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The geographic range of the zoonotic raccoon roundworm (Baylisascaris procyonis) is expanding together with the range of its host, the raccoon (Procyon lotor). This creates a new public health risk in parts of Europe where this parasite was previously absent. In the Netherlands, a raccoon population is becoming established and incidental findings of B. procyonis have been reported. To assess the risk to public health, the prevalence of B. procyonis was determined in the province of Limburg, where currently the largest Dutch raccoon population is present, as well as in the adjoining region of southern Belgium. Furthermore, genetic methods were employed to assess invasion pathways of both the raccoon and B. procyonis to aid in the development of control measures. Macroscopic analysis of intestinal content and testing of faecal samples were performed to detect B. procyonis adults and eggs. The population genetics of both B. procyonis and its raccoon host were analysed using samples from central and northwestern Europe. B. procyonis was found in 14/23 (61%, 95% C.I. 41-78%) raccoons from Limburg, but was absent from 50 Belgian raccoons. Genetic analyses showed that the majority of the Dutch raccoons and their roundworms were introduced through ex-captive individuals. As long as free-living raccoon populations originate from captivity, population control methods may be pursued. However, natural dispersal from the border regions will complicate prolonged population control. To reduce the public health risk posed by B. procyonis, public education to increase awareness and adapt behaviour towards raccoons is key. This article is protected by copyright. All rights reserved.

While fatal infections caused by the Usutu virus appeared to concern only passerines (especially the blackbird) and Strigiformes (especially the great gray owl), we report herein that the virus also naturally causes a fatal disease in an anseriforme species, the common scoter (Melanitta nigra).

Schmallenberg virus emerged in 2011 in Europe. The epicentre of primordial spreading was the region straddling Germany, the Netherlands and Belgium. One of the key questions is whether the newcomer would establish a lasting presence on the continent. The apparent seroprevalence in southern Belgium wild deer populations was followed for 6 years. Two years of intense circulation were revealed, 2012 and 2016, characterized by a peak seroprevalence in the two studied populations (Capreolus capreolus and Cervus elaphus). Between the peak years and after 2016, apparent seroprevalences declined rapidly among adults and became nil among juveniles. The general pattern of apparent seroprevalence evolution observed is consistent with a cyclic circulation of Schmallenberg virus, similar to what is observed for other Orthobunyaviruses in endemic areas. These data also suggest that wild cervids play no central role in the circulation dynamics of the virus.

African swine fever is a devastating disease of domestic pigs and wild boars caused by African swine fever virus (ASFV). ASFV originates from sub-Saharan African countries. In the last 10 years, the virus left its endemic range to spread to eastern Europe and Russia. On September 2018, Belgian authorities reported that ASFV had been detected in two wild boars in a southern area of the country. One year later, no domestic pig has been infected, with the last ASFV-positive wild boar being confirmed in mid-August 2019, suggesting that the outbreak is now controlled. However, the dispersal dynamics as well as the specific impact of ecological factors and intervention measures on the outbreak remain unknown. In total, 827 positive cases have been reported in wild boar populations. In this study, we exploit the resulting spatio-temporal distribution of occurrence data to investigate the wavefront progression. We first present the application of recently developed methods to quantify the local wavefront velocity of an invading epidemic. Second, we develop and apply a novel analytical framework that uses occurrence data to investigate the impact of ecological factors on the dispersal dynamics of a wavefront progression. Our analyses highlighted that the network of barriers, involving installed fences, had an impact on both the effective dispersal and the wavefront dispersal velocity. Furthermore, we also demonstrated that the wavefront progression was slower outside forest areas. Together, these results have concrete implications for potential future ASFV epidemics in similar regions. Synthesis and applications. We describe a novel analytical approach that exploits occurrence data to investigate the impact of ecological factors on the wavefront velocity and actual wavefront progression. This methodology has the potential to be quickly applied to outbreak datasets solely made of occurrence data, with key benefits for the epidemiological investigations of external spatial factors impacting pathogen dispersal across non-endemic areas. Our analytical workflow could also be further applied to investigate the impact of ecological factors on any kind of biological dispersions (pathogen spreads, invasive species)., SCOPUS: ar.j, info:eu-repo/semantics/published

This report describes a mixed Mycobacterium avium subspecies avium and M avium subspecies paratuberculosis infection in a free-ranging red deer (Cervus elaphus). The gross presentation was consistent with clinical paratuberculosis as previously reported in red deer and for other ruminants, with poor body condition, diarrhoea and mesenteric lymphadenitis. However, this animal presented unusual lung lesions, with necrosis and calcification similar to those reported for Mycobacterium bovis infection in wild and domestic ruminants. A mixed M avium subspecies avium and M avium subspecies paratuberculosis was shown by quantitative PCR (qPCR) performed on digestive tract samples. In addition, M avium subspecies avium was also detected in the respiratory tract (lungs and bronchial lymph nodes) by qPCR. These results were confirmed by classical bacteriology. Lesions induced by those mycobacteria cannot be differentiated from M bovis lesions. This point is particularly important with regards to increasing interactions between livestock and wild animals.

