Potential of recombinant LiHyQ, a novel Leishmania infantum protein, for the diagnosis of canine visceral leishmaniasis and as a diagnostic and prognostic marker for human leishmaniasis and human immunodeficiency virus co-infection: A preliminary study.

Laboratory diagnosis of leishmaniasis shows variable efficacy in detecting infected mammalian hosts and there is a need to identify suitable antigens to improve the accuracy of diagnostic tests. In the present study, a L. infantum hypothetical protein called LiHyQ was evaluated for the diagnosis of tegumentary (TL) and visceral (VL) leishmaniasis using canine and human samples. A collection of dog sera (n=155) were tested and contained samples from asymptomatic (n=20) and symptomatic (n=25) VL animals, from healthy dogs living in endemic (n=25) or non-endemic (n=25) areas of disease, from Leish-Tec® vaccinated dogs (n=20) or from dogs infected with Ehrlichia canis (n=15), Babesia canis (n=10) and Trypanosoma cruzi (n=15). Sensitivity (Se), Specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with L. infantum Soluble Leishmania Antigen (SLA) preparation were 60.0%, 99.0%, 96.0% and 86.0%, respectively. A collection of human sera (n=305) were tested and contained samples from TL (n=50) and VL (n=40) patients, from VL/HIV co-infected patients (n=35), from patients infected with HIV alone (n=30), Chagas Disease (n=30), malaria (n=10), tuberculosis (n=10), paracoccidioidomycosis (n=15), leprosy (n=30) or aspergillosis (n=15); and from healthy subjects (n=40). Se, Sp, PPV and NPV values of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with SLA were 58.0%, 76.0%, 50.0% and 82.0%, respectively. The antibody reactivity against the protein was compared with commercial kits, and the kappa index varied from 0.95 to 1.00 for rLiHyQ, and of 0.55 to 0.82 for the kits. In addition, the serological follow-up of treated patients showed a significant reduction in rLiHyQ-specific IgG antibody levels. All canine and human samples were tested at the same time using the same reagents, in order to reduce experimental variation and interference in data interpretation. In conclusion, our preliminary data suggest a diagnostic and prognostic role for rLiHyQ against leishmaniasis.

Serodiagnosis of canine leishmaniasis using a novel recombinant chimeric protein constructed with distinct B-cell epitopes from antigenic Leishmania infantum proteins.

Visceral leishmaniasis (VL) is an important public health problem in the world, and control measures are insufficient to avoid the spread of this neglected disease. Dogs are important domestic reservoirs of Leishmania parasites in countries where VL is a zoonosis, representing a major source of infection between sand fly vectors and humans. In this context, a precise diagnosis of canine leishmaniasis (CanL) could help to reduce the number of human cases. Distinct approaches for the diagnosis of CanL have used recombinant proteins in serological assays. However, variable results of the antigens have been found, mainly to diagnosis asymptomatic cases. The present study used bioinformatics to select specific B-cell epitopes of four Leishmania infantum proteins, which had previously been proven to be antigenic in VL, aiming to produce a novel chimeric protein and to evaluate it for the diagnosis of CanL. Seven B-cell epitopes were identified and used to construct the chimera, which was analyzed in a recombinant format through an ELISA assay against a canine serological panel. A soluble Leishmania antigenic extract (SLA) was used as an antigen control. Results showed 100 % sensitivity and specificity for chimera, while when using SLA the values were 26.0 % and 96.4 %, respectively. The performance of chimera was compared with a commercial kit using asymptomatic and symptomatic dog sera, and the data showed that no false-negative result was found when the recombinant protein was used. However, when using the commercial kit, 40.0 % and 16.0 % of the false-negative results were found, respectively. In conclusion, the recombinant chimera showed an antigenic potential to be evaluated in new studies against a larger serological panel for the diagnosis of CanL.

A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease.

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.

