Neospora caninum in sheep: a herd case report

Neospora caninum was detected by means of PCR in the brain of 4 out of 20 aborted fetuses in a flock of 117 sheep exhibiting a persistent abortion problem, and N. caninum tissue cysts were furthermore found in encephalitic lesions in one of the PCR-positive fetuses. Toxoplasma gondii was detected as aborting agent in another 3 out of 20 fetuses. Antibodies to N. caninum (by indirect fluorescence antibody test (IFAT)) were found in 10.3% of 117 ewes and antibodies for T. gondii were found in 97.4% of 117 ewes. Other organisms associated with abortion were Chlamydia psittaci in three fetuses and Pasteurella multocida in one fetus. This is the first report of N. caninum associated abortion in naturally infected sheep.     

Evaluation of an enzyme-linked immunosorbent assay for the serological diagnosis of Neospora caninum infection in sheep and determination of the apparent prevalence of infection in New Zealand

Neospora caninum has recently been shown to be a cause of abortions of sheep in New Zealand. A commercially available enzyme-linked immunosorbent assay (ELISA) was validated for use in sheep with sera from experimentally infected sheep. A cut-off threshold was established that demonstrated sero-conversion between 7 and 14 days post-infection. Higher inocula led to earlier sero-conversion. This ELISA was applied to 640 sera collected from rams across New Zealand and 0.625% (+/-0.61%) (4/640) were shown to be serologically positive. The four positive sera were also demonstrated to be positive by indirect fluorescent antibody test (IFAT). The ELISA evaluated here lends itself more readily to large-scale investigations than IFAT. The low background of N. caninum infection in the New Zealand sheep population suggests that N. caninum abortions could be more easily diagnosed by serological means than in populations with higher background sero-prevalence.     

Occurrence of Neospora caninum and Toxoplasma gondii infections in ovine and caprine abortions

Neospora caninum and Toxoplasma gondii are closely related cyst-forming apicomplexan parasites identified as important causes of reproductive failure in cattle and small ruminants, respectively. Protozoan abortion in small ruminants is traditionally associated with T. gondii, but the importance of N. caninum remains uncertain. The aim of this study was to investigate the presence of N. caninum and T. gondii infections in abortion cases in small ruminants submitted for diagnosis. For this purpose, 74 ovine and 26 caprine aborted foetuses were recovered from different areas in Spain. Foetal histopathology was used to detect the presence of protozoal-associated lesions in brain. The presence of N. caninum and T. gondii was confirmed by PCR. Protozoal infection was detected in 17 out of 100 (17%) foetuses examined by at least one of the diagnostic techniques used. Lesions suggestive of protozoal infection were observed in 10.8% (8/74) and 15.4% (4/26) of the ovine and caprine abortions respectively. N. caninum and T. gondii infection was detected by PCR in 6.8% (5/74) and 5.4% (4/74) of sheep foetuses, respectively, of which five showed protozoal-associated lesions. N. caninum DNA was detected in 11.5% (3/26) of goat foetuses, of which two showed protozoal-associated lesions, whereas T. gondii DNA was detected in one goat foetus with no lesions. The simultaneous presence of N. caninum and T. gondii DNA was detected in one sheep foetus with severe lesions. This study demonstrates that N. caninum plays a significant role in abortion in small ruminants in the studied population. In addition, our results highlight the importance of differentiating between protozoa whenever characteristic lesions are observed.     

First Confirmed Case of Neospora caninum-associated Abortion Outbreak in Portugal

In a dairy herd experiencing an abortion outbreak, 49% of the 119 cows were positive for Neospora caninum antibodies and the remaining 51% were seronegative. Using immunohistochemical analysis of foetal brain tissue, Neospora cysts and bradyzoite clusters were identified in two of the 15 aborted foetuses submitted for testing. Other agents that commonly cause abortions were not found. These results, coupled with neurological clinical symptoms in new-born calves, implicated N. caninum as the major cause of abortions in this dairy herd. This is the first report that associates N. caninum infection with bovine abortions in Portugal.     