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018–2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results.

During late summer 2016, in a northwest European region extending over Belgium, the Netherlands and the eastern border of the German state of North Rhine Westphalia, an outbreak of wild bird deaths occurred similar to those reported on the continent since 1996. Dead birds were necropsied and examined by complementary methods. Pathologic and immunohistological investigations strongly suggested an infection by Usutu virus. Subsequently, genomic segments of the said virus were detected, the virus was isolated and its complete genome was sequenced. The strain, designated Usutu-LIEGE, is a close phylogenetic relative of those isolated in Germany which form a distinct group within the USUV phylogeny, the so-called Europe_3 lineage. Should this outbreak recapitulate the characteristics of those in southwest Germany in 2011 and in/around Vienna (Austria) in 2001, it is expected that specific avian populations in the affected area will face a significant reduction in size for a few years.

Wildlife surveillance allowed the monitoring of the zoonotic mosquito-borne Usutu virus (USUV) in birds and bats (Pipistrellus pipistrellus) in southern Belgium in 2017 and 2018. USUV-RNA was detected in 69 birds (of 253) from 15 species, among which 7 species had not previously been reported to be susceptible to the infection. Similarly, 2 bats (of 10) were detected positive by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). USUV-associated lesions were mainly found in Eurasian Blackbirds (Turdus merula), in which USUV antigens were demonstrated by immunohistochemistry in the brain, heart, liver, kidney, intestine, and lung. Partial nonstructural protein 5 gene-based phylogenetic analysis showed several identical or closely related strains from 2016, 2017, and 2018 clustering together within Europe 3 or Africa 3 lineages. Further, one USUV strain detected in a common chaffinch (Fringilla coelebs) manifested a close genetic relationship with the European 1 strains circulating in Hungary and Austria. Our data provide evidence of USUV endemization in southern Belgium in local birds and bats, extension of the host range of the virus and ongoing virus introduction from abroad, likely by migratory birds. Our results highlight the need for vigilance in the forthcoming years toward new virus-associated outbreaks in birds and possible human infections in Belgium.

While fatal infections caused by the Usutu virus appeared to concern only passerines (especially the blackbird) and Strigiformes (especially the great gray owl), we report herein that the virus also naturally causes a fatal disease in an anseriforme species, the common scoter (

In a new example of pathogens hopscotching the globe, African swine fever virus hit north-western Europe's wildlife in summer 2018, marking a further spread of a disease that had invaded Central and Eastern Europe recently. The complete genomic sequence of the Belgium/Etalle/wb/2018 virus is reported, with the hope it will provide a valuable tool for tracing geographical spread and biologic evolution of the virus.

The family Parvoviridae contains diverse viruses that are capable of infecting a wide range of hosts. In this study, metagenomic sequencing of Ixodes ricinus ticks harvested in 2016 on red deer (Cervus elaphus) and European roe deer (Capreolus capreolus) in Belgium detected a new 6296-bp parvoviral genome. Phylogenetic and sequence analyses showed the new virus belongs to a new species within the Copiparvovirus genus. PCR screening of 4 pools of 10 serum samples from both deer species identified the new copiparvovirus DNA only in roe deer sera. Together, these results are the first evidence of a copiparvovirus in a deer species. Besides its potential pathogenicity to roe deers, the detection of this new virus in ticks raises questions about the possible transmission of parvoviruses by ticks. This report further increases the current knowledge on the evolution and diversity of copiparvoviruses.