Biotechnological applications from a Leishmania amastigote-specific hypothetical protein in the canine and human visceral leishmaniasis.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a rapid and precise diagnosis of the disease should be performed, mainly to treat patients as soon as possible, aiming to reduce the treatment time and the toxicity of the therapeutics. In the present study, the diagnostic role of an amastigote-specific Leishmania protein was evaluated in the canine and human VL. Results showed that the recombinant protein (called rLiHyJ) and one specific B cell epitope (called PeptJ) predicted from protein sequence presented high sensitivity and specificity values to diagnose canine and human disease, showing also a low reactivity against cross-reactive samples. The rA2 protein and a parasite antigenic extract showed variable sensitivity and/or specificity values in the ELISA experiments. A prognostic evaluation of protein and peptide in the human VL indicated that specific IgG antibodies significantly decreased after treatment, when compared to be values obtained before therapy. The in vitro immunogenicity using rLiHyJ in peripheral blood mononuclear cell (PBMC) cultures collected of such patients and healthy subjects suggested that the protein induced lymphoproliferation and high IFN-γ production in the stimulated cells. In conclusion, although preliminary, results suggest that rLiHyJ and PeptJ could present distinct biotechnological applications in the canine and human VL.

Evaluation of Leishmania infantum pyridoxal kinase protein for the diagnosis of human and canine visceral leishmaniasis.

Visceral leishmaniasis (VL) is a highly neglected disease that is present in several countries worldwide. Present-day treatments against this disease are unsuitable, mainly due to the toxicity and/or high cost of drugs. In addition, the development of vaccines is still insufficient. In this scenario, a prompt VL diagnosis was deemed necessary, although sensitivity and/or specificity values of the tests have been. In this context, new antigenic candidates should be identified to be employed in a more precise diagnosis of canine and human VL. In this light, the present study evaluated the diagnostic efficacy of the Leishmania infantum pyridoxal kinase (PK) protein, applied in its recombinant version (rPK). In addition, one specific B-cell epitope derived of the PK sequence was predicted, synthetized, and evaluated as diagnostic marker. Results in ELISA tests showed that the antigens were highly sensitive to VL identification in dogs and human sera, presenting a low reactivity with VL-related disease samples. The recombinant A2 (rA2) protein and L. infantum antigenic preparation (SLA), used as controls, also proved to be highly sensitive in detecting symptomatic cases, although a low sensitivity was found when asymptomatic sera were analyzed. High cross-reactivity was also found when these antigens were evaluated against VL-related disease samples. The post-therapeutic serological follow-up showed that anti-rPK and anti-peptide IgG antibody levels decreased in significant levels after treatment. By contrast, the presence of high levels of the anti-rA2 and anti-SLA antibodies was still detected after therapy. In conclusion, rPK and its specific B-cell epitope should be considered for future studies as a diagnostic marker for canine and human VL.

A Leishmania infantum hypothetical protein evaluated as a recombinant protein and specific B-cell epitope for the serodiagnosis and prognosis of visceral leishmaniasis.

The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.

Recombinant Leishmania eukaryotic elongation factor-1 beta protein: A potential diagnostic antigen to detect tegumentary and visceral leishmaniasis in dogs and humans.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

Diagnostic evaluation of the amastin protein from Leishmania infantum in canine and human visceral leishmaniasis and immunogenicity in human cells derived from patients and healthy controls.

The diagnosis of visceral leishmaniasis (VL) presents problems due to the toxicity and/or high cost of drugs. In addition, no vaccine exists to protect against human disease. In this study, the antigenicity and immunogenicity of amastin protein were evaluated in L. infantum-infected dogs and humans. For the diagnosis, besides the recombinant protein, 1 linear B-cell epitope was synthetized and evaluated in serological assays. Results showed high sensitivity and specificity values to detect the disease when both antigens were employed against a canine and human serological panel. By contrast, when using rA2 and a soluble Leishmania antigenic preparation, sensitivity and specificity values proved to be lower. A preliminary immunogenicity study showed that the amastin protein induced high IFN-γ and low IL-10 production in stimulated PBMC derived from treated VL patients and healthy subjects, thus suggesting a potential use of this protein as an immunogen to protect against human disease.

Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.

A conserved Leishmania hypothetical protein evaluated for the serodiagnosis of canine and human visceral and tegumentary leishmaniasis, as well as a serological marker for the posttreatment patient follow-up.

In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the serodiagnosis of canine and human leishmaniasis.

