Ovine and Bovine Congenital Abnormalities Associated With Intrauterine Infection With Schmallenberg Virus.

In December 2011, a previously unknown congenital syndrome of arthrogryposis and hydranencephaly in sheep and cattle appeared in the Netherlands as an emerging epizootic due to Schmallenberg virus (SBV). Gross lesions in 102 lambs and 204 calves included porencephaly, hydranencephaly, cerebellar dysplasia and dysplasia of the brainstem and spinal cord, a flattened skull with brachygnathia inferior, arthrogryposis, and vertebral column malformations. Microscopic lesions in the central nervous system showed rarefaction and cavitation in the white matter, as well as degeneration, necrosis, and loss of neurons in the gray matter. Brain and spinal cord lesions were more severe in lambs than in calves. Ovine and bovine cases examined early in the outbreak showed encephalomyelitis. SBV infection was confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in brain samples in 46 of 102 lambs (45%) and in 32 of 204 calves (16%). Immunohistochemistry, performed on tissue samples from 18 RT-qPCR-positive lambs, confirmed the presence of bunyaviral antigen in neurons of the brain in 16 cases. SBV antibodies were detected by enzyme-linked immunosorbent assay in fetal blood in 56 of 61 sampled ovine cases (92%). In a virus neutralization test, all tested dams of affected newborns, 46 ewes and 190 cows, were seropositive. Compared with other teratogenic viral infections, the pathogenesis and lesions of SBV in sheep and cattle fetuses are similar to those of other ruminant orthobunyaviruses. However, the loss of spinal ventral motor neurons and their tracts, resulting in micromyelia, distinguishes SBV infection from other viral central nervous system lesions in newborn ruminants.

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.

Prevalence of Coxiella burnetii infections in aborted fetuses and stillborn calves.

Coxiella burnetii infections are mostly subclinical in cattle, but can occasionally be associated with abortion. In the present study, 100 aborted fetuses or stillborn calves that were submitted for postmortem examination between September 2007 and March 2008 were examined for infection with C burnetii. Samples of both pooled fetal tissues and placental cotyledon were tested using a real-time PCR assay. In addition, the sections of placental cotyledon were examined using immunohistochemistry (IHC). The IHC of four placentas was positive. The PCR results of the IHC-positive placentas were high positive (HP); the PCR results of the organs of these four fetuses and calves varied from low positive (LP) to HP. The four IHC-positive fetuses had a gestation length of seven to nine months. All four placentas had histological signs of inflammation, but only one of four placentas had gross pathological signs of inflammation possibly due to a concomitant infection with Bacillus licheniformis. Five other IHC-negative placentas had (high) positive PCR results; the PCR results of the organs of these fetuses were LP or negative. The present study indicates that C burnetii infections are detected in a limited percentage of aborted fetuses and stillborn calves by IHC. To assess the importance of placentas with PCR-positive and IHC-negative test results, more research is needed.

Epizootic congenital hydranencephaly and abortion in cattle due to bluetongue virus serotype 8 in the Netherlands.

An outbreak of hydranencephaly in aborted foetuses and newborn calves occurred following the 2007 epidemic of bluetongue serotype 8 (BTV8\net2006) in the Netherlands. In total 35 aborted foetuses and 20 live-born calves, submitted from September 2007 to May 2008, were examined pathologically. Foetuses with gestational ages between 4 and 9 months (mean 6.8 month) showed varying stages of cerebral malformation. Initial stages were cavitations in the cerebral hemispheres with massive destruction of neuroparenchyma, calcium deposits, and a phagocytic inflammatory response. Later stages showed distinct hydranencephaly, the cerebral hemispheres being almost completely replaced by fluid-filled sacs. In seven cases the cerebellum was affected as well, but brainstem structures were intact. Newborn calves with clinical signs of abnormal behaviour ('dummy calves'), circling, head pressing, incoordination, and blindness were seen from the end of January 2008. The calves were born between 2nd January and 16th March 2008. The calves were euthanized after 1 day up to 14 weeks (mean 4-7 weeks). Brain malformations in these calves were confined to the cerebrum and consisted of varying degrees of hydranencephaly. Spleen tissue was PCR-positive for bluetongue virus (BTV) in 21 of 35 foetuses and in 1 of 20 calves. A higher percentage of PCR-positives was found in foetuses aborted in early gestation than in late gestation, suggesting clearance of BTV during gestation. Fifteen of 33 dams of PCR-negative hydranencephalic foetuses or calves could be traced and all were BTV-seropositive, indicating a previous BTV infection. The timing of hydranencephaly cases in live-born calves during the first months of 2008 was consistent with infection in early gestation during the prior transmission season. Vertical transmission and teratogenic potential have previously been described for modified-live vaccines for bluetongue but are highly unusual for field strains of BTV, which raises the issue whether BTV8\net2006 or its ancestor has been cell- or laboratory-adapted in the past.

