Exploratory investigation of Q fever in apparently healthy meat sheep flocks in Belgium.

Q fever is a cosmopolitan disease affecting both humans and many animal species. Although sheep are often implicated in human Q fever outbreaks, the disease remains largely underestimated in meat sheep flocks. In order to fulfil this gap, a preliminary study was performed aiming to investigate the serological and molecular aspects of infection with Coxiella burnetii among meat sheep flocks in Belgium. Five Belgian sheep flocks were recruited for this work. Indirect ELISA was used, and in addition, real-time PCR was performed on samples of milk, rectal and vaginal swabs, to understand the dynamics of bacterial shedding. Despite the low overall apparent seroprevalence of 1.39% (95% CI: 0.04-7.5), a high rate of bacterial shedding was found, with 27.7% of tested sheep (N = 72) with a positive result to PCR, especially through the rectal and vaginal routes and in seronegative animals. Furthermore, Coxiella burnetii DNA was detected in 26.76% of seronegative animals. It can be concluded that an overall good clinical condition of the sheep cannot be used to exclude the presence of C. burnetii in a flock. Furthermore in the diagnosis of Q fever in sheep, serology alone was not a sensitive diagnostic tool. On the contrary, molecular biology allowed to detect bacterial shedding, which is an essential element in order to assess the risk due to the contact with shedding animals. At the light of these results, the role of meat sheep flocks in the epidemiology of Q fever in Belgium needs to be better understood.

Serogroups and genotypes of Leptospira spp. strains from bovine aborted foetuses.

Leptospirosis is a global disease of animals, with potential major economic impact on livestock industry and important zoonotic capacities. The disease represents a major challenge in the developing countries as humans and animals frequently live in close association. The serovar Hardjo of Leptospira whose primary host is cattle has been studied extensively, but few data exist on other current circulating or emerging serotypes. To better understand the disease in cattle and how to prevent and/or control it, it is necessary to identify the genotype and the serotype of circulating Leptospira. This study presents results of several investigations performed on a historical Belgian collection of congenital jaundice in bovine aborted foetuses coming from the leptospirosis emerging episode of 2014 (Delooz et al., Transboundary and Emerging Diseases, 62, 2015, 124). The results revealed that L. Grippotyphosa and L. Australis were the most prevalent serogroups with, respectively, 17/42 and 13/42 positive microscopic agglutination test (MAT) during this emerging event associated with the same clinical pattern. The study also confirms that congenital jaundice is associated with L. kirscheneri and L. interrogans and provides the genotyping of DNA obtained from these two species.

Bayesian assessment of two competitive enzyme-linked immunosorbent assays for the detection of bovine viral diarrhoea virus antibodies in bovine sera.

Infections due to bovine viral diarrhoea virus (BVDV) are endemic in most cattleproducing countries throughout the world and bovine viral diarrhoea is considered a transboundary disease. The key elements of a BVDV control programme are vaccination, biosecurity, elimination of persistently infected (PI) animals and surveillance. The aim of this study was to assess the sensitivity (Se) and the specificity (Sp) of two commercial competitive enzyme-linked immunosorbent assays (ELISAs) based on selected immune-dominant BVDV proteins: the non-structural protein NS3 (p80) and the recombinant envelope glycoprotein E0(Erns). Both tests were used on individual serum samples from randomly sampled young bovines in southern Belgium in order to detect specific BVDV antibodies. The Se and Sp were assessed using a Bayesian approach and were estimated, respectively, at 97.2% (with 95% credibility interval [Cr I]: 95.1-99.8) and 98.7%(95% Cr I: 96.6-99.9) for the first test and 95.8% (95% Cr I: 91.1-99.7) and 96.1%(95% Cr I: 95.1-97.7) for the second test. The results obtained with the two tests were not significantly different. In addition, using both ELISAs, the current BVDV exposure among young bovines in southern Belgium was estimated at 23.3% (95%Cr I: 20.6-26.2). Combining virological testing of all newborns to detect PI animals with regular serological testing of young stock using ELISAs is recommended in the surveillance of BVDV.