Aspectos clínicos, epidemiológicos e diagnóstico da infecção por Trypanosoma vivax em rebanho bovino no estado do Maranhão / Clinical and epidemiological aspects and diagnosis of Trypanosoma vivax infection in a cattle herd, state of Maranhão, Brazil

O objetivo desse estudo foi investigar a ocorrência de tripanossomose em uma propriedade leiteira no município de Timon no estado do Maranhão, Brasil. O proprietário relatava histórico de abortos, nascimentos de crias fracas e mortalidade de animais adultos com perda progressiva de peso. Foram realizadas visitas à propriedade para obtenção do histórico, exame dos animais e coleta de sangue para realização do teste de Woo, hemogramas, testes sorológicos para pesquisa de anticorpos contra tripanossomose, leptospirose, e neosporose e PCR para diagnóstico molecular de Trypanosoma vivax. A identificação de animais com baixos valores no hematócrito foi a principal alteração hematológica identificada no rebanho. Dois animais foram positivos no teste de Woo, sendo visualizados tripanossomas em esfregaços sanguíneos, confirmados por meio de diagnóstico molecular como sendo T. vivax. Identificou-se que 95,23% (40/42) dos animais com hematócrito baixo foram sorologicamente positivos para T. vivax. As condições identificadas na propriedade, como ambiente propício aos vetores mecânicos, a presença de animais silvestres e a introdução de animais de estados onde já haviam sido registrados surtos de tripanossomose provavelmente estiveram associadas à introdução e disseminação do agente no rebanho. O elevado número de animais sorologicamente positivos para tripanossomose 82,51% (151/183) demonstra que praticamente todo o rebanho teve contato com o agente. O rápido estabelecimento das medidas de controle, entre elas a utilização das drogas tripanocidas, contribuiu para o controle do surto. O estudo permitiu comprovar a ocorrência de mais um surto de tripanossomíase tripanossomose no Brasil. O diagnóstico clínico da enfermidade foi dificultado pela semelhança dos sinais clínicos com outras enfermidades e pela possibilidade da associação de duas ou mais doenças no mesmo paciente, o que ressalta a importância do estabelecimento de medidas diagnósticas adequadas como forma de evitar a disseminação da enfermidade e minimizar as perdas econômicas dos produtores.(AU)

The objective of this study was to investigate the occurrence of trypanosomiasis in a dairy farm in municipality of Timon, State of Maranhão, Brazil. The owner reported abortus, births of weak calves, and mortality of adult animals with progressive weight loss. Visits to the property were carried out to obtain the history, realize animal examination and blood collection for the Woo test, hemograms, serological tests for trypanosomiasis, leptospirosis, and neosporosis and PCR for molecular diagnosis of Trypanosoma vivax. The identification of animals with low values in the hematocrit was the main hematological alteration identified in the herd. Two animals were positive in the Woo test and trypanosomes were visualized in blood smears, confirmed by molecular diagnosis as T. vivax. It was identified that 95.23% (40/42) of the animals with low hematocrit were serologically positive for T. vivax. The conditions identified in the property as an environment propitious to mechanical vectors, the presence of wild animals and the introduction of animals from states where trypanosomiasis outbreaks had already been reported were probably associated with the introduction and dissemination of the agent in the herd. The high number of serologically positive animals for trypanosomiasis 82.51% (151/183) shows that almost all the herd had contact with the agent. The rapid establishment of control measures, including the use of trypanocidal drugs, contributed to the control of the outbreak. The study allowed confirming the occurrence of another outbreak of trypanosomiasis in Brazil. The clinical diagnosis of the disease was difficult by the similarity of the clinical signs of trypanosomiasis with other diseases and the possibility of association of two or more diseases in the same patient, which emphasizes the importance of establishing adequate diagnostic measures as a way to avoid the dissemination of the disease and to minimize the economic losses of the producers.(AU)

Recombinant prohibitin protein of Leishmania infantum acts as a vaccine candidate and diagnostic marker against visceral leishmaniasis.

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.

Recombinant small glutamine-rich tetratricopeptide repeat-containing protein of Leishmania infantum: Potential vaccine and diagnostic application against visceral leishmaniasis.

Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine-rich tetratricopeptide repeat-containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein-specific production of IFN-γ, IL-12 and GM-CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4+ and CD8+ T cells contributed to the IFN-γ production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum-infected dog and human sera. No cross-reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL.

