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Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.
Abortion outbreaks caused by infection with Neospora caninum in beef cattle have not been well documented. Neospora caninum infection was confirmed in 4 fetuses that were aborted by cattle in a 350-head beef herd; an additional 58 cattle aborted during the next 2.5 months. Overall, 44.4% (157/354) of the cattle in the herd did not become pregnant or experienced fetal loss during this period. Initially, 81.3% (282/347) of the herd was seropositive for antibodies to N caninum, and seropositive cows were 6.2 times as likely to not be pregnant as were seronegative cows. Other potential causes of abortion were not identified in this herd. Following the outbreak, few cattle in the herd or in a second exposed herd seroconverted, but high antibody titers persisted in most seropositive cattle through the end of the calving season. Evidence of cow-to-fetus transmission of the organism was detected in > 82% of seropositive cows.
Toxoplasma gondii and Neospora caninum are phenotypically and phylogenetically closely related cyst-forming coccidia, both of which may cause abortion in livestock animals. T. gondii exhibits also zoonotic potential by causing diaplacental infections in the human fetus and harmful infections in immunosuppressed individuals. Humans get infected either by consuming inappropriately prepared cyst-containing meat or by ingesting oocysts originating from cat feces. Therefore, in order to assess infection risk we need to have knowledge on the prevalence of the parasite in consumable meat and thus slaughtered animals. So far, no data indicate any zoonotic potential for N. caninum. Due to its high economic impact in the bovine production in Switzerland, we included this parasite in the present study as well. The prevalence of both parasite species were investigated by PCR in muscle and brain samples of slaughtered bovines, sheep, pigs and horses. Comparatively, a serum sample from each animal was simultaneously tested serologically by a Toxoplasma-P30-ELISA and a Neospora-SA-ELISA. The prevalences determined by the T. gondii-PCR were the followings: adult cows 3%, young bulls 2%, young cows prior to gravidity 6%, calves 1%, sheep 6%, horses and pigs each 0%. For N. caninum, the PCR-prevalence was 2% for adult cows and 0% for all other animal groups. Conversely, the seroprevalences were much higher for both parasite species and all animal groups, with the exception of the fattening pigs. However, as T. gondii was principally detectable in bovine (cows and calves) as well as in sheep meat, the consumption of this meat harbours a potential infection risk for humans. In contrast, the lack of any parasite detectability in fattening pig and horse meat allows to consider this infection source as neglectable when compared to bovine and ovine meat.
The small-subunit (SSU) rDNA of the Neospora sp. NC-PV1 strain isolated in Italy from cattle has been sequenced and compared to the other five N. caninum strains SSU rDNA sequences deposited in the data bases. The NC-PV1 strain sequence is identical to three published sequences. Minor differences, respectively four nucleotide bases and one nucleotide base, have been found when comparing the NC-PV1 sequence with two other available sequences of N. caninum. According to these results, the Neospora sp. NC-PV1 strain is assigned to the species N. caninum.
The prevalence of antibodies anti-Neospora caninum in bovines was determined in 21 farms from Anápolis and Goiânia microregions, in Goiás, Brazil. Out of these 11 were dairy farms (Holstein and crossbred Holstein-Zebu), nine had beef cattle (Nelore) and one was for mixed exploitation (Simental). Blood samples were collected from 930 animals, and sera obtained were separated and stored at -20 degrees C. Serum samples were examined by the immunofluorescent antibody technique (IFAT), using tachyzoites of the NC-1 Neospora caninum strain as antigen. Antibodies anti-N. caninum were found in 283 (30.4%) of the 930 blood samples examined. All the farms were positive, with prevalence for N. caninum varying from 10.3% to 89.7% of the examined animals. The prevalence for beef herds was 29.6% (10.3% to 89.7%), for dairy herds 30.4% (14.5% to 83.3%), and for the mixed herd, the prevalence was 43.3%. No statistically significant differences were found among prevalence in animals from beef, dairy or mixed herds. Results confirmed that N. caninum is widespread in different types of bovine herds in the micro regions of Anápolis and Goiânia.
Neospora caninum was identified in a lung aspirate specimen from an adult dog with severe pneumonia. Neosporosis was tentatively diagnosed by cytologic examination of a Wright-Giemsa-stained smear of the aspirate specimen, using light microscopy. The infection was confirmed by immunohistochemical examination of a section of lung tissue obtained at necropsy. Neosporosis is usually a fatal ascending neurologic disease of dogs less than 1 year of age. Neospora caninum infections are uncommon in adult dogs and do not have a consistent clinical course. The disease in the dog of this report was unique because the dog had clinical respiratory tract disease and because preliminary diagnosis was made by cytologic examination.
