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.     

Evaluation of Neospora caninum and Toxoplasma gondii infections in alpaca (Vicugna pacos) and llama (Lama glama) aborted foetuses from Peru

The aim of this study was to investigate the participation of Neospora caninum and Toxoplasma gondii in abortion cases of Peruvian llamas and alpacas. Fifteen aborted foetuses were recovered from two main rearing areas of camelids in Peru (Central or South Andean region). Foetal histopathology was used to detect the presence of protozoal-associated lesions in target organs. N. caninum and T. gondii infections were confirmed by immunohistochemistry (IHC) combined with PCR and by PCR alone, respectively. The influence of the species (llama and alpaca), foetal age (first, second and third gestational periods) and geographical location (Central or South Andean region) of the foetuses was also studied. Thirteen of the samples (26%, 13/50) showed lesions suggestive of protozoal infection. N. caninum infection was detected by either IHC or specific PCR in 14 out of 50 foetuses (28%), of which 8 also showed protozoal-associated lesions. T. gondii DNA was not detected in any of the foetuses analysed. Protozoal infection was more frequent in the foetuses from the second gestational period (P<0.05, Fisher F-test). No significant association was observed between protozoal infection and species or geographical location (P>0.05, chi2 test). The results of the present study indicate that neosporosis should be included during the differential diagnosis of abortion in llamas and alpacas.     

Neosporosis as a cause of abortion in dairy cattle in Rio Grande do Sul, southern Brazil.

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.     

Seroepidemiology of neosporosis in dairy cattle in Uruguay

A seroepidemiological survey of Neospora caninum was done in a dairy farm in Uruguay employing indirect fluorescent antibody test (IFAT). Sera from 217 cattle (155 cows, 31 heifers and 31 calves) were tested for N. caninum antibodies in July 2000 and it was found that abortions were significantly associated with the seropositivity of the cows by chi(2)-test (P = 0.025). The cows with an IFAT titre of 1:3200 were at a significantly higher risk of abortion than seronegative cows (P = 0.0013). Neospora organisms were detected in the brains of two aborted foetuses by immunohistochemical examination. The infection of N. caninum was considered postnatal because of the even distribution of seropositive cows (60%), low seroprevalence (20%) in calves and no significant correlation in the serostatus between calves and their dams (P = 0.43). A fitted binomial generalised linear model revealed that there was an increasing risk of abortion with increasing IFAT titre and increasing age between 2 and 6 years.     

Abortions caused by protozoa in agricultural animals]

A review is given on abortions in livestock caused by Toxoplasma gondii, Neospora caninum and Sarcocystis spp. Special emphasis is put on diagnostic procedures. T. gondii is considered to be a major cause of abortions in sheep and goats. Diagnosis is based on the characteristic lesions in the cotyledons, histologic and immunohistochemical examination, and on the detection of specific antibodies in body fluids or serum of aborted fetuses. Whether abortions in swine due to T. gondii are of practical importance is still unclear. The most important causative protozoal agent of abortions in cattle is Neospora caninum. This protozoon was found in 19 per cent of cattle fetuses submitted for examination in USA. Occasionally it was found also in aborted lambs, kids and foals. Diagnosis is based on the histologic or immunohistochemical detection of the parasite. Abortions can be regularly induced experimentally in large and small ruminants and in pigs by the oral inoculation of sporocysts of certain Sarcocystis species. However, under natural conditions abortion due to Sarcocystis was diagnosed only occasionally and only in cattle and sheep. The histologic examination of the fetus and fetal membranes is the only way to diagnose Sarcocystis abortion or neonatal infection at present.     

Immunoglobulins and lesions in aborted bovine foetuses

IgG and IgM in serum from 164 aborted and 59 abattoir foetuses were measured by radial immuno-diffusion. Ig was detected in 133 (81.1%) aborted and 6 (10.2%) abattoir foetuses. The difference was statistically significant (P<0.01).

More than 20 mg/100 ml IgG was found in 90 (54.9%) aborted foetuses and 1 (1.7%) abattoir foetus. More than 20mg/100 ml IgM was present in 62 (37.8%) aborted foetuses and 2 (3.4%) abattoir foetuses. In each case the difference was statistically significant (P<0.01).