Important frequency of Anaplasma phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil / Importante frequência da infecção por Anaplasma phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil

Anaplasma phagocytophilum is responsible for granulocytic anaplasmosis in humans and various animal species. The aim of the present study was to determine the prevalence of A. phagocytophilum-infected dogs in a residential area of Belo Horizonte, Minas Gerais state, Brazil. A total of 62 dogs were submitted to serological (indirect fluorescent-antibody -IFI) and molecular (PCR) tests. Anti-A. phagocytophilum antibodies were detected in 43.8% of the dogs. Seven dogs (10.9%) were PCR-positive for the msp4 gene, six and four of these were positive for the for the msp2/p44 gene of A. phagocytophilum and 16S rRNA region of granulocytic Anaplasmataceae respectively. This study confirms a relatively high frequency of A. phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil and highlights the need for further studies on the role of Rhipicephalus sanguineus sensu lato ticks in the transmission of this bacterium to dogs in urban Brazilian areas.(AU)

Anaplasma phagocytophilum é responsável pela anaplasmose granulocítica, doença que acomete seres-humanos e várias espécies de animais. O objetivo do presente estudo foi determinar a prevalência de cães acometidos por A. phagocytophlium em uma área residencial de Belo Horizonte, MG, Brasil. Sessenta e dois cães foram submetidos a testes sorológicos (reação de imunofluorescência indireta - IFAT) e moleculares (PCR). Anticorpos anti-A. phagocytophilum foram detectados em 43,8% dos cães. Sete cães (10,9%) foram positivos no PCR para o gene msp4 de A. phagocytophilum, seis para o gene msp2/p44 A. phagocytophilum e quatro para a região 16S rRNA de Anaplasmataceae granulocíticas. Esse estudo confirma a frequência relativamente alta da infecção por A. phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil e destaca a necessidade de pesquisas para determinar o papel do carrapato Rhipicephalus sanguineus sensu lato na transmissão desse microrganismo para cães de áreas urbanas brasileiras.(AU)

An ELISA immunoassay employing a conserved Leishmania hypothetical protein for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.

In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.

In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.

Tristeza Parasitária em bovinos do semiárido pernambucano / Cattle Tick Fever in semi-arid of Pernambuco

Este estudo objetivou determinar a soroprevalência da Babesiose e Anaplasmose em bovinos dos municípios de Ouricuri e Petrolina, estado de Pernambuco, Brasil; e definir os possíveis fatores de risco para a ocorrência dessas doenças. Amostras de sangue foram coletadas para realização de teste sorológico por Imunofluorescência Indireta (RIFI). Questionários epidemiológicos sanitários foram aplicados aos produtores com o objetivo de identificar possíveis fatores de risco. Carrapatos foram coletados, identificados e testados por Reação em Cadeia da Polimerase (PCR) para o diagnóstico da infecção por Anaplasma marginale, Babesia bigemina e Babaesia bovis. O estudo foi conduzido com 861 bovinos, sendo 468 de Petrolina e 393 de Ouricuri. A soroprevalência de A. marginale, B. bigemina e B. bovis em Petrolina foi de 35,0% (164/468), 35,9% (168/468) e 32,3% (151/468), respectivamente; e em Ouricuri foi de 45,5% (179/393), 38,6% (152/393) e 54,9% (216/393), respectivamente. A co-infecção por Anaplasma spp. e Babesia spp. foi observada em 31,6% e 32,1% de amostras de Petrolina e Ouricuri, respectivamente. A detecção de DNA de Babesia spp. por PCR foi possível em 5,8% (8/137) carrapatos, dos quais em 62,5 % (5/8) foi detectada posteriormente infecção por B. bovis, e em 23,3% (32/137) por A. marginale. A presença de carrapatos, o uso de acaricidas, idade, raça, e o município de residência dos animais foram identificados como fatores de risco para TPB pela análise univariável e multivariável. Este estudo permitiu caracterizar os municípios estudados como de instabilidade enzoótica para esses hemoparasitos, e consequentemente, alertar para adoção de medidas adequadas de controle e realização de novos estudos.(AU)

