Hugues Guyot

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.

Bluetongue in captive yaks.

To the Editor: In August 2006, several northern European countries including Belgium reported cases of bluetongue (BT) (1). This noncontagious, arthropod-borne animal disease is caused by Bluetongue virus (BTV), genus Orbivirus, family Reoviridae. The genome of BTV consists of 10 segments of double-stranded RNA; 24 serotypes have been reported (2). Serotype 8 (BTV-8) was implicated in the emergence in Belgium (3). All ruminant species are thought to be susceptible to BT (2). We report laboratory-confirmed clinical cases of BT in yaks (Bos grunniens grunniens). Yaks living in captivity in a Belgian animal park showed clinical signs of BT. A clinical examination performed on 1 yak showed loss of weight associated with a progressive weakness linked to anorexia, ulcerative and necrotic lesions on the muzzle with some crusts and mucopurulent nasal discharge, and udder erythema with papules and crusts. The tongue was severely swollen and cyanotic and protruded from the mouth (Figure). The animal was reluctant to move and was recumbent (possibly as a consequence of podal lesions linked to BT); it died 7 days after examination. Necropsies were performed on carcasses of this and another yak. The main lesions found were severe diffuse congestion of the lungs with edema and emphysema, acute hemorrhagic enteritis restricted to the ileum and jejunum, and petechial hemorrhages on the abomasums. No lesions characteristic of coronitis were noted. Figure A captive yak infected with bluetongue virus. Tongue is swollen, cyanotic, and protruding from the mouth. Samples of spleen and bone marrow were taken and prepared according to the method of Parsonson and McColl (4). A real-time reverse transcription quantitative–PCR (RT-qPCR) targeting BTV segment 5 (RT-qPCR_S5) was used to detect BTV RNA in tissues samples. Each test was performed in parallel with a RT-qPCR that amplifies β-actin mRNA as an internal control (RT-qPCR_ACT). Both assays were conducted according to Toussaint and others (5), with slight modifications. Briefly, total RNA was purified from 25 mg of tissue by Trizol extraction (Invitrogen, Carlsbad, CA, USA) and denatured by heating for 3 min at 95°C with 10% dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA). Reverse transcription reactions were conducted by using the Taqman reverse transcription reagents according to the manufacturer instructions (Applied Biosystems, Foster City, CA, USA). RT-qPCR reactions consisted of 1× concentrated Taqman fast universal PCR master mix (Applied Biosystems), 375 nM (β actin) or 500 nM (BTV) of each primer, 250 nM Taqman probe, and 5 μL cDNA. Cycling conditions were as follows: 1 cycle at 95°C for 20 s, followed by 45 cycles of 1 s at 95°C, and 20 s at 60°C. The specificity of the RT-qPCR used had been previously tested against prototype strains of genetically related viruses (9 strains of epizootic hemorrhagic disease virus and 9 strains of African horse sickness virus) (5). The RT-qPCR tests confirmed BTV viremia. The yak species in its natural biotope is usually rarely exposed to competent Culicoides vectors. Antibodies against BTV have been found in many wild ruminants (6), and our results extend the number of ruminant species susceptible to BTV. In the northern European BT outbreak, lesions in cattle and sheep were mainly localized to the regions of the muzzle, mouth, and eye; clinical signs were not always obvious (7,8). As in cattle and sheep, clinical signs in yaks were observed on the muzzle, in the periocular region, and around and inside the mouth. These signs clearly reflected viral-induced endothelial damage triggering disseminated intravascular coagulation and a hemorrhagic diathesis commonly described in sheep and cattle (2). In our case, lesions depicted pronounced microvascular damage. According to the severity of the lesions and rates of illness and death observed, the yak, like sheep, appears to be highly susceptible to BTV. In the epidemiology of BT in African countries, cattle and wild ruminant species such as antelopes play a role as asymptomatic reservoir hosts of the virus (2). Some wild ruminant species in captivity could also play this role in European countries affected by the recent BT outbreak. These cases could be of particular concern for all parks and zoos that gather numerous wild ruminants. Illness, reproductive failure, and deaths usually reported with BT (9) could generate substantial losses on these premises. Moreover, the source of BTV-8 in the northern European outbreak remains unclear, and the role of wild ruminant species has to be taken into account. In the future, European authorities should consider vaccination to prevent the spread of the disease in European member states (10). All premises with wild ruminants need to be involved in BT control and prophylaxis.

