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Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.

Protozoan parasites of the genus Sarcocystis are obligatory heteroxenous cyst-forming coccidia that infect a wide variety of animals and encompass approximately 200 described species. At least four Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi and S. speeri) use opossums (Didelphis spp.) as definitive hosts, and two of them, S. neurona and S. falcatula, are known to cause disease in horses and birds, respectively. Opossums are restricted to the Americas, but their distribution in the Americas is heterogeneous. Five Didelphis spp. are distributed in South America (D. aurita, D. albiventris, D. marsupialis, D. imperfecta and D. pernigra) whereas just one opossum species (D. virginiana) is found in North America. Studies conducted in the last decades show that Sarcocystis spp., derived from South American Didelphis spp., have biological and genetic differences in relation to Sarcocystis spp. shed by the North American opossum D. virginiana. The aim of this review was to address the peculiar scenario of Sarcocystis species shed by South American opossums, with a special focus on diagnosis, epidemiology, and animal infections, as well as the genetic characteristics of these parasites., Parasitos protozoários do gênero Sarcocystis são coccídios heteroxenos formadores de cistos, que infectam variadas espécies animais e compreendem cerca de 200 espécies descritas. Pelo menos quatro Sarcocystis spp. (S. falcatula, S. neurona, S. lindsayi e S. speeri) utilizam gambás (Didelphis spp.) como hospedeiros definitivos; e duas delas, S. neurona and S. falcatula são conhecidas por causarem doença em equinos e aves, respectivamente. Gambás didelfídeos são restritos ao continente americano, contudo são distribuídos de forma heterogênea nas Américas. Cinco Didelphis spp. são distribuídos na América do Sul (D. aurita, D. albiventris, D. marsupialis, D. imperfecta e D. pernigra), enquanto somente uma espécie (D. virginiana) é encontrada na América do Norte. Trabalhos conduzidos, nas últimas décadas, mostram que Sarcocystis spp. derivados de Didelphis spp. sulamericanos possuem diferenças biológicas e genéticas, quando comparados a Sarcocystis spp. excretados pelo gambá norte-americano D. virginiana. O objetivo desta revisão é discutir a situação peculiar das espécies de Sarcocystis na América do Sul com um foco especial em diagnóstico, epidemiologia e infecções animais, assim como nas características genéticas desses parasitos., Facultad de Ciencias Veterinarias

Sarcocystis neurona and Neospora spp. are related protozoa that can cause equine protozoal myeloencephalitis (EPM). The present study aimed to determine the frequency of antibodies to these parasites in 649 equids (351 horses, 267 donkeys, and 31 mules) from six departments in the North and Northwest of Colombia. For this purpose, the indirect fluorescent antibody test (IFAT) was used for detecting antibodies against S. neurona and Neospora spp. with a cut-off point of 1:20 and 1:50, respectively. A binomial logistic regression model was selected to predict variables associated with exposure. The frequency of anti-S. neurona antibodies was 14.24% (95% CI: 10.84-18.44) for horses, 2.99% (95% CI: 1.39-6.04) for donkeys, and 16.13% (95% CI: 6.09-34.47) for mules. The risk for S. neurona infection was significantly lower in donkeys (OR: 0.18 [0.08-0.38]; p0.001) than horses and mules, and higher in animals with a poor body condition (OR: 2.82 [1.45-6.05]; p0.05). Additionally, older animals (12y) had a higher risk of seropositivity (OR: 5.26 [1.88-19.1]; p0.05), as well as animals that inhabit climatic conditions associated with tropical very dry forest (OR: 1.85 [1.01-3.51]; p0.05). Córdoba and Antioquia departments presented the highest seropositivity to S. neurona with 13.01 and 8.3%, respectively. The frequency of anti-Neospora spp. antibodies was 1.42% (95% CI: 0.52-3.48) for horses, 1.12% (95% CI:0.29-3.52) for donkeys and 0% (95%, CI: 0-0) for mules. Atlántico was the state with the highest seropositivity to Neospora spp. (10%). No risks associated with Neospora spp. infection were found. These findings allow us to conclude that equids from these regions of Colombia are exposed to S. neurona, but antibodies to Neospora spp. are uncommon. Further studies are necessary to explore the presence of these two agents in other areas of the country. In addition, we need to prove the importance of the above-mentioned risk factors over the susceptibility of horses to these protozoal agents and the epidemiological impact of these underdiagnosed coccidia.

Sarcocystis neurona and Sarcocystis falcatula are protozoan parasites endemic to the Americas. The former is the major cause of equine protozoal myeloencephalitis, and the latter is associated with pulmonary sarcocystosis in birds. The opossum Didelphis virginiana is the definitive host of these parasites in North America. Four Didelphis species are found in Brazil, and in most reports in this country, Sarcocystis species shed by opossums have been classified as S. falcatula–like. It is unknown whether reports on S. neurona–seropositive horses in Brazil are also derived from exposure of horses to S. falcatula–like. The aim of this study was to test the sera reactivity of 409 horses in Brazil using antigens derived from a Brazilian strain of S. falcatula–like (Sarco-BA1) and from a North American strain of S. neurona (SN138). Samples were examined by immunofluorescent antibody tests (IFATs) at start dilutions of 1:20, and a selected number of samples was tested by Western blot (WB). Sera from 43/409 (10.5%) horses were reactive to S. falcatula–like and 70 of 409 (17.1%) were reactive to S. neurona antigen; sera from 25 animals (6.1%) were positive for both parasites by IFAT. A poor agreement was observed between the two employed IFATs (κ = 0.364), indicating that horses were exposed to more than one Sarcocystis species. Horse sera evaluated by WB consisted of four sera reactive to S. falcatula–like by IFAT, six sera positive to S. neurona by IFAT, two sera that tested negative to both parasites by IFAT, and a negative control horse serum from New Zealand. Proteins in the range of 16 and 30 kDa were recognized by part of IFAT-positive sera using both antigen preparations. We concluded that Brazilian horses are exposed to distinct Sarcocystis species that generate different serological responses in exposed animals. Antigens in the range of 16 and 30 kDa are probably homologous in the two parasites. Exposure of the tested horses to other Sarcocystis species, such as Sarcocystis lindsayi, Sarcocystis speeri, and Sarcocystis fayeri, or Sarcocystis bertrami cannot be excluded in the current study.

The genotyping of 25 isolates of Toxoplasma gondii from free-range chickens in the state of Bahia, Brazil, was performed by PCR-restriction fragment length polymorphism using 11 genetic markers: SAG1, 5′+3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. The analysis revealed 8 genotypes, 3 of which had not been previously reported. Four genotypes were represented by single isolates, whereas the other genotypes were represented by 2 or more isolates. Five isolates showed mixed infections, and 2 of them were identical. None of the clonal types I, II, or III were found, but 2 isolates corresponded to the Brazilian clonal lineage BrIII. There was a single allele for the c22-8 marker. The CS3 marker demonstrated efficiency in the evaluation of virulence in mice. This study reaffirms the diverse genetic variability of T. gondii in Brazil.