Fatal intestinal coccidiosis in a three week old buffalo calf (Bubalus bubalus).

The water buffalo (Bubalus bubalus) is important to the economy of several countries, especially in Asia and Brazil. Little is known of the impact of coccidiosis in buffaloes. Cattle and buffaloes are considered to have common species of Eimeria but critical cross transmissions have not been made because it is difficult to raise these hosts coccidian free. Clinical coccidiosis was confirmed post mortem in a 22-day old buffalo calf that died after a 3-4 day illness. Oocysts morphologically identical to Eimeria bareillyi were found in the feces and in sections of small intestine. Oocysts were often pyriform, sometimes with asymmetrical sides. The shorter end was flattened and approximately 5-6 microm wide. Unsporulated oocysts in feces were 23.2-29.5 x 16.5-22 microm in size with an average of 27.2 x 19.3 microm . Schizonts, gamonts, and oocysts were identified in sections of small intestine and they were located in entrocytes of jejunum and ileum. No coccidian stages were seen in sections of colon. This is one of the first confirmed cases of clinical coccidiosis in water buffalo.

Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection.

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.

[Abortion and stillbirth among dairy goats as a consequence of Coxiella burnetii]. / Abortus en doodgeboorte op melkgeitenbedrijven ten gevolge van Coxiella burnetii.

The clinical signs and pathological findings of clinical Q fever in dairy goats in the Netherlands are described. The disease is characterized by endemic or epidemic abortion in late pregnancy, stillbirth, or the birth of weak kids. These consequences of Coxiella burnetii infection appear to have increased during recent years in the Dutch dairy goat population. Detection of the agent in placental tissue by immunohistochemistry has proven diagnostically useful.

[Four milk cows dead by electrocution]. / Vier melkkoeien dood door elektrocutie.

Two dairy cows were found dead in their cubicles and a third was found lying dazed in a stall but the animal stood up 5 minutes later. Two days later; two other cows were found dead in the same row of cubicles. Death was due to electrocution caused by an electric wire in a steel pipe connected to front of the cubicles. Post-mortem findings supported this diagnosis.

Eosinophilic myositis due to Sarcocystis hominis in a beef cow.

A case of eosinophilic myositis (EM) in an 8-year-old beef cow was investigated. The animal originated from a herd in which a high incidence of the disease had been observed in slaughtered adult females over a period of 2 years. Histologically, the lesions in the muscles were characterized as granulomas with a central core of degenerate eosinophils and remnants of necrotic muscle fibres, surrounded by a rim of epithelioid cells and fibrous tissue with an infiltrate consisting predominantly of eosinophils radiating outwards. Degenerate sarcocysts with a thick (7-9 microm) wall were present in the suppurative centre of most lesions. Intact sarcocysts with similar morphology were present in adjacent muscle fibres but without an associated inflammatory reaction. By transmission electron microscopy the sarcocysts were identified as Sarcocystis hominis, based on the morphology of villar protrusions of the sarcocyst wall, which were broad-based and cylindrical, with a blunt distal end, and contained numerous long microfilaments. Circumstantial evidence indicated a human source of infection, human faecal material having been spread on the pasture grazed by the cattle. The findings supported a causal relationship between S. hominis infection and EM in cattle.

[Suckling calves with symptoms of swayback]. / Het beeld van swayback bij zoogkalveren.

Within a 1-year period, three calves from the same herd developed ataxia of the hind limbs and urinary incontinence at about 6 months of age. Signs progressed and the calves were slaughtered 1-8 months after the onset of signs. The calves belonged to a suckling beef herd of 35 cattle. Blood samples from 11 cattle of different ages were collected and glutathione peroxidase and copper levels were measured. Glutathione peroxidase levels were below the normal range in all cattle and copper levels were below the normal range in 7 of ll cattle. Pathological examination of an affected calf revealed a Wallerian type of degeneration of myelinated nerve fibres in the lateral and ventral spinal cord tracts. In addition, the hepatic copper content was very low. Copper deficiency is a well-known cause of swayback in young sheep and goats. To our knowledge, this is the first report of a possible association between a swayback-like syndrome and copper deficiency in calves.