Clinical Pattern Characterization of Cattle Naturally Infected by BTV-8

Forty-one cattle from seven Belgian farms and two French farms confirmed as infected with bluetongue virus serotype 8 (BTV-8) were monitored from the onset of clinical signs to describe the disease pattern and estimate the duration of blood RT-qPCR and competitiveELISA positivity under field conditions. On each visit, blood samples were taken, and a standardized clinical form was filled in for each animal. A clinical score was calculated for every week until the end of clinical signs. A classification and regression tree (CART) analysis was conducted to determine the most important clinical signs every week for the first 7 weeks. The highest scores were recorded within 2 weeks of clinical onset. The first recorded clinical signs were quite obviously visible (lethargy, conjunctivitis, lesions of nasal mucosa, nasal discharge). Skin lesions, a drop in milk production and weight loss appeared later in the course of the disease. A biphasic pattern regarding nasal lesions was noticed: the first peak concerned mainly congestive and ulcerative lesions, whereas the second peak mainly concerned crusty lesions. The median time estimated by survival analysis to obtain negative RT-qPCR results from the onset of clinical signs was 195 days (range 166-213 days) in the 23 cattle included in the analysis. Serological results remained strongly positive until the end of the study. These results should ensure more accurate detection of an emerging infectious disease and are of prime importance in improving the modelling of BTV-8 persistence in Europe.

Vegetative Endocarditis in Equids (1994–2006)

BACKGROUND Endocarditis is a rare heart condition with variable clinical expressions in equids. Risk factors for this disease are incompletely understood. OBJECTIVE Describe risk factors for endocarditis in equids. ANIMALS One hundred and fifty-three equids admitted to Liège University, 9 diagnosed with endocarditis and 144 free from endocarditis but admitted to the hospital with a differential diagnosis including this disease. METHODS Retrospective case-control study. RESULTS Equids with endocarditis were significantly younger (mean age = 4.84 +/- 5.74 years) than control equids (mean age = 10.8 +/- 7.73 years) (P = .01). No sex or breed predisposition was observed. Animals with hyperthermia (odds ratio [OR] = 24.4; confidence interval [CI] = 1.40-428), synovial distension (OR = 13.4; CI = 3.00-59.8), lameness (OR = 6.52; CI = 1.63-26.1), hyperglobulinemia (OR = 26.4; CI = 3.03-229), hypoalbuminemia (OR = 11.4; CI = 1.34-96.8), hyperfibrinogenemia (OR = 9.81; CI = 1.16-82.7), or leukocytosis (OR = 7.12; CI = 1.40-36.4) presented a significantly higher risk of having endocarditis than control horses. The presence of two of the clinical signs mentioned above significantly increased the probability of a diagnosis of endocarditis (P< or = .05). CONCLUSIONS AND CLINICAL IMPORTANCE Age is associated with equine endocarditis. The diagnostic value of certain clinical signs and abnormalities in blood parameters in this disease are described.

Emergence of bovine ehrlichiosis in Belgian cattle herds

Bovine ehrlichiosis is a tick-borne rickettsial disease caused by Anaplasma phagocytophilum. The disease can also be transmitted to humans. Outbreaks in cattle have been described in many European countries. In Belgium, infections caused by A. phagocytophilum have been reported in humans and dogs; however, this paper details the first report of ehrlichiosis in cattle herds in Belgium. The first case described was in a dairy herd located in eastern Belgium. Clinical signs included hyperthermia, polypnea, and swelling of the limbs. The other case was diagnosed in a second, mixed purpose herd in western Belgium. Within the second herd, all of the affected animals came from the same pasture. All animals in that pasture showed recurrent hyperthermia, and some also showed signs of mastitis and late-term abortions. Blood smears and serology revealed the presence of A. phagocytophilum in the majority of animals with pyrexia. Furthermore, the presence of leptospirosis, Neospora caninum, and Q fever antibodies was tested by serological analysis, but all results were negative. Paired serology for Adenovirus, BHV-4, BHV-1, BVD, PI3, and RSV-B did not show any significant seroconversion. Milk samples from cows affected by mastitis revealed minor pathogens. Fecal testing for the presence of Dictyocaulus viviparus in the first herd was negative. Recurrent pyrexia in pastured cattle is a non-specific sign, and can be related to several different pathogens. Bovine ehrlichiosis is transmitted by the tick species Ixodes ricinus which is known to be present throughout Belgium. Belgian practitioners should include ehrlichiosis in their differential diagnosis when confronted with pastured cattle suffering from recurrent pyrexia.

Effect of a combined iodine and selenium supplementation on I and Se status of cows and their calves.