SUMMARYToxoplasma gondii, Neosporaspp.,Sarcocystisspp.,Hammondiaspp. andBesnoitia besnoitiare genetically related cyst-forming coccidia. Serology is frequently used for the identification ofT. gondii, Neosporaspp. andB. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected withT. gondiiandH. hammondi,as well as among animals infected byT. gondiiandN. caninum. Infections caused byN. caninumandN. hughesiare almost indistinguishable by serology.Neospora caninum, B. besnoitiandSarcocystisspp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity betweenNeosporaspp. andH. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected withT. gondii, Neosporaspp.,Sarcocystisspp.,Hammondiaspp. andB. besnoiti. Emphasis is laid upon antigens and serological methods forN. caninumdiagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.

Toxoplasma gondii and Neospora caninum are widespread cyst-forming coccidian parasites of the subfamily Toxoplasmatinae that infect a wide range of wild and domestic animals. Whereas T. gondii is a zoonotic disease, N. caninum is restricted to nonhuman animals. Some chiropteran species can be infected by T. gondii and present fatal toxoplasmosis. In most cases, T. gondii–infected bats are believed to remain asymptomatic and to act as an infection source to other animals. It is not known whether N. caninum can infect bats. We determined infection rates of T. gondii and N. caninum in free-living bats in the state of Bahia, Brazil. Brain samples from 97 bats of seven species, captured in 2008–15, were analyzed by PCRs for T. gondii and N. caninum. Two of the 97 samples were positive for T. gondii DNA. None of the samples were positive for N. caninum DNA, suggesting that the bats were not susceptible to N. caninum infection or that its prevalence was very low.

Neospora caninum and Toxoplasma gondii are coccidian parasites that infect a wide range of mammalian and avian species. While viable T. gondii has been in vitro isolated in natural infections from wild and domestic birds, attempts to isolate N. caninum from naturally-infected birds were unsuccessful. We speculate that body temperatures of birds, which are usually higher than those of mammals, may impair the multiplication of N. caninum. In contrast to N. caninum, T. gondii can grow in vitro at temperatures higher than 37 °C. To test the hypothesis that N. caninum tachyzoites are impaired to grow in vitro at high temperatures, three strains of N. caninum (NC-1, NC-Liverpool, and NC-Bahia) and three of T. gondii (RH, ME-49 and NED) were cultivated at gradually increasing temperatures starting at 37 °C up to 41.5 °C. A permanent chicken cell line was chosen for the study. Parasites were observed microscopically and their presence in culture was evaluated by species-specific conventional PCRs. In a second experiment, growth rates of T. gondii (RH strain) and N. caninum (NC-1 strain) were evaluated after direct passage of tachyzoites from 37 °C to 41.5 °C, and quantified by real-time PCR. In addition to comparisons between N. caninum and T. gondii, growth rates of three T. gondii strains were compared at high temperatures. Neospora caninum tachyzoites could not sustain multiplication at temperatures between 39 °C and 41.5 °C. Toxoplasma gondii tachyzoites continued to multiply at the same experimental conditions. Direct passage of N. caninum tachyzoites from 37 °C to 41.5 °C caused a significant decrease in the number of parasites during 96 h of observation, while T. gondii had a significant increase in the number of stages after the same period of time. T. gondii RH strain (clonal type I) presented a different growth rate at 41.5 °C when compared with type II and type III strains. In conclusion, multiplication of N. caninum tachyzoites in vitro was inhibited at temperatures similar to those of chickens, what may be one of the reasons that isolation of the parasite is difficult in avian species. In contrast to N. caninum, T. gondii continued to grow at 41.5 °C.

Cystoisospora felis is a coccidian parasite commonly found in feces of domestic cats. Infection in cats occurs by ingestion of sporulated oocysts or consumption of rodents infected by the parasite. Scarce information is available about extraintestinal stages of C. felis in naturally infected intermediate hosts, as well as in cell culture. The aim of the current work was to investigate the development of C. felis in Vero cells (African green monkey kidney) and MDCK cells (Madin-Darby canine kidney). Cell monolayers were inoculated with mechanically released sporozoites of C. felis, and parasite growth was daily examined using light microscopy. After cell invasion, only parasitophorous vacuoles containing a single zoite were observed. Five days post-inoculation with sporozoites, unstained cell monolayers were evaluated by differential interference contrast (DIC), and also by Romanovsky stain using conventional light microscopy. Single zoites, each surrounded by a cyst wall, were observed by both methods. Multiplication by endodyogeny did not occur in any cell monolayer. Treatment of encysted parasites with HCl-pepsin for 15 min led to dissolution of the cyst wall and release of intact and motile zoites. To our knowledge, this is the first demonstration of in vitro production of monozoic tissue cysts of C. felis. As kittens commonly shed C. felis in their feces, oocysts are easily available for in vitro production of monozoic tissue cysts of the parasite. Development of C. felis in cell culture may be employed as a model on tissue cyst formation of Cystoisospora spp. and closely related coccidia.

Sarcocystis neurona, a coccidian parasite shed by opossums (Didelphis spp.) in the Americas, is the major cause of equine protozoal myeloencephalitis (EPM) and induces disease in other domestic and wild animal species, including domestic dogs. Sarcocystis cruzi, despite its low pathogenicity for cattle (intermediate hosts), is worldwide distributed and uses mostly dogs as definitive hosts. The aims of this study were to test serological reactivities of dog sera to S. neurona and S. cruzi antigens, and to investigate potential serological cross-reactivity to these parasites. Sera from 353 Brazilian dogs were obtained from rural areas in the municipality of Ilhéus, Bahia, and examined by immunofluorescent antibody tests (IFAT). Antigens used in serological reactions consisted of S. neurona merozoites from a North American strain (SN138), and bradyzoites of S. cruzi obtained from Brazilian bovine hearts, with parasite species identity confirmed by PCR and sequencing of the 18S gene of the rDNA. Seropositivity to S. neurona and to S. cruzi were detected in 3.39% (12/353) and 4.81% (17/353) of the dogs, respectively. Ten canine sera reacted solely to S. neurona and 15 serum samples reacted only to S. cruzi. Two serum samples were simultaneously positive for both parasites. Sera from 14 dogs that tested positive by IFAT (9 for S. neurona and 3 for S. cruzi) and from two dogs that were negative by IFAT for the two parasites, were examined by Western blot using S. neurona as antigen; these sera reacted to a great number of protein bands, including antigens on the 16 and 30 KDa positions, which encompass immunodominant antigens for S. neurona in horses. Western blot did not show any specific pattern for S. neurona infection/exposure using canine sera. Dogs act as definitive hosts for several Sarcocystis spp. that infect farm animals, including horses, sheep, goats, water buffaloes and pigs, and for this reason, should contain antibodies to a broad repertoire of Sarcocystis spp. antigens. In conclusion, low percentages of dogs from rural areas of Ilhéus, Bahia, were reactive to both S. neurona and S. cruzi antigens. It is possible that other Sarcocystis species, besides S. neurona and S. cruzi, might have contributed for the seropositivity observed in this study. IFAT was more specific than Western blot to differentiate canine serological reactions to S. neurona and S. cruzi antigens.

