Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.     

Isolation, mouse pathogenicity, and genotyping of Trypanosoma cruzi from an English Cocker Spaniel from Virginia, USA

Trypanosoma cruzi was demonstrated in blood smears and heart tissue from a 5-year old, female, English Cocker Spaniel that had never been outside of the state of Virginia, USA. Plasma from the dog was positive in a commercially available immunochromatographic dipstick assay for T. cruzi and negative in an immunochromatographic dipstick assay for visceral Leishmania spp. The plasma from the dog had an indirect immunofluorescent antibody titer of 1:800 against epimastigotes of T. cruzi while the titer was 1:50 against promastigotes of L. infantum. The parasite was isolated from the blood in vitro from the dog (TcVT-1 isolate) and used to experimentally infect female C3H and ICR mice. The parasite was nonpathogenic for experimentally inoculated mice. DNA was isolated from parasites grown in vitro and used to determine that the genotype of T. cruzi present in the dog was genotype TcIV. This genotype is common in raccoons, Procyon lotor, in North America and suggests that raccoons may serve as reservoirs for canine infection.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.     

Detection of different Leishmania spp. and Trypanosoma cruzi antibodies in cats from the Yucatan Peninsula (Mexico) using an iron superoxide dismutase excreted as antigen

Although human leishmaniasis has been reported in 20 states in Mexico, no case of leishmaniasis has been reported in cats to date. In the Yucatan Peninsula, it has been found that dogs may act as reservoirs for at least three Leishmania species (Leishmania mexicana, Leishmania braziliensis, and Leishmania panamensis). In this study we identified specific antibodies against these three Leishmania spp. and Trypanosoma cruzi in the sera from 95 cats from two States on the Yucatan Peninsula, namely Quintana Roo and Yucatan, by ELISA and Western blot techniques using whole extract and an iron superoxide dismutase excreted by the parasites as antigens. As well as demonstrating the presence of trypanosomatid antibodies in the feline population on the Yucatan Peninsula, we were also able to confirm the high sensitivity and specificity of the iron superoxide dismutase antigen secreted by them, which may prove to be very useful in epidemiological studies.     

Seroepidemiology of leptospirosis, toxoplasmosis, and leishmaniosis among dogs in Ankara, Turkey

Seroprevalence of five different Leptospira interrogans serovars, Toxoplasma gondii and Leishmania infantum in stray dogs in Ankara was investigated. A total of 116 dog sera collected from apparently healthy stray dogs were tested for L. interrogans serovars by microscopic agglutination test (MAT), for T. gondii antibodies by Sabin-Feldman dye test (SFDT), and for L. infantum antibodies by indirect fluorescence antibody test (IFAT). Of the 116 dogs, 51 (43.96%) were seropositive for leptospirosis, 72 (62.06%) for T. gondii and 3 (2.58%) for L. infantum. No statistically significant difference was observed between male and female dogs in the seroprevalences of toxoplasmosis and leptospirosis (P>0.05), but statistically significant difference was observed among different age groups in the seroprevalences of toxoplasmosis and leptospirosis (P<0.05). Although the seroprevalence of L. infantum was low, asymptomatic animals should be considered as a reservoir for the spread of the disease.     

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.     

Seroprevalence of antibodies against Leishmania spp among dogs in the United States

OBJECTIVE: To determine seroprevalence of antibodies against Leishmania spp among dogs other than Foxhounds in the United States. DESIGN: Cross-sectional study. SAMPLE POPULATION: 957 serum samples from dogs throughout the United States submitted between January 2000 and August 2001 to the Diagnostic Center for Population and Animal Health at Michigan State University for serologic testing for tick-borne diseases. PROCEDURE: Samples were tested for antibodies against Leishmania spp with an immunofluorescent antibody (IFA) assay. Samples with positive results were submitted to the Centers for Disease Control and Prevention for confirmatory testing. RESULTS: Results of the IFA assay were negative for 939 of 957 samples. For 16 samples, titers were from 1:16 to 1:64, and titers in these dogs were considered likely to be a result of cross-reactivity with antibodies directed against other organisms. For the remaining 2 samples, the titers were > or = 1:128. One of these samples was from a blood donor dog that had never had any clinical signs of leishmaniasis. Follow-up samples from both dogs also had Leishmania IFA titers > or = 1:128. Both dogs had antibodies against Trypanosoma cruzi, as determined with a radioimmunoprecipitation assay. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the seroprevalence of antibodies against Leishmania spp in dogs in the United States was low. However, results further suggested that leishmaniasis may not be limited to Foxhounds in the United States.     

