Neosporose equina: ocorrência de anticorpos anti-Neospora spp. e associação entre status sorológico de éguas e de suas crias

Os protozoários Neospora caninum e N. hughesi infectam os equinos e podem provocar diferentes sinais clínicos associados a problemas reprodutivos ou a distúrbios neurológicos, respectivamente. A patogenia da neosporose é pouco conhecida nos equinos, bem como as fontes de infecção horizontal de N. hughesi. Além disso, há dúvidas quanto ao papel da transmissão vertical de Neospora spp. na sua manutenção em populações equinas. Neste estudo avaliaram-se: (1) a ocorrência da infecção por Neospora spp. na população de éguas em idade reprodutiva em um haras de cavalos da raça Crioula; e (2) a possível associação entre o status sorológico destas éguas com o de suas crias, como meio de investigar, indiretamente, a relevância da transmissão transplacentária na ocorrência da infecção por Neospora spp. nestes animais. A associação entre o status sorológico das éguas e o de suas crias foi altamente significativa. Os animais descendentes de éguas soropositivas tiveram maior ocorrência de anticorpos anti-Neospora spp. do que os descendentes de éguas soronegativas, embora expostos aos mesmos fatores de risco ambientais. A associação entre parentesco em primeiro grau e status sorológico indica a influência da infecção vertical (transplacentária) na ocorrência de Neospora spp. na população equina estudada.

Verificação da transmissão vertical de Neospora spp. em equinos

O gênero protozoário Neospora é reconhecido como causador de desordens reprodutivas e abortos em bovinos. Entre os equinos pouco se sabe sobre os efeitos da infecção por estes protozoários. Atualmente é admitido que os efeitos da infecção por Neospora hughesi em equinos possam ocorrer no sistema nervoso central e, os efeitos provocados pela infecção por Neospora caninum recaiam sobre o sistema reprodutor de éguas. O presente trabalho verificou a presença de imunoglobulinas da classe G no soro sanguíneo de uma população de éguas de cria e, em seus respectivos potros antes da ingestão do colostro. Para execução deste trabalho foi empregada técnica de imunofluorescência indireta (RIFI), utilizando como antígeno taquizoítos de Neospora caninum, a diluição inicial dos soros das éguas foi de 1:50 e a diluição do soro dos potros empregada foi de 1:16. Foram assistidos 78 partos e todos os potros tiveram seu soro sanguíneo coletado imediatamente após o nascimento. A pesquisa de anticorpos contra Neospora spp. apontou que 50 (64%) éguas e 32 (41%) potros foram positivos. Das 50 éguas que apresentaram anticorpos contra Neospora spp. 24 geraram potros positivos. Entre as 28 éguas que não reagiram, oito deram a luz a potros positivos. De posse dos resultados encontrados podemos concluir que ocorreu a transmissão vertical de Neospora spp. nos equinos pesquisados.

Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses. (c) 2005 Elsevier B.V. All tights reserved.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Equine neosporosis: search for antibodies in cerebrospinal fluid and sera from animals with history of ataxia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in swine from Northeastern Brazil

A serologic survey was conducted among 130 swine slaughtered in the public slaughterhouse of the city of Patos, Paraíba State, Northeastern Brazil, to determine the prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies, and to verify possible associations between sex of the animals and antibody prevalence. The sera were analyzed by indirect antibody tests, considering 1:64 (T. gondii) and 1:50 (N. caninum) dilutions as cut-off points. The prevalence of anti-T. gondii antibodies was 36.2% (47/130) (95% CI = 27.9 - 45.0%) with reciprocal titers ranging from 64 to 2,048, and of anti-N. caninum antibodies was 3.1% (4/130) (95% CI = 0.8 - 7.7%) with reciprocal titers ranging from 50 to 6,400. Three of the four N. caninum-positive samples were also positive for T. gondii antibodies. All Neospora and Toxoplasma IFAT-positive animals were also positive for confirmatory immunoblotting techniques using total and purified N. caninum and T. gondii tachyzoite antigens, i.e., p38 (NcSRS2) and p30 (TgSAG1). There was no association between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies. This is the first indication of N. caninum natural infection in pigs from Brazil.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT≥50) and 36 (32.7%) for T. gondii (IFAT≥16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

IgM e IgG como marcadores da infecção transplacentária por Neospora caninum em fetos bovinos

O objetivo deste estudo foi determinar a ocorrência de anticorpos anti-Neospora caninum em 260 amostras de soro coletadas de fetos bovinos de julho de 2007 a março de 2008, em abatedouro do município de Santa Maria, Rio Grande do Sul, Brasil. Para detecção de anticorpos anti-N. caninum, a técnica de imunofluorescência indireta foi utilizada tanto para a detecção de imunoglobulinas G e M. Amostras com títulos e" 25 foram consideradas positivas. Das 260 amostras testadas, 15% (39/260) foram positivas para anticorpos anti-N. caninum. Destas, em 38 (97,4%) foi detectada a presença de IgG anti-N. caninum e em seis (15,4%) de IgM. Em cinco amostras (12,8%) detectaram-se ambos, IgG e IgM. Os resultados reafirmam a habilidade do N. caninum em determinar infecção fetal. A pesquisa de IgM foi de limitada importância na detecção da infecção via transplacentária em soro fetal bovino.