West Nile Virus, Usutu virus, Bagaza virus, Israel turkey encephalitis virus and Tembusu virus currently constitute the five flaviviruses transmitted by mosquito bites with a marked pathogenicity for birds. They have been identified as the causative agents of severe neurological symptoms, drop in egg production and/or mortalities among avian hosts. They have also recently shown an expansion of their geographic distribution and/or a rise in cases of human infection. This paper is the first up-to-date review of the pathology of these flaviviruses in birds, with a special emphasis on the difference in susceptibility among avian species, in order to understand the specificity of the host spectrum of each of these viruses. Furthermore, given the lack of a clear prophylactic approach against these viruses in birds, a meta-analysis of vaccination trials conducted to date on these animals is given to constitute a solid platform from which designing future studies.

Active monitoring of avian influenza (AI) viruses in wild birds was initiated in Belgium in 2005 in response to the first highly pathogenic avian influenza (HPAI) H5N1 outbreaks occurring in Europe. In Belgium, active wild bird surveillance that targeted live-ringed and hunter-harvested wild birds was developed and maintained from 2005 onward. After one decade, this program assimilated, analyzed, and reported on over 35,000 swabs. The 2009-2014 datasets were used for the current analysis because detailed information was available for this period. The overall prevalence of avian influenza (AI) in samples from live-ringed birds during this period was 0.48% whereas it was 6.12% in hunter-harvested samples. While the ringing sampling targeted a large number of bird species and was realized over the years, the hunting sampling was mainly concentrated on mallard (Anas platyrhynchos) during the hunting season, from mid-August to late January. Even when using just AI prevalence for live-ringed A. platyrhynchos during the hunting season, the value remained significantly lower (2.10%) compared to that detected for hunter-harvested mallards. One explanation for this significant difference in viroprevalence in hunter-harvested mallards was the game restocking practice, which released captive-bred birds in the wild before the hunting period. Indeed, the released game restocking birds, having an AI-naïve immune status, could act as local amplifiers of AI viruses already circulating in the wild, and this could affect AI epidemiology. Also, the release into the wild of noncontrolled restocking birds might lead to the introduction of new strains in the natural environment, leading to increased AI presence in the environment. Consequently, the release of naïve or infected restocking birds may affect AI dynamics.

In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever–infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia.

Between 30 and 50 red foxes ( Vulpes vulpes ) are submitted each year to the University of Liege’s veterinary faculty for postmortem examination. Occasionally, other wild carnivores such as European badgers ( Meles meles ) and raccoons ( Procyon lotor ) are also submitted. A PCR-based screening for canine distemper virus (CDV)1 has been performed systematically. Since 2014, all submitted cases tested negative for CDV. On November 14, 2017, a wild raccoon with severe neurological impairment was euthanased in a revalidation centre, near the Liege veterinary faculty. …

Hepatitis E is an acute human liver disease in healthy individuals but may become chronic in immunocompromised patients. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin, particularly in high-income countries. In this study, 383 sera from wild boars were selected for serology; for virological analyses, 69 sera and 61 livers from young wild boars were used. A total of 189 and 235 sera of, respectively, red deer and roe deer were collected for serological analysis. For virological analyses, 84 and 68 sera and 29 and 27 livers from, respectively, red and roe deer were sampled. An apparent seroprevalence of 34% (95% CI 29.71-39.46) was found in wild boars, of 1% (95% CI 0-2.4) in red deer and 3% (95% CI 0.8-4.2) in roe deer. To assess the ELISA screening prevalence, Western blot (WB) analyses were carried out, a receiver operating characteristic curve analysis was performed and different scenarios with varying ELISA specificities relative to WB were analysed. Seroprevalence remained high whatever the scenario in the wild boar population. In wild boar, 4 of 69 sera and 4 of 61 livers were detected as positive for HEV RNA. All sequences obtained from sera belonged to genotype HEV-3. HEV RNA, belonging to genotype HEV-3, was detected in one of 29 red deer livers. Wild boar can be considered as a host reservoir of the virus in Belgium. However, in contrast to the epidemiological role played by them in other countries, the low prevalence in deer makes these species an unlikely reservoir. This evidence needs further investigation to determine in which situation deer can serve as reservoir. These results also raise the question of the dynamics of HEV infection between wild fauna, domestic pigs and humans.

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6–12 copies of ISMbov1, 11–15 copies of ISMbov2 and 4–10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4–8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.