Seroepidemiological survey on canine leishmaniasis among dogs from an urban area of Brazil

A cross-sectional seroepidemiological survey on canine leishmaniasis among pet dogs was carried out in an urban area in the State of Pernambuco (Brazil) where human cases of visceral leishmaniasis have sporadically been reported. Using an indirect fluorescent antibody test, anti-Leishmania antibodies were detected in 130 out of 322 dogs, confirming previous exposure to Leishmania parasites. The overall seroprevalence found was 40.3% (95% confidence interval: 34.9-45.9). Data analysis revealed that serological positivity was statistically associated with male (chi2=20.60, P-value=0.000) and juvenile dogs (chi2=4.24, P-value=0.039). Furthermore, it was observed that 85.3% of all seropositive dogs showed no clinical signs of leishmaniasis. The results showed a high seroprevalence of anti-Leishmania antibodies among dogs from an urban area of Pernambuco - with a large proportion of asymptomatic seropositive dogs - indicating that the prevalence of Leishmania infection in this area has been underestimated.     

The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis

Leishmania parasites cause a group of diseases collectively known as leishmaniases. The primary hosts of Leishmania are sylvatic mammals of several orders (Rodentia, Marsupialia, Carnivora, etc.). Under certain circumstances, particularly in peridomestic and domestic transmission foci, synanthropic and domestic animals can act as source of infection for phlebotomine sand fly vectors. Dogs have long been implicated as the main domestic reservoirs of Leishmania (Leishmania) infantum, the aetiological agent of zoonotic visceral leishmaniasis, and there exists an increasing trend to regard dogs as the main domestic reservoirs of Leishmania (Viannia) braziliensis, the most widespread aetiological agent of American tegumentary leishmaniasis. However, insights derived from recent research indicate that not dogs but humans are probably the most important domestic reservoirs of L. (V.) braziliensis. In the present article, the role of dogs as reservoirs of Leishmania parasites, with emphasis on L. (L.) infantum and L. (V.) braziliensis, is reviewed.     

Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran

An epidemiological study to examine the sero-prevalence of zoonotic visceral leishmaniosis (ZVL) among domestic and wild canines in endemic foci of Iran was carried out during 1999-2003 to assess the distribution of the disease and the possible association between infection in dogs, wild canines and people. Anti-leishmanial antibodies were detected by the direct agglutination test (DAT). Parasitological study was performed for all captured wild canines and were detected in some of the seropositive dogs with specific clinical signs (n=107). Serum samples (n=1568) were collected from domestic dogs in villages that are known endemic foci of human visceral leishmaniosis (HVL). Wild canine sera were collected from jackals (Canis aureus, n=10), foxes (Vulpes vulpes, n=10) and wolves (Canis lupus, n=10). Of the 1568 serum sampled collected from domestic dogs, 222 (14.2%) were positive by DAT (1:320 and above). No statistically significant difference was found between male (15.2%) and female (11.8%) sero-prevalence (P=0.083). Dogs of 8 years and above showed the highest sero-prevalence (40.6%). Only 23.9% of the seropositive domestic dogs had clinical signs. Parasitology and serology tests that were performed in 30 wild canines showed 10% these animals were infected by Leishmania infantum. Ten out of 11 Leishmania spp. isolated from the dogs and wild canines were identified as L. infantum and one other as L. tropica by molecular and biochemical techniques. For the first time in Iran, L. infantum and L. tropica were isolated from viscera of both a wolf and a domestic dog.     

Visceral leishmaniasis in a New York foxhound kennel

Although endemic throughout much of the world, autochthonous visceral leishmaniasis has been reported on only 3 previous occasions in North America. After diagnosis of visceral leishmaniasis in 4 foxhounds from a kennel in Dutchess County, New York (index kennel), serum and ethylenediamine-tetraacetic acid (EDTA)-anticoagulated blood were collected from the remaining 108 American or cross-bred foxhounds in the index kennel and from 30 Beagles and Basset Hounds that were periodically housed in the index kennel. Samples were analyzed for antibodies to or DNA of tickborne disease pathogens and Leishmania spp. Most dogs had antibodies to Rickettsia spp., Ehrlichia spp., Babesia spp., or some combination of these pathogens but not to Bartonella vinsonii (berkhoffi). However, DNA of rickettsial, ehrlichial, or babesial agents was detected in only 9 dogs. Visceral leishmaniasis was diagnosed in 46 of 112 (41%) foxhounds from the index kennel but was not diagnosed in any of the Beagles and Basset Hounds. A positive Leishmania status was defined by 1 or more of the following criteria: a Leishmania antibody titer > or = 1:64, positive Leishmania polymerase chain reaction (PCR), positive Leishmania culture, or identification of Leishmania amastigotes by cytology or histopathology. The species and zymodeme of Leishmania that infected the foxhounds was determined to be Leishmania infantum MON-1 by isoenzyme electrophoresis. Foxhounds that were > 18 months of age or that had traveled to the southeastern United States were more likely to be diagnosed with visceral leishmaniasis. Transmission of Leishmania spp. in kennel outbreaks may involve exposure to an insect vector, direct transmission, or vertical transmission.     

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 methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.     