The mean concentration of IgG was 128 mg/100 ml in the aborted and 13 mg/100 ml in abattoir foetuses. The mean concentration of IgM was 34 mg/100 ml in aborted foetuses and 1.4 mg/100 ml in abattoir foetuses.

Tissues from 143 aborted foetuses were examined histologically. Serum from 86 of the 143 had more than 20 mg/100 ml IgG, IgM or both; of these, 79 (91.9%) had well defined or possible lesions. No lesions were detected in 7 (8.1%).

Serum from 32 of the 143 foetuses had 1 to 20 mg/100 ml IgG, IgM or both. Well-defined or possible lesions were seen in 24 (75.0%) of these. No lesions were detected in 8 (25.0%).

Serum from 25 of the 143 foetuses had no detectable Ig. Of these, 11 (44.0%) had well defined or possible lesions. No lesions were seen in 14 (56.0%).

If results of further investigation indicate the foetus does not frequently produce excess Ig in response to stimuli other than intrauterine infection, then measuring foetal Ig by RID may prove valuable as a first step in diagnosing infectious bovine abortion.     

First identification of Neospora caninum infection in aborted bovine foetuses in China

The first identification of Neospora caninum infection in the tissues of aborted bovine foetuses in China is reported. Aborted foetuses were collected from 16 dams, and 12 of the dams had high serum antibody titres to N. caninum determined using an ELISA test kit. The Nc-5 gene of N. caninum was amplified from DNA samples extracted from brains of four aborted foetuses using a Neospora-specific PCR assay, confirming N. caninum infection in the aborted foetuses. Histology and immunohistochemistry showed thick-walled (3 microm) tissue cyst in 25 microm diameter in the brain of one foetus. Non-suppurative encephalomyelitis, focal haemorrhage, hepatic lesions consisted of lymphocyte infiltration and haemorrhage were also found in the heart and lung of the foetus. Thus, we have confirmed for the first time the infection of N. caninum in aborted foetuses of cattle in the People's Republic of China.     

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.     

Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain

Eighty foetuses from some of the main cattle-producing regions in Spain were analysed to investigate the participation of Neospora caninum in cases of bovine abortion. Diagnosis of the infection was determined by histopathological analysis complemented with immunohistochemistry, serology (IFAT and ELISA) and PCR tests. A total of 38.8% of the bovine foetuses analysed were considered to be infected by at least one of the diagnostic techniques used. Microscopic lesions consistent with Neospora infection in brain were identified in 31.3% of the samples, whereas only 10.7 and 15.3% were positive using serological and PCR analysis, respectively. Perfect agreement was shown between IFAT and ELISA, although there was little agreement among results of the other diagnostic techniques. Gestational age of aborted foetuses checked ranged from <3 to 9 months, with a mean of 5.9 months, and no difference in age was found between infected and non-infected foetuses (P>0.05). This study confirms the importance of N. caninum as a cause of abortion in Spain and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted foetuses.     

Neospora abortion epidemic in a dairy herd

Neospora caninrm is a protozoan closely related to Toxoplasma gonclii. It was discovered only a few years ago, but it is now recognised as one of the most common and important causes of abortion in cattle. Brain lesions characteristic of Neospora infection have been found in about 25% of aborted foetuses submitted to diagnostic laboratories in New Zealand and overseas⁽¹⁾⁽²⁾. Average abortion rates due to Neospora on New Zealand farms have been estimated at 7% of pregnancies compared with 4% due to other causes⁽¹⁾. The upper range of abortion rates for Neospora was 17–30% compared to only 7–10% for other causes, showing that the economic effects of Neospora abortions on the more severely affected farms can be devastating.     

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.     

A survey of causes of bovine abortion occurring in the San Joaquin Valley, California

The causes of abortion in cattle in the San Joaquin Valley of California were surveyed from submissions to the California Veterinary Diagnostic Laboratory at Tulare. Four hundred sixty-eight abortion cases were examined. Most submissions (89%) were from large drylot dairies, milking an average of 814 cows. Abortion evaluations included necropsy, histopathology, bacteriology, virology, and other immunologic and serologic tests. A specific cause was identified in 29.5% of the abortions. Bacterial infections, most of which were sporadic, accounted for 16% of all abortions. Viral causes and protozoal infections were diagnosed in 5.6% and 3.2% of the abortions, respectively. Fetuses with protozoal infection had histologic lesions of focal nonsuppurative necrotizing encephalitis, and protozoa were detected. Similar histologic lesions were seen in 80 additional fetuses (17.1%), and although an etiologic agent was not identified for these cases, a protozoal infection was suspected.     

