Prevalence of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="Italic">Neospora caninum</Emphasis> infections in sheep from Federal District,central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%<CI 0.95<49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%<CI 0.95<10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

<Emphasis Type="Italic">Eimeria bovis</Emphasis> meront I-carrying host cells express parasite-specific antigens on their surface membrane

Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.     

First identification of Sarcocystis tenella (Railliet, 1886) Moulé, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil

Sarcocystis tenella is a dog–sheep protozoan parasite, causing a widespread enzootic muscle parasitosis and neurological disease mainly in lambs. This parasite is pathogenic to sheep and important to the economical production of sheep. The present study was initially aimed to determine Toxoplasma gondii infection and the occurrence of co-infection with other Apicomplexa parasites in 602 Brazilian sheep. Twenty of these sheep were positive with antibodies to T. gondii by MAT and IFAT-IgG tests, positive with PCR-RFLP genotyping at multiple loci, and parasites were isolated from mice infected with sheep tissue samples. Two additional sheep born in Brazil, a 2-year-old female Polwarth (Ideal) sheep, a breed originated from Australia (#1), and a 1-year-old male Corriedale sheep, a breed originated from New Zealand and Australia (#2) were positive to T. gondii antibodies by serum tests, and PCR, but negative for bioassay in mice. In genotyping at 12 loci, sheep #1 sample and #2 presented positive results only for some markers. PCR-RFLP of 18S ribosomal RNA (18S rRNA) was performed in all 22 animals to identify the possibility of co-infection of T. gondii with other Apicomplexa parasites, such as S. tenella, Neospora caninum and Hammondia hammondi, resulting in a T. gondii profile for the first 20 animals and a unique genotyping profile for sheep #1 and #2, identical to S. tenella. The 18S rRNA PCR products (310 bp) were sequenced and blasted to GenBank database at NCBI. Both samples were identical to S. tenella 18S rRNA gene (GenBank accession number L24383-1). These results suggest the existence of co-infection of S. tenella with T. gondii in ewes from Brazil.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain)

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.     

In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells

The growth of Sarcocystis neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 microM was found to release intracellular merozoites with a 40 min treatment at 37 degrees C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.     

Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds

Neospora caninum infections in chickens have been recently described by epidemiological and experimental approaches, and these birds may be considered natural intermediate hosts of the parasite. It has been postulated that other bird species might perform this role in wildlife as well. To better understand the sylvatic life cycle of N. caninum, further studies are required. In that sense, this work aimed to observe infection kinetics in pigeons experimentally infected with N. caninum. Experimental infections were conducted in parallel with a related protozoan, Toxoplasma gondii, which has been already described as able to infect pigeons in nature. Our results demonstrated that N. caninum disseminated through various tissues of this host and induced parasite-specific IgG seroconversion. Infection parameters were similar to that observed in the T. gondii infected group, although N. caninum-infected pigeons presented lower IgG titers during acute phase. The results herein described demonstrate that pigeons are a suitable model for N. caninum infection, considering that these data are in agreement with those observed in chickens experimentally infected with this parasite. As pigeons may be revealed as important reservoirs for N. caninum infection in nature, future studies are necessary to determine the real prevalence of this parasite in this and other birds in wildlife.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in sheep intended for human consumption in the Rondônia state,Western Brazilian Amazon

The aim of this study was to carry out a seroepidemiological study of anti-T. gondii and anti-N. caninum antibodies in serum samples from sheep intended for human consumption in the Rondônia state, in the Western Brazilian Amazon, and identify possible risk factors associated with seropositivity. The analysis of the 616 sheep serum samples revealed animal-level seroprevalence of T. gondii in the order of 52.4 % and of N. caninum of 60.6 %. Prevalence of co-infections was 33.4 %. The herd-level seroprevalence was 100 % for both the parasites. Applying of multivariate logistic regression analysis, test of the association between seroprevalence of T. gondii infection and the potential predictors showed that feed, fetal malformation, abortion, neurological problems (lambs), presence of rodents were the significant (P < 0.05) predictors. The variables presence of rodents, cats, wild animals and main activity showed statistical association (P < 0.05) with the seroprevalence of N. caninum. Due to the high prevalence of T. gondii and N. caninum found in this study, the absence of specific slaughterhouses for sheep and raw or undercooked sheep meat for human consumption we concluded that sheep can represent an important source of infection for humans.     

