Canine leishmaniosis (CanL) caused by Leishmania infantum remains a major public health concern in endemic regions, where dogs are the primary domestic reservoir. Despite high accuracy, the invasiveness of current diagnostic methods limits their practical application. This study evaluated the diagnostic potential of hair as a non-invasive sample for molecular detection of L. infantum using a nested PCR targeting the ITS1 region (HAIR-test). All PCR-based tests in this study, including those performed on lymph node aspirates (LN-test), blood (BL-test), and conjunctival swabs (CS-test), employed the same nested ITS1-PCR protocol. A cohort of 44 naturally infected dogs, defined by parasitological confirmation or LN-test positivity, was evaluated using the HAIR-test, with hair samples independently collected by two different operators. The combined sensitivity of the HAIR-test was 59.1 %, outperforming the BL-test and matching the performance of the CS-test. Sequencing confirmed L. infantum DNA in hair samples, but also revealed co-amplification of non-Leishmania trypanosomatids in a subset, underscoring the need for high-specificity assays. Inter-collector agreement was moderate, indicating potential variability in parasite distribution or inconsistencies in sample collection and handling. No correlation was observed between hair mass and PCR-positivity, suggesting that L. infantum DNA is present in follicular bulbs rather than in hair shafts. Beyond suboptimal sensitivity, limitations in assay reproducibility and uncertainties regarding the precise origin of the detected DNA underscore the need for further methodological refinement and validation. The HAIR-test offers a viable, non-invasive alternative in settings where invasive sampling is impractical, but its routine diagnostic application requires methodological refinements and validation.

In this study, an evaluation was made of three treatments against feline leishmaniosis (FeL) and their impacts on the transmission of Leishmania infantum to its vector, Lutzomyia longipalpis. A cat with clinical signs of FeL was examined and L. infantum diagnosed. Subsequently, the cat was subjected to xenodiagnosis and L. infantum detected in the vectors. The cat was then treated with three different drugs and the clinical improvement and parasite transmissibility to the vector were evaluated. Promastigotes were observed in 21/52 female sandflies (40.38%) in a xenodiagnosis prior to the treatments. Clinical signs persisted after the first treatment with marbofloxacin, and the cat remained positive in serological, molecular, and parasitological tests. Therefore, the cat was treated with miltefosine but remained sick and tested positive. A second xenodiagnosis was performed a month after treatment with miltefosine, and promastigotes were observed in 5/9 females (55.55%). Lastly, the cat was treated with allopurinol, which led to good clinical improvement, but it remained positive, and a final xenodiagnosis revealed Leishmania in 2/29 (6.89%) females. The results showed that only treatment with allopurinol produced a good clinical response, but none of the treatments succeeded in eliminating L. infantum infection or preventing transmission to the vector.

Abstract Canine monocytic ehrlichiosis (CME) is one of the most important tick-borne diseases worldwide, with multisystemic presentations. Immune dysregulation has been proposed as the primary mechanism involved in its pathogenesis and in tissue injury in dogs with CME. Experimental infection of German Shepherd dogs in the present study demonstrated that CME caused marked pathological changes in their lymph nodes and spleen, and also gave rise to mononuclear infiltration in organs and tissues. Immunophenotyping of cells in lymph nodes, spleen and injured tissues highlighted differences in lymphocyte subsets, local expression of immunoglobulin subclasses and MHCII molecules between infected and control dogs. These findings suggest that the immunophenotypic and immunopathological changes in dogs with acute experimental CME are related to Th1 bias and compartmentalized immune response.

A Leishmaniose Visceral Canina (LVC) e uma doenca cronica cujos dados epidemiologicos sao intrigantes visto que muitos animais infectados apresentam doenca grave que culmina na morte do animal, enquanto outros sao assintomaticos. As opcoes de sorodiagnosticos da LVC incluem testes de deteccao direta e indireta. Em 2012, o DPP®, passou a ser o teste de triagem e o ELISA confirmatorio como diagnostico oficial para a LVC, os quais apresentam vantagens por ser de facil execucao e de custo acessivel e, por isso sao empregados em inqueritos sorologicos em municipios considerados endemicos. Contudo, diversos estudos relatam reatividade cruzada com outros patogenos e, por outro lado, existem pesquisas que relatam a existencia da nao reatividade cruzada e sim, da coinfeccao entre os agentes. Por esse motivo, objetivou-se comparar os resultados do DPP® em caes naturalmente infectados por L. infantum em area endemica para LVC com a soropositividade para E. canis pelo teste ELISA. O presente projeto visa um estudo experimental in vitro, aprovado pelo CEUA, Protocolo no 129/2018, no dia 17 de maio de 2018. As amostras sericas de 35 caes foram submetidas ao DPP® para deteccao de anticorpos anti- Leishmania infantum e ELISA para a confirmacao da infeccao nas amostras soropositivas no DPP® e deteccao de anticorpos anti- Ehrlichia canis . Pesquisa de morulas de E. canis em papa leucocitaria e pesquisa de formas amastigotas de L. infantum em citologia de linfonodos tambem foram realizadas. Das 35 amostras sericas submetidas aos testes sorologicos, 13 (37,2%) amostras apresentaram soropositividade no DPP®. Posteriormente, as amostras foram submetidas ao Ensaio Imunoenzimatico, no qual 6 (17,2%) amostras apresentaram sororreatividade frente ao antigeno de L. infantum e 21 (60%) amostras apresentaram sororreatividade frente ao antigeno de E. canis . Apenas 3 amostras de puncao aspirativa de linfonodo apresentaram formas amastigotas de L. infantum e nenhuma morula de E. canis foi encontrada no esfregaco de papa leucocitaria. Contudo, concluiu-se que a especificidade permitiu identificar caes soropositivos para o diagnostico da LVC, sendo que a co-positividade em alguns caes deve-se a uma infeccao previa pelos dois agentes.