A cranial abdominal mass on the left side was detected upon palpation in a two-and-a-half-year-old male wirehaired dachshund. No other clinical signs were recorded except exhaustion following exertion, and soft faeces. Abdominal echography showed hepatomegaly with extensive histological alterations; microscopical examination of smears from hepatic mass and fluid punction revealed a low cellularity and protoscolex-like structures. Following those findings, euthanasia was requested by the owner. Necropsy revealed a severe multifocal chronic active hepatitis with some degree of right ventricular dilatation. Based on histopathology and PCR, a diagnosis of alveolar echinococcosis due to Echinococcus multilocularis was established. The dog was living in the Ardennes region in Belgium known as being endemic for E multilocularis . The present observation is in agreement with a similar canine case previously published and the high prevalence of E multilocularis in the local fox population.

The complete coding sequences of four avian influenza A viruses (two H7N7, one H7N1, and one H9N2) circulating in wild waterfowl in Belgium from 2009 to 2012 were determined using Illumina sequencing. All viral genome segments represent viruses circulating in the Eurasian wild bird population.

In southern Belgium, a brown hare ( Lepus europaeus ) was found moribund and killed for ethical reason. The animal was transmitted for postmortem examination. Major histopathological findings consisted of multifocal subacute necrotising meningitis and multifocal subacute necrotising orchitis. Infection with Francisella tularensis was confirmed by both bacteriological isolation and detection by real-time PCR. Further, subtyping of F tularensis colonies stated that it was F tularensis subspecies holarctica biovar I . It is the first case of tularaemia detected in wildlife in Belgium since 2003. The event pushed health professionals to communicate with hunters and other groups with outdoor activities about the Francisella risk and the ways to take care of it, such as wearing gloves to handle found-dead or hunted hares and taking protective measures against tick bites.

Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli.

Bovine tuberculosis (BTB) is a chronic and contagious disease that affects domestic animals, wildlife, and humans. Caused by Mycobacterium bovis, BTB causes major economic losses and poses a serious constraint to international livestock trade. Moreover, in developing countries where BTB controls are lacking, M. bovis is a public health concern. In most developing countries, the prevalence of BTB in livestock is unknown because the information is either not reported or not available. In Ecuador, there is no national BTB control program. This article reviews the BTB situation in Ecuador by examining exhaustive data from tuberculin testing surveys and slaughterhouse surveillance studies conducted in 1972-2008 in a variety of the country's geographic areas. In Ecuador, several factors, including the dairy industry's expansion (preempted by the high demand for milk and its by-products), intensified efforts to increase the cattle population, the presence of M. bovis, and a lack of BTB controls, have caused a rise in BTB prevalence, and consequently, a growing push for the implementation of a national BTB control program.La tuberculosis bovina es una enfermedad contagiosa crónica que afecta a los animales domésticos, los animales salvajes y los seres humanos. Es producida por Mycobacterium bovis; causa grandes pérdidas económicas y plantea una grave limitación para el comercio ganadero internacional. Por otro lado, en los países en desarrollo donde no hay controles de la tuberculosis bovina, la infección por M. bovis representa un problema de salud pública. En la mayoría de los países en desarrollo, la prevalencia de tuberculosis en el ganado se desconoce porque la información no se comunica o no se consigue. En el Ecuador no hay un programa nacional de control de la tuberculosis bovina. En este artículo se revisa la situación de la tuberculosis bovina en el Ecuador, sobre la base de un análisis de los datos exhaustivos obtenidos de encuestas sobre pruebas de tuberculina y de los estudios de vigilancia llevados a cabo en mataderos entre 1972 y 2008 en varias zonas geográficas del país. En el Ecuador varios factores, como el crecimiento de la industria láctea (impulsado por la alta demanda de leche y sus derivados), los intensos esfuerzos para aumentar la población bovina, la presencia de M. bovis y la falta de controles de la tuberculosis bovina, han ocasionado un aumento de la prevalencia de esta y, en consecuencia, representan una motivación creciente para llevar a cabo un programa nacional de control de la tuberculosis bovina.

Aims: The aim of this study was to investigate the presence of enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli strains in free-ranging wild ruminants in Belgium and to characterize the positive isolates (serogroups and virulence-associated factor-encoding genes). Methods and Results: Escherichia coli strains isolated from faeces of wild cervids were characterized by PCR targeting genes coding for the main virulence properties of EPEC, EHEC and VTEC strains. The prevalence rate of these pathogenic strains in faecal samples obtained from the wild ruminants was found to be 15%. No pathogenic isolate was found to belong to the O157, O26, O111, O103 or O145 serogroups. Moreover, a new gene, eibH, showing 88% identity with eibG was detected in VTEC strains. Conclusions: The results reveal that wild ruminants could be considered as a potential source of VTEC and EPEC infection for humans and possibly also for domestic ruminants. Significance and Impact of the Study: Our study suggests the potential risk of transmission of VTEC, EHEC and EPEC strains from wild ruminants to humans via the consumption of venison and to domestic ruminants because of sharing of the same pasture. Indeed, many serogroups other than O157 EHEC have also been shown to be responsible for outbreaks in humans in several countries, and studies focusing solely on O157:H7 EHEC tend to underestimate this risk of transmission.

