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.     

Survey of ovine and caprine toxoplasmosis by IFAT and PCR assays in Sardinia, Italy

During the period 1999-2002, we have analyzed 9639 serum samples and 815 aborted samples (670 fetuses and 145 placenta) from 964 ovine and caprine farms distributed over all Sardinia island. After abortion notification, sera collected at random from adult animals were examined to detect simultaneously IgG and IgM antibodies specific to Toxoplasma gondii by indirect immunofluorescence assay, whereas fetuses and placenta were analyzed by a single tube nested PCR assay. Specific IgG antibodies were detected in 2048 (28.4%) sheep and 302 (12.3%) goats, specific IgM antibodies were found in 652 (9%) sheep and 139 (5.6%) goats. From a total of 2471 ovine and 362 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 271 (11.1%) ovine and 23 (6.4%) caprine samples were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. On the one hand, these results indicate that the seroprevalence of T. gondii infection in sheep and goats is relatively high, on the other PCR results demonstrate that T. gondii has a significant role in ovine and caprine abortion. Adequate management might be useful and essential to control the toxoplasmosis in the sheep and goats herds of Sardinia.     

Abortion in small ruminants in the Netherlands between 2006 and 2011

During five successive lambing seasons between 2006 and 2011, 453 submissions of abortion material, 282 of ovine and 171 of caprine origin, were examined at the Animal Health Service in the Netherlands. Infectious agents as the most plausible cause of the abortion were found in 48 percent of the ovine submissions and in 34 percent of the caprine submissions. Submission of both aborted fetus and placental membranes increased the diagnostic yield of laboratory investigations (17 percent and 21 percent for ovine and caprine submissions, respectively). The main infectious causes of abortion in sheep were Chlamydia abortus, Campylobacter spp., Toxoplasma gondii, Listeria spp., and Yersinia pseudotuberculosis. The main infectious causes of abortion in goats were Coxiella burnetii, Chlamydia abortus, Listeria spp., Toxoplasma gondii, and Campylobacter spp. In 42 percent of the ovine and in 56 percent of the caprine submissions a causal agent was not identified. Furthermore, in 12 percent of the ovine and 10 percent of the caprine submissions evidence of placentitis, indicative of an infectious cause of the abortion, was found, but no infectious agent was identified. Most infectious causes of ovine and caprine abortion have zoonotic potential. Humans, especially pregnant women, who are in close contact with lambing sheep or goats should be aware of the importance of precautionary hygiene measures.     

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.     

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.     

Role of Chlamydophila abortus in ovine and caprine abortion in Sardinia, Italy

Between 1999-2003, 14321 sera and 646 abortion samples (498 foetuses and 148 placentae) were analysed from 807 sheep and goat farms distributed all over the island of Sardinia. After notification of abortion in a flock, sera collected at random from adult animals were examined to detect antibodies specific to Chlamydophila (C.) abortus by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 611 (4.8%) sheep and 106 (5.8%) goats. From a total of 2050 ovine and 151 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 29 (1.4%) ovine and 1 (0.6%) caprine samples were C. abortus PCR-positive. Placenta was the tissue with the highest detection rate. These results indicate that the seroprevalence of C. abortus infection in sheep and goats is very low in Sardinia, and PCR results demonstrate that C. abortus has no significant role in abortion, especially in goats.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

Molecular detection of Coxiella burnetii and Neospora caninum in equine aborted foetuses and neonates

Abortion, stillbirth and neonatal death are major causes of equine mortality and cause severe economic loss to the equine industry. The present study was based on a complete necropsy protocol associated with classical microbiological examinations and molecular biology on 407 cases of abortion, stillbirths and neonate death. Based on this retrospective survey, "less common" abortive infectious agents were characterised by molecular tools in nine independent cases of abortion or neonate mortality. Among others, Chlamydophila abortus (1 case), Coxiella burnetii (6 cases) and Neospora caninum (3 cases) were detected by real-time PCR; one of these samples being co-infected by N. caninum and C. burnetii. DNA detection of this latter bacterium is reported here for the first time in equine abortion samples. C. burnetii should, along with other common pathogens, probably be taken into account in equine abortion.     

Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004

The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.     

Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions

Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

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.     

Occurrence, distribution, and role in abortion of Coxiella burnetii in sheep and goats in Sardinia, Italy

Between 1999 and 2002, 9349 sera and 517 aborted samples (422 foetuses and 95 placenta) were analysed from 675 sheep and 82 goat farms distributed all over the island of Sardinia. After abortion notification, sera collected at random from adult animals were examined to detect antibodies specific to Coxiella burnetii by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 255 (38%) sheep farms and in 39 (47%) goat herds whereas 40 ovine (10%) and 3 (6%) caprine foetuses were C. burnetii PCR-positive. Although C. burnetii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. Seroprevalence analysis indicates that C. burnetii distribution in sheep and goats is very high, but PCR results demonstrate that C. burnetii has a relatively low role in abortion, especially in goats.     

