Transplacental transmission of Neospora caninum to lambs in successive pregnancies of naturally infected sheep in Southern Brazil.

This study aims to report the detection of N. caninum DNA in a newborn lamb (1) with neurological signs and congenital neosporosis and in a stillborn lamb (2), both born from the same ewe in a herd of Southern Brazil. The lambs were born during different pregnancies of a Suffolk ewe seropositive to N. caninum and seronegative to T. gondii. Histopathological lesions were observed only in the central nervous system of the lambs. The newborn lamb (1) showed mild and focal gliosis in the frontal lobe. In the hippocampal region of the stillborn lamb (2), lymphoplasmacytic perivascular cuffs and N. caninum cysts were observed in the cytoplasm of neurons and confirmed by IHC. PCR was performed using brain samples to detect the protozoa N. caninum and Toxoplasma gondii. The infection with N. caninum was confirmed in the newborn lamb (1) by PCR and in the stillborn lamb (2) by histopathology, immunohistochemistry, and PCR tests.

Risk factor analysis associated with Neospora caninum in dairy cattle in Western Brazilian Amazon / Análises de fatores de risco associado a Neospora caninum em gado leiteiro na Amazônia Ocidental Brasileira

Abstract Neospora caninum is considered to be one of the main causes of abortion among cattle. The present survey was conducted in the municipality of Rolim de Moura, Rondônia State, Brazil. A questionnaire that investigates the epidemiological aspects of neosporosis was used in the analysis of risk factors associated with the animal-level and herd-level prevalence in dairy cattle. A total of 416 bovine blood samples were collected from 30 farms, and N. caninum antibody levels were measured by Indirect Fluorescent Antibody Test (IFAT). Analysis of dairy cattle serum samples revealed the presence of anti-N. caninum antibodies to be 47.36% (n = 197). Risk factors associated with N. caninum infection were the management system and access locations of dogs. The results of the present survey indicated that infection of dairy cattle with N. caninum is widespread in the studied region of Western Amazon, which has implications for prevention and control of neosporosis in this region. Therefore, integrated control strategies and measures are recommended to prevent and control N. caninum infection in dairy cattle. In addition, direct contact between dairy cattle, dogs and wild animals, which can influence the epidemiology of neosporosis, should be investigated further.

Resumo A infecção por Neospora caninum é considerada uma das principais causas de aborto entre bovinos. Esta pesquisa foi realizada no município de Rolim de Moura, estado de Rondônia, Brasil. Um questionário que investiga os aspectos epidemiológicos da neosporose foi utilizado na análise dos fatores de risco associados à prevalência em animais e em rebanhos. Um total de 416 amostras de sangue bovino foi colhido em 30 fazendas, e os níveis de anticorpos de N. caninum foram mensurados pela reação de Imunofluorescência Indireta (RIFI). A análise das amostras mostrou prevalência de anticorpos contra N. caninum de 47,36% (n = 197). Os fatores de risco associados à infecção por N. caninum foram o sistema de manejo e os locais de acesso dos cães. Os resultados da presente pesquisa indicam que a infecção de bovinos leiteiros com N. caninum está disseminada na região estudada da Amazônia Ocidental, o que tem implicações para a prevenção e controle da neosporose nessa região. Portanto, estratégias e medidas de controle integrado são recomendadas para prevenir e controlar a infecção por N. caninum em gado leiteiro. Além disso, o contato íntimo entre gado leiteiro, cães e animais selvagens, pode influenciar a epidemiologia da neosporose e deve ser investigada mais detalhadamente.

Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re-emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re-examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non-infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.

Presumptive vertical transmission of Neospora caninum in related Bernese Mountain dogs.

Neospora caninum is a tissue cyst-forming coccidium capable of causing spinal cord or skeletal muscle disease in dogs. Infected bitches can transmit the parasite to their pups in utero. Seroprevalence of N. caninum was studied in naturally-infected, privately owned Bernese Mountain dogs, using antibody detection via an indirect fluorescent antibody test (IFAT) to identify infected individuals. A retrospective study was undertaken on available dogs from 14 litters. Five of eight dams tested seropositive. The index case was a bitch with a titre of 1:3200 by IFAT. Only one offspring from her first litter was seropositive. The frequency of putative congenital transmission in the breeding kennel was variable. Our results reiterate the importance of serological testing of all dams and littermates in a breeding kennel when clinical neosporosis is suspected in neonatal puppies.

Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi.

Bovine besnoitiosis, caused by Besnoitia besnoiti, is an economically important disease of cattle in many countries but its transmission remains a mystery. Wild felids are suspected to be its definitive hosts. The domestic cat (Felis catus) is known experimental definitive host for Besnoitia species of rodents. Here, we report for Besnoitia darlingi the first identification of a natural definitive host, the bobcat (Lynx rufus). Oocysts resembling Toxoplasma gondii (unsporulated; 10.9±0.8×12.1±0.2µm; n=5) were detected microscopically in the feces of two of 25 free ranging wild bobcats from Mississippi, USA. After detailed investigation, we identified these oocysts as B. darlingi and not T. gondii. The IFN-γ gene knockout (KO) mice fed oocysts from bobcats died of acute besnoitiosis and tachyzoites were found in their tissues. Oocysts were also mildly pathogenic to outbred Swiss Webster mice (SW) (Mus musculus). The SW mice fed oocysts became ill but generally survived and developed characteristic thick-walled Besnoitia tissue cysts in their tongue and heart muscles and brains. Two laboratory-raised domestic cats (Felis catus) excreted B. darlingi oocysts after ingesting murine tissues infected with bobcat-derived oocysts. The parasite was successfully cultivated in African green monkey kidney fibroblast cells (CV-1 cell line) seeded with infected murine tissue homogenate. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, and ITS-1) from culture-derived tachyzoites confirmed the parasite as B. darlingi. Our results suggest that bobcats may be an important link in the sylvatic cycle of Besnoitia species and bioassay or molecular tests are needed to differentiate Toxoplasma gondii-like oocysts in feces of felids, both domestic and wild cats.

Transplacental transmission of Neospora caninum in naturally infected small ruminants from northeastern Brazil / Transmissão transplacentária de Neospora caninum em pequenos ruminantes infectados naturalmente do Nordeste do Brasil

Toxoplasma gondii and Neospora caninum are causative agents of abortion in sheep and goats. Thus, the present study aimed to describe the transplacental transmission of these protozoans in small ruminants of northeastern Brazil. Seventeen fetuses (6 goats and 11 sheep) from farms with history of abortion were necropsied and samples were collected from different tissues (brain, liver, lung, kidney and heart). The samples were analyzed by PCR, histopathology (HP) and immunohistochemistry (IHC) to evaluate whether T. gondii and/or N. caninum infection were the cause of abortion. None of the samples was positive for T. gondii according to PCR and IHC results. Some brain, liver, lung, kidney and heart samples of goat fetuses were positive for N. caninum by PCR. In the histopathology, mild mononuclear infiltration and necrosis with calcification were observed in the liver and brain of one goat fetus, respectively, that also was positive for N. caninum by PCR and IHC. The results confirmed vertical transmission of N. caninum in naturally infected goats of northeastern, Brazil.(AU)

Toxoplasma gondii e Neospora caninum são reconhecidos como protozoário causadores de aborto em ovinos e caprinos. Desta forma, objetivou-se descrever a transmissão transplacentária desses agentes em pequenos ruminantes na região Nordeste do Brasil. Foram examinados seis fetos caprinos e onze fetos ovinos, totalizando 78 amostras de diferentes tecidos (cérebro, fígado, pulmão, rim e coração) provenientes de propriedades rurais com histórico de aborto. As amostras foram analisadas por Reação em Cadeia da Polimerase (PCR), Histopatologia (HP) e Imunohistoquímica (IHQ), com a finalidade de associar o abortamento a T. gondii e/ou N. caninum. Nenhuma amostra foi positiva na PCR e IHQ para T. gondii. Algumas amostras de cérebro, fígado, pulmão, rim e coração de fetos de caprinos e ovinos foram positivas na PCR para N. caninum. Na histopatologia foi observado leve infiltrado mononuclear no fígado e necrose com calcificação no SNC de um caprino, associada à imunomarcação positiva para N. caninum na IHQ e PCR positiva. Os resultados confirmam a transmissão vertical de N. caninum em caprinos naturalmente infectados na região nordeste do Brasil.(AU)