Freqüência de anticorpos anti-Neospora caninum em soros de caprinos do estado de São Paulo e sua relação com o manejo dos animais

Com o objetivo de se avaliar a freqüência de caprinos leiteiros soropositivos para Neospora caninum, no estado de São Paulo, e se verificarem possíveis associações com idade, sexo e problemas reprodutivos, nos capris, e, também, presença de cães, nas propriedades, foram obtidos soros de 923 caprinos de ambos os sexos e idade acima de 3 meses. Os animais eram provenientes de 17 propriedades de diferentes municípios. Para o diagnóstico, foi utilizado o teste de aglutinação para Neospora (NATe"25), e, em todos os capris, aplicou-se um inquérito a partir do qual se obtiveram informações epidemiológicas e de esfera reprodutiva. Todos os resultados estatísticos foram discutidos no nível de 5% de significância. Assim, chegou-se à conclusão de que a freqüência percentual de positividade para N. caninum foi de 19,77%, e, em apenas uma propriedade, não houve registro de animal soropositivo, o que revela difusão do agente, no Estado. Não foram verificadas diferenças significativas entre freqüências de positividade quanto ao sexo, idade ou problemas reprodutivos. Porém, ressalta-se que a presença de cães, nos capris, foi associada a uma maior freqüência de caprinos soropositivos a N. caninum. A representação geográfica da distribuição de caprinos soropositivos para o protozoário, em mapa coroplético em hachuras, pode implicar em um ganho considerável para estudos da epidemiologia geográfica, na elaboração de um planejamento de controle da enfermidade.

Presence of antibodies against Toxoplasma gondii, Neospora caninum and Leishmania infantum in dogs from Piauí

This study aimed to evaluate the presence of antibodies against Neospora caninum, Toxoplasma gondii and Leishmania infantum in dogs attended at the Veterinary Hospital of the Federal University of Piauí, Northeastern Brazil, where there are no reports of the occurrence of N. caninum and T. gondii in dogs. Serum samples from 530 dogs of genders, different ages and breeds from the municipality of Teresina and nearby towns were analyzed using three indirect fluorescent antibody tests, each one targeting one of the three agents. The associations between the parasites and gender, breed and age of the dogs were assessed by the chi-square test (p > 0.05). The occurrence of antibodies to N. caninum, T. gondii and L. infantum was 3.2, 18.0 and 78.1%, respectively. Toxoplasma gondii was more frequently found in older dogs (p < 0.05) whereas L. infantum was more common in animals aged between 1 to 3 years (p < 0.05). In order to evaluate potential associations between the presence of anti-N. caninum and anti-T. gondii antibodies and Leishmania infection, 240 dogs were selected (120 positive and 120 negative for Leishmania spp.), based on serological and parasitological diagnoses. No association was found between Leishmania spp. and the coccidian parasites (p > 0.05). The results confirm the exposure of dogs to these parasites in the State of Piauí.

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88(-/-)) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88(-/-) mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88(-/-) mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-gamma responses by NKT, CD4(+) and CD8(+) T lymphocytes, and production of high levels of IL-10. MyD88(-/-) mice replenished with IL-12 and IFN-gamma abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-gamma-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.

PREVALENCE OF ANTIBODIES TO TRYPANOSOMA CRUZI, LEISHMANIA INFANTUM, ENCEPHALITOZOON CUNICULI, SARCOCYSTIS NEURONA, AND NEOSPORA CANINUM IN CAPYBARA, HYDROCHOERUS HYDROCHAERIS, FROM SAO PAULO STATE, BRAZIL

Little is known about the importance of capybara. Hydrochoerus hydrochaeris, as reservoirs for parasites of zoonotic or veterinary importance. Sera from 63 capybaras, from 6 counties in the state of Sao Paulo, Brazil, were examined for antibodies to Trypanosoma cruel, Leishmania infantum, Encephalitozoon cuniculi. Sarcacystis neurona, and Neospora caninum using an indirect immunofluorescent antibody test. Five (8%) of the 63 capybaras had antibodies to T cruzi epimastigotes. None of the samples from capybara reacted positively with L. infantum promastigotes or with spores of E. cuniculi. Two (3%) of the serum samples were positive for antibodies to S. neurona merozoites, and 2 (3%) of the serum samples were positive for antibodies to N. caninum tachyzoites. A serum sample from 1 capybara was positive for antibodies to both T cruzi and N. caninum. None of the remaining 62 samples reacted with more than 1 parasite.

Occurrence of Neospora caninum antibodies in capybaras (Hydrochaeris hydrochaeris) from Sao Paulo State, Brazil

Capybara (Hydrochaeris hydrochaeris) is a large rodent distributed throughout tropical America. Antibodies to Neospora caninum in 213 feral capybaras from 11 counties of the State of Sao Paulo, Brazil. were assessed using the indirect immunofluorescent antibody test (titer >= 1:25) and found in 20 (9.4%), with titers of 1:25 in 4, 1:50 in 7, and 1:100 in 9 animals. This is the first report of occurrence of N. caninum antibodies in capybaras.

VIABILITY OF SPORULATED OOCYSTS OF NEOSPORA CANINUM AFTER EXPOSURE TO DIFFERENT PHYSICAL AND CHEMICAL TREATMENTS

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for I hr; 70% ethanol for I hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G(i) protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.