Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

Therapeutic success and failure in using miltefosine to treat dogs naturally infected with Leishmania infantum.

In urban environments, domestic dogs (Canis familiaris) are a major reservoir for the parasite Leishmania infantum. Miltefosine has been used as the standard treatment for canine visceral leishmaniasis in Brazil. However, therapeutic failures have been reported. In the present study, two dogs (CG03 and CG06) with a diagnosis of infection by L. infantum underwent two cycles of treatment with miltefosine (Milteforan™ - Virbac®). Analyses showed increases in the parasite load of both CG03 and CG06, even after treatment. The clinical score of CG03 dropped from 1 to 0 (after one round of treatment), such that this dog became asymptomatic. CG06 showed clinical worsening, such that its score increased from 1 to 2. After the second therapeutic round, the parasite load in CG03 was found to have decreased, but it was still higher than before drug treatment even though this dog was physically asymptomatic. There was no decrease in the parasite load in CG06 and there was clinical worsening. The clinical response of these dogs to the treatment differed, but the parasite load remained high in both cases, which poses a risk to public health, making it essential take measures to prevent the sandfly vector from accessing the dog.

Correction: Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

[This corrects the article DOI: 10.1371/journal.pone.0247560.].

A case of canine visceral leishmaniasis of unknown origin in Curitiba (state of Paraná, Brazil) treated successfully with miltefosine.

There are no records of autochthonous cases of canine visceral leishmaniasis in the city of Curitiba, Paraná state, Brazil. In 2020, a male French bulldog (CW01), approximately 2 years old was taken by its owners to a private veterinarian clinic. The suspicion of CVL was confirmed by means of a serology test (ELISA/IFAT reagent), rapid chromatographic immunoassay (DPP®) (ELISA - Biomanguinhos®), parasitological culture and quantitative polymerase chain reaction (qPCR). The animal routinely frequented parks in Curitiba and was taken on several trips to the municipalities of Bombinhas and Balneário Camboriú (Santa Catarina) and to Matinhos (Paraná) where CVL had not previously been reported. Treatment was initiated orally with Milteforan™ which resulted in a significant reduction in the parasitic load. The suspicion of autochthony was investigated through entomological research. A total of 10 traps were installed, one at the animal's home, seven in adjacent city blocks and two in a forest edge. No sandflies were trapped in the dog's home and adjacent houses. The traps in the forest edge caught one Migonemyia migonei female and five Brumptomyia spp. females. This case serves as a warning of the possible introduction of CVL in the city of Curitiba.

Serosurvey of anti-Leishmania (Leishmania) infantum antibodies in hunting dogs and hunters in Brazil.

BACKGROUND AND AIM: Although wild boar hunting activities and the hunting dog trade in the Atlantic Forest and Cerrado biomes of Brazil overlap both with endemic and with non-endemic areas for visceral leishmaniasis, no study to date has focused on Leishmania spp. exposure among hunting dogs and hunters. The aim of the present study was to assess the presence of Leishmania spp. antibodies in hunting dogs and hunters in different anthropized areas of two Brazilian biomes. MATERIALS AND METHODS: Blood samples were collected from 170 hunting dogs and 46 hunters between October 2016 and May 2018. The presence of antibodies against Leishmania spp. in hunting dogs was screened through a dual-path platform immunochromatographic test (DPP rapid test; Bio-Manguinhos/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil) and in hunters through an rK39-based rapid immunochromatographic test. Both tests were used in accordance with Brazilian Ministry of Health recommendations. RESULTS: Overall, although antibodies were detected through the immunochromatographic test in 3/170 (0.02%) of these female asymptomatic hunting dogs, all living in anthropized areas of the Atlantic Forest biome in South Brazil, no sample was confirmed through the enzyme-linked immunosorbent assay. All the hunters were non-reactive in the rapid immunochromatographic test. CONCLUSION: Our study on three suspicious hunting dogs has suggested that Leishmania (Leishmania) infantum may circulate both in endemic and non-endemic areas in Brazil. In addition, a high rate of hunting dog replacement due to death and trade may have led to less chance of infection and transmission between animals and between animals and humans, which would corroborate the outcomes reported here. Further studies should be conducted to fully establish whether hunting dogs and hunters may be used as sentinels in other areas endemic for Leishmania spp.

Increased Leishmania infantum resistance to miltefosine and amphotericin B after treatment of a dog with miltefosine and allopurinol.

BACKGROUND: Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. METHODS: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. RESULTS: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. CONCLUSION: The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated.

Characterization of a municipality as free of canine visceral leishmaniasis in the context of One Health.