In a previous study in Brazil, six isolates of Sarcocystis spp. recovered from budgerigars fed sporocysts excreted by opossums of the genus Didelphis were characterized by means of sequencing fragments of gene coding cytochrome B (CYTB), internal transcribed spacer 1 (ITS1), and surface antigen genes (SAG2, SAG3 and SAG4). The isolates shared identical ITS1 and CYTB sequences, but differed at SAG2, SAG3 and SAG4: three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in three multilocus genotypes (MLGs) (MLG1, MLG2, and MLG3). At ITS1 and CYTB, all the isolates from budgerigars were identical to the Sarcocystis falcatula-like isolate 59-2016-RS-BR that was detected in a barefaced ibis (Phimosus infuscatus) causing necrotizing meningoencephalitis in Brazil. At ITS1 locus, all the above isolates were clearly distinct from Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis lindsayi, and Sarcocystis speeri, the four known species of Sarcocystis that use opossums of the genus Didelphis as definitive hosts. Here, we replicated the experiment above to identify additional MLGs or other species of Sarcocystis. Fifteen budgerigars were experimentally infected with sporocysts of Sarcocystis spp. from 12 opossums of the genus Didelphis. All the birds died 9–19 days after infection and tissue samples containing merozoites and schizonts of Sarcocystis spp. were recovered. Fractions of sequences coding for 18S ribosomal RNA gene (18S), CYTB, ITS1, SAG2, SAG3 and SAG4 were PCR amplified and sequenced from the infected lungs. In addition, fractions of 18S, SAG2, SAG3 and SAG4 were sequenced from the isolate 59-2016-RS-BR and fractions of 18S were sequenced from the six isolates from budgerigars described above. From the results, all the isolates shared identical 18S, ITS1 and CYTB sequences. Among the 15 new isolates from budgerigars, three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in five MLGs, of which four were novel (MLG1, MLG4, MLG5, MLG6 and MLG7). Isolate 59-2016-RS-BR was assigned to an eighth MLG (MLG8). Molecular data pointed that Sarcocystis assigned to MLGs 1 to 8 are variants of the same species, but the SAG-based trees of the isolates conflicted, which supports genetic admixture among them. The sarcocystinae studied have high diversity of SAG alleles per locus and the correlation of such an abundant variety of SAG alleles to host specificity and pathogenicity needs to be assessed. Remains to be elucidated if the parasites studied here and S. falcatula are variants of the same species that have diverged to the point of possessing differences at ITS1 level, but that are still capable of exchanging genes.

A Brazilian fox (Lycalopex vetulus) was rescued from a highway, and 16 days after maintained in captivity, the fox shed oocysts with sizes compatible with Hammondia sp. and Neospora caninum. DNA extracted from oocysts were initially tested in two PCRs targeting the internal transcribed spacer 1 (ITS-1) of the rDNA of Hammondia heydorni and the Nc-5 gene of N. caninum. A 270-bp product was visualized in the PCR for H. heydorni. No amplification was observed for N. caninum PCR. Since ITS-1-based PCR is not sufficient to differentiate Hammondia species derived from canids, oocyst DNA was examined using multilocus sequence analysis of five genetic fragments [intron 1 of the alpha tubulin gene (intron 1), internal transcribed spaces 1 and 2 (ITS-1 and ITS-2) of the rDNA, 28S rRNA gene (D2/D3 domain), and heat shock protein 70 (Hsp70)]. The Hammondia sp. oocyst from the Brazilian fox, referred here as H-FOXBR isolate, is closely related to H. heydorni and Hammondia triffittae, but differs from these parasites in three genetic markers (alpha tubulin gene, ITS-2, and 28S rRNA). As reported by other research groups, Hammondia spp. excreted by canids are genetically diverse and may encompass additional species besides H. heydorni and H. triffittae. In this study, we confirmed that H-FOXBR has significant genetic differences in comparison to H. heydorni and H. triffittae and may represent a separate species. Further studies are needed to identify the life cycle of this parasite and to characterize the parasite stages in the intermediate and definitive hosts.

Hammondia heydorni is a coccidian parasite believed to be nonpathogenic for naturally-infected animals, but it is biologically and genetically related to Neospora caninum, a worldwide cause of abortion in cattle. The major aim of the present work was to determine whether dogs shed H. heydorni oocysts after consuming in vitro generated tissue cysts of the parasite. In addition, we investigated cross-immunity between H. heydorni and N. caninum in mice. Two dogs were fed cultured cells containing tissue cysts of H. heydorni mixed with canned dog food, and a third dog (negative control) received only non-infected cells mixed with canned food. The two dogs that consumed in vitro produced tissue cysts shed high numbers of oocysts, which were induced to sporulate and tested positive for H. heydorni by a species-specific PCR. The third uninfected dog did not shed H. heydorni oocysts in the feces. Oocysts shed by the dogs induced the formation of encysted bradyzoites of H. heydorni on KH-R cells. Nineteen BALB/c mice were employed in the cross-immunity study. Nine mice were orally inoculated with 1 × 105 sporulated oocysts of H. heydorni and challenged with N. caninum tachyzoites 30 days after infection with H. heydorni. Other ten mice, which did not receive H. heydorni oocysts, were infected with 2 × 105 N. caninum tachyzoites. Thirty days after challenging with N. caninum, all mice were euthanized and N. caninum DNA in their tissues was quantified by real time PCR. No statistically significant difference in N. caninum DNA concentrations were observed between the two groups. We concluded that in vitro generated cysts of H. heydorni are biologically active, because they induced oocyst shedding in dogs. As no cross-protection occurred in mice inoculated with H. heydorni and challenged with N. caninum, it is suspected that these parasites do not express significant numbers of homologous proteins during infection, or the immune response of BALB/c mice after H. heydorni infection was not sufficient.