Molecular and immunodiagnostic studies of bovine neosporosis in Switzerland

Cyst-forming coccidia may cause significant losses in livestock, primarily due to abortion, loss of young animals and neuromuscular diseases. Rather recently, Neospora caninum has been recognized as one of the major protozoal abortion-inducing parasites in cattle. The present study addressed the performance of different diagnostic tools (in vitro-cultivation; histology; immunohistochemistry; serology; PCR) suitable for the direct or indirect detection of N. caninum. By PCR, Neospora-DNA was detected in 24 brains (29%) from 83 bovine abortion, many of these brains were simultaneously characterized by histopathological findings typical for a protozoal, cerebral parasitosis. The diagnostic methods were furthermore assessed using samples of different tissues and body fluids from three experimentally Neospora-infected pregnant cows and their foetuses. The diaplacental passage of N. caninum to the foetus was successful in two of the three cases. In these two cases, PCR was positive for different foetal organs and, additionally, for the abomasal and amniotic fluid. The successfully infected cows developed anti-Neospora serum antibodies between 10 and 17 days post infection, foetuses remained serologically negative in all cases. The results obtained in the present study demonstrated the usefulness of PCR, complemented by serology, for the specific diagnosis of bovine neosporosis. Such tests may prove suitable to perform epidemiological investigations. Taken together, our data indicated that prenatal neosporosis may be an important cause of infectious bovine abortion in Switzerland.     

A Swiss case-control study to assess Neospora caninum-associated bovine abortions by PCR, histopathology and serology.

Neospora caninum is one of the most frequent infectious organisms causing abortion in cattle worldwide. The present case-control study was designed to assess the importance of bovine neosporosis for causing abortion in Swiss cattle and to identify selected risk factors. Infection was primarily diagnosed by a N. caninum-specific PCR and serology, complemented with histopathology and immunohistochemistry. A total of 113 case and 113 corresponding control-farms were studied for 1.5 year. During this time period, 242 abortions were reported and referred for bacteriological, virological, parasitological and pathohistological examinations. N. caninum was detected by PCR in the brains of 21% of all aborted fetuses. Microscopic lesions indicative for cerebral protozoa infection were detected in 84% of PCR-positive fetal brains. Bovine viral diarrhea virus (BVDV) was demonstrated in 7% of the cases, and bacterial infections were detected in 4% of the abortions. One or more N. caninum-abortions occurred in 20% of the herds (41 case-farms and 3 control-farms). Serological examination of aborting mother cows revealed a significantly higher percentage of N. caninum-seropositive animals (44%) in comparison to the prevalence in a randomly selected population (12%). However, in eight cases (4% of all investigated abortions) seronegative cows aborted N. caninum PCR-positive fetuses, and in 50 cases the fetus remained negative although the respective mother cow was N. caninum-seropositive. Repetitive serological investigations (at a 3-12 months interval) of 3551 cows from case- and control-farms showed a decrease of the overall N. caninum-seroprevalence from 17 to 12%. Ninety out of 3008 seronegative animals were converted to N. caninum-seropositivity. Conversely, 212 out of 543 initially seropositive animals became seronegative for their second serum sample. The obtained data underlined the importance of N. caninum as a causative agent for abortion in Swiss cattle. Furthermore, PCR was confirmed to be a valuable diagnostic tool for the primary diagnosis of N. caninum in aborted fetuses. On the other hand, the value of serology appears to be hampered by the temporal instability of N. caninum antibody concentrations in adult cattle, including especially seronegativity of some individual animals. Thus, seronegativity in a mother cow or heifer does not exclude N. caninum-associated abortions.     

Neosporosis in bovine dairy herds from the west of France: detection of Neospora caninum DNA in aborted fetuses, seroepidemiology of N. caninum in cattle and dogs.

Neospora caninum is considered one of the major causes of abortion in cattle in most parts of the world. In this study, the role of N. caninum was investigated in groups of aborted cattle and dairy herds from the west of France. Good correlation was found between parasite DNA detection in fetuses and serologic statuses of dams. In groups with documented abortion status and no antibodies to other pathogens, 17-45% of aborted animals were seropositive for N. caninum, and significant relationship between prevalence of Neospora antibodies and frequency of abortions was found. Neospora-associated abortions were observed all the year round, with a peak in summer. Higher ratios of seropositive abortions were found before the 6th month of gestation. In 12 herds studied in the field, serologic prevalence ranged 6-47%. No difference in age was found between seropositive and seronegative cows. Results indicate that N. caninum is an important and stable cause of abortion in cattle in France.     