All about neosporosis in Brazil / Tudo sobre neosporose no Brasil

Abstract Neospora caninum is protozoan parasite with domestic and wild dogs, coyotes and grey wolves as the definitive hosts and many warm-blooded animals as intermediate hosts. It was cultivated and named in 1988. Neosporosis is a major disease of cattle and has no public health significance. Since 1990's N. caninum has emerged as a major cause of abortion in cattle worldwide, including in Brazil. N. caninum also causes clinical infections in several other animal species. Considerable progress has been made in understanding the biology of N. caninum and there are more than 200 papers on this subject from Brazil. However, most of the reports on neosporosis from Brazil are serological surveys. Overall, little is known of clinical neosporosis in Brazil, particularly cattle. The few reports pertain to sporadic cases of abortion with no information on epidemics or storms of abortion. The objective of the present review is to summarize all reports from Brazil and suggest topic for further research, including prevalence of N. caninum oocysts in soil or in canine feces, and determining if there are additional definitive hosts, other than the domestic dog. There is need for a national survey in cattle using defined parameters. Future researches should focus on molecular characterization of N. caninum strains, possibility of vaccine production and relationship between wildlife and livestock epidemiology.

Resumo Neospora caninum é um protozoário parasita que possui os canídeos domésticos e selvagens, coiotes e lobos cinzentos como hospedeiros definitivos e vários animais de sangue quente como hospedeiros intermediários. Foi cultivado e nomeado em 1988. A neosporose é uma das principais doenças em bovinos e não tem significância em saúde pública. Desde 1990, N. caninum tem emergido como uma das principais causas de aborto em bovinos em todo o mundo, inclusive no Brasil. N. caninum também causa infecções clínicas em várias outras espécies animais. Consideráveis avanços foram feitos na compreensão da biologia desse parasita e há mais de 200 trabalhos sobre o assunto no Brasil. No entanto, a maioria dos relatos de neosporose do Brasil são relacionados a sorologia. Em geral, pouco se sabe sobre a neosporose clínica no Brasil, particularmente em bovinos. Os poucos relatos referem-se a casos esporádicos de aborto sem informações sobre epidemias ou surtos de aborto. O objetivo da presente revisão é resumir todos os relatos sobre N. caninum no Brasil e sugerir tópicos para pesquisas futuras, incluindo a prevalência de oocistos de N. caninum no solo ou em fezes caninas e determinar se há hospedeiros definitivos adicionais, exceto o cão doméstico no país. Uma pesquisa nacional em bovinos usando parâmetros definidos seria de grande importância. Pesquisas futuras deveriam ser focadas na caracterização de cepas de N. caninum, possibilidade de produção de vacinas e a relação epidemiológica entre a vida selvagem e o gado.

Assessment of transplacental transmission of Neospora caninum in dairy cattle in the Agreste region of Pernambuco / Avaliação da transmissão transplacentária do Neospora caninum em bovinos de leite na região Agreste de Pernambuco

Abstract The transplacental transmission is the primary route of Neospora caninum infection in bovine herds around the world. This study aimed to determine the frequency of transplacental transmission of the parasite in dairy cattle of Agreste region of Pernambuco through serological tests (IFAT and ELISA). Three hundred sixteen serum samples from cows and heifers and their offspring were analyzed. The transplacental transmission rate was 72.22% (13/18) for cows and 69.23% (9/13) for heifers by IFAT. ELISA test showed transplacental transmission rate of 43.58% (17/39) for cows and 50% (9/18) for heifers. The transplacental transmission rates were similar, in both groups in test, but a higher seropositivity was found in cows by IFAT. Data were statistically analyzed using the chi-square and Fisher’s exact test. A significant relationship of dependence between seropositivity of mothers and their offspring was found. The more frequent IFAT antibody titers and ELISA levels for N. caninum were, respectively, 200 and between four (cows) and five (heifers and offspring). In the Spearman correlation, no association was found between the magnitude of antibody titers for N. caninum between mothers and their offspring. The kappa test showed an index of 0.35, indicating a mild correlation between the serological tests used. The study suggests that cows and heifers are the main transmitters of N. caninum in the studied region and that vertical transmission is the major form of transmission in dairy herds of the Agreste region of Pernambuco.

