Daniela Farias Larangeira

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG, and subclasses, parasitism, and parasite transmission by xenodiagnosis

BACKGROUND The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use. PURPOSE To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL). METHODS From 2010 to 2012, healthy dogs were vaccinated with Leishmune(®) (50 animals) or Leish-Tec(®) (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis. RESULTS Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune(®) and Leish-Tec(®) groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune(®) and on the 21st day after the second dose of Leish-Tec(®). The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune(®) and Leish-Tec(®) groups, respectively. The Leishmune(®) group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec(®) group (p<0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p<0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune(®) group, and 7.9% (3/38) of the Leish-Tec(®) group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively. CONCLUSIONS No significant differences were observed in dogs vaccinated with Leishmune(®) or Leish-Tec(®), with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune(®)-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec(®) exhibited adverse reactions with greater frequency and severity.

Investigação de áreas de risco como metodologia complementar ao controle da leishmaniose visceral canina

Risk areas of canine visceral leishmaniasis in the city of Camacari, Bahia, Brazil, were investigated. A total of 278 dogs from 141 homes pertaining to 20 investigated risk areas was serologically screened (ELISA). The general seroprevalence was 21.7% (56/258) after exclusion of 20 dogs used at the beginning of the survey to limit the study area. The respective results of the univariated and multivariated analysis of factors related to infection of dogs by Leishmania chagasi, to vector distribu-tion pattern in the area and to the methodology used to localize the canine focuses are discussed.

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors

The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector.

Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp.

In this work, we investigated the association between the disruption of splenic lymphoid tissue and the severity of visceral leishmaniasis in dogs. Clinical and laboratory data from 206 dogs were reviewed. Spleen sections collected during the euthanasia of these animals were analyzed, and the splenic lymphoid tissue samples were classified as well organized (spleen type 1), slightly disorganized (spleen type 2), or moderately to extensively disorganized (spleen type 3). Of 199 dogs with evidence of Leishmania infection, 54 (27%) had spleen type 1, 99 (50%) had spleen type 2, and 46 (23%) had spleen type 3. The number of clinical signs associated with visceral leishmaniasis was significantly higher in the animals with evidence of Leishmania infection and spleen type 2 or 3 than in the animals with spleen type 1. Alopecia, anemia, dehydration, dermatitis, lymphadenopathy, and onychogryphosis were all more frequent among animals with evidence of Leishmania infection and spleen type 3 than among the dogs with evidence of Leishmania infection and spleen type 1. The association between the severity of canine visceral leishmaniasis and the disorganization of the splenic lymphoid tissue was even more evident in the group of animals with positive spleen culture. Conjunctivitis and ulceration were also more common in the animals with spleen type 3 than in the animals with spleen type 1. The serum levels (median, interquartile range) of albumin (1.8, 1.4–2.3 g/dL) and creatinine (0.7, 0.4–0.8 mg/dL) were significantly lower and the serum levels of aspartate aminotransferase were significantly higher (57, 39–95 U) in animals with spleen type 3 than in animals with spleen type 1 (2.8, 2.4–3.4 g/dL; 0.9, 0.7–1.2 mg/dL and 23, 20–32 U, respectively). Our data confirm the hypothesis that disruption of the splenic lymphoid tissue is associated with a more severe clinical presentation of canine visceral leishmaniasis.

Xenodiagnosis on dogs with visceral leishmaniasis: Canine and sand fly aspects related to the parasite transmission.