Evaluation of vaccination with Neospora caninum protein for prevention of fetal loss associated with experimentally induced neosporosis in sheep

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing fetal loss associated with experimentally induced neosporosis in sheep. ANIMALS: 30 Dorset ewes. PROCEDURE: Ewes were randomly allocated to receive vaccination on days 1 and 60 of the study with a killed N caninum tachyzoite preparation in a commercially available adjuvant or a saline-adjuvant mixture. A ram was placed on pasture with the ewes from days 15 to 60. Blood was collected from ewes before primary and booster vaccinations and prior to experimental challenge with N caninum tachyzoite performed on day 90; sera were assessed via Neospora agglutination (NA) and immunofluorescence antibody (IFA) assays. Blood was collected from lambs before they suckled, and sera were tested for antibodies against N caninum. RESULTS: Of the 14 vaccinated ewes that became pregnant, 12 gave birth to live-born lambs; in contrast, 5 of 11 pregnant control ewes gave birth to live-born lambs. Whereas vaccination improved fetal survival in pregnant ewes challenged with N caninum tachyzoites, it did not appear to have any appreciable effect on transmission of N caninum to offspring, as indicated by results of NA and IFA assays. CONCLUSIONS AND CLINICAL RELEVANCE: The N caninum tachyzoite vaccine used in this study appeared to provide protection against fetal loss associated with experimentally induced neosporosis in a high proportion of pregnant ewes.     

Prevalence of antibodies to Neospora caninum in dogs of rural or urban origin in central New Zealand

AIM: To investigate the seroprevalence of Neospora caninum infection in populations of dogs from dairy farms, sheep/beef farms and urban areas in the central part of New Zealand. It was postulated seroprevalence would be higher for farm dogs than urban dogs if the life-cycle of this parasite involves transmission between dogs and cattle. METHODS: Serum samples were obtained from dogs that lived on dairy farms (n=161), sheep/beef farms (n=154) and in urban situations (n=150). The relative risk of detecting antibodies to N. caninum using an immunofluorescent antibody test (IFAT) was compared between farm and urban dogs. RESULTS: The relative risk of having a titre of > or = 1:200 to N. caninum was 2.43 (95% CI=1.88-3.14) for dairy-farm dogs and 3.16 (95% CI=2.48-4.02) for sheep/beef-farm dogs, compared with urban dogs. At this titre, which is currently used in New Zealand to indicate seropositivity, seroprevalence of N. caninum infection was 30.7% in urban dogs, 74.5% in dairy-farm dogs and 96.8% in sheep/beef-farm dogs. CONCLUSION: This observation is consistent with a cycling of this disease between cattle and dogs on farms in New Zealand and with higher exposure of dogs to N. caninum on farms than occurs in urban environments. The prevalence of antibodies in all three groups of dogs tested in this study (dairy-farm dogs, sheep/beef-farm dogs and urban dogs) is higher than has generally been reported elsewhere. New Zealand farm dogs have a higher serological prevalence of N. caninum infection than urban dogs. CLINICAL RELEVANCE: Management and disease control practices that break the life-cycle of transmission between cattle and dogs should assist in controlling cattle abortion due to N. caninum.     

Neospora caninum seropositivity and association with abortions in dairy cows in Mexico

The seroprevalence of Neospora caninum infection was estimated from a sample of 813 cattle from 20 dairy herds in five regions in Mexico. The true prevalence of infection was estimated to be 42% (95% CI: 39, 46). Seropositivity was associated with abortion (odds ratio (OR)=2.0) and was higher among cows raised on-farm (41%), than among replacement cattle purchased outside the farm (28%). The ORs relating abortion to seropositivity in individual herds ranged from 1.3 to 10. Overall, 26% of the abortions were attributed to N. caninum.     

Neospora caninum infection associated with stillbirths in captive antelopes (Tragelaphus imberbis)

Neosporosis was diagnosed in full-term stillborn twin calves of captive antelopes (Tragelaphus imberbis) in a German zoo. In both calves a multifocal non-suppurative encephalitis was present and infection with Neospora (N.) caninum was confirmed by foetal serology and PCR. N. caninum-DNA was detected in brain, heart, lung, liver and spleen of a third full-term stillborn calf. Histopathological lesions were absent in this calf and antibodies against N. caninum were not detected in pleural fluid. Immunohistochemistry failed to demonstrate N. caninum antigen in all three calves.     