Hypodermosis in cattle slaughtered in Nigde province,Turkey<Superscript>*</Superscript>

This study was carried out to investigate the prevalence of hypodermosis in cattle between January and June 2005 in Nigde province, which is located in the middle of Turkey. A total of 1336 cattle, which were slaughtered in Nigde, were investigated for Hypoderma larvae. The 68 out of 1336 cattle (%5.08) were found positive for Hypoderma larvae. A total of 536 Hypoderma larvae were found in the skin and subcutaneous tissue of the back of infested cattle. The 489 out of 536 larvae (%91.23) were identified as Hypoderma bovis and 47 out of 536 (%8.77) as H. lineatum. Number of Hypoderma larvae counted on single infested cattle varied between 1–45 and the mean number of Hypoderma larvae per cattle was 7.88 (536/68). Hypodermosis was recorded for the first time in cattle from Nigde. ^*This study was supported by the Scientific Research Projects Unit of Nigde University (FEB 2004/07).     

Molecular assessment of the transplacental transmission of Toxoplasma gondii,Neospora caninum,Brucella canis and Ehrlichia canis in dogs

Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents.     

Factors influencing the adherence of strains of Streptococcus bovis and Escherichia coli isolated from ruminal epithelium

Two strains of Streptococcus bovis (A1 and A5) and one strain of Escherichia coli (0141: H28) isolated from the surface of bovine ruminal mucous epithelium were examined for adherence to isolated and cultured ruminal epithelial cells. The E. coli adhered to the target cell by means of fimbriae, which had several common properties with type 1 common fimbriae and caused mannose-sensitive haemagglutination. The A1 strain of S. bovis was devoid of fimbriae and its adherence to the epithelial surface was not inhibited by treatment with sugars or phenol-treated bacterial membrane from the same organism. It was therefore postulated that the bacterial glycocalyx of the S. bovis organisms acted as ligand. The extent of bacterial adherence depended on the state of differentiation of the target cell in both the isolated and the cultured ruminal cell systems. The receptors for both adherent bacterial species were in all probability associated with the glycocalyx of the target cells.     

Seroprevalence of <Emphasis Type="Italic">Neospora caninum</Emphasis> antibodies in beef cattle in three southern states of Mexico

The aims of the present study were to examine the seroprevalence of neosporosis in beef herds from three southern states of Mexico and determine the association with several risk factors. A cross-sectional serological survey for Neospora caninum was carried out by sampling a total of 596 animals from 31 herds in Chiapas, Veracruz, and Yucatan States and tested using an ELISA assay (IDEXX) to detect anti-N. caninum antibodies. The overall prevalence was 11.6%, (95% CI: 0.93–0.14), however, the prevalence for Chiapas was 15% (30/200) (95% CI: 0.11–0.21), in Yucatan 11.3% (21/186) (95% CI: 0.07–0.17) and in Veracruz 8.6% (18/210) (95% CI: 0.05–0.13). Of the 596 serum samples taken, 578 were from females and 18 were bulls., Only one bull sample was found seropositive from one herd in Veracruz State. The overall herd-prevalence was 70.9% (22/31) (95% CI: 0.53–0.84), and by State was: Chiapas 90% (9/10) (95% CI: 0.60–0.98); Yucatan 72.7% (8/12) (95% CI: 0.39–0.86); and Veracruz 50% (5/10) (95% CI: 0.24–0.76), respectively. The highest age-prevalences were found in animals 1 yr old (19.4% [95% CI: 0.01–0.35]) and 4 yrs old (19.6% [95% CI: 0.12–0.29]), and the lowest in animals 3 –yrs of age (6.2%,[95% CI: 0.02–0.20]). No association was found among all the variables tested in this study since most of the ranches have similar management conditions. In conclusion, N. caninum infection is common among beef herds in the Mexican tropics.     