To address the question whether N. caninum-infections occur in German cattle, 388 sera from dairy farms with abortions and fecundity problems in North Hesse were tested for antibodies to N. caninum and seropositive farms epidemiologically analyzed. 16 sera (4.1%) with titres of > or= 1:400 from a total of 10 farms (45.5% of the farms) were considered Neospora-positive. There was a conspicuous clustering of seropositive and indifferent animals in three farms. A history of abortion could not be established for all seropositive cows, a finding which may indicate that some animals mount an immune response that can protect against abortions. The validity of the indirect immunofluorescent assay for the diagnosis of the Neospora caninum-infection of cattle is discussed.
A protocol was developed for the separation, concentration enumeration, and cryopreservation of Neospora caninum tissue cysts from mouse brains. Brains from chronically infected mice were homogenized and tissue cysts counted in 10-microliters aliquots. Tissue cysts were separated from brain homogenates by centrifugation at 4,400 g on 35% (v/v) Percoll/phosphate-buffered saline (PBS) continuous-density gradients. After removal of the brain layer, the separated tissue cysts were concentrated by diluting the remaining solution with PBS and centrifuging at 500 g. The pellet was resuspended in PBS and tissue cysts were enumerated. Fifty percent of tissue cysts were recovered from brains centrifuged once and 64% from brains centrifuged twice. Tissue cysts were preserved with 7.5% dimethyl sulfoxide in horse serum at -60 C. After thawing, bradyzoites were digested in an acid/pepsin solution and placed onto Vero cell cultures. Neospora caninum tachyzoites were recovered from cell cultures, indicating that bradyzoites retained viability after concentration and cryopreservation. Separated tissue cysts ranged in diameter from 107 microns to 15 microns (average = 31 microns), and the average bradyzoite dimensions were 2 x 7.5 microns. These methods make it possible to store viable N. caninum tissue cysts for oral-infectivity trials and other studies.
Neospora caninum, an apicomplexan parasite closely related to Toxoplasma gondii, causes abortion, stillbirths, and congenital neurologic disease in multiple animal species. The present study focuses on the development of encephalitis and intracerebral parasite load that occurs 6 wk postinfection (PI). Utilizing BALB/c, C57BL/6, and B10.D2 mice, an initial investigation was undertaken to determine the relative resistance of inbred strains to N. caninum-induced encephalitis. Relative resistance was defined in terms of central nervous system lesion development and parasite load. Based on other protozoal infections in mice, it was hypothesized that BALB/c and C57BL/6 should be contrasting in their relative resistance to N. caninum, with BALB/c and congenic B10.D2 mice less susceptible than C57BL/6 mice. Contrary to expectation, BALB/c and C57BL/6 were both highly susceptible to the development of N. caninum-induced encephalitis, whereas B10.D2 mice were resistant. Both BALB/c mice and C57BL/6 mice had significantly higher numbers of brain lesions and intracerebral tachyzoites than B10.D2 mice. Resistance in B10.D2 was associated with a high interferon (IFN)-gamma: interleukin (IL)-4 ratio from antigen-stimulated splenocytes, whereas susceptibility in C57BL/6 and BALB/c mice corresponded with a low splenocyte IFN-gamma: IL-4 ratio. In vivo measurement of Neospora-specific isotype antibodies demonstrated predominately IgG2a in serum from B10.D2 mice and IgG1 in serum from BALB/c and C57BL/6 mice. In conclusion, susceptibility of mice to N. caninum is unique compared to other protozoal diseases. The present study also demonstrates that parasite load is a fundamental measurement for evaluating disease induced by N. caninum and that a type 1 cytokine response may be necessary for regulation of this parameter.