Chronic Neospora caninum infection and repeat abortion in dairy cows: a 3-year study

The serological status of Neospora was monitored in animals older than 6 months in a dairy herd with a 3-year history of prevalent N. caninum and N. caninum-associated abortions. The numbers of animals in the herd tested each year of the study period were 259, 222 and 231, respectively. A separate analysis was performed on the 122 animals persisting in the herd for the 3 years. The overall seroprevalence of N. caninum in the herd decreased from 31.7% in the first year to 24.8% in the second year and to 19.9% in the third year of the study, while the overall abortion rate decreased from 20.6% in the first year to 5.5% in the second year, and 9.9% in the third. These decreases occurred in response to control measures adopted from the second year onwards, such as culling Neospora-seropositive aborted animals and inseminating Neospora-seropositive dams with beef bull semen. Of the total number of abortions recorded in seropositive animals, 51% were repeat abortions that occurred in 36.8% of the animals with a previous history of abortion. The initial seroprevalence of Neospora in the 122 animals followed for the 3 years was 18%, increasing to 21.3% in the second and third years. Seroconversion only occurred in four animals during the second and third years of the study and abortion occurred only in seropositive individuals. Of the total number of abortions recorded in the 122 animals, 61.5% were repeat abortions that occurred in 26.7% of the animals with a previous history of abortion. These results indicate that Neospora seropositivity can be very stable through time and N. caninum infected cows can show a high rate of repeat abortions. The present data reinforce the idea that annual serological screening for Neospora can be an effective and rapid method of detecting N. caninum infection, such that control measures can be established at the farm level.     

Tunicamycins, a class of nucleoside antibiotics similar to corynetoxins of the Rathayibacter toxicus, increase susceptibility of mice to Neospora caninum

Neosporosis is the leading cause of abortion in cattle. Neospora caninum-associated abortion may exhibit both endemic and epidemic patterns. It was reported that the epidemic outbreaks took place in the form of "abortion storms" and were not significantly correlated with seasonal changes or consumption of any particular feeds; and thus, the mechanisms by which the epidemic "abortion storms" are triggered remain unclear. Annual ryegrass toxicity (ARGT) is a severe or fatal neurological disorder of livestock of Australia and South Africa. This disorder is caused by the ingestion of several plant genera (Lolium, Polypogon, and Agrostis) colonized by a nematode (Anguina sp.) and a bacterium, Rathayibacter toxicus. Corynetoxins (CTs) produced by R. toxicus contaminate plants which are in turn ingested by sheep and cattle, causing severe or often fatal hepatocerebral disorders in affected animals. We hypothesize that N. caninum-associated fetal deaths and abortions in cattle may be potentiated by pre-exposure of sublethal levels of CT-contaminated plants prior to N. caninum infection or reactivation of a latent infection. The exposure of sublethal CTs may not cause clinical diseases, but may increase susceptibility to pathogens such as N. caninum. Indeed, CT poisoning surviving pregnant sheep can have up to 10% abortions. The present study investigated whether animals pre-exposed to tunicamycins (TMs), a functional substitute for CTs, had a lowered resistance to sublethal experimental infection by N. caninum tachyzoites in mice. The results showed that sublethal doses of TMs or N. caninum alone did not cause significant deaths. Sublethal doses of N. caninum induced high mortality in TM-treated mice in a dose dependent manner. When mice were treated with a low dose of TMs (5 μg/mouse), as few as 2.5 × 10(6) tachyzoites were needed to induce more than 30% of mortality which is equivalent to the mortality rate caused by 40 × 10(6) tachyzoites. Spleen cells of mice treated with TMs had reduced (p<0.05) Neospora antigen-induced interferon-gamma (IFN-g) production and Con A-stimulated proliferation. The results suggest that CT contaminated plants, when exposed to animals under natural conditions, may contribute to lowered host resistance and increased N. caninum-associated fetal morbidity and mortality in affected animals.     

Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.     

Neosporosis in water buffalo (Bubalus bubalis) in Southern Italy

A study was carried on 1377 water buffalo serum samples from 50 farms in southern Italy to test the presence of Neospora caninum antibodies by indirect fluorescence antibody test (IFAT). Rabbit anti-buffalo immunoglobulins conjugated to fluorescein were used in the test. Fluorescence in sera dilutions above 1:200 was considered as indicative of the presence of N. caninum antibodies. The overall prevalence of infection in the animals was 34.6%. The prevalence increased in relation to the age of subjects and most of the herds examined (82%) were found infected. In two farms abortions and neurological signs were reported. No suppurative inflammatory lesions were seen, but few protozoan-like cysts were observed on foetal tissues by histology.     