Detection of an Ehrlichia bovis-like Organism in Cultured Buffalo Monocytes

Monocytes from a buffalo were cultured in RPMI 1640 medium following separation of plasma by the erythrocyte sedimentation technique and subsequent separation of mononuclear cells by density gradient centrifugation. Growth of an organism considered to be Ehrlichia bovis was noticed in the cultured monocytes after 10 days. The inclusions were considered to be those of E. bovis from their morphology, staining characteristics and growth characteristics in culture, and by indirect immunofluorescence examination with an anti-E. canis serum. The utility of peripheral blood monocyte cultures opens the possibility of diagnosing the carrier status of ehrlichiosis in animals.     

Serological survey of Neospora caninum and Toxoplasma gondii in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in ten provinces of Brazil

The objective of this study was to determine the prevalence of antibodies to Neospora caninum and Toxoplasma gondii among 500 cattle (Bos indicus) and 500 buffaloes (Bubalus bubalis) using the indirect fluorescent antibody test (IFAT) technique. Blood samples from were collected from water buffalo and cattle in 10 municipalities in the northern region of Brazil. The frequency of cattle and water buffaloes seropositive for Neospora caninum in Pará state, Brazil, was 55% and 44%, respectively, and the frequency of cattle and water buffaloes seropositive for Toxoplasma gondii was 52% and 39%, respectively. Seropositivity for both N. caninum and T. gondii was detected in 10.6% of the cattle samples and 14.8% of the buffalo samples. The frequency of cattle positive for N. caninum and T. gondii was significantly (p < 0.05) higher than that of buffalo in two and three provinces, respectively. Buffaloes had a lower seroprevalence for N. caninum or T. gondii in all of the provinces studied. These results suggest that both species, when exposed to the same risks for N. caninum and T. gondii infection, have a high serological prevalence. Cattle showed a higher probability of being seropositive when exposed to the same risks for N. caninum and T. gondii. Our study, which included an extensive number of blood samples, provides important epidemiological information pertinent to buffalo production in tropical countries that can be used as a basis for disease-management practices in Latin America.     

Epidemiology of Leishmania infantum,Toxoplasma gondii,and Neospora caninum in Rattus rattus in absence of domestic reservoir and definitive hosts

Isolated environments are privileged settings to study transmission of infection. Montecristo is a small island where no wild or domestic carnivores are present. Invasive Black rats Rattus rattus (n = 78) were captured and tested by PCR for Leishmania infantum, Toxoplasma gondii and Neospora caninum. We wanted to test, for these parasites, the existence of a sylvatic cycle independent of reservoir or definitive hosts. None of the rats tested positive by PCR for either T. gondii or N. caninum. We recorded a 15.5% prevalence (CI95% 8–26%) of L. infantum in the rats and Phlebotomus mascittii was captured in Montecristo, leading us to identify it as possible vector of the parasite.     

Neospora caninum and Ehrlichia canis co‐infection in a dog with meningoencephalitis