Sarcocystis neurona and Neospora spp. are protozoan parasites that induce neurological diseases in horses and other animal species. Opossums (Didelphis albiventris and Didelphis virginiana) are definitive hosts of S. neurona, which is the major cause of equine protozoal myeloencephalitis (EPM). Neospora caninum causes abortion in cattle and infects a wide range of animal species, while N. hughesi is known to induce neurologic disease in equids. The aims of this study were to investigate S. neurona and N. caninum in tissues from opossums in the northeastern Brazil, and to isolate Brazilian strains of Sarcocystis spp. from wild opossums for comparison with previously isolated strains. Carcasses of 39 opossums from Bahia state were available for molecular identification of Sarcocystis spp. and N. caninum in their tissues, and for sporocyst detection by intestinal scraping. In addition, Sarcocystis-like sporocysts from nine additional opossums, obtained in Sao Paulo state, were tested. Sarcocystis DNA was found in 16 (41%) of the 39 opossums' carcasses; N. caninum DNA was detected in tissues from three opossums. The sporocysts from the nine additional opossums from Sao Paulo state were tested by bioassay and induced infection in nine budgerigars, but in none of the gamma-interferon knockout mice. In vitro isolation was successful using tissues from all nine budgerigars. The isolated strains were maintained in CV-1 and Vero cells. Three of nine isolates presented contamination in cell culture and were discarded. Analysis of six isolates based on five loci showed that these parasites were genetically different from each other and also distinct from S. neurona, S. falcatula, S. lindsayi, and S. speeri. In conclusion, opossums in the studied regions were infected with N. caninum and Sarcocystis spp. and represent a potential source of infection to other animals. This is the first report of N. caninum infection in tissues from black-eared opossum (D. aurita or D. marsupialis) and white-eared opossum (D. albiventris). Brazilian opossums are probably infected by different Sarcocystis spp. distinct from S. neurona and S. falcatula, or present a high level of genetic recombination.

Rearing cattle and goats is an important economic activity for smallholder farmers in Mozambique where animals are raised in traditional production systems in communal pastures. Despite its importance, there is little information on their health conditions, particularly the occurrence of infections by parasites that can cause economic losses, especially Neospora caninum and Besnoitia besnoiti. A serological study to estimate the prevalence of Neospora caninum and Besnoitia besnoiti was undertaken in Angónia district of Central Western Mozambique between April and May 2010. Animals were randomly selected at acaricide dip tanks upon the consent of the owners, and all the selected animals were regarded as one flock in each locality. A serological survey for these protozoa was undertaken using the Indirect Fluorescent Antibody Test. A prevalence of anti-N. caninum IgG was estimated in 297 cattle serum samples and 8.4% were positive at the dilution of 1:200 to 1:800, while in goats from the 132 samples 3.8% were positive at 1:50 and 1:100 dilutions. Ninety four cattle samples were examined and 39.4% were positive for anti-B. besnoiti antibodies, which represents a high prevalence. This is the first evidence of seroprevalence of N. caninum in cattle and goats, and of B. besnoiti in cattle in Angónia, Mozambique.

Sarcocystis neurona is the major agent of equine protozoal myeloencephalitis. It infects several mammalian species in the Americas, where the definitive hosts, marsupials of the genus Didelphis (D. virginiana and D. albiventris) are found. Domestic cats are one of the confirmed intermediate hosts of the parasite; however, antibodies against S. neurona had never before been demonstrated in Brazilian cats. The aim of this study was to determine whether cats in Bahia, Brazil, are exposed to the parasite. A total of 272 feline serum samples (134 from feral and 138 from house cats) were subjected to an indirect fluorescent antibody test using cultured merozoites of S. neurona as antigen. Positivity was detected in 4.0% (11/272) of the tested samples, with titers ranging from 25 to 800. The feline sera were also tested for antibodies against the protozoan Neospora caninum, with an observed antibody frequency of 2.9%. To the author's knowledge, this is the first study to report antibodies against S. neurona in Brazilian cats. We conclude that cats are exposed to the parasite in the region of this study. Further investigations are needed to confirm the role of cats in the transmission cycle of S. neurona in Brazil. Sarcocystis neurona é o principal agente da mieloencefalite protozoária equina. Esse parasito infecta várias espécies de mamíferos nas Américas, onde são encontrados os hospedeiros definitivos, os marsupiais do gênero Didelphis (D. virginiana and D. albiventris). O gato doméstico é um dos hospedeiros intermediários do parasito. Contudo, anticorpos contra S. neurona ainda não tinham sido demonstrados em gatos brasileiros. O objetivo deste trabalho foi determinar se gatos da Bahia, Brasil, são expostos ao parasito. Amostras séricas de 272 felinos (134 de gatos errantes e 138 de gatos domiciliados) foram testadas pelo teste de imunofluorescência indireta, utilizando-se como antígeno, merozoítos produzidos em cultura celular. Entre as amostras testadas, 4,0% (11/272) foram positivas com títulos entre 25 e 800. Os soros dos felinos foram também testados para anticorpos contra o protozoário Neospora caninum, cuja frequência de anticorpos foi de 2,9%. Esse é o primeiro relato de anticorpos contra S. neurona em gatos brasileiros. Conclui-se que os gatos da região estudada são expostos a S. neurona. Estudos futuros são necessários, a fim de se confirmar o papel dos gatos no ciclo de transmissão de S. neurona no Brasil.

Besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. Besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥ 200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other Besnoitia species in Brazilian cattle.

The Toxoplasmatinae parasites Toxoplasma gondii, Neospora caninum and Hammondia spp. have carnivores as definitive hosts that shed the parasite oocysts in their feces. Birds that feed directly from the soil, such as chickens, are exposed to infection and may serve as indicators of the presence of the parasite in the environment and as a source of infection for other animals. The aims of this study were to determine the frequency of infection by these parasites in free ranging chickens, to test whether chickens are intermediate hosts of Hammondia spp., and to isolate N. caninum from chickens. One hundred chickens, which were raised in contact to cattle and dogs, were bought in five towns located in Bahia, Brazil. Blood and tissues (brain and heart) were used for serology, molecular tests and bioassay in mice for parasite isolation. T. gondii DNA was detected in 29 chickens, and N. caninum DNA was observed in six animals. Hammondia spp. DNA was not detected in tissues from any chicken. Tissues from eight N. caninum seropositive chickens were bioassayed in interferon-gamma gene knockout mice, but the mice did not become infected; T. gondii was isolated from six of 14 seropositive chickens after bioassay in outbreed Swiss mice. The authors concluded that: chickens seem to be better hosts for T. gondii when compared to N. caninum, based on the molecular and bioassay results; Hammondia spp. probably does not infect chickens or is rarely found in this animal species.