The protozoan parasite Toxoplasma gondii is a serious cause of fetal mortality in sheep and goats. Oocysts, the parasite stage responsible for initiating infection, are produced following a primary infection in cats. A primary infection in pregnant sheep and goats can establish a placental and fetal infection which may result in fetal death and resorption, abortion or stillbirth. Diagnosis is aided by the clinical picture, the presence of characteristic small white necrotic foci in placental cotyledons, the possible presence of a mummified fetus and on fetal serology and histopathology. Development of the polymerase chain reaction (PCR) specific for T. gondii may also provide a valuable diagnostic tool. Measures to control abortion include improved management of farm cats, fodder and water. Vaccination of sheep with the live vaccine is an effective preventive measure and the use of decoquinate in feed may be useful in some situations. Neospora caninum is related to T. gondii and while its asexual life cycle is similar to that of the latter it is currently not known whether it has a similar sexual life cycle in a definitive host. Neospora is an important cause of fetal loss in cattle and parallels that of T. gondii infection in sheep and goats. While it does not appear to cause frequent losses in these latter animals, experimental infection is readily induced in them and if initiated during pregnancy provides a very good model of the bovine infection. Furthermore clinical signs and pathological lesions in sheep and goats are similar to those induced in them by T. gondii, although there are subtle histopathological differences. These changes will aid possible diagnosis as will specific serological tests such as the indirect immunofluorescent antibody test and the enzyme linked immunosorbent assay and the PCR. Sarcocystis, which exists as numerous species, undergoes a coccidian-like life cycle with each having a distinctive definitive (usually carnivore) host which excretes sporocysts into the environment. Clinical sarcocystiosis is much less commonly diagnosed than toxoplasmosis and neither is it normally associated with fetal infection or abortion in either sheep or goats. However, infection is extremely common throughout the world and follows ingestion of food or water contaminated with sporocysts. Clinical signs, when seen, include fever, anaemia, inappetance and weight loss or reduced weight gain. Central nervous signs (hind limb weakness, ataxia, paresis), acute myopathy and death may occur. Diagnosis is difficult as infection is so common and clinical signs absent, mild or non-specific. Serology may be useful in some situations and histopathology/immunohistochemistry is valuable for confirming the cause of death. Control relies on preventing contamination of pasture and water with faeces of dogs, foxes and cats or by controlling access of young susceptible stock to contaminated land. Relatively little is known of the immunity induced by infection with Sarcocystis spp. but research indicates that protective immunity does develop and that cell-mediated mechanisms are probably important. It is likely that sarcocystiosis is underdiagnosed as a problem and that better diagnostic methods are needed to show the true extent of the losses caused. Neosporosis on the other hand would appear not to be so common in sheep and goats. The value of experimental infections in these animals may be to provide a comparative model of the infection in cattle in the same way that our understanding of toxoplasmosis in sheep provides a superior model of human toxoplasmosis.
Dogs were investigated to determine if they are definitive hosts of Neospora caninum. Four dogs were fed N. caninum tissue cysts in infected mouse tissue, and two negative control dogs were fed uninfected mouse tissue. Dog faeces were examined daily for 30 days using a sucrose flotation technique. Three challenged dogs shed spherical to subspherical unsporulated oocysts, measuring 10 to 11 microns in diameter. Oocysts sporulated within 3 days and contained two sporocysts, each with four sporozoites. Outbred, inbred, and gamma-interferon knockout mice were inoculated with canine faecal extracts and monitored for evidence of neosporosis using a variety of morphologic, immunohistologic, serologic, and genetic analyses. Mice that received faeces from each dog observed to shed oocysts were demonstrated to have neosporosis by two or more techniques. One mouse was demonstrated to be infected with N. caninum by immunohistochemistry, ultrastructural analysis, and a species-specific PCR test. No evidence of neosporosis was observed in control animals. Based on this study, dogs are a definitive host of Neospora caninum.
Protozoal abortion in cattle was retrospectively studied by examining material submitted to the Batchelar Animal Health Laboratory in the years 1987-90. Only cases in which foetal brain had been submitted were examined. Histologically, protozoal lesions were seen in the brains of 28% of 320 aborted foetuses. Lesions were also seen in 10% of the hearts and 34% of the placentas examined, but these were not as characteristic as those in the brains. Protozoa, confirmed immunohistochemically as being Neospora caninum-like in two brains, were seen in 5% of aborted foetuses with lesions. No protozoa or associated lesions were seen in 57 late term foetuses or neonatal controls. A farm survey supported the hypothesis that Neospora is an important cause of multiple abortions in cattle. Laboratory and farm data suggested that protozoal abortion was more common in early gestation. No predisposing causes of abortion were found and there was no evidence of point infection of affected herds.
Of 163 dogs, randomly selected from those examined at the University of Liverpool Small Animal Hospital, 12.9 per cent had antibody titres > or = to 1/200 to Neospora caninum in an indirect fluorescent antibody test. None was apparently suffering clinical neosporosis. There was no association between the occurrence of neospora antibodies and either toxoplasma antibodies measured by the dye test, sex, age, type of feeding or the presence of other dogs in the household. Antibody was detected at titres > or = to 1/200 in nine breeds, suggesting that there is a substantial level of subclinical infection in British dogs.
Eighty bovine fetuses with presumed protozoal infections from a previous 2-year retrospective study were examined by immunohistochemistry using antisera against Neospora caninum. In 66 (83%) of the fetuses, protozoa were found that reacted positively with anti-N. caninum sera. In three (4%) additional fetuses, protozoa identified as Sarcocystis species did not react, and in two fetuses (3%) single protozoal clusters were found only in hematoxylin and eosin-stained slides. A group of 20 fetuses were chosen for further evaluation. They included 14 fetuses from the first group of 80 fetuses plus six additional fetuses that had large numbers of protozoa in the fetal brain. The 20 fetuses were examined immunohistochemically with antisera to N. caninum, Hammondia hammondi, and Toxoplasma gondii. Protozoa from 3/20 fetuses, identified as Sarcocystis species, failed to react with any antisera. In 16/20 fetuses protozoa reacted positively to antisera against N. caninum, and in most cases reacted to H. hammondi, and weakly to one or more of the antisera against T. gondii. Thick-walled protozoal tissue cysts were found in the brain of four of these 16 fetuses by transmission electron microscopy. The cyst wall morphology was comparable to N. caninum. The results suggested that a single protozoal parasite of unknown identity was responsible for most of the bovine abortions. By immunohistochemistry, the unknown protozoon reacted most strongly and consistently to N. caninum antisera, but was antigenically distinct from N. caninum. Ultrastructurally, tissue cysts found in four fetuses most closely resembled Neospora caninum.