Neospora-associated abortion episode over a 1-year period in a dairy herd in north-east Spain

This report describes a retrospective study based on serological screening, performed on a Neospora-associated abortion episode over a 12-month period in a dairy herd in north-east Spain. During the 1-year study period, the overall abortion rate for the herd was 23.2% (38 abortions of 164 diagnosed pregnancies). The data analysed were those derived from blood samples collected from the whole herd (n = 237) and from diagnosed pregnancies. Antibodies to Neospora caninum were found in 35.4% of the cattle with 44% of seropositive pregnant animals aborting over 1-year period. Based on the odds ratio, the risk of abortion was 12.2 times higher (P < 0.0001) in the Neospora-seropositive animals than in seronegative animals and significantly higher during the second term of gestation (P < 0.01) than during the first and third terms. Abortions were not found to be associated with parity status or season of pregnancy, and the common risk factors associated with pregnancy loss in the geographical area of the study. Age-related differences in N. caninum seroprevalence were not statistically significant, indicating vertical transmission as the main route of infection. Indeed, a high percentage of congenitally infected offspring was observed (90.6%) and the farm had been free of dogs for the last 7 years. Our results suggest that, when a dairy herd shows an increased incidence of abortions due to Neosporosis, maternal serology can be a good indicator of the abortion risk in individual cows, and that the effects of factors normally related to abortion, such as parity and pregnancy season, may be masked.     

Recent advances in Neospora and neosporosis.

Neospora caninum has emerged as a major cause of abortion in cattle in many countries. This paper reviews recent advances in the life cycle and biology of Neospora with the emphasis on bovine neosporosis. The role of the recently discovered oocyst stage of N. caninum in the epidemiology of neosporosis is discussed. Progress made in serologic diagnosis of N. caninum infection is discussed. There is no vaccine for preventing Neospora-induced abortions in cattle or to prevent oocyst shedding in dogs.     

Survey of nine abortifacient infectious agents in aborted bovine fetuses from dairy farms in Beijing, China, by PCR

Abortion in dairy cattle causes considerable economic losses to the dairy industry. Aborted fetuses and samples from the corresponding aborting dams from 12 dairy herds in Beijing were tested for 9 abortifacient infectious pathogens by PCR between 2008 and 2010. From a total of 80 abortion cases collected during this period, infectious agents were detected in 45 (56.3%) cases, 22 (48.9%) of which represented co-infections with two or three infectious agents. The detected pathogens included infectious bovine rhinotracheitis virus (36.3%) and Neospora caninum (31.3%), followed by bovine viral diarrhoea virus (7.5%), Brucella abortus (6.3%), Tritrichomonas foetus (5%) and Toxoplasma gondii (1.3%). Campylobacter fetus, Coxiella burnetii and Chlamydophila psittaci were not detected in any abortion case. Findings from this study indicated that infectious bovine rhinotracheitis virus and Neospora caninum were the main potential causes of abortions in Beijing dairy herds, whereas the bacterial pathogens were not, in contrast to reports from other countries. This is the first study to test nine abortifacient infectious agents by PCR at the same time, and it is also the first time to report the involvement of a variety of infectious agents in bovine abortion cases in China.     

Placental thrombosis in acute phase abortions during experimental Toxoplasma gondii infection in sheep

After oral administration of ewes during mid gestation with 2000 freshly prepared sporulated oocysts of T. gondii isolate M4, abortions occurred between days 7 and 11 in 91.6% of pregnant and infected ewes. Afterwards, a further infection was carried out at late gestation in another group of sheep with 500 sporulated oocysts. Abortions happened again between days 9 and 11 post infection (pi) in 58.3% of the infected ewes. Classically, abortions in natural and experimental ovine toxoplasmosis usually occur one month after infection. Few experimental studies have reported the so-called acute phase abortions as early as 7 to 14 days after oral inoculation of oocysts, and pyrexia was proposed to be responsible for abortion, although the underline mechanism was not elucidated. In the present study, all placentas analysed from ewes suffering acute phase abortions showed infarcts and thrombosis in the caruncullar villi of the placentomes and ischemic lesions (periventricular leukomalacia) in the brain of some foetuses. The parasite was identified by PCR in samples from some placentomes of only one sheep, and no antigen was detected by immunohistochemical labelling. These findings suggest that the vascular lesions found in the placenta, and the consequent hypoxic damage to the foetus, could be associated to the occurrence of acute phase abortions. Although the pathogenesis of these lesions remains to be determined, the infectious dose or virulence of the isolate may play a role in their development.     

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.