Objetivou-se, no presente estudo, pesquisar a prevalência de anticorpos anti-Neospora caninum em 812 amostras de soros sangüíneos de bovinos leiteiros procedentes de propriedades rurais de sete municípios das microrregiões de Itapecuru-Mirim, Médio Mearim e Presidente Dutra, estado do Maranhão, Brasil. Para o cálculo do tamanho da amostra, considerou-se um soroprevalência de 34,7% para N. caninum, com erro máximo de 9,5% e intervalo de confiança de 95%. Para a detecção da presença de anticorpos da classe IgG, utilizou-se a técnica de Imunofluorescência Indireta (IFI), com ponto de corte 1:200, usando como antígeno, taquizoítos da cepa NC-1, mantida em cultura celular no Laboratório de Diagnóstico das Parasitoses dos Animais da Escola de Medicina Veterinária da UFBA. Do total de amostras analisadas, encontrou-se uma prevalência de 50,74%. Os títulos variaram de 1:200 a 1:6400, assim distribuídos: 108 (26,21%) amostras de soro apresentaram título de 1:200; 132 (32,04%) 1:400; 94 (22,81%) 1:800; 46 (11,16%) 1:1600; 23 (5,58%) 1:3200 e nove (2,18%) com títulos de 1:6400. Dentre as microrregiões, a Itapecuru-Mirim apresentou o menor percentual de animais soropositivos (20,69%) e Presidente Dutra o maior (47,66%). Com relação à variável sexo, observou-se maior prevalência de sororreagentes nas fêmeas (46,80%) do que nos machos (52,46%). Não se verificou diferença significativa (P>0,05) para as variáveis microrregiões, sexo e idade. Conclui-se que os bovinos leiteiros das regiões estudadas estão expostos à infecção por N. caninum. The objective in the present study was to research the prevalence of anti-Neospora caninum in 812 samples of blood serum of dairy cattle from farms of seven municipalities of microrregions of Itapecuru-Mirim, Middle Mearim and President Dutra, state of Maranhão, Brazil. For the calculation of sample size, it was considered a seroprevalence of 34.7% for N. caninum, with a maximum error of 9.5% and a confidence interval of 95%. To detect antibodies, it was used the technique of Indirect Immunofluorescence (IFI), with the cut-off of 1:200, using as antigen, tachyzoites strain NC-1, maintained in cell culture in the Laboratory of Diagnosis of Parasitism of the Animals, School of Veterinary Medicine of the Federal University of Bahia, Brazil. Of the total samples, it was obtained a prevalence of 50.74%. The titles ranged from 1:200 to 1:6400, distributed as follows: 108 (26.21%) serum samples showed title of 1:200; 132 (32.04%) 1:400; 94 (22.81%) 1:800; 46 (11.16%) of 1:1600; 23 (5.58%) of 1:3200 and nine (2.18%) with titers of 1:6400. Among the microrregiões the Itapecuru-Mirim showed the lowest percentage of animals seropositive (20.69%) and President Dutra the largest (47.66%). It was observed higher prevalence of seropositives in females (46.80%) than in males (52.46%). There was no significant difference (P> 0.05) for the microrregions variables, sex and age. Concluded that the dairy cattle of the regions studied are exposed to infection by N. caninum.

This study investigated the epidemiology of canine ehrlichiosis in Northeastern Brazil, focusing the identification of the Ehrlichia species and vectors involved. Samples were collected from 472 domestic dogs residing in the health districts of Cajazeiras and Itapuã of Salvador city. The average prevalence of antibodies reactive to E. canis by immunofluorescent antibody test (IFAT) (titer > 1:80) was 35.6% (168/472). Blood samples from the E. canis-seropositive animals were tested by nested PCR in order to identify the Ehrlichia species responsible for the infection. Among the seropositives, 58 (34.5%) were found to be PCR-positive for E. canis. Ticks were found in 32 dogs. Nested-PCR analysis showed that 21.9% (7/32) of the Rhipicephalus sanguineus were infected by E. canis. In both dogs and Rhipicephalus sanguineus, nested-PCR for E. ewingii and E. chaffeensis was negative, with no amplification of DNA fragment. Este estudo objetivou pesquisar a epidemiologia da erliquiose canina no Nordeste do Brasil, com especial atenção na identificação da espécie de Ehrlichia envolvida nas infecções caninas e vetoriais detectadas. Para isso foram coletadas amostras de 472 cães domiciliados nos distritos sanitários de Cajazeiras e Itapuã. A prevalência de anticorpos anti-E. canis, pela imunofluorescência indireta (título > 1:80), em cães foi de 35,6% (168/472). Os animais soropositivos foram analisados por uma nested-PCR para identificação da espécie de Ehrlichia responsável pela infecção. Dentre os positivos, 58 (34,5%) cães foram PCR-positivos para E. canis. Foram coletados e classificados os carrapatos em 32 cães. A nested-PCR de Rhipicephalus sanguineus resultou em 21,9% (7/32) de infecção por E. canis. A nested-PCR de amostras de sangue de cães e Rhipicephalus sanguineus para E. chaffeensis e E. ewingii foi negativa, não havendo amplificação de fragmento de DNA.

Neospora caninum, Hammondia sp., and Toxoplasma gondii are parasites with morphological and genetic similarities. N. caninum and T. gondii are important abortive agents of cattle and sheep, respectively, and may infect numerous animal species. Hammondia sp. is not known to induce disease in animals, but may cause confusion in the identification of closely related coccidia. The aim of this study was to investigate infection rates caused by N. caninum, Hammondia sp., and T. gondii in beef cattle using a nested PCR for Toxoplasmatinae rDNA, followed by sequencing of the PCR products. Antibodies to N. caninum and T. gondii were also investigated in the tested animals. Brains and hearts were obtained from 100 beef cattle in a slaughterhouse in Bahia. Seven samples from brain tested positive for Toxoplasmatinae DNA. No positive reactions were found in heart tissues. After sequencing of the PCR products from all positive tissues, five sequences matched with N. caninum and two matched with T. gondii. Antibodies to N. caninum and T. gondii were found in 20% and 26% of the animals, respectively. The confirmation of N. caninum and the absence of Hammondia heydorni in the tested animals is suggestive that cattle are not efficient intermediate hosts of H. heydorni; however further studies need to be performed using a greater variety of tissues and a higher sample size. The detection of T. gondii DNA in bovine tissues reinforces the potential risk of transmission of this parasite to humans and other animals through the consumption of bovine meat.

A serological survey was carried out to assess the occurrence of anti- Neospora caninum antibodies in dogs from the State of Pernambuco. A total of 625 serum samples of dogs (289 from Paulista, 168 from Amaraji and 168 from Garanhuns) were tested by an immunofluorescence antibody assay for the detection of anti- N. caninum antibodies. A total of 177 (28.3%; IC 95%, 24.9–32.1) samples were positive. The seropositivity rates found in Paulista, Amaraji and Garanhuns were 26% (IC 95%, 21–31.4), 26.2% (IC 95%, 19.7–33.5) and 34.5% (IC 95%, 27.4–42.2), respectively. Of the 177 serum samples positive to anti- N. caninum antibodies, 170 were additionally tested for the presence of anti- Toxoplasma gondii antibodies and out of these 57.6% (IC 95%, 49.8–65.2) were positive. The results indicate that dogs from Amaraji, Paulista and Garanhuns are exposed to both N. caninum and T. gondii infections. The presence of dogs infected by N. caninum in Pernambuco represents a potential risk factor for the occurrence of outbreaks of abortion in cattle and small ruminants in this state. This study is the largest serological survey on the presence of anti- N. caninum antibodies in dogs carried out in Brazil and reports for the first time the exposure to N. caninum and T. gondii in dogs from Pernambuco.

