Dominique Cassart

Comparative pathogenesis of acute and latent infections of calves with bovine herpesvirus types 1 and 5

Summary. This study was conducted to compare the pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 (BHV-1) and 5 (BHV-5) in their natural host. Two groups of eight calves were inoculated intranasally with BHV-1 or BHV-5. Although BHV-1 and BHV-5 similarly replicate in the nasal mucosa after inoculation, both viruses differ markedly in their ability to cause disease, BHV-5 being responsible of some fatal encephalitis while BHV-1 inducing rhinotracheitis. Virus isolation and immunohistochemistry demonstrated that BHV-5 replicates extensively in neurons of the central nervous system (CNS) and in respiratory cells of lungs, tracheal and nasal mucosae. Invasion of the CNS likely occurs through the trigeminal and olfactory pathways. Both groups developed cross-neutralising antibodies during this experiment suggesting partial clinical cross-protection afforded by the two infections. Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves. Moreover, laboratory findings suggested that BHV-5 could also persist in the tracheal and nasal mucosae. These results indicate that, after primary infection, BHV-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BHV-1 infection.

Schmallenberg virus: a new Shamonda/Sathuperi-like virus on the rise in Europe

In the summer-fall of 2011, a nonspecific febrile syndrome characterized by hyperthermia, drop in milk production and watery diarrhea was reported in adult dairy cows from a series of farms located in North-West Europe. Further, in November 2011, an enzootic outbreak of abortion, stillbirth and birth at term of lambs, kids and calves with neurologic signs and/or head, spine or limb malformations emerged throughout several European countries. Both syndromes were associated with the presence in the blood (adults) or in the central nervous system (newborns) of the genome of a new Shamonda-Sathuperi reassortant orthobunyavirus provisionally named Schmallenberg virus after the place where the first positive samples were collected. The clinical, pathological, virological and epidemiological facts that were made publicly available during the first 6 months after the emergence are presented here. Current knowledge of the epidemiology of the phylogenetically closest relatives of the newcomer (Shamonda, Sathuperi, Aino and Akabane viruses) is not exhaustive enough to predict whether the current outbreak of Schmallenberg virus is the prelude to endemicity or to a 2 years long outbreak before the infection burns out when serologically naïve animals are no longer available. In the future, cyclic epizootic reemergences are a possibility too, either synchronized with a global decrease of herd immunity or due to antigenic variants escaping the immunity acquired against their predecessors. The latter hypothesis seems unlikely because of the wide array of biologic constraints acting on the genome of viruses whose life cycle requires transmission by a vector, which represses genetic drift. The remarkable stability of the Shamonda virus genome over the last forty years is reassuring in this regard.