Dogs are the main urban reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL), which is transmitted by sand flies. In the state of Paraná, the first detection of a positive dog for VL was in 2014, this year Paraná lost free status for this disease (VL). The objectives of this study were to determine the prevalence of canine visceral leishmaniasis in Palotina, the occurrence of vectors that may transmit Leishmania infantum, and the number of notifications of human visceral leishmaniasis cases from period 2010 to 2020. To determine the occurrence of canine visceral leishmaniasis, blood samples from 204 dogs were analyzed using the rapid test DPP® to detect anti-L. infantum antibodies. To investigate the occurrence of potential vectors, monthly collections were made at 18 points within the urban area of the municipality. The number of human visceral leishmaniasis cases was investigated from Epidemiological Surveillance records. None of the serologically tested dogs showed positive titration. Only two specimens of Lutzomyia neivai, one of Lutzomyia sp. and four of Brumptomyia brumpti specimens were collected. No human visceral leishmaniasis cases were reported. These results suggest that there is no evidence of circulation of L. infantum in Palotina.

A comparative approach on the activation of the three complement system pathways in different hosts of Visceral Leishmaniasis after stimulation with Leishmania infantum.

Visceral Leishmaniasis is an infectious disease that affects mainly humans and dogs, with the latter being important reservoirs of the parasite. Conversely, cats are naturally resistant. The immune system can offer important explanation to this problematic as there is no evidence on the role that the complement system plays in cats. In this context, effect of the complement system from human, dog and cat sera on Leishmania infantum was evaluated. Activation of the classical, alternative and lectin pathways was assessed through hemolytic and ELISA assays. Lytic activity of the complement on the parasite's viability was investigated by Transmission Electron Microscopy and Flow Cytometry. Complement proteins were more consumed in dog serum on the classical and alternative pathways, leading to less hemolytic activity, and only in cat serum they were consumed on the lectin pathway when incubated with L. infantum. Lytic activity on the parasite's surface was more accentuated in human serum, and varied throughout the parasite's developmental stages.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

First description of parasite load and clinicopathological and anatomopathological changes in a dog naturally coinfected with Dioctophyme renale and Leishmania infantum in Brazil.

This article reports the case of a domestic dog naturally coinfected with the nematode Dioctophyme renale and with the protozoan Leishmania infantum. The dog exhibited no clinical signs but had normocytic hypochromic anemia, hyperproteinemia, hyperglobulinemia, hypoalbuminemia, and hematuria. Necropsy revealed eight D. renale specimens in the abdominal cavity and in right kidney whose parenchyma was atrophied. Histopathological analysis showed glomerular atrophy, fibrosis and a marked diffuse pyogranulomatous inflammatory infiltrate in the right kidney. Moderate multifocal granulomatous peritonitis was observed in the greater omentum. Several Dioctophyme renale eggs were present amidst the inflammatory infiltrate of the right kidney and greater omentum. Leishmania infantum parasites were detected in perirenal adipose tissue of the right kidney, greater omentum, spleen, bone marrow, and popliteal lymph node. The high D. renale load and the severe and uncommon histological alterations associated with the eggs of this parasite may have been influenced by coinfection with L. infantum.

Corrigendum: Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

[This corrects the article DOI: 10.3389/fimmu.2018.01690.].

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs.

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Impact of 4% Deltamethrin-Impregnated Dog Collars on the Prevalence and Incidence of Canine Visceral Leishmaniasis.

In Brazil, visceral leishmaniasis is caused by the protozoan Leishmania infantum, primarily transmitted by Lutzomyia longipalpis and with the dog as its main urban reservoir. This study aims to evaluate the effectiveness of 4% deltamethrin-impregnated dog collars (DMC) DIC, Scalibor® 65 cm model and MSD manufacturer, on the prevalence and incidence of canine visceral leishmaniasis (CVL) and on the rate of infection of sandflies by L. infantum. The research was conducted in two areas of the municipality Mossoró, State of Rio Grande do Norte in Northeast of Brazil. Two semiannual serosurveys, followed by culling seropositive dogs, and searches for phlebotominae were performed in the control area (CA), whereas in the collar intervention area (IA), aside from those procedures, DMC were fitted to dogs every 6 months. CVL was diagnosed by the Dual Path Platform rapid test (TR-DPP®) and the Immunoenzymatic assay (EIE). The sandflies were collected monthly, identified, and the females were submitted to Quantitative Real-Time Polymerase Chain Reaction for detection of L. infantum DNA. The use of collars was associated with a 53-59% reduction in the incidence of CVL. The most abundant phlebotomine species were L. longipalpis (81.8%). Positive pools of L. longipalpis were obtained in the IA only in the first survey, whereas the presence of the DNA of the parasite in the vector was observed in the CA in both surveys. We conclude that the continuous use of these collars may have the potential to reduce both the incidence of CVL and the rate of infected phlebotomine sandflies.