Serological survey of Neospora caninum and Leishmania infantum co-infection in dogs

A seroprevalence survey and risk analysis of Neospora caninum and Leishmania infantum was conducted in dogs from an area of the Campania region of southern Italy, in order to investigate the co-infection of these two protozoa.Blood samples were collected from 1058 asymptomatic dogs over a 18 months period. Serum samples were tested for antibodies to N. caninum and to L. infantum using the indirect fluorescent antibody test.Epidemiological data (breed, age, sex, and utilization) were collected and statistically analysed in relation to N. caninum and to L. infantum seropositivity and antibody titres.Out of the 1058 sera samples tested, 68 (6.4%) were found to have antibodies to N. caninum, and 222 (21.0%) to have antibodies to L. infantum. The co-presence of antibodies to N. caninum and to L. infantum was found in 46 (4.3%) dogs. Thus, 67.6% of the dogs positive for N. caninum also had antibodies to L. infantum.The major risk factor for N. caninum seropositivity was the presence of antibodies to L. infantum, and the major risk factor for L. infantum seropositivity was the presence of antibodies to N. caninum. In addition, high N. caninum seroprevalence was closely correlated to Boxer breed, and high L. infantum seroprevalence was correlated to masculine gender and Setter and Pit bull breeds. Low L. infantum seroprevalence was closely correlated to Yorkshire breed.The findings of this survey indicate that in the Campania region of southern Italy the co-presence of antibodies to N. caninum and to L. infantum is very common in dogs, and that infection by one protozoan seems to enhance the susceptibility to the other one. This is probably due to the immunological status of the tested dogs.     

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MTT) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p>0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p>0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p>0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells.     

Trypanosoma cruzi infection in a dog from Oklahoma

A dog with signs of weakness, labored breathing, and generalized edema was examined. It was heavily infested with fleas and had wounds resulting from a recent fight. Hematologic findings were not remarkable, except for parasites in the blood. The dog was treated for fleas and given antibiotics, but was euthanatized when it failed to respond. Blood and tissue specimens were found to contain Trypanosoma cruzi, and the serum contained antibodies to the organism. We believe this is the first confirmed case of T cruzi infection in dogs from Oklahoma. The public health implications of this finding are underscored by a report on the detection of T cruzi in raccoons in the same season and geographic area.     

Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania infantum infection in dog as compared with immunofluorescence assay and Western blot

In this study we compared a commercial enzyme linked immunosorbent assay (ELISA) rapid test (Snap CLATK Canine Leishmania Antibody Test Kit, IDEXX-Snap) with indirect immunofluorescence assay (IFA) and Western blot (WB) for the detection of Leishmania infantum antibodies in dogs. In total sera from 234 dogs were collected: 59 positives and 51 doubtful sera (IFA 1:40-1:80) from an L. infantum endemic area and 124 negative sera from a non-endemic area were tested. To evaluate the Snap CLATK's performances on whole blood, blood in EDTA and sera from 37 dogs were tested in parallel with Snap CLATK. Snap CLATK sensitivity and specificity compared to IFA were 91.1% and 99.2%, while compared to WB were 93.4% and 98.3%, respectively. When IFA doubtful sera (titers of 1:40 or 1:80) were tested Snap CLATK, using WB as reference, sensitivity and specificity were 90.9% and 100%, respectively. Moreover, a complete concordance was observed when Snap CLATK rapid assay was carried out on whole blood or sera from 37 dogs. The Snap CLATK has demonstrated simplicity and performance and can be considered a quick and reliable alternative for the diagnosis of L. infantum infection in dogs.     

Serosurvey of antibodies against zoonotic pathogens in free-ranging wild canids (Cerdocyon thous and Lycalopex gymnocercus) from Southern Brazil

The expansion of urbanization on natural areas is increasing contact between human populations with wild animals. Wild carnivores can act as sentinel hosts or environmental health indicators. Thus, the aim of this work was to investigate the exposure of two major species of wild canids from Southern Brazil to selected pathogens. For that, we live-trapped free-ranging Cerdocyon thous and Lycalopex gymnocercus in five localities and determined the frequency of animals with antibodies against Toxoplasma gondii, Trypanosoma cruzi, Leishmania infantum, Neospora caninum, and Leptospira spp. Among the canids sampled, 23% (12/52) (95%CI: 13–36%) had antibodies against T. gondii, with titers ranging from 64 to 512. For T. cruzi, 28% (15/52) (95%CI: 18–42%) of sampled canids were seropositive, with titers ranging from 8 to 64. Concerning the protozoan pathogen N. caninum, a total of 5% (3/52) (95%CI: 2–15%) of wild canids had antibodies against it. None of the sampled canids showed the presence of antibodies against L. infantum. On the other hand, 44% (23/52) (95%CI: 31–57%) of the wild canids showed antibodies against Leptospira spp. The set of results presented here, show that free-ranging neotropical wild canids are exposed and have antibodies against to T. gondii, T. cruzi, Leptospira spp., and to a lesser degree to N. caninum. We found no evidence of L. infantum circulation among the studied populations. These results highlight some of the major pathogens which may represent risks for populations of these wild canids.Data Availability StatementThe data that support the findings of this study are available from the corresponding author upon reasonable request.     

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.