Summary The purpose of the present work is to report the feasibility, humaneness and reliability of a method for measurement of diaphragm performance in intact, awake horses. Phrenic nerves were activated through the vena cava by repeated transvenous stimulations (40 to 80 V, 100 to 200 μs square-wave pulses), using an electrode-catheter advanced from the jugular vein. Twitches were produced that caused changes in gastric and oesophageal pressures, abdominal and rib cage motion and airflow tracings, suggesting that the diaphragm was activated. Four sites were selected over each hemithorax for placement of surface electrodes able to detect and to ensure constancy of evoked compound diaphragmatic action potentials (CDAP). Trains of repeated pulses applied to nerves (10 to 100 Hz) gave repeatable diaphragmatic force-frequency relationships. Nerve stimulation produced no visible signs of discomfort and increases in Cortisol and β-endorphins similar to those produced by nasogastric intubation. Transvenous single and repeated activations of the phrenic nerves in ponies is feasible, free of iatrogenic risks and ethically acceptable, recording reliable evoked potentials via surface electrodes is possible, and force-frequency characteristics of the diaphragm can be conveniently obtained from simultaneous oesophageal and gastric pressures measurements.

Summary This Case Report describes a periarticular abscess and a suppurative arthritis of the atlanto-occipital joint in an adult horse. The horse showed a painful swelling localised on the atlanto-occipital region and ataxia in all 4 limbs. During hospitalisation, the horse developed recumbency, dysphagia, facial paralysis and seizures. Ultrasonography of the atlanto-occipital region enabled a tentative diagnosis to be made, which was later confirmed at autopsy. Culture of the abscess revealed a Staphylococcus aureus. However, the horse had a history of recurrent fever, hypertrophy and abscessation of the submandibular lymph nodes, neck pain and dyspnoea for 2 months, which was suggestive of strangles.

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The so-called beta2 toxin (CPB2) is the most recently described major toxin produced by C. perfringens. In this study, the cpb2 ORF (cpb2FM) from a cattle C. perfringens-associated enterotoxaemia was cloned and sequenced. The cpb2FM and its deduced nucleotide sequence clearly corresponded to the cpb2 allele considered as "consensus" and not to "atypical" allele, despite its "non-porcine" origin. Expression assays of the recombinant toxin CPB2FM were performed in Escherichia coli and Bacillus subtilis with the expression vector pBLTS72, and by genomic integration by double recombination in B. subtilis. Highest level of production was obtained with the expression vector in B. subtilis 168 strain. The recombinant CPB2FM protein was purified and a specific rabbit polyclonal antiserum was produced. Polyclonal antibodies could detect CPB2 production in supernatants of C. perfringens from enterotoxaemic cattle.

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2 , is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves ( n = 14, 87 isolates) and from a group of enterotoxaemic calves ( n = 8, 41 isolates). PCR results revealed the exclusive presence of the typical “consensus” cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.

Chronic wasting disease (CWD) has not been reported in Europe, whereas it is considered to be enzootic in free-ranging mule deer, Rocky mountain elk and white-tailed deer in the area of Colorado, Wyoming, and Nebraska, and new foci of CWD have been detected in other parts of the United States. However, no large-scale active epidemiosurveillance of European wild cervids has been installed in Europe. In accordance with the opinion of the European Scientific Steering Committee, a preliminary (active) surveillance scheme was installed, in order to improve the knowledge of the CWD status of the Belgian free-ranging cervids (roe deer and red deer). Spleen samples (n=866) of roe deer and red deer collected in the south-eastern part of Belgium, were examined for CWD using a enzyme-linked immunosorbent assay of Bio-Rad. Afterwards, the ELISA was systematically confirmed by immunohistochemistry using three antibodies, namely R524, 2G11 and 12F10. There were no indications on the occurrence of transmissible spongiform enncephalopathy (TSE) in any of the samples. A Bayesian framework was used for the estimation of the true prevalence of CWD in south-eastern part of Belgium that was estimated to have a median value of zero with a 95% percentile value of 0.00115.