One of the main limitations for the effective control of canine leishmaniasis in endemic areas is the difficulty in identifying infectious dogs. The objective of this study was to determine factors, related to dogs and to parasite detection in sand flies, which are associated with the positive xenodiagnosis of Leishmania infantum using the vector Lutzomyia longipalpis. The xenodiagnosis was performed in 50 owned dogs residing in endemic areas, which were divided into three different groups: G1-26 dogs proved to be infected and classified by severity of VL clinical signs on physical examination; G2-15 dogs proved to be infected and classified by severity of clinical signs and intensity of laboratory abnormalities; G3-nine dogs that were seropositive for anti-Leishmania IgG in ELISA tests. Parasite search in the sand flies after having fed on dogs in the xenodiagnosis was performed by both methodologies, PCR and dissection followed by microscopy. In G1, 58% (15/26) of dogs were able to transmit Leishmania to the vector, when parasite detection in sand flies were performed by PCR technique, 5 days after blood meal, whereas in G2, 53% (8/15) transmitted the parasite to the vector, however, confirmation was performed by direct observation of parasite through optical miscroscopy held 10 days after blood meal. Rate of infectiousness of dogs to sand flies was positively associated to severity of disease (p=0.042 and p=0.040), regardless the method used for clinical classification or for parasite detection in sand flies after xenodiagnosis. In G1 30% (3/10) of dogs with subclinical infection were infectious to the vector, while 80% (12/16) of dogs with clinical disease were also infectious. Even more, 17% (1/6) of dogs that had moderate disease were infectious to the sand flies, while 78% (7/9) of dogs with severe disease were infectious in G2. Still in G2, the proportion of sand flies infected (grade of infectiousness) was significantly lower (p=0.0098) when they fed on dogs with moderate disease (1%) in comparison with dogs with severe disease (38%). The dogs from G3 presented a rate of infectiousness of 11% (1/9), demonstrating that the indirect ELISA is not a good indicator of infectiousness and, therefore, should not be used as a confirmatory test for the euthanasia of dogs, as it is currently done in Brazil.

Ocorrência de Leishmania infantum em fezes de cão

Este e o primeiro relado sobre a ocorrencia de Leishmaniasp. em fezes de cao. Foram encontradas formas amastigotas intra e extracelulares por meio de citologia de amostra fecal de um cao apresentando hematoquezia recorrente associada a leishmaniose visceral canina. O diagnostico de Leishmania infantumfoi confirmado por PCR de fezes e por cultura e PCR em amostras de baco.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.

Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dogs body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.

Association between Leishmania infantum DNA in the hair of dogs and their infectiousness to Lutzomyia longipalpis

Diagnosis of infection with Leishmania infantum by DNA detection in the hair has been recently demonstrated in dogs and wild animals. Our objective was to investigate if polymerase chain reaction (PCR) in hair might be used to identify infectious dogs. Thus, we assessed the infectiousness to Lutzomyia longipalpis by xenodiagnosis in comparison with the detection of L. infantum DNA by PCR in the hair, and with serology for anti-Leishmania IgG by ELISA in 15 positive dogs for L. infantum infection. Eight healthy dogs were included as negative controls. Among the 15 infected dogs, 13 were found positive in the ELISA (87%), 12 were PCR positive in the hair (80%), and 10 were positive in xenodiagnosis (67%). Positivity in the hair was associated with positivity in spleen (p=0.0003), seropositivity for antibodies (p=0.0006) and parasite transmission to L. longipalpis (p=0.0028). Considering the benefits to animal welfare and feasibility of hair sampling method, studies in larger and more diverse populations of naturally infected dogs from endemic areas should be conducted to evaluate the sensitivity, specificity, and predictive values of PCR using hair as a possible biomarker of infectiousness in dogs.

Ectopia ureteral unilateral congênita em uma cadela Teckel Dachshund com pelagem arlequim: relato de caso

The present work reports the first description of congenital ureteral ectopy in the canine breed Teckel Dachshund, diagnosed in a female dog with merle colour pattern. The two month old animal, presented with continuous dribbling of urine and bacterial cystitis, was subjected to a diagnostic plan for ectopic ureter. The excretory urography showed a right hidroureter, which had an orifice located caudally to the trigone of the bladder. The animal also presented a bilateral inguinal hernia, which confirmed the case description as a multiple congenital anomaly condition. Animal welfare aspects are also discussed.