Schmallenberg Virus in Calf Born at Term with Porencephaly, Belgium

To the Editor: From the end of August through the end of October 2011, a clinical syndrome involving adult cattle and the fetuses of pregnant cows emerged in the border area between the Netherlands and North Rhine-Westphalia, Germany (1). The syndrome was characterized by nonspecific clinical signs (fever, decreased milk production), severe diarrhea, and some abortions. A metagenomic analysis was conducted on pooled samples from cattle with acute signs on a farm in the city of Schmallenberg, Germany. The analysis detected nucleotide sequences homologous to arthropod-borne Akabane, Aino, and Shamonda viruses, all belonging to the family Bunyaviridae, genus Orthobunyavirus, and Simbu serogroup (1). Real-time PCR detected the genomic RNA of the new and emerging virus, tentatively designated Schmallenberg virus (SBV), in the blood of adult cattle, abdominal fluid of a stillborn calf, and brains of lambs born with birth defects on dozens of farms in the Netherlands, Germany, and Belgium. No data are yet available to predict how the emerging virus might affect the cattle industry. We report the case of a 1-week old calf with severe central nervous system (CNS) lesions probably caused by in utero infection with the new virus. In Belgium in January 2012, a Belgian Blue multiparous cow gave birth to a 45-kg female calf that was morphologically normal but hypertonic and hyperreflexic. Pregnancy had proceeded uneventfully and lasted 9 months and 4 days. Spontaneous reflexes such as sucking, swallowing, micturition, defecation, and crying were completely preserved, but the calf was unable to stand, and its consciousness alternated from mild to severe depression. It was obviously blind and showed ventrolateral strabismus, but the pupils functioned normally. Muscle tone was permanently increased, as indicated by tetanus-like erection of the ears and by a violent but brief startle response to the slightest acoustic or tactile stimulation (Figure). When the calf was placed upright, loss of conscious proprioception was obvious; it maintained its position only a few seconds before collapsing. Altogether, the clinical signs suggested severe dysfunctions of the cerebral cortex, basal ganglia, and mesencephalon. The calf drank from a bottle twice a day for a week, but then was euthanized for humane reasons (infected decubital ulcers). Figure A 7-day old, female, Schmallenberg virus–positive calf showing severe central nervous system dysfunctions (A–C) and lesions (D–E). A) Spontaneously lying down; B–C) standing with assistance; D–G) porcencephaly, ... At necropsy, the cerebellum, brainstem, and diencephalon appeared normal in shape and volume (Figure). However, the cerebral hemispheres were replaced by 2 thin-walled, fluid-filled cysts with some floating islets and peninsulae corresponding to preserved cortex. There was variable preservation of the cerebrum, total liquefaction of occipital lobes, and irregular preservation of the outer layers of some parts of the temporal and frontal lobes. Altogether, the picture was compatible with severe porencephaly or hydranencephaly. The spine showed no sign of scoliosis, and movement of the limb joints was not restricted (i.e., no arthrogryposis). Samples were removed from the remnants of the cerebrum, diencephalon, and organs (thymus, lung, myocardium, jejunum, ileum, mesenteric lymph node, liver, spleen, kidney, and striated muscle), and 3 independent real-time PCR protocols were conducted to detect genomes of bovine viral diarrhea/mucosal disease virus, bluetongue virus serotype 8, and the novel SBV. Initial retrotranscription of the RNA genomes was followed by quantitative (real-time) PCR. The process was conducted by using our procedures (2) and, for SBV, by following the protocol and using recently developed control reagents as described (1). The SBV genome was detected in only CNS samples (quantification cycle value 28.8); bovine viral diarrhea/mucosal disease virus and BTV-8 genomes were not detected. The new virus genome load was 1.61 × 104 copies per gram of cerebrum sample. Taken together, the above data suggest that, like other Simbu serogroup viruses, the new virus crosses the placenta, contaminates the bovine fetus, infects the fetus’ CNS, and causes necrosis and/or developmental arrest of the cerebral cortex. Unlike the viruses mentioned above (3,4), and provided this case is not an exception, the SBV genome seems to persist in the infected fetus and is detectable after birth by real-time reverse transcription PCR, despite gestation length. Although reliable reagents for detecting seroconversion are temporarily unavailable, the persistence of the new virus in fetal tissue should greatly facilitate the epidemiologic monitoring of the emergence and spread of the new virus. When calves from experimentally infected dams are infected with the closest phylogenetic relative to SBV, Akabane virus, porencephaly develops during gestational days 62–96 (5). If the same is true for the new virus, the above calf was probably infected during June 9–July 13, 2011. Therefore, it is hypothesized that infected arthropods were already circulating in the village of Hamois-in-Condroz (50°24′56′′N, 5°8′7′′E), which is ≈240 km southwest of Schmallenberg (51°8′42′′N, 8°17′18′′E), ≈2 months before the emergence of the clinical syndrome that led to the identification of the new virus.