Resumo A transmissão transplacentária é a principal via de infecção do Neospora caninum nos rebanhos bovinos em todo o mundo. O presente estudo teve como objetivo determinar a frequência da transmissão transplacentária do parasita em bovinos leiteiros do Agreste Pernambucano, por meio de testes sorológicos (RIFI e ELISA). Foram analisadas 316 amostras de soro de fêmeas bovinas (vacas e novilhas) e de suas crias. A taxa de transmissão transplacentária pela RIFI foi de 72,22% (13/18) para vacas e 69,23% (9/13) para as novilhas. O ELISA teste mostrou taxa de transmissão transplacentária de 43,58% (17/39) para as vacas e 50% (9/18) para as novilhas. As taxas de transmissão transplacentária foram similares para os dois testes em geral, porém uma maior soropositividade foi encontrada nas vacas pela RIFI. Os dados foram estatisticamente analisados pelo teste de qui-quadrado e teste exato de Fischer. Foi encontrada uma relação significativa de dependência entre a soropositividade das mães e de suas crias. Os títulos de anticorpos anti- N. caninum foi de 200 na RIFI e posicionados entre o nível quatro (vacas) e cinco (novilhas e bezerros) pelo ELISA. Pela correlação de Spearman, não foi observada associação entre a magnitude de títulos de anticorpos anti- N. caninum de fêmeas com o de suas crias. O teste de concordância kappa revelou um índice de 0,35, indicando uma concordância leve entre os testes sorológicos utilizados. O estudo sugere que vacas e novilhas são as principais transmissoras do N. caninum na região estudada, sendo a transmissão vertical, a principal forma de transmissão do agente em rebanhos leiteiros do Agreste de Pernambuco.

Hepatozoon spp. infections in wild rodents in an area of endemic canine hepatozoonosis in southeastern Brazil.

Hepatozoon canis is a tick-borne parasite that occurs worldwide. In rural areas of Brazil, H. canis vectors remain unknown, which has led to speculation about alternative routes of transmission. Small rodents can play a role in the transmission (via predation) of Hepatozoon americanum, which led us to question whether predation might be an alternative mode of transmission for H. canis. Thus, this study investigated whether Hepatozoon spp. are present in wild small rodents in forest fragments that surround rural areas in Botucatu County, São Paulo, Brazil, where canine hepatozoonosis is endemic. The study included blood samples from 158 dogs, which were screened by microscopy and molecular analysis. Blood samples and tissues from 67 rodents were obtained for histopathology and molecular detection. The prevalence of H. canis was high (66.45%) in dogs from rural areas of Botucatu, São Paulo, Brazil. The molecular analysis showed that wild rodent species in Brazil were infected with Hepatozoon spp. other than H. canis. Therefore, although the hypothesis that sylvatic rodents act as reservoirs for H. canis was not supported, the presence of monozoic cysts in the rodents suggests that, in addition to intermediate hosts, wild small rodents in Brazil might act as paratenic hosts of Hepatozoon spp. because they harbor infective stages for intermediate host predators.

Vector-borne transmission of Besnoitia besnoiti by blood-sucking and secretophagous flies: epidemiological and clinicopathological implications.

BACKGROUND: Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease. FINDINGS: Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents. CONCLUSIONS: Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

Developmental biology of Cystoisospora (Apicomplexa: Sarcocystidae) monozoic tissue cysts.