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy.Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.     

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.     

An abortion storm in cattle associated with neosporosis in Taiwan

An abortion storm associated with acute neosporosis involving 18 cattle was observed in a dairy farm in Taiwan. Aborted fetus age ranged from 3 to 8 months. Of the 38 cattle in that farm examined during the abortion storm, 52.6% (20/38), 13.2% (5/38) and 10.5% (4/38) contained both IgG and IgM, only IgG and only IgM antibodies to Neospora caninum, respectively. No antibody to N. caninum was detected prior to the abortion storm. Follow-up study conducted a year later showed that 23 out of 28 cattle had sero-converted. Since some cattle were positive to either only IgG or IgM, we suggest that both IgG and IgM should be tested for diagnosing neosporosis. Neosporosis surveillance of naive cattle herd is recommended.     

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.     

Neospora-like protozoan infection as a major cause of abortion in California dairy cattle

Ninety-five aborted bovine fetuses received from California dairies over a 4.5-year period had histologic lesions of focal encephalitis. Protozoa that reacted with Neospora caninum antiserum were detected in the brain of 88 of these fetuses and in the heart of 1 fetus. Sarcocystis spp schizonts were seen in the vascular endothelium of 1 fetus. It was concluded that a Neospora-like cyst-forming coccidian may be a major cause of abortion in California dairy cattle.     

Aetiology of bovine abortion in Switzerland from 1986 to 1995--a retrospective study with emphasis on detection of Neospora caninum and Toxoplasma gondii by PCR

In a retrospective study, covering the period from 1986 to 1995, tissues of aborted fetuses were re-examined. A total of 347 cases were tested immunohistochemically, among them samples of 223 brains were examined for Neospora caninum, Toxoplasma gondii and Bovine Virus Diarrhoea Virus (BVDV), and 249 placentae for Chlamydiaceae. Two real-time PCR assays, one for N. caninum, and one for T. gondii, were developed. These potential abortion-inducing agents were detected - and confirmed by PCR, except for BVDV - in 16.1% (N. caninum), 0% (T. gondii), 9.9% (BVDV) and 0.8% (Chlamydiales) of the cases examined. Immunohistochemistry proved to be inadequate for the detection of the protozoal epitopes, whereas it was confirmed as a very useful tool for the detection of BVDV. In abortion material, PCR is considered to be more suitable for the detection of protozoa and Chlamydophila abortus, an adequate sampling presupposed.     

Evaluation of ovine abortion associated with Toxoplasma gondii in Spain by different diagnostic techniques

A total of 173 aborted ovine foetuses and seven aborted caprine foetuses, submitted from different points of north and central Spain, were analysed to determine the role of T. gondii in abortion and to compare the utility of the most widely used techniques in diagnosis of the congenital infection (histopathology, serology--IFAT and ELISA--and a nested-PCR). Parasite infection was diagnosed in 40 (23.1%; n = 173) ovine foetuses by at least one of the diagnostic techniques used. A higher percentage of foetuses were diagnosed using serological techniques (IFAT and ELISA) (28.3%; n = 106) than by histologic examination (8.7%; n = 173) or PCR (6.9%; n = 173). No significant association between infection and the foetal age categories was found (P > 0.05). In this study, 106 aborted foetuses were analysed by all of the three diagnostic techniques. When we compared serological results, perfect agreement between ELISA and IFAT was obtained. On the contrary, slight to fair agreements were observed when histology results were compared with those obtained by serology and PCR techniques. All the positive foetuses were aborted in the mid (60%) or last (40%) term of pregnancy, but no significant differences were found between ages of the infected and non-infected foetuses (P > 0.05). This report indicates that toxoplasmosis may be a common cause of small ruminant abortion and neonatal death in Spain and points out the necessity of using different and complementary techniques to increase the probability of detecting Toxoplasma infection in an aborted foetus.