Les infections par le virus de la diarrhée virale bovine (VDVB) sont endémiques dans la plupart des pays d'élevage du monde et la diarrhée virale bovine estune maladie transfrontalière. Les éléments d'un programme de contrôle de la VDVB sont la vaccination, la biosécurité, l'élimination d'animaux porteurs d'une infection persistante et la surveillance. L'objectif de cette étude était d'évaluer la sensibilité (Se) et la spécificité (Sp) de deux kits commerciaux ELISA (épreuve immuno-enzymatique) par compétition basés sur une sélection de protéines immunodominantes du VDVB, la protéine non structurale NS3(p80) et la glycoprotéine d'enveloppe recombinante E0 (Erns). Les deux kits étaient testés sur des échantillons individuels de sérum collectés demanière aléatoire chez de jeunes bovins dans le sud de la Belgique afin de détecter les anticorps VDVB spécifiques. L'analyse bayésienne montrait une Se de 97,2 % (intervalle de crédibilité de 95 % [ICr] de 95,1 à 99,8) et une Sp de 98,7 (ICr 95 % de 96,6 à 99,9) pour le premier kit et une Se de 95,8 (ICr 95 % de91,1 à 99,7) et une Sp de 96,1 (ICr 95 % de 95,1 à 97,7) pour le deuxième. Les différences n'étaient pas significatives. De même, l'application des deux ELISA montrait que l'exposition actuelle des jeunes bovins du sud de la Belgique au VDVB s'élevait à 23,3 % (ICr 95 % de 20,6 à 26,2). L'association de tests virologiques effectués chez tous les nouveau-nés afin de détecter des animaux à infection persistante et de tests sérologiques de routine par ELISA chez les jeunes animaux est recommandée pour la surveillance de VDVB.

Las infecciones por el virus de la diarrea viral bovina (BVDV), considerada enfermedad transfronteriza, son endémicas en la mayoría de los países del mundo que albergan producción bovina. Los principales elementos de todo programa de lucha contra este virus son la vacunación, la seguridad biológica, la eliminación de los animales con infección persistente y la vigilancia. Los autores describen un estudio encaminado a evaluar la sensibilidad y especificidad de dos ensayos inmunoenzimáticos (ELISA) comerciales basados en sendas proteínas inmunodominantes del virus: la proteína no estructural NS3 (p80) y la glucoproteína recombinante de envoltura E0 (Erns). Ambas pruebas fueron aplicadas a sueros procedentes de una muestra aleatoria de bovinos jóvenes del sur de Bélgica con el fin de detectar anticuerpos específicos contra el virus. Empleando un método de estadística bayesiana se calcularon la sensibilidad y la especificidad, que resultaron, respectivamente, de un 97,2% (intervalo de credibilidad [I Cr] al 95%: 95,1­99,8) y un 98,7% (I Cr 95%: 96,6­99,9) en el caso dela primera prueba y de un 95,8% (I Cr 95%: 91,1­99,7) y un 96,1% (I Cr 95%: 95,1­97,7) en el caso de la segunda. Los resultados obtenidos con una y otra prueba no diferían significativamente entre sí. Además, utilizando ambas técnicas ELISA se calculó que la exposición actual al virus de los bovinos jóvenes del sur de Bélgica se cifraba en un 23,3% (I Cr 95%: 20,6­26,2). Para las tareas de vigilancia del BVDV se recomienda combinar el análisis virológico de todos los recién nacidos ­ para detectar animales con infección persistente ­ con la realización periódica de pruebas serológicas en el ganado joven con la técnica ELISA.

Three Different Routes of Inoculation for Experimental Infection with Schmallenberg Virus in Sheep.

Schmallenberg virus (SBV) is an emerging Orthobunyavirus affecting European domestic ruminants. In this study, three groups of ewes (n = 3) were inoculated with 1 ml of an SBV infectious serum, via the subcutaneous (SC), intradermal (ID) or intranasal (IN) route. The ewes were monitored for 10 days and no clinical signs were reported. IN inoculation failed to generate any detectable RNAemia. SC and ID inoculation induced typical SBV RNAemia and seroconversion upon day 6 post-inoculation in 3/3 and 2/3 sheep, respectively. In all the animals that showed RNAemia, the viral genome could be detected in spleen and mesenteric lymph nodes. Both the SC and ID routes seem suitable to properly reproduce field conditions, as comparable observations were reported regarding RNAemia, seroconversion and viral genome detection in organs.

Q Fever Serological Survey and Associated Risk Factors in Veterinarians, Southern Belgium, 2013.