Tissue cyst stages are an intriguing aspect of the developmental cycle and transmission of species of Sarcocystidae. Tissue-cyst stages of Toxoplasma, Hammondia, Neospora, Besnoitia, and Sarcocystis contain many infectious stages (bradyzoites). The tissue cyst stage of Cystoisospora (syn. Isospora) possesses only 1 infectious stage (zoite), and is therefore referred to as a monozoic tissue cyst (MZTC). No tissue cyst stages are presently known for members of Nephroisospora. The present report examines the developmental biology of MZTC stages of Cystoisospora Frenkel, 1977 . These parasites cause intestinal coccidiosis in cats, dogs, pigs, and humans. The MZTC stages of C. belli are believed to be associated with reoccurrence of clinical disease in humans.

Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: a descriptive study.

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 10(8) tachyzoites, group 3 contained two cows inoculated with 2.5 × 10(8) tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG1/IgG2 ratios were ≤ 1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.

Assessment of experimental infection for dogs using Gallus gallus chorioallantoic membranes inoculated with Neospora caninum / Avaliação da infecção experimental de cães com membranas córion-alantóides de Gallus gallus inoculadas com Neospora caninum

The aim of this study was to evaluate parasitism kinetics and tissue lesions in the first week of infection by Neospora caninum in dogs fed Gallus gallus chorioallantoic membranes (CMs) previously infected in ovo. Five two-month-old pups were used. Each dog was given five CMs that were previously infected with N. caninum via the oral route. Four animals were euthanized in the first week of infection. All four dogs had their stools examined one week prior to and up to the day they were euthanized. The stools of the uneuthanized dog were collected for 30 days. After euthanasia, organ sections were utilized for histopathology, immunohistochemistry, indirect immunofluorescent tissue reactions, PCR and real-time PCR to detect parasites. Necropsy revealed that the small and large intestines, spleen, and lungs were affected. No oocysts or N. caninum DNA were identified in the stool samples. Real-time PCR was the most sensitive technique used to detect the protozoa in tissues, which were identified in 41% of the analyzed samples. Our results indicate that an experimental model using previously infected CMs appears to be a useful model for the study of the host-parasite relationship during the infection's acute phase.

O objetivo deste estudo foi avaliar a cinética de parasitismo e lesões teciduais, na primeira semana de infecção por Neospora caninum, em cães alimentados com membranas corioalantóicas (MCs) de Gallus gallus, previamente infectadas in ovo. Foram utilizados cinco filhotes de dois meses de idade. Cada cão recebeu cinco MCs previamente infectadas com N. caninum, por via oral. Quatro animais foram eutanasiados na primeira semana de infecção. Todos os quatro cães tiveram suas fezes examinadas uma semana antes e até o dia em que foram eutanasiados. O cão que não foi eutanasiado teve suas fezes colhidas durante 30 dias. Depois da eutanasia fragmentos de órgãos foram processados para histopatologia, imuno-histoquímica, reação de imunofluorescência indireta em tecidos, PCR e PCR em tempo real para detecção do parasito. A necropsia revelou que os intestinos delgado e grosso, baço e pulmões foram os órgãos afectados. Oocistos de N. caninum não foram identificados nas amostras de fezes. A PCR em tempo real foi a técnica mais sensível para detectar o protozoário nos tecidos, sendo identificados em 41% das amostras analisadas. Os nossos resultados indicam que o modelo experimental utilizando MCs evidenciou ser um bom modelo para estudar a relação parasito-hospedeiro durante a fase aguda da infecção.

Neospora caninum: evaluation of vertical transmission in slaughtered dairy cows (Bos taurus) / Neospora caninum: avaliação da transmissão vertical em vacas leiteiras (Bos taurus)