Avaliação dos padrões imunológicos de cães imunizados contra leishmaniose visceral / Evaluation of immunological patterns of dogs immunized against visceral leishmaniasis

No Brasil, a Leishmaniose visceral (LV) é causada por Leishmania infantum e tem como seu principal vetor Lutzomyia longipalpis, e o cão como reservatório no ambiente doméstico e peridoméstico. Na tentativa de interromper o ciclo de transmissão e diminuir a prevalência da LV, são recomendados o recolhimento e a eutanásia dos cães infectados por L. infantum. No entanto, essa prática parece não estar sendo efetiva para a diminuição do número de casos e para a preservação de áreas não endêmicas, o que causa grande incomodo social. Autores sugerem que uma das medidas eficazes para o controle da leishmaniose visceral canina (LVC) seria o desenvolvimento de uma vacina efetiva. No Brasil, existem duas vacinas anti- LVC (Leishmune® e a Leish-Tec®). Porém, o Ministério da Saúde não recomenda o seu uso como forma de controle da LV, uma vez que os estudos de eficácia ainda são preliminares. O objetivo deste trabalho foi avaliar os padrões imunológicos de 28 cães imunizados com as vacinas Leish-Tec® ou Leishmune®, acompanhados durante um ano após o protocolo vacinal e comparar com a resposta de 18 cães naturalmente infectados. A avalição de resposta humoral foi realizada por meio do estudo da soroconversão dos cães imunizados utilizando-se os testes DPP® LVC e ELISA LVC fornecidos pelo Ministério da Saúde, além da avaliação da indução de IgG total, IgG1 e IgG2 específicas para as proteínas indutoras de cada vacina FML (Leishmune®) e A2 (Leish Tec®) e ao extrato total de promastigota de L. infantum nos cães imunizados e naturalmente infectados

Por meio da técnica de citometria de fluxo foram avaliados os fenótipos de linfócitos T e linfócitos B ex vivo, no sangue periférico dos cães em diferentes tempos após a vacinação em comparação com cães naturalmente infectados. Foi avaliada também a produção de citocinas pelas células mononucleares do sangue periférico após estimulo com extrato total de promastigota de L. infantum pela técnica de Luminex®. Observamos que os cães vacinados, seja com Leishmune® ou Leish-Tec®, não soroconverteram com os testes do Ministério da Saúde, sugerindo que a metodologia com DPP- LVC e ELISA é sensível e específica, sendo capaz de discriminar os cães verdadeiramente positivos para LVC daqueles imunizados. O nível de reatividade no soro dos animais vacinados com Leishmune® ou Leish-Tec® frente ao FML e a proteína A2, respectivamente foram altos. Com relação a avaliação dos fenótipos celulares observamos que, os animais naturalmente infectados com sinais clínicos, apesar de possuírem um percentual mais elevado de linfócitos T CD8+ no sangue periférico, apresentam uma diminuição no fenótipo de células CD8+ ativadas (CD8+CD25+) quando comparados aos grupos imunizados. Quanto à produção de citocinas, foi observado que os cães imunizados ou naturalmente infectados produziram citocinas pró e anti-inflamatórias, mas que não se mantiveram por 12 meses. O padrão de resposta imunológica celular ou humoral foi semelhante nos cães vacinados com a Leishmune® ou Leish-Tec®, entretanto não há indícios suficientes, até o momento, para o uso das vacinas como controle da LV. Estudos com cães de áreas não endêmicas, com as vacinas estudadas e novas proteínas vacinais são fortemente recomendadas. (AU)

Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

Can vaccines against canine visceral leishmaniasis interfere with the serological diagnostics recommended by the Brazilian Ministry of Health? / Vacinas anti-leishmaniose visceral canina podem interferir no diagnóstico sorológico preconizado pelo Ministério da Saúde brasileiro?