Iodine (I) and selenium (Se) deficiencies are commonly reported in cattle, however, there are also studies regarding a very high iodine supply. The aim of the study was to determine the long-term effect of I and Se supplementation on non-pregnant cows, pregnant cows and their calves. The hypothalamus pituitary axis was investigated (TSH, T4, T3 assays) during a TRH challenge on non-pregnant cows. Twenty-four cows, half of them pregnant, were assigned into 2 diet-groups, one group with a low I (0.45 ppm) and Se (0.15 ppm) diet (LISe), the other with a high I (5.45 ppm) and Se (0.45 ppm) diet (HISe), for a period of 120 days. Nutritional (plasma iodide, urinary I, plasma Se, I content in colostrum and foetal fluids) and functional (thyrotropin, thyroid hormones, glutathione-peroxidase activity in erythrocytes) markers of I and Se status were assayed in dams at regular intervals for 120 days and in their calves at birth. A TRH challenge was performed on 8 non-pregnant cows at day 110 of the trial. At the end of the study, I and Se nutritional markers were higher in dams in the HISe group, compared to the LISe group, except for plasma Se. At birth, I nutritional markers in calves in the HISe group were higher compared to the LISe group. Reactivity of the pituitary-thyroid-axis was not influenced by I and Se supplementation. I and Se supplementation is efficient in improving newborn status.

Clinical differentiation of malignant catarrhal fever, mucosal disease and bluetongue.

THE 2006/07 outbreak of bluetongue virus infection in northern Europe has been characterised by a higher proportion of cattle showing clinical signs (Thiry and others 2006, Guyot and others 2007). This is not often associated with outbreaks of the disease as it is more usual for cattle to be asymptomatic. Adult cattle have mainly been affected, showing ulcerative and necrotic lesions on the muzzle and in the oral cavity, conjunctivitis, lacrimation and periocular dermatitis, photosensitivity-type skin lesions around the midline (later in the course of disease), a drop in milk yield, mild pyrexia and transient loss of body condition, ulcerative and necrotic skin lesions on the udder, teats and vulva, and oedema of the distal limbs (Guyot and others 2007). Bluetongue was first reported in the UK in September 2007, and there is a real threat of the disease affecting more animals in the country, both from infected midges in the UK and those arriving from continental Europe in wind currents (Gloster and others 2007). This, alongside foot-and-mouth disease (FMD), has led to severe economic consequences in terms of both animal movement restrictions and the export market, and animal welfare. Among the differential diagnoses for bluetongue are malignant catarrhal fever (MCF) and mucosal disease, which are endemic to the UK. The clinical differentiation of these three conditions is therefore highly relevant. The aim of this study was to characterise the clinical findings typical of cases of MCF and mucosal disease, and to compare these with clinical findings characteristic of the recent bluetongue outbreak in northern Europe, to assess their value in differentiating these three conditions. Thirty-eight cases of bluetongue were selected from the database of records of the ambulatory clinic of the Faculty of Veterinary Medicine, University of Liège, from August 2006 to February 2007. The criteria for inclusion were a clinical diagnosis of bluetongue and a positive test result either for detection of antibody by a competitive ELISA or detection of viral nucleic acid by real-time reverse transcriptase-PCR, as described by Toussaint and others (2007). Sixty-six cases of mucosal disease and 11 cases of MCF were selected from the database of records of cattle admitted to the University of Glasgow Veterinary School (UGVS) from January 1989 to June 2007. Cases of mucosal disease were included on the basis of typical pathological changes at postmortem examination and on being antigen-positive (HerdChek BVDV Antigen; Idexx) and antibody negative (Svanovir BVDV-Ab; Svanova) for bovine viral diarrhoea virus (BVDV) by ELISA. Cases of MCF were included on the basis of postmortem examination findings and on being positive for antibody to ovine herpesvirus type 2 (OvHV-2) by an immunofluorescent antibody test (Reid 2004) or for OvHV-2 nucleic acid by PCR (Hussy and others 2001). All cases diagnosed as MCF were antigen-negative for BVDV. All the animals had been subjected to a thorough clinical examination according to standard protocols, albeit with slight differences between the two institutions. Cases of bluetongue were examined on-farm in Belgium, whereas cases of MCF and cases of mucosal disease were examined after being referred to the UGVS clinic. Case data were summarised to determine the most common presenting signs for each disease. A Freeman-Halton extension of Fisher’s exact probability test was performed (VassarStats 2007) to test for significant differences in the frequency of clinical signs between the three conditions (Freeman and Halton Veterinary Record (2007) 161, 858-859