Sarcocystis spp. are cyst-forming coccidia that infect numerous animals species, including several livestock species. Despite the importance of sheep and goat production in Brazil, little it is known about the Sarcocystis species that infect small ruminants in the country and their potential impact on meat condemnation due to the presence of macroscopic cysts of the parasite. The aims of the present study were to determine the frequency of infection by Sarcocystis spp. in goats and sheep intended for human consumption in Bahia State, Brazil, as well as to identify the parasite species in selected samples. The entire tongue, esophagus, and heart were collected from 120 goats and 120 sheep. Tissues were examined for Sarcocystis spp. by macroscopic evaluation, light microscopy, electron microscopy, and molecular tests. Microscopic cysts of Sarcocystis spp. were detected in 95.8 % of sheep and 91.6 % of goats. Using either transmission electron microscopy or partial sequencing of the 18S region of the ribosomal DNA (rDNA) for species identification, Sarcocystis tenella and Sarcocystis arieticanis were observed in sheep and Sarcocystis capracanis in goats. Macroscopic cysts were not detected in the analyzed samples. We concluded that goats and sheep destined for human consumption in Bahia possess high frequencies of Sarcocystis infection. Carcass condemnation due to Sarcocystis macrocysts seems to be rare in the studied region. S. arieticanis and S. capracanis were confirmed for the first time by electron microscopy or by molecular tests in small ruminants from Brazil.

Donkeys (Equus asinus) are closely related to horses and are known to be infected by several equine pathogens. Neospora caninum and Neospora hughesi are protozoan parasites that infect horses, but they were not confirmed in donkeys up to this date. The aim of this study was to evaluate the exposure of donkeys (Equus asinus) to Neospora spp. using tachyzoites of N. caninum as antigen and employing two common serologic methods, IFAT and immunoblot. Sera from 500 donkeys were obtained from 30 municipalities in Bahia state and tested by IFAT. Two of 500 sera were positive for Neospora spp. by IFAT with antibody titers of 100, and recognized a 37kDa antigen in immunoblot. Approximately 22% of the samples showed strong apical reactions and/or incomplete fluorescence, what may cause confusion in the interpretation of IFAT. We concluded that Neospora spp. are possibly of minor importance for Brazilian donkeys. Future studies are necessary to prove that Neospora spp. can naturally infect donkeys. Asininos (Equus asinus) são próximos filogeneticamente a equinos e podem ser infectados por vários patógenos de cavalos. Neospora caninum e Neospora hughesi são parasitos protozoários que infectam equinos, porém não foram confirmados em asininos até o momento. O objetivo deste estudo foi avaliar a exposição de asininos (Equus asinus) a Neospora spp., usando-se taquizoítos de N. caninum como antígeno e empregando-se duas técnicas sorológicas comuns para esta finalidade, reação de imunofluorescência indireta (RIFI) e immunoblot. Soros de 500 asininos, obtidos em 30 municípios no Estado da Bahia, foram testados por meio da RIFI. Dois dos 500 soros foram positivos para Neospora spp. pela RIFI com títulos de anticorpos de 100, e reconheceram um antígeno de 37kDa no immunoblot. Aproximadamente, 22% das amostras apresentaram fortes reações apicais e/ou fluorescência incompleta, o que pode causar confusão na interpretação da RIFI. Conclui-se que Neospora spp. são, possivelmente, de pouca importância para asininos brasileiros. Estudos posteriores são necessários para provar que Neospora spp. podem causar infecção natural em asininos.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

Hammondia heydorni was in vitro isolated from oocysts shed by three dogs using a finite cell line from embryonal bovine heart (KH-R). The oocysts were purified and suspended in 2% potassium dichromate or 2% sulphuric acid for sporulation for 2-5 days at room temperature. The parasites were confirmed as H. heydorni by PCR using specific primers (JS4/JS5) and by negative reaction for Neospora caninum employing the primers Np6+/Np21+. H. heydorni sporulated oocysts (1 × 10(6)) from each dog were initially treated with sodium hypochlorite. For excystation of sporozoites, oocysts from one dog were lysed by ultrasound followed by incubation with 0.75% taurocholate. Excystation of sporozoites from the other two dogs was achieved by oocyst fragmentation with glass beads with no further chemical treatment. Tachyzoites were clearly seen in the cultures at three days post inoculation (dpi). Bradyzoite conversion and cyst formation were evaluated at different time points by using a polyclonal rabbit serum against a bradyzoite-specific antigen (anti-BAG1), and a rat monoclonal antibody (mAbCC2) against a cyst wall protein. Bradyzoites were firstly detected at 7 dpi. Between 18 and 21 dpi most of cultured parasites consisted of encysted bradyzoites. The H. heydorni cysts increased in size during cultivation and reached a length of up to 135 μm. The parasite was maintained in the bovine heart cells up to 4.5months. Sera from mice and sheep experimentally infected with H. heydorni oocysts reacted with H. heydorni by IFAT, but did not cross-react with N. caninum antigens using IFAT or immunoblot. These findings suggest that serological cross-reactivity between H. heydorni and N. caninum seems to be of minor importance.

Neosporosis, which is caused by the coccidian parasite Neospora caninum, is recognized as a major disease of domestic animals that causes high abortion rates in cattle and fatal neurological disease in dogs. A life cycle of N. caninum in wild animals (i.e. sylvatic) has long been suspected because neosporosis has been detected in several wildlife species. Recently, the transmission of N. caninum has been confirmed in coyotes and white-tailed deer. The newly confirmed wild hosts and other wild animals are probably involved in the sylvatic cycle of the parasite. Control measures for neosporosis could now become more complicated, given the participation of wildlife in the life cycle of N. caninum.

Three-hundred and eighty-five serum samples were taken from dairy cows on 90 farms in 12 counties from the northern region of the Paraná State, Brazil. The samples were analyzed by IFAT for the detection of anti-Neospora caninum and anti-Toxoplasma gondii IgG antibodies. Forty-five (12%) samples were seropositive to N. caninum, while 102 (26%) samples were seropositive to T. gondii. Only four animals were seropositive to both coccidia. No significant difference was observed between the N. caninum serology and any of the variables studied, such as dairy cattle management, milk production, reproductive problems, feeding, and presence of dogs, cats and rodents. These data suggest that neosporosis is present among dairy cattle in the studied geographic region and the simultaneous detection of serum positive animals to both types of coccidian parasite demonstrates the independent occurrence of these coccidia in dairy cows.

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was ≤35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 ( n = 3) or week 9 ( n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1,500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.

Small differences have been reported in the internal transcribed spacer 1 (ITS1) region among strains of Neospora caninum. We compared ITS1 sequences among 6 N. caninum strains analyzed in our laboratory, including 2 strains that have not been examined previously (NC-Illinois and NC-Bahia). Five sequences showed 100% similarity and also were identical to 7 of 11 sequences that were previously reported by others. In contrast, initial attempts to sequence the ITS1 of NC-Bahia generated 12 nucleotide differences compared with the other 5 strains, and several ambiguous bases. However, the single band containing the ITS1 region, as observed after electrophoresis on a 2% agarose gel, became divided into 2 distinct bands when reanalyzed using 5 or 10% polyacrylamide gel electrophoresis (PAGE), and the ITS1 within these separate bands were sequenced without ambiguity. The other 5 N. caninum strains were also reexamined using PAGE, and in each strain 2 distinct bands were discovered. In comparison, 2 strains of Toxoplasma gondii continued to show only 1 band when examined using PAGE. The ITS1 sequence of NC-Bahia, from Brazil, differs in several base pairs from those of North American and European strains of N. caninum. Intrastrain variation of the ITS1 region appears to be common in N. caninum, in contrast to T. gondii.