ABSTRACT: The objective of the current research was to assess seroconversion in dogs immunized with Leishmune® and Leish Tec® vaccines using rapid chromatographic immunoassay DPP® (Dual Path Platform) (DPP CVL) and enzyme immunoassay (EIE) up to one year after the vaccination protocol. The study sample comprised 28 dogs divided into two groups, each group immunized with an anti-CVL vaccine and clinically monitored for one year through clinical evaluation and laboratory tests. 22 (78.5%) dog were monitored. During the evaluation time (T1-30 days, T2-6 months, and T3-1 year after vaccination) the results for all dogs were negative for CVL, except for one animal vaccinated with Leish tec® that seroconverted in the DPP CVL test at T2. Subsequent examinations of this dog were negative. Our results showed that in a non-endemic area, even at different evaluation times, dogs vaccinated against CVL with Leishmune® or Leish tec® did not seroconvert in the serological protocol used by the Brazilian Ministry of Health (DPP/EIE).

RESUMO: O objetivo deste trabalho foi avaliar a soroconversão em cães imunizados com as vacinas Leishmune® e Leish tec®, através do teste imunocromatográfico rápido DPP® (Dual Path Platform) (DPP LVC) e do ensaio imunoenzimático (EIE) durante um ano após o protocolo vacinal. Trata-se de um estudo onde 28 cães divididos em dois grupos foram imunizados cada um com uma vacina anti - LVC e acompanhados durante um ano através de avaliação clínica e exames laboratoriais. Foi possível acompanhar 22 (78.5%) cães. Nos exames dos tempos 1, 2 e 3 (respectivamente 30 dias, 6 messes e 1 ano após a vacinação) os resultados de todos os cães também foram negativos para LVC, exceto de um cão que recebeu a vacina Leish tec® e soroconverteu no DPP LVC no T2, após 6 meses a vacina. Os exames posteriores deste cão foram negativos. Os resultados do presente estudo demostraram que, em área não endêmica e mesmo em diferentes tempos de avaliação, cães vacinados contra LVC, independente da vacina utilizada, não foram capazes de soroconverter no protocolo utilizado pelo Ministério da Saúde brasileiro (DPP/EIE).

Cytokine and iNOS profiles in lymph nodes of dogs naturally infected with Leishmania infantum and their association with the parasitic DNA load and clinical and histopathological features.

In South America, visceral leishmaniasis is a zoonotic disease with severe evolution characteristics in humans, and dogs are its main reservoir. In this context, this study aimed to evaluate the clinical status of dogs from a Brazilian endemic area naturally, at Barra Mansa municipality, infected with Leishmania infantum, in conjunction with their histopathological profile and, in order to determine possible markers of susceptibility or resistance to the disease, parasitic DNA load, cytokine and iNOS mRNA expression profiles were investigated in lymph nodes. High levels of IFN-É£ and IL-6 mRNA were detected. Both IFN-É£ and IL-6 mRNA were associated with disorganization of the corticomedullary region. IFN-É£ and TNF-α mRNA were associated with the absence of follicular hyperplasia. The regulatory pathway was remarkable with IL-10 mRNA detection and its significant association with the severity of the disease. Plasmacytosis and sinus histiocytosis were associated with high loads of parasitic DNA, but there was no significant association between the parasite DNA load and animal clinical alterations. Since high parasitic loads were found in animals with or without symptoms, clinical examination cannot be considered as a criterion for disease susceptibility assessment.

Comparação entre amostras de pele íntegra parafinada e congelada através da reação em cadeia da polimerase - quantitativa (qPCR) no diagnóstico da leishmaniose visceral canina / Comparison between samples of paraffin and frozen intact skin by polymerase chain reaction - quantitative (qPCR) in the diagnosis of canine visceral leishmaniasis

A leishmaniose visceral (LV) representa um importante problema de saúde pública no Brasil.Podem acometer os seres humanos e mamíferos silvestres e domésticos, sob a forma de doenças infecciosas crônicas com uma ampla gama de manifestações clínicas. O diagnóstico laboratorial é requerido para confirmar a suspeita clínica. Atualmente as técnicas moleculares baseadas na reação em cadeia da polimerase (PCR) têm demonstrado alta especificidade e sensibilidade. Na literatura existem diversos relatos da utilização da PCR com diferentes tipos de material clínico, alvos moleculares, conservação das amostras. Alguns autores consideram amostras congeladas mais vantajosas em relação às amostras fixadas em formol e em bebida sem parafina (FFPE). No entanto, muitos pesquisadores defendem a importância das amostras FFPE devido à facilidade de conservação e possibilidade de utilização da PCR em estudos retrospectivos. O objetivo desse projeto foi avaliar a acurácia da qPCR realizada em amostras de pele íntegra congelada e parafinada. Trata-se de um estudo de validação, com 50 amostras de tecido congelado e 50 de tecido parafinado, provenientes de cães da cidade de Belém-PA.De cada animal foram coletados fragmentos de pele íntegra, sendo um dos fragmentos congelado e outro conservado por parafinização. As amostras de pele íntegra analisadas foram coletadas da região escapular utilizando punch de 3 mm. A qPCR foi orientada para alvos dokDNA da espécie Leishmania infantum...