Pathogenesis of bovine neosporosis.

The protozoan parasite Neospora caninum is a major pathogen of cattle and dogs, being a significant cause of abortion in cattle in many countries. It is one of the most efficiently transmitted parasites, with up to 90% of cattle infected in some herds. The pathogenesis of abortion due to Neospora is complex and only partially understood. Losses occur after a primary infection during pregnancy but more commonly as the result of recrudescence of a persistent infection during pregnancy. Parasitaemia is followed by invasion of the placenta and fetus. It is suggested that abortion occurs when primary parasite-induced placental damage jeopardises fetal survival directly or causes release of maternal prostaglandins that in turn cause luteolysis and abortion. Fetal damage may also occur due to primary tissue damage caused by the multiplication of N. caninum in the fetus or due to insufficient oxygen/nutrition, secondary to placental damage. In addition, maternal immune expulsion of the fetus may occur associated with maternal placental inflammation and the release of maternal pro-inflammatory cytokines in the placenta. Thus N. caninum is a primary pathogen capable of causing abortion either through maternal placental inflammation, maternal and fetal placental necrosis, fetal damage, or a combination of all three. The question of how N. caninum kills the fetus exposes the complex and finely balanced biological processes that have evolved to permit bovine and other mammalian pregnancies to occur. Defining these immunological mechanisms will shed light on potential methods of control of bovine neosporosis and enrich our understanding of the continuity of mammalian and protozoal survival.

Supranational comparison of Neospora caninum seroprevalences in cattle in Germany, The Netherlands, Spain and Sweden.

Herd, within-herd and animal prevalences for Neospora caninum in beef and dairy cattle were compared between four countries. In randomly selected herds from regions of Germany, The Netherlands, Spain and Sweden that were representative for the cattle production of these countries, all animals > or = 2 years were examined serologically by enzyme-linked immunosorbent assays (ELISAs) with high test specificity (> 98.0%). In a previous study, the ELISAs had been validated against each other. Single reacting animals within a herd were confirmed by immunobloting. At the time of sampling, animal (age, breed, herdtype, sex, lactation stage) and herd data (region) were collected. Considerable differences in N. caninum herd, within-herd, and overall animal prevalence estimations were observed between countries, regions, herdtype, age categories and breeds. Herd prevalences, based on confirmation of single reactors, for dairy herds were estimated to be 16% (95%CI: 10-24%) in Sweden, 49% (95%CI: 39-59%) in Germany, 63% (95%CI: 57-69%) in Spain and 76% (95%CI: 67-84%) in The Netherlands and for beef herds 41% (95%CI: 31-50%) in Germany, 46% (95%CI: 41-51%) in Spain and 61% (95%CI: 50-72%) in The Netherlands. No beef herds were examined in Sweden. The lowest animal true prevalence was estimated in dairy cattle in Sweden (0.5% (95%CI: 0.1-0.8%)) while the highest animal true prevalence was estimated for dairy cattle in Spain (16.2% (95%CI: 14.9-17.5%)). Within-herd prevalences varied greatly, with very few farms in Sweden having more than 10% seropositive animals while in Spain more than 10% of the herds had within-herd prevalences between 50 and 100%. Seropositivity was significantly associated with herdtype (beef versus dairy), age, breed and region within countries. The results of this supranational comparative study showed that the importance of N. caninum infection varied greatly within in Europe. Estimates of prevalence can be used to calculate the economic impact of N. caninum infection as well as to evaluate the effect of prevention and control strategies over time.

Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk.

