Immunogenic mapping of rDyn-1 and rKDDR-plus proteins and selection of oligopeptides by immunoblotting for the diagnosis of Leishmania infantum-infected dogs.

Endemic in Brazil, visceral leishmaniasis (VL) is a zoonotic infection that is among the most important parasitic diseases transmitted by vectors. Dogs are the main reservoirs of canine leishmaniasis (CanL) and their identification is used in some countries as part of disease prevention and control measures in the canine and human population. In this context, serological tests are necessary, composed of antigens capable of correctly identifying infected dogs, minimizing the number of false-negative cases. This study aimed to identify more immunoreactive peptides derived from two previously described whole proteins (rDyn-1 and rKDDR-plus) and compare their performance to the control antigens rK39 and the crude extract for the detection of dogs infected with L. infantum, especially the asymptomatic ones. The three selected peptides and a mixture of them, along with the rDyn-1, rKDDR-plus, rK39, and crude extract antigens were evaluated using indirect ELISA with sera samples from 186 dogs with CanL, being asymptomatic (n = 50), symptomatic (n = 50), co-infected (n = 19), infected with Babesia sp. (n = 7), Ehrlichia sp. (n = 6), T. cruzi (n = 20) and uninfected (n = 34). The results showed that the rDyn-1 protein and the peptide mixture had the highest sensitivity (100% and 98.32%, respectively) and specificity (97.01 and 98.51, respectively). A high degree of kappa agreement was found for rDyn-1 protein (0.977), mixed peptides (0.965), rKDDR-plus protein (0.953), K-plus peptide 1 (0.930) and Dyn-1 peptide (0.893). The mixture of peptides showed the highest likelihood (65.87). The ELISA using the mixture of peptides and the rDyn-1 protein showed high performance for CanL serodiagnosis. More mix combinations of the peptides and additional extended field tests with a larger sample size are recommended.

Molecular detection of vector-borne pathogens in semen from dogs in southeastern Brazil.

Vector-borne pathogens (VBPs) are primarily transmitted by arthropod vectors, but secondary ways of transmission have been described, including via venereal route. Nonetheless, there is still limited research on possible sexual transmission of VBPs in dogs. We molecularly investigated the presence of vector-borne pathogens in semen from dogs living in an area where these agents are endemic. Upon PCR testing, seven out of 22 (31.8%) semen samples tested positive for at least one VBP, whereas simultaneous positivity to two or more pathogens was detected in three (13.6%) dogs. Among pathogens detected in semen, Trypanosoma cruzi (n = 1) and Leishmania infantum (n = 3) were identified to species level by restriction fragment length polymorphism analysis. Attempts to sequence PCR products from other pathogens were unsuccessful, but coupled epidemiological and molecular data suggest the presence of Anaplasma platys (n = 5), Babesia vogeli (n = 1) and Ehrlichia canis (n = 1) in semen from dogs. Further experimental studies would be needed to confirm the sexual transmission hypothesis for these VBPs and also the possible implications of these findings for canine reproduction.

Report of the presence of Leishmania infantum in the milk of a naturally infected female dog in Brazil.

Dogs are the most important reservoir of Leishmania infantum, the causal agent of visceral leishmaniasis in Brazil. Although lymphoid tissue is the most important biological tissue where amastigotes can be found, this paper describes the presence of L. infantum DNA in the milk of a lactating naturally infected female dog. This finding suggests the need for further studies to elucidate whether breastfeeding can be a route of infection.

Molecular epidemiology and prevalence of babesial infections in dogs in two hyperendemic foci in Brazil.