Scarce information is available about Neospora caninum oocysts and sporozoites, in part because only small numbers of oocysts have typically been produced by experimentally infected dogs. We hypothesized that I reason for low experimental production of oocysts is that dogs have been fed tissues from experimentally infected mice instead of tissues from cattle (which are natural intermediate hosts of N. caninum). In this study, 9 dogs were fed tissues from N. caninum-infected calves, and oocyst production was compared with 6 dogs that were fed infected mouse carcasses. The number of oocysts produced by dogs that ingested infected calf tissues (mean = 160,700) was significantly greater (P = 0.03) than the number of oocysts shed by dogs that ingested infected mice (mean = 5,400). The second goal of our experiment was to demonstrate cyclical oral transmission of N. caninum between dogs and cattle. As few as 300 oocysts were used to successfully infect calves, and tissues from these calves induced patent infections in 2 of 3 dogs; oocysts from I of these dogs were administered to another calf, and tissues from this calf subsequently induced a third dog to shed oocysts. Oocysts were confirmed to be N. caninum using a species-specific polymerase chain reaction technique. In addition, sporulated oocysts were used to recover N. caninum in vitro after digestion in an acid-pepsin solution and inoculation of cell monolayers.

Forty-six aborted bovine fetuses submitted to the Faculty of Veterinary, Department of Clinical Pathology, Universidade Federal do Rio Grande do Sul, were examined histopathologically. Non-suppurative inflammation was observed mainly in the brain and heart of 22 fetuses. Brain lesions consisted primarily of mononuclear inflammatory cell infiltrates with occasional foci of necrosis. Protozoa that reacted with Neospora caninum antisera were seen in 18 of the 22 (81.8%) brain specimens from fetuses with encephalitis. Blood samples collected from 223 Holstein cows on five dairy herds were tested for N. caninum antibodies by an immunofluorescent antibody technique. These samples were obtained from aborting cattle and normally calving cattle (control group). Overall, 11.2% of cows sampled had N. caninum antibodies at a dilution of 1:200. Seroprevalence was higher (P = 0.0053) in aborting (23.3%) than in non-aborting cows (8.3%). Association between seropositivity to N. caninum and abortion was found, with seropositive cows being 3.3 times more likely to abort than seronegative cows (OR = 3.33; 95% CI: 1.38, 8.062). Additionally, N. caninum antibodies were detected in sera from seven cows that had aborted fetuses with lesions suggestive of protozoal infection. These results suggest that N. caninum is an important cause of abortion in dairy cattle in Rio Grande do Sul State, Brazil.

Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.

Texto completo: acesso restrito. p. 257-259 Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2014-01-21T15:53:29Z No. of bitstreams: 1 1-s2.0-S0921448806000976-main.pdf: 104706 bytes, checksum: 7d32cd410373f016c947b37fe722b333 (MD5) Approved for entry into archive by Alda Lima da Silva (sivalda@ufba.br) on 2014-01-21T21:11:58Z (GMT) No. of bitstreams: 1 1-s2.0-S0921448806000976-main.pdf: 104706 bytes, checksum: 7d32cd410373f016c947b37fe722b333 (MD5) Made available in DSpace on 2014-01-21T21:11:58Z (GMT). No. of bitstreams: 1 1-s2.0-S0921448806000976-main.pdf: 104706 bytes, checksum: 7d32cd410373f016c947b37fe722b333 (MD5) Previous issue date: 2007 Sera from 384 dairy goats in nine herds from Bahia State, Brazil, were assayed for Neospora caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Animals were selected and divided in groups by age and breed. Antibodies against N. caninum were found in 15% (58/384) of goats. Serum titers ranged from 1:100 to 1:3200. There was no significant difference between the age groups. Seroprevalence was higher in Alpine breed (24%) than in Saanen (14%) and Nubian (3%) breeds. The 58 N. caninum-positive sera were also tested for Toxoplasma gondii and 71% of the samples reacted solely to N. caninum. These results indicate that N. caninum infection is common in goat herds in Bahia.

The frequency of antibodies to Neospora caninum (Protozoa: Apicomplexa) in dog sera was investigated. Blood samples from 100 mongrel dogs, captured in the streets of Sao Luis, State of Maranhao, were analized using imunofluorescent antibody test. Forty five percent of the dogs were positive, and the titers ranged from 1:50 to 1:800. No sex difference was observed for frequency of N. caninum (60% in males and 40% in females). This is the first report of antibodies to N. caninum in dogs from Maranhao.

Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12 + 4.11.5, II: 1/24-12 + 4.15.15, III: 4.15.15 + 4.11.5, IV: 1/24-12 + 4.11.5 + 4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum.

Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to 28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.

Neospora caninum is regarded as one of the most important infectious causes of abortions in cattle worldwide, yet the global economic impact of the infection has not been established. A systematic review of the economic impact of N. caninum infections/abortions was conducted, searching PubMed with the terms 'cattle' and 'Neospora'. This yielded 769 publications and the abstracts were screened for economically relevant information (e.g. abortion prevalence and risk, serological prevalence). Further analysis was restricted to countries with at least five relevant publications. In total, 99 studies (12.9%) from 10 countries contained data from the beef industry (25 papers (25.3%)) and 72 papers (72.8%) from the dairy industry (with the remaining two papers (2.0%) describing general abortion statistics). The total annual cost of N. caninum infections/abortions was estimated to range from a median US $1.1 million in the New Zealand beef industry to an estimated median total of US $546.3 million impact per annum in the US dairy population. The estimate for the total median N. caninum-related losses exceeded US $1.298 billion per annum, ranging as high as US $2.380 billion. Nearly two-thirds of the losses were incurred by the dairy industry (US $842.9 million). Annual losses on individual dairy farms were estimated to reach a median of US $1,600.00, while on beef farms these costs amounted to just US $150.00. Pregnant cows and heifers were estimated to incur, on average, a loss due to N. caninum of less than US $20.00 for dairy and less than US $5.00 for beef. These loss estimates, however, rose to ∼US $110.00 and US $40.00, respectively, for N. caninum-infected pregnant dairy and beef cows. This estimate of global losses due to N. caninum, with the identification of clear target markets (countries, as well as cattle industries), should provide an incentive to develop treatment options and/or vaccines.