Development and validation of a radioimmunoassay for thyrotropin in cattle

In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and reproducibility. The physiologic validation included a thyrotropin-releasing hormone (TRH) challenge performed on euthyroid cows and a follow-up of bTSH concentration over a 24-hour period. Furthermore, bTSH concentration was assayed in a large population of healthy dairy and beef cows to define reference interval. The pathologic validation was made by assaying bTSH and thyroid hormones on healthy and goitrous newborn calves. The minimum detection limit (MDL) for bTSH assay was 1.3 μU/ml. The recovery was 101% to 106%. The intra- and interassay coefficients of variation (CVs) ranged from 5% to 11% and 11% to 15%, respectively. The RIA covered the whole range of physiologic bTSH values, as shown by bTSH values induced by TRH-challenge. A pulsatile secretion of bTSH was observed, accompanied by a diurnal variation with lower night values than day values. Reference intervals of bTSH ranged from 1.3 to 13.0 μU/ml for beef and dairy breeds. Finally, bTSH easily discriminated goitrous newborn calves from healthy ones, leading to the definition of a cutoff value of 35 μU/ml. The bTSH assay positively reacted to physiologic and pathologic conditions. The accuracy and precision of the RIA were satisfying.

Comparison of Various Indices of Energy Metabolism in Recumbent and Healthy Dairy Cows

Background Downer cow syndrome (DCS) is often diagnosed in dairy cattle during the early post-partum period. The etiology of this condition is not completely understood, as it can be related to the energetic or electrolyte metabolism, as well as to infectious diseases or to trauma. Hypothesis/Objectives The aim of this study is to compare energy metabolism and insulin sensitivity indices and various biochemical parameters between recumbent and healthy dairy cows. Animals A prospective study has been undertaken on 361 recumbent and 80 healthy Holstein cows. Methods Plasmatic glucose, insulin, non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHB) were assayed in all cows in order to calculate the insulin sensitivity indices but also minerals (Calcium, Phosphorous and Magnesium), thyroxin and creatine kinase. Body Condition Scores (BCS) was assessed. Results Significant differences in NEFA, and the glucose and insulin sensitivity indices (“Homeostasis Model Assessment” HOMA, “Revised Quantitative Insulin Sensitivity Check Index” RQUICKI, RQUICKI-BHB) were observed between healthy and recumbent cows in the early post-parturient period indicating disturbances of glucose and insulin homeostasis in the recumbent cows. In the same manner, mineral concentrations were significantly different between healthy and recumbent cows. Glucose, insulin NEFA, and HOMA, were different between early post-partum downer cows and the DCS-affected cows later in lactation. Conclusion and clinical importance Results indicate disturbances in energy homeostasis in DCS-affected dairy cows. Further research should determine a prognostic value of the indices in cows suffering from recumbency of metabolic origin.

Effects of three methods of oral selenium-enriched yeast supplementation on blood components and growth in Holstein dairy calves

The objective of this study was to investigate the effect of three methods of oral selenium-enriched yeast (Se-yeast) supplementation on the blood characteristics and growth of Holstein dairy calves. The three methods were: (1) maternal (dams supplemented with 0.3 mg Se per kg of dietary DM during 3 weeks pre-calving), (2) post-calving (calf starter enriched with 0.3 mg Se per kg of DM), and (3) maternal and post-calving Se-yeast supplementation. These three methods were compared with a control group (no supplementation). Maternal supplementation with Se-yeast successfully increased Se concentration in the colostrum and the serum of calves 24 h after calving (P < 0.05). At day 56, the Se concentration in the blood of calves supplemented with Se-yeast pre- (maternal) or post-calving (via starter) was greater than the control group (P < 0.05), but maternal plus post-calving supplementation was not more effective than either maternal or post-calving supplementation. Globulin and total protein concentration in the blood of calves at day 0 increased and albumins : globulins decreased with maternal supplementation (P < 0.05), but these variables were similar for all treatments at day 56. Immunoglobulins in the blood of calves and colostrum were not affected by maternal Se-yeast supplementation. Maternal supplementation increased the concentration of urea in the blood of calves at 0 days (P < 0.05), but not at 56 days. Calf birthweight was increased with maternal supplementation of Se-yeast (P < 0.01), but growth to day 56 was not affected by the methods of Se supplementation. Maternal supplementation with Se-yeast may improve growth and health characteristics of calves at calving time.