Babesial parasites are some of the most ubiquitous blood pathogens and consequently have considerable worldwide veterinary impact. Dogs living in the tropics are highly exposed to babesial parasites, particularly to Babesia vogeli. Limited data on the seroprevalence and molecular prevalence of Babesia spp. in dogs are available in Latin America. We conducted a cross-sectional study combining serological and molecular tests to estimate the seroprevalence and molecular epidemiology of Babesia spp. infections in dogs in two hyperendemic foci in Brazil. A total of 630 privately owned dogs (417 from Goiana municipality, Pernambuco state, north-eastern Brazil, and 213 from São Joaquim de Bicas municipality, Minas Gerais state, south-eastern Brazil) were sampled and molecularly and serologically tested for Babesia spp. Overall, 519 dogs (82.4%) presented detectable IgG antibodies against Babesia spp., and seropositivity was significantly higher in dogs older than 1 year. Molecularly, 34 dogs (5.4%) were positive for a ~ 200 bp fragment of the 18S rRNA gene of Babesia spp. and 88 (14.0%) for a longer fragment (~ 450 bp) of the same gene of Babesia spp. and other protozoa. The 18S rRNA gene sequences generated herein corresponded to B. vogeli (n = 52) or Hepatozoon canis (n = 20). This study confirms a high level of exposure to B. vogeli in two areas of Brazil and highlights that most of the dogs living in these areas are infected during the course of their life, reflected by increased seroprevalence in older dogs. Increased awareness and prevention of tick-borne protozoa infections in dogs from Brazil and Latin America are urgently needed.

Prevalence and incidence of vector-borne pathogens in unprotected dogs in two Brazilian regions.

BACKGROUND: Various vector-borne pathogens (VBPs) affect dogs worldwide, with their diversity and force of infection being usually higher in the tropics. Cross-sectional studies have been conducted to investigate the prevalence of VBPs in dogs, but data from longitudinal studies are scarce. Herein, we assessed the prevalence and the year-crude incidence (YCI) of Leishmania spp. and other VBPs in privately-owned dogs from two geographical regions of Brazil. METHODS: A total of 823 dogs were initially screened for Leishmania spp. by both serology and polymerase chain reaction (PCR). From the negatives, 307 (103 from São Joaquim de Bicas, Minas Gerais, and 204 from Goiana, Pernambuco) were randomly selected for the longitudinal study. These dogs were tested for various VBPs at baseline, after 8 and 12 months. RESULTS: Out of 823 dogs initially screened, 131 (15.9%) were positive for Leishmania spp. Out of the 307 dogs enrolled in the longitudinal study, 120 (39.1%) were lost for different reasons (e.g. animal death, owner decision, and lost to follow-up). In São Joaquim de Bicas, the baseline prevalence and YCI were as follows: 16.5% and 7.1% for Anaplasma spp.; 81.6% and 100% for Babesia spp.; 0% and 1.3% (only one faint positive) for Dirofilaria immitis; 37.9% and 22.9% for Ehrlichia spp.; 19.5% and 43.8% for Leishmania spp. In Goiana, the baseline prevalence and YCI were as follows: 45.1% and 38.3% for Anaplasma spp.; 79.9% and 96.0% for Babesia spp.; 36.3% and 39.8% for D. immitis; 64.7% and 58.5% for Ehrlichia spp.; 14.7% and 19.6% for Leishmania spp. Anti-Borrelia burgdorferi antibodies were not detected in any of the samples tested herein. The prevalence and YCI of Anaplasma spp., D. immitis and Ehrlichia spp. were significantly higher in Goiana. In contrast, the YCI of Leishmania spp. infection was significantly higher in São Joaquim de Bicas. CONCLUSIONS: We confirmed a high prevalence and YCI of various VBPs among privately-owned dogs in two geographical regions of Brazil. Our data also indicate that the risk of infection varies significantly for individual VBPs and between the regions, which may be related to several factors that are still poorly understood.

Vaccination against canine leishmaniasis in Brazil.

