in Azarshahr county of Iran, as a new endemic focus of disease. &nbsp; Key words:&nbsp;Visceral leishmaniosis, Seroprevalence,&nbsp;Polymerase chain reaction, Leishmania infantum, Azarshahr.

Cytokine Gene Expression in the Tissues of Dogs Infected by Leishmania infantum

Canine leishmaniosis (CanL) caused by the protozoan parasite Leishmania infantum is a chronic systemic disease that is endemic in certain parts of the world. The domestic dog is the most important reservoir of L. infantum and is the main source of infection for other animals and for the human population. The aim of this study was to evaluate and compare the level of expression of genes encoding particular cytokines (interleukin [IL]-12, interferon [IFN]-γ, IL-2 and IL-4) in different tissues and organs of 53 adult dogs with or without clinical signs of leishmaniosis and after treatment for the disease. Asymptomatic dogs showed high expression of genes encoding IL-4 in blood leucocytes and of genes encoding IL-12 and IL-2 in lymph nodes. Blood leucocytes from symptomatic dogs had a mixed Th1 and Th2 cytokine gene expression profile, but lymph nodes from these animals had dominant IL-2 and IFN-γ gene expression, while bone marrow appeared to be unresponsive. The predominance of IL-4 gene expression in the blood of asymptomatic dogs may favour parasite replication, while the balance between Th1 and Th2 cytokine gene expression in the blood of symptomatic dogs may be important in reducing parasite replication and delaying the dissemination of Leishmania to other organs. The drugs used to treat CanL do not completely eliminate the parasite, so the high expression of the gene encoding IL-4 in blood leucocytes and the high expression of IL-12 and IL-4 mRNA in lymph nodes may reflect the persistence of residual Leishmania amastigotes. L. infantum appears able to regulate the host immune response in order to ensure its survival, but also to prevent the host from succumbing to infection. This guarantees its transmission and the completion of its life cycle.

Recent advances in leishmaniosis in pet animals: Epidemiology, diagnostics and anti-vectorial prophylaxis

The leishmanioses are diseases caused by protozoa of the genus Leishmania, parasites infecting numerous mammal species, including humans, and transmitted by the bite of phlebotomine sand flies. They are a large group of diseases ranging over inter-tropical zones of America and Africa, and extend into temperate regions of Latin America, Europe and Asia. Pet animals are found infected with different Leishmania species but Leishmania infantum is the most widespread being dogs the main reservoir of zoonotic visceral leishmaniosis (ZVL). Dogs are very susceptible to this parasite and may suffer from a complex syndrome, canine leishmaniosis (CanL), one of the major zoonoses globally causing severe fatal disease in this animal. Infections in cats and horses have also been reported in areas where CanL is diagnosed. In Europe dogs and cats are common companion animals and their health is of great concern, therefore management of leishmaniosis in pets generally follows that of human ZVL. The recent spread of Leishmania infections in non-endemic territories has been monitored by means of canine surveys, which represent a suitable approach because of the dog's role as a sentinel host. New tools have been developed for the surveillance and control of ZVL. A number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites, with elevated efficacy for both individual and mass protection.

Canine leishmaniosis and its relationship to human visceral leishmaniasis in Eastern Uzbekistan

BackgroundThe Namangan Region in the Pap District, located in Eastern Uzbekistan is the main focus of visceral leishmaniasis (VL) in Uzbekistan. In total, 28 cases of human VL were registered during 2006-2008 in this region. A study on the epidemiology of VL in this area was carried out in 2007-2008 in the villages of Chodak, Oltinkan, Gulistan and Chorkesar located at elevations of 900-1200 above sea level.ResultsA total of 162 dogs were tested for Leishmania infection. Blood was drawn for serology and PCR. When clinical signs of the disease were present, aspirates from lymph nodes and the spleen were taken. Forty-two dogs (25.9%) had clinical signs suggestive of VL and 51 (31.5%) were sero-positive. ITS-1 PCR was performed for 135 dogs using blood and tissue samples and 40 (29.6%) of them were PCR-positive. Leishmanial parasites were cultured from lymph node or spleen aspirates from 10 dogs.Eight Leishmania strains isolated from dogs were typed by multi-locus microsatellite typing (MLMT) and by multilocus enzyme electrophoretic analysis (MLEE), using a 15 enzyme system. These analyses revealed that the strains belong to the most common zymodeme of L. infantum, i.e., MON-1, and form a unique group when compared to MON-1 strains from other geographical regions.ConclusionsThe data obtained through this study confirm the existence of an active focus of VL in the Namangan region of Uzbekistan. The fact that L. infantum was the causative agent of canine infection with typical clinical signs, and also of human infection affecting only infants, suggests that a zoonotic form of VL similar in epidemiology to Mediterranean VL is present in Uzbekistan.

Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus sanguineus

BackgroundLeishmania infantum is a widespread parasite that affects dogs and humans worldwide. It is transmitted primarily by phlebotomine sand flies, but recently there has been much discussion on the role of the brown dog tick, Rhipicephalus sanguineus, as a potential vector for this protozoan. Recent laboratory and field investigations have contributed to this hypothesis, but a proof of the vector capacity of R. sanguineus has yet to be provided. Following a recent study suggesting that L. infantum passes transovarially from the female tick to her progeny the current study provides new evidence of the transovarial transmission of L. infantum in R. sanguineus.MethodsEngorged females of R. sanguineus were collected from the environment in a dog shelter of southern Italy, where canine leishmaniosis is endemic. In the laboratory, 97 females that successfully laid eggs, their eggs and the originated larvae were subjected to DNA extraction and then tested by a TaqMan-based real time PCR targeting a fragment of the kinetoplast DNA (kDNA) of L. infantum.Results and conclusionsL. infantum kDNA was detected in engorged females, their eggs and originating larvae, with a parasite load ranging from 1.8 × 10-4 to 10.0 × 100. Certainly, the current study provides further evidence on the passage of L. infantum from R. sanguineus females to their offspring. The observation of promastigote forms in larvae is necessary to definitively confirm this hypothesis, which would raise interesting questions about the possible role of ticks in the maintenance of L. infantum infection among dogs in certain areas.

The immunopathology of canine vector-borne diseases

The canine vector-borne infectious diseases (CVBDs) are an emerging problem in veterinary medicine and the zoonotic potential of many of these agents is a significant consideration for human health. The successful diagnosis, treatment and prevention of these infections is dependent upon firm understanding of the underlying immunopathology of the diseases in which there are unique tripartite interactions between the microorganism, the vector and the host immune system. Although significant advances have been made in the areas of molecular speciation and the epidemiology of these infections and their vectors, basic knowledge of the pathology and immunology of the diseases has lagged behind. This review summarizes recent studies of the pathology and host immune response in the major CVBDs (leishmaniosis, babesiosis, ehrlichiosis, hepatozoonosis, anaplasmosis, bartonellosis and borreliosis). The ultimate application of such immunological investigation is the development of effective vaccines. The current commercially available vaccines for canine leishmaniosis, babesiosis and borreliosis are reviewed.

An immunohistochemical study in cases with usual and unusual clinicopathological findings of canine visceral leishmaniosis

The present study describes pathologic findings and immunohistochemical diagnosis of canine visceral leishmaniasis (CVL) in 22 dogs who died naturally in the Aegean region of Turkey. At necropsy, lymphadenomegaly, hepatosplenomegaly, hepatic, and nephrosclerotic lesions were conspicuous. Histopathologically, chronic inflammatory reactions of the spleen, lymph nodes, bone marrow, liver, and skin were marked findings. Cytological and histological examinations showed macrophages loaded with Leishmania amastigotes in these organs. Immunohistochemistry revealed that immunolabeling of amastigotes and/or parasite antigen, especially in the lymph nodes, spleen, bone marrow, liver, and skin, and occasionally, in the kidneys, intestines and lungs. Our laboratory results showed that immunohistochemistry should be included, along with cytological and histological examinations, in the diagnosis of CVL.

Acute phase protein response in experimental canine leishmaniosis

Acute phase proteins (APPs) have been proposed as useful markers for the diagnosis and monitoring of treatment of dogs infected by Leishmania infantum. However, the kinetics and behavior of these proteins in canine leishmaniosis is still unknown. The aim of this study was to monitor the kinetics of APPs in dogs experimentally infected with L. infantum, before, during and after therapy against canine leishmaniosis. Levels of serum haptoglobin, serum amyloid A and C-reactive protein from 6 infected beagles, positive by both PCR and parasite culture, were monitored for 7 months post-infection. The dogs were then treated for 3 months with allopurinol (20mgmg/kg/day PO), and their response to therapy was followed for 11 additional months. Levels of Immunoglobulins G and M were recorded during these 21 months and compared. Experimental infection with L. infantum amastigotes induced an increase in all APPs studied which was statistically significant 2 months after infection for all proteins. Clinical recovery was accompanied by a significant decrease of all APPs 1 month after the beginning of treatment. However, differences were found between the APPs in both magnitude and duration of serum level elevations. The increase in total IgG and IgM was delayed in comparison to APPs and contrarily to the APPs, these immunoglobulins did not significantly decrease with treatment. In conclusion, the results of this study suggest that APPs could be used as early markers for disease as well as for monitoring the response to treatment in canine leishmaniosis.