Neospora caninum is a worldwide parasite recognized as one of the main parasites responsible for abortion in cattle. The objective of this study was to evaluate vertical transmission of N. caninum in dairy cows (Bos taurus) that were slaughtered at an abattoir in the state of Santa Catarina, southern Brazil. Blood samples (with and without EDTA) from 60 pregnant dairy cows and blood and tissue samples (brain, lung, heart and liver) from their fetuses were collected and used for PCR and serological evaluation. Blood samples from 60 non-pregnant cows were collected and used to detect antibodies. Anti-N. caninum antibodies were detected by indirect ELISA. Antibodies against N. caninum were observed in 41.6% (25∕60) of the pregnant cows and in 43.3% (26∕60) of the non-pregnant cows. Antibodies against the parasite were detected in sera from three fetuses (5.5%). PCR analysis revealed that 3.3% (2∕60) of the cows and 6.6% (4∕60) of the fetuses evaluated were positive for specific N. caninum primers. These positive fetuses were between 4-6 months of age. Therefore, considering PCR and serological tests to be indicative of vertical transmission in fetuses, 11.6% (7∕60) of the fetuses were infected by N. caninum during gestation.

Neospora caninum é um parasita de distribuição mundial reconhecido como um dentre os principais parasitas, responsável por abortamento em bovinos. O objetivo deste estudo foi avaliar a transmissão vertical de N. caninum em vacas leiteiras (Bos taurus) que foram submetidas ao abate em matadouro no Estado de Santa Catarina, sul do Brasil. Sangue (com e sem EDTA) de 60 vacas leiteiras prenhas e amostras de sangue e tecidos (cérebro, pulmão, coração e fígado) de seus fetos foram coletados e utilizados para PCR e avaliação sorológica. Amostras de sangue de 60 vacas não-gestantes foram obtidas e utilizadas para detecção de anticorpos. A detecção de anticorpos séricos anti-N. caninum foi avaliada pelo ELISA-teste indireto. Anticorpos anti-N. caninum foram observados em 41,6% (25∕60) das vacas prenhas e em 43,3% (26∕60) das vacas não-gestantes. Três fetos (5,5%) foram soros positivos para N. caninum. Análise pela PCR revelou que 3,3% (2∕60) das vacas e 6,6% (4∕60) dos fetos avaliados foram positivos paraN. caninum. As idades dos fetos positivos eram de 4 a 6 meses. Portanto, considerando a PCR e a sorologia como indicativo de transmissão vertical em fetos, 11,6% (7∕60) dos fetos foram infectados por N. caninum durante a gestação.

[Advances in the development of vaccines for bovine neosporosis]. / Avances en el desarrollo de vacunas contra la neosporosis bovina.

Neosporosis, a disease caused by the obligate intracellular protozoan Neospora caninum, produces abortions in cattle. The severe economic losses in cattle industry justify the need to develop control measures for preventing bovine abortion. Apicomplexan parasitic resistance is associated with T helper 1 immune response mediated by CD4 cytotoxic T lymphocytes, the production of interferon-gamma, interleukin-12, tumor necrosis factor and immunoglobulin G2. The reduction of vertical transmission in subsequent pregnancies and the low levels of abortion repetition suggests the existence of protective immune mechanisms. Inoculation with live tachyzoites before mating protects against infection and abortion. Antecedents of the development of live vaccines against other protozoa stimulate research to develop a live vaccine against N. caninum. On the other hand, an inactivated vaccine with low efficacy against neosporosis is useful in the prevention of abortion in farms with epizootic disease. A neosporosis vaccine should avoid abortion, transplacental transmission and infection persistence. In the present work, advances in vaccine development including lysate of tachyzoites, live parasites, recombinant antigens and vaccine vectors are reviewed.

Experimental infection with Neospora caninum in pregnant bitches / Infecção experimental com Neospora caninum em cadelas prenhes

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

Neste estudo a transmissão transplacentária de Neospora caninum foi avaliada em fêmeas em diferentes estágios de gestação. Três cadelas foram inoculadas na 3ª semana e três na 6ª semana de gestação com 10(8) taquizoítos de N. caninum (cepa Nc-1). Todas as cadelas infectadas, e pelo menos um de seus filhotes, apresentaram anticorpos anti-N. caninum por imunofluorescência indireta (RIFI > 400). Os filhotes e suas mães foram sacrificados e tecidos de sistema nervoso central (SNC), linfonodo poplíteo, músculo esquelético, cérebro, pulmões, coração e fígado foram analisados para a presença de N. caninum pela reação em cadeia da polimerase (nested PCR), polimorfismo de comprimento de fragmentos de restrição (RFLP) e imunoistoquímica (IHQ). O parasita foi encontrado em filhotes em linfonodo, SNC, coração e fígado pela nested PCR. Mortalidade perinatal não apresentou diferença entre os filhotes das cadelas infectadas na 3ª semana (60%) ou na 6ª semana de gestação (53,8%).