Prevention of canine Leishmania infantum infection is critical to management of visceral leishmaniasis in people living in endemic areas of Brazil. A bill (PL 1738/11), currently under consideration, proposes to establish a national vaccination policy against canine leishmaniasis in Brazil. However, there is no solid scientific evidence supporting the idea that this could reduce transmission from infected vaccinated dogs to sand flies to a level that would significantly reduce the risk of L. infantum infection or visceral leishmaniasis in humans. Thus, we advocate that insecticide-impregnated collars should the first line protective measure for public health purposes and that vaccines are applied on a case-by-case, optional basis for individual dog protection.

Performance of different serological tests in the diagnosis of natural infection by Leishmania infantum in dogs.

Visceral leishmaniasis (VL) is a zoonosis caused by the parasite Leishmania infantum and the dog is its main reservoir in rural and urban areas. The diagnosis of infection is mainly based on the presence of anti-Leishmania IgG antibodies in the serum of infected dogs. In this study, the sensitivity and specificity of qualitative rapid tests (RTs) dual path platform (DPP) Bio-Manguinhos, rapid enzyme-linked immunosorbent assay (ELISA) IDEXX, Kalazar Detect and ALERE, as well as quantitative ELISA Bio-Manguinhos and in-house indirect immunofluorescence assay (IFA) tests were analyzed in sera from infected and uninfected dogs. Serial dilutions of the in-house IFA were compared with RTs and ELISA Bio-Manguinhos. The results showed that none of the tests reached 100% sensitivity and specificity. There was no statistical difference between the analyzed RTs. The most sensitive test was the DPP Bio-Manguinhos (97.9%), while the rapid ELISA IDEXX showed higher specificity (100%). In the treatment setting of infected and/or diseased animals, quantitative tests for monitoring the evolution of antibody titers are required, which indicates the maintenance of in-house IFA in animal handling. Furthermore, we demonstrate that the RTs present higher sensitivity in serum samples with superior antibody titers obtained in the in-house IFA. However, the RTs exhibited false negatives in samples with low titers of antibodies. Among the RTs, only the DPP Bio-Manguinhos presented better performance in this situation. Therefore, the use of RTs for the diagnosis of VL in dogs with low titers of antibodies, such as asymptomatic, should be carefully evaluated.

Serological evidence of Leishmania infection by employing ELISA and rapid tests in captive felids and canids in Brazil.

Visceral leishmaniasis (VL) is a zoonosis and the dog is considered the most important urban reservoir. Cases in cats have been reported, but little is known about Leishmania infection and disease in wild felids and canids kept in captivity in endemic areas. Thus, the serological pattern of wild felids and canids kept in captivity at the Belo Horizonte Zoological Garden was investigated using two primary antigens for conventional ELISA: k39 and rKDDR, as well as three serological rapid kits: Dual Path Platform (DPP®) immunochromatographic test, rKDDR Immunochromatographic assay and ELISA SNAP Leishmania IDEXX®. A total of 21 serum samples, 13 of wild felids and 8 wild canids of varying age and sex were evaluated. The results obtained in the tests were analyzed by agreement using Kappa coefficient, and between ELISA antigens all the analysis performed had showed significant agreement among both of them, as well between the three immunochromatographic tests. The results demonstrated that there is serological evidence of wild animals seropositive for Leishmania antibodies at the Belo Horizonte Zoological Garden, and that all the antigens and rapid tests used can be employed in serological screening for VL in wild felids and canids.

Control of visceral leishmaniasis in Brazil: recommendations from Brasileish.

On 26 October 2012, veterinary medicine clinicians and researchers, members of Brasileish - Study Group about Animal Leishmaniasis - met at the Regional Council of Veterinary Medicine of Minas Gerais, in the city Belo Horizonte, in order to discuss many aspects of the situation of canine visceral leishmaniasis (CVL) in Brazil. In the face of recent national and international scientific evidence, we, the members of Brasileish, have elaborated some recommendations for the management and control of CVL in Brazil.