Schmallenberg Virus in Domestic Cattle, Belgium, 2012

To determine prevalence of antibodies against Schmallenberg virus in adult cows and proportion of infection transmitted to fetuses, we tested serum samples from 519 cow/calf pairs in Belgium in spring 2012. Of cattle within 250 km of location where the virus emerged, ≈91% tested positive for IgG targeting nucleoprotein. Risk for fetal infection was ≈28%.

Bluetongue in Eurasian lynx.

To the Editor: Bluetongue is an infectious disease of ruminants; it is caused by bluetongue virus (BTV), has 24 known serotypes, and is transmitted by several species of Culicoides biting midges. The disease mainly affects sheep and occurs when susceptible animals are introduced to areas where BTV circulates or when BTV is introduced to naive ruminant populations. The natural host range is strictly limited to ruminants, although seroconversion without disease has been reported in carnivores (1). We report BTV infection, disease, and death in 2 Eurasian lynx (Lynx lynx) and the isolation of BTV serotype 8 (BTV-8) from this carnivorous species. The 2 Eurasian lynx, held in the same cage in a zoo in Belgium, became lethargic in September 2007; animal 1 died after 2 days, and animal 2 died in February 2008. Both had been fed ruminant fetuses and stillborns from surrounding farms in an area where many bluetongue cases had been confirmed (2). Necropsy findings for animal 1 were anemia, subcutaneous hematomas, petechial hemorrhages, and lung congestion with edema. Necropsy findings for animal 2 were emaciation, anemia, enlarged and gelatinous lymph nodes, petechial hemorrhages, and pneumonia. For each animal, microscopic examination showed edematous vascular walls; enlarged endothelial cells; and evidence of acute to subacute vasculitis in muscle, myocardium, peritoneum, and lung. Tissue samples (spleen, lung, intestine) were analyzed by using 2 real-time reverse transcriptase–quantitative PCR techniques targeting BTV segment 5 and host β-actin mRNA as a control. BTV RNA was found in all samples from animal 1; cycle threshold values (3) ranged from 28.6 to 36.2. Tissues from animal 2 were negative for BTV RNA. Although the internal control was originally designed to detect β-actin mRNA of bovine or ovine species, clear positive signals were noted in all lynx samples, which indicated that this was a reliable control procedure. Infectious virus was subsequently isolated from the lung sample of animal 1 after inoculation of embryonated chicken eggs and amplification in baby hamster kidney–21 cell cultures (4). The specificity of the cytopathic effect, observed 48 hours after passage on baby hamster kidney–21 cells, was confirmed by real-time reverse transcriptase–quantitative PCR. Virus neutralization using specific reference serum (5) proved that the isolated virus was BTV-8. Anti-BTV antibodies were detected in lung tissue fluid from animal 2 (ID Screen Bluetongue Competition assay, ID VET, Monpellier, France) (6). We describe a natural, wild-type infection of a carnivorous species. Although deaths have been documented in dogs accidentally infected with a BTV-contaminated vaccine (7), the 2 lynx in this report were neither vaccinated nor medically treated by injection. BTV-8 was first introduced to northern Europe in 2006 and has subsequently spread rapidly to many countries on that continent. During 2007, a total of 6,870 bluetongue cases were reported in Belgium (2); animal 1 died in September 2007, which corresponded to the peak of bluetongue outbreaks in that region. No deaths were reported during that period among other animals, including ruminants, held in the same zoo as the 2 lynx reported here. The time lapse between initial clinical signs and death could explain the failure to detect BTV-8 RNA in animal 2. Although speculative, the suspicion of bluetongue in this animal is based on the presence of anti–BTV-8 antibodies, vasculitis, and pneumonia, which have been found in dogs accidentally infected with BTV (7). This report raises questions about the current knowledge of the epidemiology of bluetongue. Bluetongue in lynx indicates that the list of known susceptible species must be widened, at least for serotype 8. Although infection of a susceptible host by an insect vector is the only proven natural transmission mechanism for wild-type BTV, transplacental transmission of BTV-8, resulting in the birth of seropositive (8) or virus-positive calves (9), has recently been described in cattle. Although infection by an insect vector cannot be excluded, transmission by the oral route must be strongly suspected because the lynx described in this report had been fed ruminant fetuses and stillborn animals from surrounding farms. This possibility is supported by a previous suspicion that seroconversion to BTV in carnivores was a result of oral infection (1). The possibility of oral transmission is also supported by evidence of lateral transmission of BTV infection to cattle having occurred, in the absence of insect vectors, as a result of direct contact with newborn viremic calves born to infected dams that had been imported to Northern Ireland from a bluetongue-infected region of continental Europe (S. Kennedy, unpub. data). The role of wildlife, especially carnivores, in the epidemiology of bluetongue deserves further study to elucidate their role as either dead-end hosts or new sources of infection for livestock and to help determine the risks for wildlife populations. Our findings clearly indicate that a novel transmission pathway enables the virus to cross species. Consequently, transmission to other species, including domestic animals, can no longer be excluded. Moreover, oral transmission is likely to have considerable implications for disease control, including vaccination, because BTV-8 is a fast-emerging virus with major financial consequences.