Anurans as paratenic hosts in the transmission of Hepatozoon caimani to caimans Caiman yacare and Caiman latirostris.

Prevalence of Hepatozoon caimani has been reported in 76% of caimans Caiman yacare from the Pantanal region. Culex (Melanoconion) spp. mosquitoes were recently identified as natural vectors of this parasite. However, culicids are not typically eaten by crocodilians, suggesting that the main transmission route is through ingestion of insectivorous vertebrates, such as anurans. The susceptibility of wild frogs Leptodactylus chaquensis, Leptodactylus podicipinus and Scinax nasicus to infection by H. caimani was verified. Wild-caught anurans were force fed with sporulated oocysts from laboratory-bred Culex (Melanoconion) mosquitoes. Frogs were killed 30 days postinfection, and their internal organs were fed to caimans C. yacare and Caiman latirostris. Cystozoites were identified in fresh liver impression smears of L. chaquensis. C. yacare fed on anuran organ presented gametocytes in peripheral blood circulation between 74 and 80 days postinoculation (dpi). Gametocytes were also verified in C. latirostris fed on the internal organs of L. podicipinus and S. nasicus between 60-70 and 69-75 dpi, respectively. Since frogs used in experiment are sympatric with C. yacare and C. latirostris and may occur in the diet of these caimans, the results suggest these amphibians are paratenic hosts in the natural transmission cycle of H. caimani in Pantanal.

Molecular pathology, taxonomy and epidemiology of Besnoitia species (Protozoa: Sarcocystidae).

Until recently, besnoitiosis has been a neglected disease of domestic animals. Now, a geographic expansion of the causing protozoan parasite Besnoitia besnoiti in livestock has been recognized and the disease in cattle is considered emerging in Europe. Bovine besnoitiosis leads to significant economic losses by a decline in milk production, sterility, transient or permanent infertility of bulls, skin lesions and increase of mortality in affected cattle population. Phylogenetically, the Besnoitia genus is closest related to the well studied and medically important protozoans, Toxoplasma gondii and Neospora caninum. In contrast, discriminative molecular markers to type and subtype large mammalian Besnoitia species (B. besnoiti, B. caprae, B. tarandi, B. bennetti) on a relevant level of species and strains are lacking. Similarly, these cyst-forming parasites may use two hosts to fulfill their life cycle, but this has not been proven for all large mammalian Besnoitia species yet. Most important though, the final hosts and transmission routes of these Besnoitia species remain mysterious. Here, we review aspects of parasite's pathology, speciation, phylogeny, epidemiology and transmission with a special focus on recent molecular studies of all to date known Besnoitia species. Using an integrated approach, we have tried to highlight some promising directions for future research.

Implications of wild dog ecology on the sylvatic and domestic life cycle of Neospora caninum in Australia.

Neospora caninum is transmitted either transplacentally or horizontally by ingestion of tissue cysts present in tissues or oocysts shed by dogs. Neosporosis is a significant disease, causing cattle abortion at 5-7 months of pregnancy. Infected cows may remain infective for life transmitting the infection in several consecutive or non-consecutive pregnancies. A great deal is known about the epidemiology of neosporosis, although only limited information is available on the main routes of horizontal transmission. In Australia, the presence of the dingo as the top-order predator suggests a potential sylvatic route of transmission between dingoes and as yet unknown native wildlife in addition to the domestic route via dogs with access to infected tissue on farms. This review article critically evaluates the overlap between the domestic and sylvatic routes, taking into account canine ecology, and summarises current understanding of the transmission of N. caninum to provide a foundation for epidemiologists, farmers and conservation biologists dealing with neosporosis and wild dog control programs.