Mapping the Current Distribution and Predicted Spread of the Leishmaniosis Sand Fly Vector in the Madrid Region (Spain) Based on Environmental Variables and Expected Climate Change

Leishmaniosis caused by Leishmania infantum is a widespread zoonotic disease that is endemic in the Mediterranean basin. Based on prior point abundance data for the two sand fly vectors of leishmaniosis in the Madrid region (Phlebotomus perniciosus and Phlebotomus ariasi), models were constructed to predict the spatial distribution patterns of these vectors. The models were obtained by negative binomial regression of several environmental variables and then used to map vector distributions. To validate the maps, we used serological prevalence data of Leishmania infection in dogs and incidence data were obtained through questionnaires completed by veterinarians in the region. Seropositive dogs and veterinary clinics registering a higher incidence of canine leishmaniosis appeared closer to our modeled vector foci. In the face of climate change, we simulated the future distributions of the sand flies for each third of the 21st century and predicted their spread in the region.

Susceptibility and Resistance to Canine Leishmaniose is Associated to Polymorphisms of the Canine TNF-α Gene

The prevalence of canine leishmaniosis (CanL) infection in an enzootic area is considerably higher than the overall prevalence of the disease, suggesting a role of host genetics related to the outcome of the disease. It is accepted that one determining factor for the outcome of CanL is the type of the triggered immune response, which seems to be genetically determined. TNF-α is a cytokine which plays a crucial role during the immune response against Leishmania parasites. In the present study a case-control study with 20 resistant and 20 susceptible dogs was performed. The distribution of breeds was equal in both groups. By Sanger method the nucleotide sequence upstream the Open Reading Frame of the canine TNF-α gene was determined and four polymorphisms were identified (−40 C/A, −1134 T/G, −1150 T/C κα −1243 C/G). Statistical analysis showed that the polymorphism TNF-α −40 C/A is correlated with susceptibility to CanL, while the polymorphism TNF-α −1243 C/G is correlated with resistance to CanL. Further statistical analysis, regarding the possible correlation of gender as well as clinical manifestations of the disease with the above-mentioned polymorphisms of the TNF-α gene, showed no significant findings. Further analysis of the above polymorphisms, as well as identification of more polymorphisms in candidate genes, is required to provide a better understanding of the complex underlying immune response in CanL.

Canine leishmaniosis and euthanasia in Italy: a critical legal-ethical analysis

The increasing awareness that control of zoonotic visceral leishmaniosis (ZVL) depends on effective control of canine leishmaniosis has promoted research into leishmanial infection in dogs, but has also encouraged the indiscriminate elimination of seropositive dogs. Public health campaigns in various parts of the world (e.g. in Brazil and China) are designed to reduce the incidence of human ZVL by targeting/killing dogs. However, not all situations are the same; in Italy, for example, there would be little support for this type of control measure because attitudes towards animals are different. Moreover, research has suggested that the destruction of seropositive dogs is a relatively ineffective control measure. In this context, the authors reflect on some epidemiological, ethical and juridical aspects, aiming to contribute to the discussion and to find more feasible solutions.

Claw histopathology and parasitic load in natural cases of canine leishmaniosis associated with Leishmania infantum

Histological lesions and the presence of Leishmania spp. amastigotes in claw tissues were investigated in 40 dogs with leishmaniosis, with (16/40--group A) or without (24/40--group B) generalized onychogryphosis. Following euthanasia, the entire third phalanx with intact claw was amputated, formalin fixed, decalcified in a formic acid solution, embedded in paraffin, sectioned longitudinally and stained with haematoxylin and eosin, and acid orcein-Giemsa. Nested polymerase chain reaction (PCR) was used for the detection of Leishmania amastigotes. Lichenoid mononuclear infiltration (all dogs in group A, 21 of 24 dogs in group B), basal keratinocyte vacuolation (nine of 16 dogs in group A, 15 of 24 dogs in group B) and dermoepidermal clefting (13 of 16 dogs in group A, 18 of 24 dogs in group B) were the most prominent histopathological findings. There was no difference in the frequency and severity of these lesions between the two groups. Leishmania amastigotes could not be visualized in the dermis of any of the H&E sections, but their presence was demonstrated by nested PCR in three of 16 dogs in group A and two of 24 dogs in group B. However, the frequency of positive nested PCRs was not significantly different between the two groups. In conclusion, claw histopathology in symptomatic dogs with leishmaniosis, either with or without onychogryphosis is mainly characterized by mononuclear lichenoid dermatitis with or without interface dermatitis and dermoepidermal clefting, and is not accompanied by substantial local parasitism.