Three ELISAs for the detection of antibodies against Neospora caninum in bulk milk were evaluated in 162 Dutch dairy herds. The first ELISA was the Dutch Animal Health Service (AHS) in-house ELISA, developed from the routine in-house serum ELISA. The other two ELISAs were commercial milk ELISAs from IDEXX and LSI. Blood samples of all lactating cows in 162 dairy herds were tested using the AHS in-house serum ELISA. Based on previous studies in the Netherlands a within-herd N. caninum seroprevalence of 15% was associated with increased risk for reproductive losses. This percentage was therefore used as positive seroprevalence cut-off value. Repeatability of the ELISAs was evaluated by testing on three different days. The AHS in-house ELISA lacked specificity, probably due to use of a different batch of antigen on the second and third test-day. Cut-off values were determined using misclassification costs term calculations. At cut-off values 0.6 for the IDEXX and 0.2 for the LSI, a herd sensitivity of 61% (95% CI: 49--73%) and 47% (95% CI: 35--60%) was estimated. Herd specificity at these cut-off values was 92% (95% CI: 87--98%) for the IDEXX and 94% (95% CI: 90--99%) for the LSI ELISA. The positive and negative predictive values were 84% (95% CI: 68--100%) and 86% (95% CI: 79--94%) for the IDEXX ELISA, and 85% (95% CI: 67--100%) and 82% (95% CI: 74--90%) for the LSI ELISA. The agreement between all possible combinations of test-days was expressed by kappa values. These were found to be slightly higher for the IDEXX than for the LSI ELISA. It is concluded that both commercial ELISAs performed satisfactorily to detect a within-herd seroprevalence of N. caninum in lactating cows of at least 15%.

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues.

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.

Comparison and standardisation of serological methods for the diagnosis of Neospora caninum infection in bovines.

Various existing serological tests were compared with a standard panel of 523 sera in a multicentred study across Europe. Well characterised sera from animals that were experimentally or naturally infected with Neospora caninum as well as sera from cattle deemed uninfected with N. caninum were provided by the participants of the study and analysed in several commercial (CHEKIT Dr. Bommeli/Intervet, CIVTEST BOVIS NEOSPORA Hipra, Cypress Diagnostics C.V., Herd Check IDEXX, Mastazyme MAST Diagnostics, P38-ELISA Animal Welfare and Food Safety GmbH (AFOSA)) as well as in-house assays (five ELISAs and one IFAT). Most tests showed a high level of agreement in the interpretation of the test results (positive or negative). A further distinct increase in agreement between tests was obtained after the application of standardised cut-offs offered by a two-graph receiver operating characteristic analysis. This procedure allows a standardised interpretation of results obtained with different tests used in independent, parallel seroepidemiological studies.

Evaluation of a single serological screening of dairy herds for Neospora caninum antibodies.

Twenty-one dairy herds with a history of Neospora caninum-associated abortions were used for a longitudinal serological study. A total of 1,676 animals were blood sampled 3 times and used to evaluate a single serological screening for N. caninum antibodies. The results of the first serological screening were compared with the results based on three consecutive samples, whereby two or more positive or negative test results per animal were considered to determine its serological status as positive or negative, respectively. In both test regimes 95.3% of the animals had the same interpretation, of which 33.9% were seropositive, and 61.3% seronegative. Relative sensitivity of one-time sampling compared to three consecutive samplings was 94.7%, while relative specificity was 95.6%. Relative specificity differed between herds. Predictive values positive and negative of one-time sampling were 92.4 and 97%, respectively. The agreement between one-time sampling and three consecutive samplings, kappa, was 0.90. For evaluation of discrepant results age distribution and pedigree data were used to provide clues regarding likelihood of transmission. Age clustering of seropositive animals was interpreted to indicate a point source infection. Daughter-mother relationships were used for the interpretation of congenital infections. The proportion of congenital infection decreased with increasing parity of the mother. Seropositive heifers had 80% congenitally infected offspring, while in older cows 66% of the offspring was congenitally infected, possibly due an increased immunity to transplacental infection with age. It is concluded that a single serological screening of a whole herd in connection with an analysis of age distribution and pedigree data is a rapid and valid method to interpret the serologic status of individual animals and to study the mode of transmission of N. caninum.

A high rate of seroconversion for Neospora caninum in a dairy herd without an obvious increased incidence of abortions.

The purpose of this paper is to investigate the high rate of seroconversion for Neospora caninum in a dairy herd. Forty-five of 95 (47%) of the seronegative animals seroconverted within a period of 6 months. Seropositive animals were not equally distributed among age-groups. A high seroprevalence age-group of 8-30 months was housed together during a period of 4 months, indicating a point source infection within this period. A lack of association between the serological status of daughters and mothers also indicated horizontal transmission of the infection. Low avidity indices in the seroconverted animals confirmed a recent infection in this herd. However, there was no increased abortion rate in connection with the seroconversion, which indicates that mass seroconversions in dairy herds may remain unnoticed. There was circumstantial evidence that the farm dog played a role in the transmission of the infection to the cattle. However, also evidence was found for an unexplained ongoing horizontal transmission after the initial point source exposure.