Protozoal encephalomyelitis was diagnosed in a 3-day-old calf that was stunted, weak, and recumbent. Grossly, the calf had contracted tendons in the forelegs, a slightly doomed skull, a porencephalic cyst in the cerebellum, ulcerative esophagitis, and abomasitis. Histologically, there was a multifocal nonsuppurative encephalomyelitis with clusters of protozoal tachyzoites and numerous protozoal cysts. The porencephalic cyst and gastrointestional lesions appeared to be unrelated to the protozoal infection and were suggestive of a concurrent bovine virus diarrhea infection. A few groups of protozoal tachyzoites and numerous tissue cysts were found in neuropile, particularily in neurons of the spinal cord. By light microscopy, smaller tissue cysts were found in the brain (majority from 14 to 20 microns) than in the spinal cord (majority from 20 to 48 microns). The cyst walls ranged in thickness from less than 1 micron to a maximum of 2 microns wide. Bradyzoites contained PAS-positive slender bradyzoites (5-8 x 1-2 microns). Tissue cysts reacted positively to anti-Neospora caninum sera; but unlike N. caninum, they were positive to 2 of 4 antisera against Toxoplasma gondii and to antisera to H. hammondi. Ultrastructurally, tissue cysts closely resembled a Neospora-like organism, including the finding of interneuronal protozoal cysts, thick cyst walls, a lack of micropores in the bradyzoites, and the presence of numerous micronemes oriented perpendicular to the pellicle. Ultrastructural features in the calf protozoan that have not been reported for N. caninum in dogs included the presence of numerous tubulovesicular structures in the cyst ground substance and bradyzoite vesicles that contained small vesicular structures and short, flat membrane segments.(ABSTRACT TRUNCATED AT 250 WORDS)
BACKGROUND: Neospora caninum is an important cause of infectious abortion and stillbirth in cattle world-wide. Infection is common and may frequently be passed from mother to calf (vertical transmission) with no signs of disease. Based on our previous observation that N. caninum-infection can be efficiently controlled with toltrazuril-sulfone (ponazuril) in experimentally infected mice, we addressed the question if efficacy could also be obtained in experimentally infected calves. MATERIAL AND METHODS: The study included 19 calves and represents an initial explorative approach to document a basic effectiveness at first. Fifteen animals received each 2 x 10(8) N. caninum trophozoites, half of the dose being injected intravenously and the other half subcutaneously. Efficacy of treatment was assessed using molecular detection of parasite DNA with PCR and pathological alterations by immunohistochemistry in different organs of the animals. Assessment included also clinical, serological and pathophysiological parameters. RESULTS: In those calves medicated with ponazuril (one, or six consecutive days, respectively, starting one day after infection), a complete abrogation of the parasite detectability was obtained in the brain and other organs, while 50% of non-treated calves became PCR-positive in brain and muscles. Clinically, ponazuril chemotherapy of infected calves--in comparison to non-treated infected animals--reduced symptoms (fever), but no differences were observed between treated and non-treated animals with regard to serum enzymes and metabolites. Efficacy of a six-day treatment was also reflected by significantly lower anti-Neospora antibody concentrations developed after infection, when compared to non-treated animals. CONCLUSION: Based on our findings in this initially explorative approach that indicate a basic effectiveness of ponazuril against experimental N. caninum infection in calves, we plan to follow our chemotherapeutical intervention strategy to control bovine neosporosis with a subsequent more extensive field study with naturally infected calves.
Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.
Naturally-occurring neosporosis with multiple organ involvement was identified in a 10-year-old neutered male Basset hound dog. Clinical signs were first noticed 3 weeks prior to referral and consisted of crouched stance and mild pelvic limb ataxia. Dexamethasone administration had provided transient improvement. On presentation to the teaching hospital, clinical signs included depression, pelvic limb ataxia, inability to stand without assistance, and pain on deep palpation of the cervical and lumbar vertebral column. Lesions were found in the myocardium, liver, spleen, adrenal glands, brain, and spinal cord. Tachyzoites of Neospora caninum were found in the myocardium and adrenal glands. Organisms stained with anti-Neospora caninum, but not to anti-Toxoplasma gondii serum in an immunohistochemical test.