Birds of the psittaciform order, composed by the Psittacidae and Loridae family have several characteristics making them more frequently kept as companion animals, promoting the increase of breeding sites in Brazil. The present study aimed to analyze the specificity and sensitivity of three different coproparasitological tests, Willis, Hoffman and Direto de feces, through statistical tests: Chi-Square and Kappa. 70 fecal samples of exotic parrots were collected from a commercial breeding site and these were submitted to the three tests, totaling 210 coproparasitological exams. Among the tests performed, 29,5% were positive for nematode eggs, cestodes and oocysts. Coproparasitological exams are inexpensive, have clinical importance, indicating the population of endoparasites and therapeutic treatments.

This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88% (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.A proteína de superfície eritrocitária, Antígeno 1 do Merozoíta de Theileria equi (EMA-1), é um potencial candidato para o desenvolvimento de antígenos de valor diagnóstico para a piroplasmose equina. Com o objetivo de estabelecer um método de diagnóstico efetivo e prático, o gene EMA-1 da amostra Jaboticabal - SP de T. equi foi clonado e expresso em Escherichia coli contendo uma cauda de poli-histidina (His6-EMA1). O EMA-1 expresso reagiu com anticorpos específicos no Western blot e apresentou peso molecular aparente de 34 kDa, sendo altamente consistente com seu valor teórico. A sequência nucleotídica do gene EMA-1 da amostra Jaboticabal foi analisado comparativamente com outras sequências públicas, e os resultados indicam elevado grau de homologia com amostras de diversos países. A proteína recombinante purificada His6-EMA1 foi testada no ensaio imunoenzimático (ELISA) para a detecção de anticorpos anti-T. equi em equinos. O teste de ELISA diferenciou-se claramente entre soros de equinos infectados por T. equi, soros de animais infectados por Babesia caballi e soro normal de equino. Amostras de soros coletadas de equinos do Estado de São Paulo, sudeste do Brasil, foram examinadas para o diagnóstico da infecção por T. equi pelo ELISA. Das 170 amostras analisadas, 95,88% (163/170) foram positivas para T. equi. Os resultados sugerem que a proteína His6-EMA1 expressa em E. coli pode ser um antígeno confiável para diagnóstico imunológico pelo teste de ELISA, e que T. equi merece grande atenção no Estado de São Paulo.

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania)infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L. (L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.A leishmaniose visceral (LV) é uma zoonose amplamente disseminada, causada no Brasil pela Leishmania (Leishmania) infantum chagasi. Flebotomíneos vetores adquirem o agente etiológico, alimentando-se do sangue de animais contaminados, como cachorros ou animais selvagens. A doença é endêmica no Brasil, e focos de epidemia são relatados em cidades densamente povoadas por todo o país. Muitas manifestações clínicas relacionadas à infecção por Leishmania estão ligadas à relação parasito-hospedeiro, e vários possíveis fatores de virulência dos parasitas, que causam a LV, são alvos de estudo, tais como os genes A2. O gene A2 foi isolado pela primeira vez em 1994 e, em seguida, em 2005, três novos alelos foram descritos em Leishmania (Leishmania) infantum. No presente estudo, um fragmento do gene A2 de uma população clonal de L.(L.) infantum chagasi foi amplificado por PCR e sua sequência de nucleotídeos determinada. O fragmento mostrou 90% de similaridade com alelos do gene A2 de Leishmania (Leishmania) donovani e de L. (L.) infantum, descritos na literatura. Entretanto, a tradução da sequência de nucleotídeos mostra diferenças na sequência de aminoácidos da proteína, que podem ser essenciais em determinar a variabilidade do gene A2 em espécies do complexo L. (L.) donovani e representa uma ferramenta adicional na compreenssão do papel dessa família de genes na virulência e imunidade da leishmaniose visceral. O conhecimento dessa variação é importante para o desenvolvimento de testes diagnósticos mais precisos e ferramentas mais eficazes no controle da doença.

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

Blood and serum samples from 170 horses raised in the Jaboticabal microregion, São Paulo State, Brazil, were collected and tested by microscopic examination of blood smears, indirect fluorescent antibody test (IFAT) and nested polymerase chain reaction (nPCR) for Theileria equi infections. The association among the test results was verified by the McNemar test. During the examination of thin blood smears, parasites were detected in six (3.52%) horses. Anti-T. equi antibodies were detected in 100% sera samples, with titers ranging between 1:80 and 1:5120. The nPCR based on the T. equi merozoite antigen gene (EMA-1) allowed the visualization of species-specific amplified product in 108 (63.53%) horses. All six samples judged positive microscopically were also positive for nPCR. Statistical analysis indicated general disagreement (p < 0.0001) between IFAT and nPCR; IFAT and blood smear; and nPCR and blood smear on the detection of parasite carriers. The results of the present study indicate that T. equi is widely spread among horses in the Jaboticabal microregion, Northeast region of São Paulo State, Brazil.

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates.In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA.IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI).Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively.IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples.The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative.Sixty animals were positive for both T. gondii and N. caninum.Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed.Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed.The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3% foram co-negativas, mas 27,6% foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.