Visceral leishmaniasis (VL) is a significant public health problem in Brazil. It affects humans,wild and domestic mammals, in the form of chronic infectious disease with a wide range of clinical manifestations. Laboratory diagnosis is required to confirm the clinical suspicion ofVL. Currently, molecular techniques based on polymerase chain reaction (PCR) have shownhigh specificity and sensitivity. Some authors consider more advantageous the use of frozensamples in PCR due to the better quality of DNA, in relation to that of fixed in formalin andembedded in paraffin (FFPE) samples. However, many researchers argue describe the importance of FFPE samples, due to the facility of conservation and the possibility of usingPCR in retrospective studies. The aim of this study was to evaluate the accuracy of the quantitative polymerase chain reaction (qPCR) performed on intact frozen and FFPE skin samples. This is a validation study with 50 fresh tissue samples and 50 FFPE tissues of dogsfrom Belém-PA. From each animal fragments of intact skin were collected, being one of the frozen fragments and another saved by paraffinization. The samples analyzed were collectedfrom intact skin of the scapular region using a 3 mm punch. The qPCR was oriented targetskDNA of Leishmania infantum...

First autochthonous case of canine visceral leishmaniasis in Volta Redonda, Rio de Janeiro, Brazil.

In Brazil, American visceral leishmaniasis (AVL) is caused by Leishmania (Leishmania) chagasi and its main vector is Lutzomyia longipalpis. Cases of canine visceral leishmaniasis (CVL) in non-endemic areas have been reported over the last few years throughout the country. The objective of this research note is to describe an autochthonous case of CVL that occurred in the municipality of Volta Redonda, state of Rio de Janeiro, an area where the disease is not endemic, alerting veterinarians and the scientific community to the expansion of this important zoonosis and advising veterinary practitioners on how to deal with a suspicion of CVL. Canine visceral leishmaniasis can be misdiagnosed within a broad spectrum of canine diseases based on clinical and laboratory findings. Therefore, knowledge of its clinical manifestations, specific and sensitive laboratory diagnostic tests and parasitological procedures are of the utmost importance for rapid confirmation and notification of a case, thus contributing directly to the control of a focus.

First autochthonous case of canine visceral leishmaniasis in Volta Redonda, Rio de Janeiro, Brazil / Primeiro caso autóctone de leishmaniose visceral canina em Volta Redonda, Rio de Janeiro, Brasil

In Brazil, American visceral leishmaniasis (AVL) is caused by Leishmania (Leishmania) chagasi and its main vector is Lutzomyia longipalpis. Cases of canine visceral leishmaniasis (CVL) in non-endemic areas have been reported over the last few years throughout the country. The objective of this research note is to describe an autochthonous case of CVL that occurred in the municipality of Volta Redonda, state of Rio de Janeiro, an area where the disease is not endemic, alerting veterinarians and the scientific community to the expansion of this important zoonosis and advising veterinary practitioners on how to deal with a suspicion of CVL. Canine visceral leishmaniasis can be misdiagnosed within a broad spectrum of canine diseases based on clinical and laboratory findings. Therefore, knowledge of its clinical manifestations, specific and sensitive laboratory diagnostic tests and parasitological procedures are of the utmost importance for rapid confirmation and notification of a case, thus contributing directly to the control of a focus.

No Brasil a leishmaniose visceral americana (LVA) é causada por Leishmania (Leishmania) chagasi e tem como seu principal vetor Lutzomyia longipalpis. Nos últimos anos vêm sendo relatados casos de leishmaniose visceral canina (LVC) em áreas não endêmicas em todo país. O objetivo desta nota é descrever um caso autóctone de LVC no município de Volta Redonda, Estado do Rio de Janeiro, área não endêmica para essa doença e assim, chamar a atenção dos clínicos veterinários e da comunidade científica para a expansão dessa importante zoonose, além de orientar os médicos veterinários, como proceder frente a um caso suspeito de LVC. A LVC pode ser clínica e laboratorialmente confundida com uma ampla gama de patologias caninas e o conhecimento de suas manifestações clínicas e de procedimentos laboratoriais específicos e sensíveis para esse diagnóstico, são de grande importância para uma rápida confirmação e notificação do caso, contribuindo assim diretamente para o controle do foco.