An 8‐year‐old mixed‐breed dog was presented for acute, progressive weakness and ataxia, inappetence, and weight loss. The patient was mentally normal, but nonambulatory, with a right head tilt, right positional ventral strabismus, and slight head tremors. A neurologic lesion was localized to the cerebellum and right brainstem. Cerebrospinal fluid (CSF) analysis showed a markedly increased protein concentration and mixed pleocytosis, with eosinophil predominance (44%), intracytoplasmic inclusions within eosinophils, consistent with Ehrlichia canis (E canis) morulae, and Toxoplasma gondii (T gondii) or Neospora caninum (N caninum) tachyzoites within eosinophils and monocytes. A serum indirect immunofluorescent antibody test was positive for N caninum (titer 1:12 800) and negative for T gondii. Both blood and CSF PCR results were N caninum‐ and E canis‐positive and T gondii‐ and Anaplasma phagocytophilum‐negative, and blood PCR, but not CSF PCR, was Hepatozoon canis‐positive. The dog was treated for 30 days with clindamycin, sulfamethoxazole‐trimethoprim, doxycycline, prednisone, and cephalosporin, but did not improve neurologically, and was euthanized. Brain histopathology showed moderate multifocal, subacute meningoencephalitis with necrosis and gliosis. The neurologic disease was mostly attributed to central nervous system (CNS) neosporosis, with the possible contribution of ehrlichiosis, which was likely a manifestation of blood‐brain barrier disruption. Hepatozoonosis was probably a result or cause of underlying immunosuppression. To our knowledge, this is the first report of CNSN caninum and E canis co‐infection detected by both CSF PCR and cytology and E canis morulae identified within CSF eosinophils.     

Neospora caninum infection as a cause of reproductive failure in a sheep flock

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.     

Effects of crossbreed pregnancies on the abortion risk of Neospora caninum-infected dairy cows

Previous studies have shown that the use of beef bull semen significantly reduces the rate of abortions due to Neospora caninum in artificially inseminated (AI) seropositive dairy cows. In addition, certain beef breeds could be more resistant to N. caninum infection and abortion than others. The aim of the present study was to determine whether different crossbreed pregnancies, those derived from Limousin, Charolais, Piedmontese or Belgian Blue semen, carry different risks of abortion in Neospora-infected dairy cows. The effects of possible interactions between maternal levels of N. caninum antibodies and the different breed crosses were also evaluated. The study was performed on five commercial Holstein–Friesian dairy herds in Northeast Spain with previously confirmed diagnoses of N. caninum infection in aborted foetuses. The study population was comprised of 1115 pregnancies: 633 pregnancies recorded after AI using Holstein–Friesian semen from 18 bulls and 482 after AI using beef semen from 27 bulls (304 inseminations using semen from Limousin bulls, 191 from Belgian Blue bulls, 89 from Piedmontese bulls and 49 from Charolais bulls). Abortion rates were 32.2% (155/482) and 15.2% (96/633) for seropositive cows inseminated with Holstein–Friesian and beef breed semen, respectively. Logistic regression analysis revealed the herd and the interaction between maternal N. caninum antibody titre and the different crossbreeds as significant factors affecting the abortion rate. Lowest abortion rates, similar to that shown by seronegative animals in the analysed herds (3.2%, 239/7432), were observed in dams AI using Limousin semen that had low (<30 relative index (RI) units) N. caninum antibody titres (2.1% abortion, 3/145) and these cows were used as reference. Compared to the cows used as reference, cows with low N. caninum antibody titres (<30 RI units) showed a similar risk of abortion when inseminated with Piedmontese or Charolais bull semen, but higher risk of abortion when inseminated with Holstein (17.9 times) or Belgian Blue (7.2 times) bull semen. All cows with high N. caninum antibody titres (≥30 RI units) had a higher risk of abortion, ranging from 8.9 times (cows inseminated with Limousine semen) to 37.8 times (cows inseminated with Piedmontese semen), compared to the cows used as reference. In conclusion, different crossbreed pregnancies carried different abortion risks in Neospora-infected dairy cows. The use of beef bull semen dramatically reduced the risk of abortion in dairy cows, especially if Limousin breed semen was used. Moreover, this reduction was found to be dependent on the N. caninum antibody titre such that the lowest incidence of abortions was recorded in Limousin semen inseminated cows with low antibody titres. Insemination of Neospora-seropositive cows with beef bull semen could both reduce the risk of abortion and avoid breeding replacements for infected cattle.