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells.

An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a frameshift that causes premature truncation of the translated protein, indicating that p40* exists as a pseudogene in M. bovis.

The susceptibilities of 40 recent Belgian field isolates of Mycoplasma bovis to 10 antimicrobial agents were assessed. Tiamulin was the most active antimicrobial agent against M bovis, with an initial inhibitory concentration (IIC50) of 0.06 microg/ml, but it is not licensed for the treatment of cattle. All three fluoroquinolones tested (danofloxacin, enrofloxacin and marbofloxacin) were effective against strains of M bovis, and had a minimum mycoplasmacidal concentration (MMC50) less than or equal to 1 microg/ml. Gentamicin was poorly effective, having an IIC50 of 8 microg/ml. Many strains of M bovis were resistant to tylosin, spectinomycin, lincomycin, tetracycline and oxytetracycline.

Mycoplasma bovis is responsible for considerable economic losses in cattle due to pneumonia, arthritis and mastitis. As the agent was shown to be capable of adhering to neutrophils and embryonic bovine lung (EBL) cells and invading the respiratory epithelium it is highly desirable to improve our understanding of cytadherence processes. Although several surface proteins likely to be directly involved in this initial stage of interaction between pathogen and host cells have been identified, these findings mainly referred to type strain PG45 adhering to the continuous EBL cell line. The present study provides new and complementary data about cytadherence of M. bovis based on adherence of various radiolabeled strains to a primary culture of bovine bronchial epithelial (BBE) cells using a standardized adherence assay. M. bovis was shown to adhere specifically to the primary culture of BBE cells. Inhibition of adherence was observed upon addition of monoclonal antibodies (MAbs), trypsin treatment of mycoplasmas, and competition with non-radiolabeled mycoplasma cells. Interestingly, three MAbs against proteins involved in adherence to EBL cells failed to inhibit significantly the adherence to BBE cells. On the other hand, significant reduction of adherence rates by MAbs 2A8 and 9F1 directed against epitopes of variable surface lipoproteins VspC and VspF, respectively, demonstrated the involvement of these proteins in adherence of M. bovis to primary culture of BBE cells.

Schmallenberg virus was detected in cattle and sheep in northwestern Europe in 2011. To determine whether wild ruminants are also susceptible, we measured antibody seroprevalence in cervids (roe deer and red deer) in Belgium in 2010 and 2011. Findings indicated rapid spread among these deer since virus emergence ≈250 km away.

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis.

Three sampling procedures were compared to determine the optimal technique for isolating mycoplasmas in cattle with respiratory diseases. The prevalence of mycoplasmas isolated from these animals is also reported. In the first group, bronchoalveolar lavage (BAL) and nasal swab cultures were compared with the corresponding lung cultures from cattle necropsied for fatal respiratory diseases (n = 20). In a second group, nasal swabs were compared with corresponding BAL cultures in living animals with recurrent respiratory pathologies (n = 49). There was complete agreement between the paired BAL and lung cultures. In contrast, nasal cultures were not representative of the mycoplasmas present in the lower respiratory airways. The relative sensitivity and specificity of the nasal swab technique compared to BAL in living animals confirmed that the nasal swab cultures were not predictive of lower respiratory airway pathogens, such as Mycoplasma bovis. BAL is considered to be the best method for isolating M. bovis in cattle with respiratory diseases as it combines reliability and feasibility under field sampling conditions. In the present study, Mycoplasma dispar (43%) and M. bovis (29%) were mainly isolated in mixed infections. This confirms the need to search for mycoplasmas in routine examinations and to take them into account in therapeutic strategies for respiratory diseases in cattle.

Background: Anaplasma phagocytophilum is a tick-borne pathogen of veterinary and human importance. Both ticks as vectors and vertebrates as reservoir hosts are essential for the cycle maintenance of this bacterium. Currently, the whole range of animal species reservoirs for A. phagocytophilum in natural environment is still unknown. Therefore, the aim of this study was to estimate the prevalence of infection with A. phagocytophilum in the wild boar population in southern Belgium. Results: In the frame of a targeted surveillance program, 513 wild boars were sampled during the hunting season 2011. A nested 16S rRNA PCR was used to screen the presence of A. phagocytophilum DNA in spleen of boars. Within 513 samples, 5 (0,97%) were tested PCR positive and identification was confirmed by sequencing. Conclusions: This study gives the first insight of presence of A. phagocytophilum in wild boars in southern Belgium.