Caracterização clínica e epidemiológica das leishmanioses em cães no Estado de São Paulo / Clinical and epidemiological characterization of leishmaniasis in dogs in São Paulo State

Caracterizou-se retrospectivamente, a partir do ano de 1997 até março de 2008, a ocorrência de casos de leishmaniose, visceral ou tegumentar, dentre os cães dermatopatas atendidos em serviço especializado de hospital-escola veterinário de Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Para o diagnóstico em 427 animais, foram utilizados testes sorológicos (Ensaio Imunoenzimático Indireto, Imunofluorescência Indireta e/ou Fixação de Complemento), parasitológicos diretos e moleculares (Reação em Cadeia da Polimerase), que resultaram na confirmação de 117 (27,4%) casos positivos. Da amostragem, 96 (82,1%) eram cães de raça definida, os demais 21 (17,9%) não dispunham de plena definição racial, sendo 57 (48,7%) fêmeas e 60 (51,3%) machos. Quanto à faixa etária, 26 (22,2%) apresentavam idade inferior a 36 meses, 48 (41%) entre 36 e 84 meses e 43 (36,8%) ultrapassavam o sétimo ano de vida. Considerou-se 64 (54,7%) cães como oligossintomáticos, 44 (37,6%) sintomáticos e os nove (7,7%) remanescentes como assintomáticos. As principais alterações clínicas observadas foram, em ordem decrescente, lesões tegumentares, linfonodomegalia, anemia, disorexia, prostração e esplenomegalia. Quanto às alterações hematológicas, a mais importante foi a linfopenia relativa e absoluta. Dos animais positivos para a enfermidade, 96 (82,1%) correspondeu a casuística alóctone ao Município de São Paulo.

The occurrence of visceral or cutaneous canine leishmaniasis cases among dermatophatic dogs admitted to the veterinary teaching hospital School of Veterinary Medicine and Animal Husbandry - University of São Paulo from 1997 to 2008 was retrospectively characterized. For the diagnosis of 427 animals, serological (Enzyme-Linked Immunosorbent Assay, Indirect Immunofluorescence and/or Complement Fixation), parasitological and molecular (Polymerase Chain Reaction) tests were performed, which resulted in 117 (27.4%) confirmed positive cases. Of these, 96 (82.1%) were pure breed dogs and the others 21 (17.9%) were cross breed, being 57 (48.7%) females and 60 (51.3%) males. In terms of age, 26 (22.2%) were younger than 36 months, 48 (41%) were between 36 and 84 months, and 43 (36.8%) were older than seven years old. Sixty-four (54.7%) dogs were considered oligosymptomatic, 44 (37.6%) symptomatic, and the remaining nine (7.7%) asymptomatic. The main clinical findings were, in descending order, dermal lesions, lymphadenopathy, anemia, dysorexia, prostration and splenomegaly. As for hematological disorders, relative and absolute lymphopenia was the most important findings. Ninety-six (82.1%) of the positive animals correspond to allochthonus cases from São Paulo municipality.

Canine leishmaniosis in the Old and New Worlds: unveiled similarities and differences.

Canine leishmaniosis is a potentially life-threatening disease which is spreading geographically in the Old and New Worlds, where different diagnostic procedures, treatments, and control strategies are currently in place. This Opinion article outlines the similarities and differences between canine leishmaniosis in the Old and New Worlds, with emphasis on South America and Europe. Finally, it calls the attention of veterinary and public health authorities to standardize and improve practices for diagnosing, treating, and preventing the disease.

First report of infection of Lutzomyia longipalpis by Leishmania (Leishmania) infantum from a naturally infected cat of Brazil.

In recent years, cases of feline visceral leishmaniasis (FVL) have been described in different countries. In urban areas, domestic cats are suggested as possible alternative reservoirs of Leishmania (L.) infantum, the causal agent of visceral leishmaniasis (VL). This paper reports the first case of infection of Lutzomyia longipalpis by L. infantum of a naturally infected cat from Brazil through xenodiagnosis. The presence of a cat with FVL and its infectivity to the natural vector in Belo Horizonte city, an endemic area of VL in Brazil, suggests the need for further studies to determine the rate of occurrence of FVL among domestic cats and the infectivity ratio of L. longipalpis in endemic areas, and what is the role of these animals in the epidemiology of the disease.