This paper aimed to identify Toxoplasma gondii infection in house sparrows (Passer domesticus, Linneaus 1758) coming from poultry farms in the "agreste" region of the Brazilian state of Pernambuco. 151 sparrows (Passer domesticus) captured in eight broiler, egg layer and commercial laying poultry farms, were used. Indirect hemagglutination test was used to research anti-T. gondii antibodies. Animals that presented titration of 1:16 were destined to DNA research through Polymerase Chain Reaction (PCR) technique, followed by Nested-PCR. It was observed that, from 151 analyzed samples. 91 (60.3%) were reagents and 60 (39.7%) were not reagents. It was verified, through analysis of the distribution of infected animals frequency per farm, that in only one farm (12.5%) no animal reagent to T. gondii was captured. It was also observed that three (30.00%) of the ten samples destined to DNA research for T. gondii were positive to PCR and four (40.00%) were positive to Nested-PCR. Anti-T gondii antibodies occurrence and the molecular identification of the agent confirmed natural T. gondii infection in sparrows from poultry farms in Brazil. Other studies must be carried out to highlight the real importance of these animals in the epidemiological chain and their efficiency in the transmission of the parasite to felines. Therefore, researches that use parasite isolation and molecular techniques to determine genomic profile of the agent present in these poultry farms are needed. Objetivou-se com este trabalho identificar a infecção por Toxoplasma gondii em pardais domésticos (Passer domesticus, Linneaus 1758) procedentes de granjas avícolas no agreste do estado de Pernambuco. Foram utilizados 151 pardais (Passer domesticus) capturados em oito granjas de frango de corte, matrizes e poedeiras comerciais. Para a pesquisa de anticorpos anti-T. gondi utilizou-se o teste de hemaglutinação indireta, aqueles animais que apresentaram titulação 1:16 foram encaminhados para pesquisa do DNA por meio da técnica de Reação em Cadeia da Polimerase (PCR) seguida do Nested-PCR. Das 151 amostras analisadas observou-se que 91 (60,3%) foram reagentes e 60 (39,7%) não reagentes. Na análise da distribuição de freqüência dos animais infectados por granja constatou-se que em apenas uma (12,5%) não foi capturado animal reagente para T. gondii. Das dez amostras que foram encaminhadas para pesquisa do DNA do T. gondii, observou-se que três (30,00%) foram positivas ao PCR e quatro (40,00%) ao Nested-PCR. A ocorrência de anticorpos anti-T. gondii e a identificação molecular do agente confirmam a infecção natural por T. gondii em pardais em granjas avícolas no Brasil. Outros estudos devem ser conduzidos para elucidar a real importância destes animais na cadeia epidemiológica e sua eficiência da transmissão do parasito para felinos. Para tal serão necessárias pesquisas que utilizem técnicas de isolamento do parasito e molecular para determinar o perfil genômico do agente presente nestas granjas.

Texto completo: acesso restrito. p. 156-159 Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-10-11T11:38:27Z No. of bitstreams: 1 1-s2.0-S0304401709001071-main.pdf: 132359 bytes, checksum: f3ecaf49ad6e7ac9f2d103d90d3da1ec (MD5) Approved for entry into archive by Alda Lima da Silva(sivalda@ufba.br) on 2013-10-15T18:26:02Z (GMT) No. of bitstreams: 1 1-s2.0-S0304401709001071-main.pdf: 132359 bytes, checksum: f3ecaf49ad6e7ac9f2d103d90d3da1ec (MD5) Made available in DSpace on 2013-10-15T18:26:02Z (GMT). No. of bitstreams: 1 1-s2.0-S0304401709001071-main.pdf: 132359 bytes, checksum: f3ecaf49ad6e7ac9f2d103d90d3da1ec (MD5) Previous issue date: 2009 Hammondia heydorni is a coccidian parasite with an obligatory two host life cycle, with dogs and foxes as definitive hosts, and a number of intermediate hosts, including goats. While infection by this parasite seems to be unassociated with any clinical signs, infection by the closely related parasites Neospora caninum and Toxoplasma gondii can result in abortion, stillbirths and low yielding in caprine herds. The aim of this work was to investigate the frequency of goats infected with H. heydorni using a nested PCR, specific to Toxoplasmatinae internal transcribed spacer 1 (ITS1) of the rDNA, followed by sequencing of the purified PCR fragments. The same molecular techniques were used to determine the frequencies of N. caninum and T. gondii-infected animals. A total frequency of 13.72% (14/102) was obtained for Toxoplasmatinae DNA in goat tissues. After sequencing the PCR products from all positive tissues, a frequency of 3.92% (4/102), 1.96% (2/102) and 7.84% (8/102) were obtained for H. heydorni, N. caninum and T. gondii, respectively. All sequences shared 98–100% identity with sequences from other strains of these coccidia present in GenBank. To the authors’ knowledge, this is the first report of H. heydorni DNA in tissues from naturally infected intermediate hosts.

The aim of this study was to evaluate the prevalence of anti-Toxoplasma gondii antibodies and to identify risk factors associated to the infection in the three meso-regions of the State of Alagoas, Brazil. A total count of 23 towns and 27 meat sheep farms were visited where blood samples were collected in order to perform the indirect immunofluorescence test to evaluate the antibodies presence. Questionnaires exploring the production system and nutritional, sanitary, and reproduction handling were handed out. The prevalence rate was 32.9% and the number of foci was 100%. In the multivariate statistical analysis, there was a significant association for the following variables: age (OR = 4.01; C.I. 2.03-7.94), size of the property (or the farm; OR = 0.48; C.I. 0.26-0.90), semi-intensive rearing system (OR = 3.17; C.I. 1.24-8.13), running water source (OR = 3.13; C.I.-1.66-5.87), and presence of cats (OR = 1.72; C.I. 1.08-2.75). It is concluded that sheep of the three meso-regions of the State of Alagoas are exposed to the infection caused by T. gondii with high prevalence. Control and prophylactic measures must be adopted seeking the improvement of the rearing system and the implantation of health promoting programs in cooperation with sheep farmers in order to elucidate the transmission means of this disease.

Whilst it is presumed that infection of pregnant cattle with Neospora caninum oocysts can provoke abortion and is the likely cause of epidemic abortion outbreaks, only two previous experiments have involved inoculation of pregnant cows with oocysts (and only one abortion was provoked in 22 pregnancies). Here, we describe the oral oocyst challenge of 18 cows synchronously bred and inoculated precisely at 70 (n = 6), 120 (n = 6) and 210 (n = 6) days in pregnancy with a nominal dose of 40,000 oocysts. Only one abortion occurred (at the 120 days challenge) which could be definitively ascribed to N. caninum and no transplacental infection (TPI) was detected in any of the other 11 calves born in the 70 and 120 day challenge groups. In contrast, 4/5 live calves born to cattle challenged at 210 days were transplacentally infected. When cows which had transplacentally infected their calves in the first pregnancy were rebred, no TPI occurred. The results show that the timing of challenge influences clinical and parasitological outcomes and that cattle in late pregnancy are exquisitely sensitive to oocyst challenge leading to exogenous TPI and congenitally infected calves. However, cattle which were indisputably systemically infected in their first pregnancy did not induce endogenous TPI in their subsequent pregnancy. This confirms previous results with experimental tachyzoite challenge and suggests that post-natal infection does not lead to persisting infections which can recrudesce in pregnancy.

To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deerl oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.