This study aimed to determine the seroprevalence of Babesiosis and Anaplasmosis in cattle from the municipalities of Ouricuri and Petrolina, state of Pernambuco, Brazil, and to define the risk factors for the occurrence of the diseases. Blood samples were collected for serologic testing by Indirect Immunofluorescence Assay (IFA). Sanitary epidemiological questionnaires were applied to the producers aiming to identify possible risk factors. Ticks were collected, identified and tested by Polymerase Chain Reaction (PCR) for the diagnosis of infection by Anaplasma marginale, Babesia bigemina and Babesia bovis. The study was conducted with 861 cattle, being 468 in Petrolina and 393 in Ouricuri. The seroprevalence of A. marginale, B. bigemina and B. bovis in Petrolina was of 35.0% (164/468), 35.9% (168/468) and 32.3% (151/468), respectively; and in Ouricuri was 45.5% (179/393), 38.6% (152/393), and 54.9% (216/393), respectively. Co-infection for Anaplasma spp. and Babesia spp. was observed in 31.6% and 32.1% samples of Petrolina and Ouricuri, respectively. The detection of DNA of Babesia spp. by PCR was possible in 5.8% (8/137) ticks; which 62.5% (5/8) was detected later infection with B. bovis; and 23.3% (32/137) with A. marginale. The presence of ticks, the use of acaricide, age, race, and county of residence of the animals were identified as risk factors for TBD by univariate analysis and multivariate. This study allowed the characterization of the municipalities studied as enzootic instability areas for these hemoparasitic, and consequently alert for adoption of adequate control measures and new studies.(AU)

Tristeza Parasitária em bovinos do semiárido pernambucano / Cattle Tick Fever in semi-arid of Pernambuco

This study aimed to determine the seroprevalence of Babesiosis and Anaplasmosis in cattle from the municipalities of Ouricuri and Petrolina, state of Pernambuco, Brazil, and to define the risk factors for the occurrence of the diseases. Blood samples were collected for serologic testing by Indirect Immunofluorescence Assay (IFA). Sanitary epidemiological questionnaires were applied to the producers aiming to identify possible risk factors. Ticks were collected, identified and tested by Polymerase Chain Reaction (PCR) for the diagnosis of infection by Anaplasma marginale, Babesia bigemina and Babesia bovis. The study was conducted with 861 cattle, being 468 in Petrolina and 393 in Ouricuri. The seroprevalence of A. marginale, B. bigemina and B. bovis in Petrolina was of 35.0% (164/468), 35.9% (168/468) and 32.3% (151/468), respectively; and in Ouricuri was 45.5% (179/393), 38.6% (152/393), and 54.9% (216/393), respectively. Co-infection for Anaplasma spp. and Babesia spp. was observed in 31.6% and 32.1% samples of Petrolina and Ouricuri, respectively. The detection of DNA of Babesia spp. by PCR was possible in 5.8% (8/137) ticks; which 62.5% (5/8) was detected later infection with B. bovis; and 23.3% (32/137) with A. marginale. The presence of ticks, the use of acaricide, age, race, and county of residence of the animals were identified as risk factors for TBD by univariate analysis and multivariate. This study allowed the characterization of the municipalities studied as enzootic instability areas for these hemoparasitic, and consequently alert for adoption of adequate control measures and new studies.

Este estudo objetivou determinar a soroprevalência da Babesiose e Anaplasmose em bovinos dos municípios de Ouricuri e Petrolina, estado de Pernambuco, Brasil; e definir os possíveis fatores de risco para a ocorrência dessas doenças. Amostras de sangue foram coletadas para realização de teste sorológico por Imunofluorescência Indireta (RIFI). Questionários epidemiológicos sanitários foram aplicados aos produtores com o objetivo de identificar possíveis fatores de risco. Carrapatos foram coletados, identificados e testados por Reação em Cadeia da Polimerase (PCR) para o diagnóstico da infecção por Anaplasma marginale, Babesia bigemina e Babaesia bovis. O estudo foi conduzido com 861 bovinos, sendo 468 de Petrolina e 393 de Ouricuri. A soroprevalência de A. marginale, B. bigemina e B. bovis em Petrolina foi de 35,0% (164/468), 35,9% (168/468) e 32,3% (151/468), respectivamente; e em Ouricuri foi de 45,5% (179/393), 38,6% (152/393) e 54,9% (216/393), respectivamente. A co-infecção por Anaplasma spp. e Babesia spp. foi observada em 31,6% e 32,1% de amostras de Petrolina e Ouricuri, respectivamente. A detecção de DNA de Babesia spp. por PCR foi possível em 5,8% (8/137) carrapatos, dos quais em 62,5 % (5/8) foi detectada posteriormente infecção por B. bovis, e em 23,3% (32/137) por A. marginale. A presença de carrapatos, o uso de acaricidas, idade, raça, e o município de residência dos animais foram identificados como fatores de risco para TPB pela análise univariável e multivariável. Este estudo permitiu caracterizar os municípios estudados como de instabilidade enzoótica para esses hemoparasitos, e consequentemente, alertar para adoção de medidas adequadas de controle e realização de novos estudos.