Histopathological and parasitological investigations of ear healthy skin of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

Although 90% of clinical cases of American visceral leishmaniasis (AVL) occur in the northeastern region of Brazil, the incidence of cases in recent years has increased in southeastern states such as Minas Gerais (MG), where the disease has been reported in several cities, including Belo Horizonte, the state capital. Some studies have shown a strong correlation between the incidence of AVL and canine visceral leishmaniasis (CVL) in Belo Horizonte. A study of 108 dogs with parasite Leishmania chagasi detected by immuno-histochemistry in healthy ear skin was obtained from two distinct geographical areas: 55 from a metropolitan area of the municipality (Santa Luzia, MG) and 53 dogs from a central area of Belo Horizonte. In parallel, a group of 10 beagles were experimentally infected with L. chagasi. Considering the clinical aspects of all naturally infected dogs, symptomatic dogs were more frequent than asymptomatic ones, especially animals from the metropolitan area compared with the central area (79.6% and 20.3%, respectively). A chronic exudate was observed in the ear of 51 out of 55 dogs naturally infected from the metropolitan area (92.7%) and 45 out of 53 dogs naturally infected from the central area (84.9%). Importantly, asymptomatic dogs from the central area harbor more parasites in the skin than the asymptomatic ones from the metropolitan area. In addition, a profound difference was noted in the intensity of the inflammatory reaction and parasite load in the skin of experimental infected dogs.

Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy.

The goal of the present study was to characterize the semen quality of dogs naturally infected with Leishmaniachagasi, and treated with Allopurinol and Amphotericin B. Eight naturally infected and eight non-infected dogs were selected. Following semen collection, progressive motility, vigor, concentration and sperm morphology were evaluated. The seminal patterns in the treated animals were evaluated at the beginning (d0) and at days 30 (d30), 60 (d60) and 150 (d150) of treatment. The progressive motility at d0 (35.7+/-22.3%) was less than that of the control group (77.8+/-7.1%) (P<0.05). The vigor was similar to the control group throughout the treatment (P>0.05). The number of sperm/mL, sperm/ejaculate and sperm/kg of body weight was similar among groups (P>0.05). The percentages of normal spermatozoa of infected and treated animals were similar throughout the treatment and to the control group (69.1+/-8.7%) at d60 (37.5+/-11.2%) and d150 (48.3+/-10.8%) (P>0.05), but smaller at d0 (22.7+/-10.5%) and d30 (28.8+/-15.9%) (P<0.05). A greater percentage of acrosome damage was observed in the control group (3.1+/-2.3%) compared to the d60 (0.1+/-0.2%) (P<0.05). The infected dogs had a greater percentage of principal piece defects at d60 (37.0+/-6.3%) than the control group (16.8+/-7.3%) (P<0.05); and greater percentages of detached normal heads at d0 (28.7+/-19.7%) and d30 (18.5+/-18.5%) than the control group (0.4+/-0.5%) (P<0.05). This reduction in semen quality of the infected animals is suggestive of an epididymal dysfunction. Due to this poor semen quality, caution is recommended when using infected male dogs for reproductive purposes.

First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil.

Dogs are the most important reservoir of Leishmania (L.) infantum, the causal agent of visceral leishmaniasis (VL) in Brazil. Vectorial infection is the main route of transmission of the parasites. This paper reports the first case of vertical transmission of L. infantum in Brazil, confirmed by PCR and immunohistochemistry techniques in samples from spleen and liver of two stillborn pups from a bitch naturally infected with L. infantum in Belo Horizonte city, endemic area of VL. This result confirms the existence of transplacental transmission of Leishmania between dogs, and suggests the need for further studies to determine the rate of occurrence of this fact in endemic areas and what is their role in the epidemiology of the disease.