A sero-epidemiological survey was organized among veterinarians working in Southern Belgium to estimate the seroprevalence of Q fever and the risk factors associated with exposure. A total of 108 veterinarians took part to this cross-sectional study, with a majority practicing with livestock animals. The overall seroprevalence was 45.4%, but it increased to 58.3% among veterinarians having contact with livestock. Three main serological profiles were detected (relatively recent, past and potentially chronic infections). The contact with manure during the prior month was the risk factor associated with seropositivity after multivariate logistic regression analysis. Classification and regression tree analysis identified the age as the most predictive variable to exclude potentially chronic infection in apparently healthy seropositive veterinarians. In conclusion, livestock veterinarians practicing in Southern Belgium are highly exposed to Q fever, a neglected zoonosis for which serological and medical examinations should be envisaged in at risk groups.

Laboratory Findings Suggesting an Association Between BoHV-4 and Bovine Abortions in Southern Belgium.

Abortions cause heavy economic losses for the bovine sector. The use of a standardized panel of analyses covering a large spectrum of pathogens responsible of abortion in cattle allowed demonstrating the direct involvement of at least one pathogen in 57% of analysed abortions in the southern part of Belgium. This result suggests a margin of improvement in the diagnostic efficacy. In order to evaluate the interest to broaden the list of pathogens included in the panel of analyses, the implication of bovine herpesvirus 4 (BoHV-4) in abortion was assessed by two different studies. In the first study, coupled serology was performed after abortion on 714 dams to identify specific seroconversion against BoHV-4. The overall seroconversion in cows was 19.5%, with a higher frequency in primiparous compared to multiparous females. In addition, the type of breed (beef cattle) and the time period from the fourth quarter 2008 until the last quarter 2009 were significantly related to the seroconversion of cows. The second study investigated the virus ability to infect the foetus. In this study, 368 cases of bovine abortions were specifically tested for BoHV-4, using PCR on foetus tissues and ELISA on dam and foetus sera. The results showed a maternal seroprevalence of 64.7%, a foetal seroprevalence of 0.8% and a PCR prevalence in foetuses of 1.1%, demonstrating the ability of BoHV-4 to infect the foetus.

Clinical Sentinel Surveillance of Equine West Nile Fever, Spain.

West Nile fever (WNF) is a viral zoonotic infection caused by a mosquito-borne flavivirus of the Flaviviridae family. According to a comparative study, the passive surveillance of horses by equine veterinarians appeared to be the most cost-effective system in the European context of WNF. Clinical data issued from a passive epidemiosurveillance network from September 2010 to December 2011 on horses in Spain were statistically compared and used to develop a predictive diagnostic decision tree, both with the aim to improve the early clinical detection of WNF in horses. Although clinical signs were variable in horses affected by WNF, four clinical signs and the month of occurrence were identified as useful indicators to distinguish between WNF-related and WNF-unrelated cases. The signs that pointed out a presumptive diagnosis of WNF in horses were cranial nerves deficits, limb paralysis, photophobia and nasal discharge. Clinical examination of horses with neurological signs that are not vaccinated against WNV could provide important clues for the early clinical detection of WNF and therefore serve as an alert for possible human viral infections. The study of the clinical pattern of WNF in horses is of importance to enhance awareness and better understanding and to optimize surveillance designs for clinical detection of WNF in horses in advance of epidemic activity affecting humans.

Clinical indicators of exposure to Coxiella burnetii in dairy herds.

Q fever is a zoonosis occurring worldwide in livestock. Often neglected in differential diagnoses, Q fever can persist in herds causing financial losses in the long run. In ruminants, well-known manifestations of Q fever are abortion, stillbirth, delivery of weak offspring and premature delivery. In cattle, Q fever is frequently asymptomatic and/or under-reported. The use of new methodologies in veterinary clinical epidemiology is of prime importance to find accurate clinical indicators of exposure to C. burnetii at herd level. A retrospective randomly cross-sectional survey was conducted to estimate the seroprevalence of Q fever in southern Belgium by means of an ELISA test performed on the bulk tank milk (n = 206 cattle herds). At the same time, a questionnaire was accomplished allowing the investigation of presumptive clinical signs observed at herd level during the previous twelve months for dairy cows. A multivariate logistic regression analysis was used to identify abortion and irregular repeat breeding as two indicators associated with Q fever exposure in dairy herds. In addition, a bootstrapped quantile regression revealed that the average score of putative clinical signs related to Q fever was significantly more important in exposed versus non-exposed herds. A classification and regression tree (CART) analysis confirmed the importance of the average clinical score and the irregular repeat breeding as main splitters, considering or not each clinical sign separately. Considering herd clinical patterns, instead of taking each clinical sign separately, seems to be more useful to differentiate herds at risk of Q fever exposure.