Isolation and attempted cultivation of an Anaplasma marginale strain from Brazilian brown brocket deer (Mazama gouazoubira, Fisher, 1814) in the tick cell line IDE8.

The aim of the study was to isolate and establish an Anaplasma marginale strain from Brazilian brown brocket deer, Mazama gouazoubira, in the Ixodes scapularis cell line IDE8. Blood from a free-living adult female M. gouazoubira naturally infected with A. marginale (MGI5) was inoculated intravenously into a splenectomized calf. When A. marginale rickettsemia was 2.5%, blood was collected and cryopreserved in liquid nitrogen with dimethylsulfoxide (DMSO). IDE8 cell cultures were infected with calf blood inoculated with the A. marginale (MG15) isolate. The cultures were monitored by examination of Giemsa-stained cytocentrifuge smears. Light microscopy of stained IDE8 samples revealed the first inclusions of A. marginale (MGI5) at 48days post-inoculation (d.p.i). The IDE8-infected cells contained parasitophorous vacuoles with amorphous material and a few cocci-like organisms. A sample from IDE8-infected cells from the 16th subculture (336 d.p.i.) was analyzed by nPCR, nucleotide sequencing, electron microscopy, and an indirect fluorescent antibody test (IFAT). The IFAT highlighted some IDE8-infected cells with intense fluorescence in the parasitophorous vacuole, while in other cells, fluorescence was observed only at the periphery. DNA from a culture of the MG15 isolate was amplified with A. marginale msp4 gene primers, and nucleotide sequencing of the PCR product and BLAST software analysis further confirmed 100% identity with the MGI5 blood isolate (GenBank no. JN022558.1). Electron microscopy revealed increased numbers of lysosomes in the cytoplasm of IDE8 cells. Several cells exhibited large vacuoles containing cellular debris and amorphous material. After the 29th subculture, it was not possible to detect compatible Anaplasma structures by light microscopy, and subculture samples tested negative in nPCR. Despite the failure of the attempt to establish A. marginale (MGI5) in IDE8 cells, the results demonstrated the isolate's ability to infect, survive and multiply, although in limited numbers, in IDE8 cells.

Molecular assays reveal the presence of Theileria spp. and Babesia spp. in Asian water buffaloes (Bubalus bubalis, Linnaeus, 1758) in the Amazon region of Brazil.

Approximately 50% of buffalo herds in Brazil are located in Pará state in northern Brazil. There are several properties where cattle and buffalo live and graze together, and thus, buffalo pathogens may threaten the health of cattle and vice versa. Therefore, knowledge of infectious agents of buffalo is essential for maintaining healthy livestock. Clinical disease caused by Theileria and Babesia parasites in the Asian water buffalo is not common, although these animals may act as reservoir hosts, and the detection of these hemoparasites in buffaloes is as important as it is in cattle. Studies of the infection of buffaloes by hemoparasites in Brazil are scarce. The objective of the present study was to investigate the occurrence of Piroplasmida parasites in Asian water buffaloes in the state of Pará in the Amazon region of Brazil using nested PCR assays and phylogenetic analysis. The 18S rRNA gene and ITS complete region were amplified from DNA extracted from blood samples collected from 308 apparently healthy buffaloes bred on six properties in the state of Pará, Brazil. The prevalence of positive buffalo samples was 4.2% (13/308) for Theileria spp., 3.6% (11/308) for Babesia bovis and 1% (3/308) for Babesia bigemina. Animals infected with Theileria were detected in 50% (3/6) of the assessed properties. Phylogenetic analyses indicated that the Theileria species detected in this study were closely related to Theileria buffeli, Theileria orientalis and Theileria sinensis. To our knowledge, this is the first report of Theileria in Asian water buffaloes in the Americas. The majority of Theileria-positive buffaloes (11/13) belong to a property that has a history of animals presenting lymphoproliferative disease of unknown etiology. Therefore, the present research suggests that this disorder can be associated with Theileria infection in this property. Our results provide new insights on the distribution and biological aspects of hemoparasites transmissible from buffaloes to cattle.