Evidence for transplacental transmission of the current wild-type strain of bluetongue virus serotype 8 in cattle.

DURING the winter 2007/08, an outbreak of unprecedented development lesions of the central nervous system was detected in newborn or stillborn calves and lambs among the routine submissions to the Faculty of Veterinary Medicine, Liege, Belgium, for postmortem examination. The congenital

Evaluation of a protocol for fast localised abdominal sonography of horses (FLASH) admitted for colic

The aim of this prospective study was to establish a protocol for fast localised abdominal sonography of horses (FLASH) admitted for colic. The FLASH protocol was then presented to clinicians without extensive ultrasound (US) experience to determine whether they could learn to use it in less than 15 min. The clinical subjects comprised 36 horses that had been referred for colic over a 2 month period. Each horse was examined at admission and FLASH findings at seven topographical locations were compared to serial clinical examinations, surgical and non-surgical outcomes, or with post-mortem reports. FLASH was able to show free abdominal fluid and abnormal intestinal loops, with a mean time of 10.7 min required to complete the protocol. The positive and negative predictive values of requirement for surgery of dilated turgid small intestinal loops using FLASH were 88.89% and 81.48%, respectively. The results suggested that FLASH is a technique that can be used in an emergency setting by veterinarians without extensive US experience to detect major intra-abdominal abnormalities in horses with colic.

European outbreaks of atypical myopathy in grazing equids (2006-2009). Spatiotemporal distribution, history and clinical features

REASONS FOR PERFORMING STUDY Improved understanding of the epidemiology of atypical myopathy (AM) will help to define the environmental factors that permit or support the causal agent(s) to exert toxicity. OBJECTIVES This European survey of AM aimed to describe spatiotemporal distribution, survival, clinical signs, circumstances in which AM develops and its different expressions between countries and over time. METHODS The spatiotemporal distribution, history and clinical features of AM cases reported to the Atypical Myopathy Alert Group from 2006 to 2009 were described. Comparisons of data from the most severely affected countries and from the large outbreaks were made with Fishers exact and Welchs tests with Bonferroni correction. RESULTS Of 600 suspected cases, 354 met the diagnostic criteria for confirmed or highly probable AM. The largest outbreaks occurred during the autumns of 2006 and 2009 in Belgium, France and Germany. For the first time, donkeys, zebras and old horses were affected, and clinical signs such as gastrointestinal impaction, diarrhoea, penile prolapse, buccal ulceration and renal dysfunction were observed. Affected horses spent >6 h/day on pastures that almost always contained or were surrounded by trees. The latency period was estimated at up to 4 days. Overall survival rate was 26%. Although differences between countries in affected breeds, body condition, horse management and pasture characteristics were recognised, the common presenting clinical signs and mortality were similar between countries. CONCLUSIONS AND POTENTIAL RELEVANCE This study describes new data on case details, history and clinical course of AM that is of preventive, diagnostic and therapeutic value. However, the true impact of the findings of this study on the development of or severity of AM should be tested with case-control studies.