Toxoplasma gondii and Chlamydophila abortus in caprine abortions in Tobago: a sero-epidemiological study

A sero-epidemiological study was conducted on a goat farm that experienced an abortion epidemic in the 2005 breeding season in Tobago. Serum samples of goats (aborting and non-aborting) and cats were collected, in addition to the use of stored sera from the farm sampled in 2003 and 2004. Farm records on the reproductive and mortality rates for year 2003, 2004 and 2005 were also reviewed. The sera were screened for Toxoplasma gondii antibodies using the latex agglutination test (LAT), Chlamydophila abortus with an enzyme-linked immunosorbent assay (ELISA) and Brucella abortus using the buffered plate agglutination test (BPAT). Farm records revealed that for the period 2003-2005, the average kid per doe rate decreased from 2.1 to 1.5, the mortality rate increased from 6.3% in 2002 to 19.4% in 2004 and the fertility rate decreased from 98-99% (2002-2004) to 89% (2005). There was a dramatic increase in the abortion rate from <1% (2002, 2003 and 2004) to 29.2% (2005). Of a total of 161 sera tested comprising 12 from 2003, 89 from 2004 and 70 from 2005, 0 (0.0%), 21 (23.6%) and 45 (64.3%) were positive for T. gondii agglutinins (i.e. titres > or =1 : 64) and the differences were statistically significant (P < 0.05; chi(2)). Of all serum samples tested, only 1 (1.1%) of 89 from 2004 was positive for C. abortus while all the sera tested were negative for B. abortus. Amongst the 24 does which aborted in 2005 and were available for testing in mid-2005, 15 (62.5%) had reciprocal titres of > or =1 : 2048, three (12.5%) each had titres of 1 : 1024, 1 : 256 and < or =1 : 16 i.e. negative. The seroprevalence and titres of does that aborted, 20 (87.0%) of 23, all with titres > or =1 : 256 suggesting current infection, were statistically significantly (P < 0.05; chi(2)) higher than was detected amongst does that delivered normal kids, 25 (53.25) of 47 with 22 (48.8%) having titres of > or =1 : 256. One (50.0%) of two cats caught and tested was seropositive with a reciprocal titre of 128. This is considered the first documentation of T. gondii agglutinins in caprine abortion as well the detection of C. abortus antibodies from livestock in Trinidad. It is concluded that of the three zoonotic abortifacient pathogens tested for, T. gondii appeared to have played some aetiological role in the abortion epidemic investigated.     

Abortion in humans caused by Chlamydophila abortus (Chlamydia psittaci serovar 1)

On a farm housing cattle and goats an abortion storm occurred affecting 50% of the goats during the lambing season 2000/2001. In one of three investigated caprine abortions Chlamydophila abortus could be identified as etiology. During this time a pregnant woman (pregnancy week 19/20) had contact with aborting goats. She developed a severe generalized infection and aborted. The placenta contained Chlamydophila abortus shown by immunohistochemistry and PCR. Aim of the present case report is to alert veterinarians about the potential zoonotic risk of ovine/caprine abortions.     

Application of touchdown enzyme time release (TETR)-PCR for diagnosis of Chlamydophila abortus infection

Chlamydophila abortus-DNA was detected using a touchdown enzyme time-release (TETR)-polymerase chain reaction (PCR) assay as an improved test for sensitive and rapid diagnosis of abortion in small ruminants. Two hundred and fifty two placentae, liver or spleen tissue samples from aborting ewes and goats or aborted lambs and kids in which C. abortus infection was suspected were examined by TETR-PCR and the results were compared with cell culture. Sixty-five tissue samples were found to be TETR-PCR positive while only 56 samples were cell culture-positive. After resolution of discrepant samples with a confirmatory nested PCR assay, TETR-PCR had a sensitivity of 97% and a specificity of 99.5% while culture had a sensitivity of 84.8% and a specificity of 100%. The analytical sensitivity of the TETR-PCR assay was determined with DNA extracted from 4-fold serial dilution of C. abortus B577 culture and found to be 0.25 inclusion-forming unit per PCR. No reduction in the analytical sensitivity was noted when the assay was tested with mouse liver samples spiked with 4-fold serial dilution of C. abortus B577 culture. No target product was amplified when DNA from Chlamydophila pecorum was tested. TETR-PCR used in this study is a practical, rapid, sensitive and specific assay that could be used for the detection of C. abortus in infected tissue samples. We recommend the use of this assay as a supplemental diagnostic tool for detection of C. abortus in infected tissue samples.     

Simple technique for the direct isolation of toxoplasma tissue cysts from fetal ovine brain

A simple technique is described for the isolation of Toxoplasma gondii tissue cysts from fetal ovine brain by centrifugation on a discontinuous density gradient of 30 per cent and 90 per cent Percoll (colloidal silica solution). Brain samples from 51 aborted ovine fetuses were examined by both the Percoll and mouse inoculation techniques; eight infections were detected by the Percoll technique compared to 12 by mouse inoculation. Possible reasons for this discrepancy and the scope for improving the Percoll technique are discussed.     

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.     

Toxoplasmosis and other causes of abortions in sheep from north central United States

Between 1983 and 1989, we examined 1,201 aborted fetuses and dead lambs from the north central United States. Toxoplasmosis was diagnosed in 17.5%, campylobacteriosis in 9.9%, chlamydiosis in 4.7%, and miscellaneous infections in 15.1%. Inflammatory lesions suggestive of infectious causes were seen in 13%. Noninfectious causes were identified in 6.1%, and a diagnosis was not reached in 33.3%. An agglutination test was used to detect Toxoplasma gondii-specific antibodies in ovine fluids. Toxoplasma gondii antibodies were detected in 223 of 1,064 (20.9%) fluids from fetuses and dead lambs. Of 201 seropostive (greater than or equal to 16) fetuses and lambs, T gondii antibody titers (reciprocal) were 16 (21 fetuses and lambs), 32 (10 fetuses and lambs), 64 (2 fetuses and lambs), 128 (7 fetuses and lambs), 256 (9 fetuses and lambs), 512 (5 fetuses and lambs), 1,024 (15 fetuses and lambs), 2,048 (13 fetuses and lambs), 4,096 (13 fetuses and lambs), 8,196 (13 fetuses and lambs), 16,392 (19 fetuses and lambs), and greater than or equal to 32,784 (74 fetuses and lambs).     

Comparison of enzyme-linked immunosorbent assay, indirect fluorescent antibody test, and direct agglutination test for detecting Toxoplasma gondii antibodies in naturally aborted ovine fetuses

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.