Dose-dependent effect of experimental Schmallenberg virus infection in sheep.

Schmallenberg virus (SBV) is an orthobunyavirus affecting European domestic ruminants. In this study, the dose-dependent effect of experimental infection of sheep with SBV was evaluated. Four groups of three ewes were each inoculated subcutaneously with 1 mL of successive 10-fold dilutions of an SBV infectious serum. The ewes were monitored for 10 days, but no clinical signs were observed. The number of productively infected animals within each group, as evidenced by viraemia, seroconversion and viral RNA in the organs, depended on the inoculated dose, indicating that a critical dose has to be administered to obtain a homogeneous response in infected animals under experimental conditions. In the productively infected animals, no statistical differences between the different inoculation doses were found in the duration or quantity of viral RNA circulating in blood, nor in the amount of viral RNA present in virus positive lymphoid organs.

Preliminary survey on the impact of Schmallenberg virus on sheep flocks in South of Belgium.

Between late February and May 2012, a preliminary anonym survey was conducted among sheep farmers in south of Belgium in order to contribute to future estimations of the economic losses caused by Schmallenberg virus (SBV). Based on clinical signs consistent with SBV infection, this survey involved 13 meat sheep flocks considered as positive flocks with subsequent SBV detection by RT-qPCR [SBV-positive flocks (PF); total of 961 animals], and 13 meat sheep flocks considered as negative flocks (NF; total of 331 animals). These preliminary results indicated several significant characteristics that were more present in PF than in NF. These include an increased rate of abortions (6.7% in PF versus 3.2% in NF), of lambs born at term but presenting malformations (10.1% in PF versus 2.0% in NF) and of dystocia (10.1% in PF versus 3.4% in NF). Lamb mortality during the first week of life was reported more frequently in PF (8 of 13 PF, 61.5%) than in NF (1 of 13 NF, 7.7%). In PF, the observed prolificacy rate was 2-fold lower (93%) than expected (186%). The implementation of a survey at larger scale, including a high number of breeders, is necessary to allow a more detailed analysis of the SBV impact in the sheep sector.

Field veterinary survey on clinical and economic impact of Schmallenberg virus in Belgium.

We implemented a questionnaire-based methodology targeting veterinary field practitioners to evaluate clinical and economic impact of Schmallenberg virus in Belgium. First suspicious cases were detected as soon as July 2011. The mean cost for individual symptomatic treatment was 65 or 107 Euros, in case of fatal outcome or apparent recovery, respectively.

Antiviral chemotherapy in veterinary medicine: current applications and perspectives.

The current situation in the use of antiviral drugs in veterinary medicine is characterised by a novel and optimistic approach.Viruses of veterinary importance are still used as animal models in the developmentof human therapeutics, but there is growing interest in many of these viruses in the identification of antiviral molecules for use in both livestock and companion animals. The use of antiviral drugs in livestock animals is envisaged for the treatment or control of disease on a large scale (mass treatment), whereas in companion animals an individual approach is favoured. An overview of the most recent examples of research in the use of antivirals in veterinary medicine is presented, with particular emphasis on their in vivo applications.

Original findings associated with two cases of bovine papular stomatitis.

Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

Experimental reproduction of bluetongue virus serotype 8 clinical disease in calves.

Cattle are commonly subclinically infected following natural or experimental infection with bluetongue virus (BTV). The introduction of BTV serotype 8 (BTV-8) in Europe has been characterized by the manifestation of clinical signs in infected cattle. In order to study the pathogenesis of BTV-8 in this host, an animal model able to reproduce the clinical manifestations of the disease is required. In this work, two calves were subcutaneously and intravenously injected with a low passage cell-adapted strain of BTV-8. Both calves showed typical bluetongue clinical signs, including pyrexia, ocular discharge, conjunctivitis, oral mucosal congestion, development of ulcers and necrotic lesions on the lips and tongue, submandibular oedema, coronitis and oedema of the coronet and pastern region. A score was assigned depending on the severity of the lesions and a total clinical score was calculated for each animal daily and at the end of the experiment. Both calves became viraemic 24h post-infection and seroconversion occurred between 7 and 11 days P.I. In this study we present the development of a protocol of infection in calves able to reproduce the severity of the lesions observed with BTV-8 in field conditions.

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [corrected] (HPMPC, cidofovir, CDV, Vistide) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir [corrected] and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved [corrected] more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.