Fetal infection with Neospora caninum in dairy and beef cattle in Belgium

Neospora caninum is a protozoan parasite, which causes fetal and neonatal mortality in livestock and companion animals. In 224 abortions in Belgian cattle, different diagnostic methods were used to demonstrate infection, and the presence of N. caninum. An indirect fluorescent antibody test (IFAT) was used to analyze fetal and maternal sera and immunohistochemistry (IHC) was performed when lesions consistent with neosporosis were observed in the brain, heart or liver. Twenty dairy cattle sera out of 70 (29%) and 13 beef cattle sera out of 93 (14%) were positive by IFAT. A positive titer to N. caninum was found in seven and three fetuses born to beef and dairy cows, respectively. Lesions consistent with N. caninum infection were observed in 17 fetuses. Of nine positive beef fetuses, five were confirmed by IHC while, all but one dairy fetus were confirmed using the same technique. Age had no influence on the serological status of the mother (P = 0.486) whereas husbandry system had a borderline influence (P = 0.082). However, a strong association (P = 0.004) between the level of antibodies in the dam and the occurrence of lesions in the fetus was observed and lesions were more prominent in dairy than in beef fetuses. Additionally, the distribution of intra-cerebral lesions was more extensive in dairy than in beef fetuses (P < 0.0001). Age and serological status of the fetus were found to influence the occurrence of lesions in beef fetuses (both P < 0.001) but no such significant relationships could be demonstrated in dairy fetuses. The study indicated that N. caninum must be considered as an important cause of bovine abortion in Belgium.

European outbreaks of atypical myopathy in grazing horses (2006–2009): determination of indicators for risk and prognostic factors

REASONS FOR PERFORMING STUDY Appropriate management of atypical myopathy (AM) requires the establishment of an accurate diagnosis and prognosis. Furthermore, preventive measures to avoid AM need to be refined. OBJECTIVES The aims of the study were as follows: 1) to improve the diagnosis of AM; 2) to identify prognostic predictors; and 3) to refine recommended preventive measures based on indicators of risk factors. METHODS An exploratory analysis of cases in Europe between 2006 and 2009 reported to the Atypical Myopathy Alert Group was conducted. Based on clinical data, reported cases were allocated into 2 groups: confirmed or highly probable AM (AM group; further divided into survivors and nonsurvivors); and cases with a low probability of having AM or with another final diagnosis (non-AM group). Using Welchs test and odds ratios corrected for multiple comparisons, the AM vs. non-AM groups were compared to identify indicators for diagnosis and risk factors, and survivors vs. nonsurvivors in the AM group were compared to identify prognostic factors. Sensitivity, specificity and positive and negative predictive values were calculated for specific clinical signs related to final diagnosis and outcome. RESULTS From 600 reported cases, 354 AM cases (survival rate of 26%) and 69 non-AM cases were identified, while there were insufficient data to categorise the remainder. Variables valuable for diagnosing AM compared with similar diseases were as follows: presence of dead leaves and wood and/or trees on pastures; sloping pastures; full-time pasture access; no food supplementation; normal body condition; pigmenturia; normothermia; and congested mucous membranes. Nonsurvival was associated with recumbency, sweating, anorexia, dyspnoea, tachypnoea and/or tachycardia. Survival was associated with remaining standing most of the time, normothermia, normal mucous membranes, defaecation and vitamin and antioxidant therapy. CONCLUSIONS AND POTENTIAL RELEVANCE This study refines the list of risk factors for AM. Clinical signs valuable for diagnosis and prognosis have been identified, enabling clinicians to improve management of AM cases.