The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.     

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.     

Bayesian estimation of flock-level sensitivity of detection of Salmonella spp., Enteritidis and Typhimurium according to the sampling procedure in French laying-hen houses

A study was carried out to estimate the prevalence of flocks infected by Salmonella spp., S. Enteritidis and S. Typhimurium in 521 French laying-hen farms from October 1st 2004 to September 30th 2005 as part of a European Union-wide baseline study to define targets for Salmonella reduction in member states. The sampling scheme prescribed and financed by the European Commission to detect Salmonella in laying-hen flocks was based on 2 dust-samples and 5 faeces-samples per farm. A latent-class Bayesian approach for correlated tests was used to estimate the sensitivity of detection of reduced sampling schemes corresponding to the 16 combinations of 2 dust- and 5 faeces-samples. For each model the full sampling scheme (7 samples) and the reduced protocol were considered as two correlated tests, the biological principle being identical and the reduced protocol being a subset of the full sampling scheme. As the observed apparent prevalence in cage flocks was higher than in other systems (barns, outdoor, or organic) these two sub-populations were considered separately. Bayesian estimation of posterior medians with 95% probability intervals for true prevalence in cage flocks were 0.34 (0.29; 0.39) and 0.13 (0.10; 0.18) for Salmonella spp. and Salmonella Enteritidis+Typhimurium respectively. In alternative flocks posterior medians with 95% probability intervals for true prevalence were 0.09 (0.06; 0.13) and 0.05 (0.03; 0.08) for Salmonella spp. and Salmonella Enteritidis+Typhimurium, respectively. In cage flocks Bayesian estimation of posterior distributions for sensitivity indicated that at least 5 samples, including 2 dust samples were necessary to attain comparable sensitivity levels to the full sampling scheme. In alternative flocks and for Salmonella spp. 6 samples were required to ensure a comparable sensitivity level to the full sampling scheme. Detection sensitivity was improved by increasing the number of dust samples in cage farms and by increasing the total number of samples whatever their type in alternative farms.     

Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.     

Thromboelastographic tracings in retired racing greyhounds and in non-greyhound dogs

BACKGROUND: Bleeding disorders in patients with normal coagulation test results are frequently reported in Greyhounds. The purpose of this study was to compare Greyhounds to non-Greyhounds by thromboelastography (TEG). HYPOTHESIS: TEG parameters in Greyhounds are different from those in non-Greyhounds. ANIMALS: Forty-three healthy dogs (28 Greyhounds and 15 non-Greyhounds) based on the results of physical examination, CBC, activated partial thromboplastin time, prothrombin time, fibrinogen, and platelet count. MATERIALS AND METHODS: Recalcified citrated native TEGs were performed in both groups; data were compared using Student's, Mann-Whitney, and Pearson's statistical tests. RESULTS: In Greyhounds, mean +/- SD were as follows: R-time 4.3 +/- 1.7 minutes, K-time 3.8 +/- 1.4 minutes, angle (alpha) 50.0 +/- 8.0 degrees , maximum amplitude (MA) 47.6 +/- 5.6 mm, clot strength (G) 4,647 +/- 1,097 dyn/cm2, and percent lysis at 60 minutes (LY60) 2.8 +/- 5.0%. In the non-Greyhounds they were R-time 3.7 +/- 1.6 minutes, K-time 2.5 +/- 0.9 minutes, angle 59.8 +/- 7.0 degrees , MA 53.1 +/- 5.6 mm, G 5,811 +/- 1,256 dyn/cm2, and LY60 3.1 +/- 2.5%. All parameters were significantly different between the groups, except for R-time and LY60. CONCLUSION: In Greyhounds, clotting kinetics are slower and clot strength are weaker than in non-Greyhounds, supporting the increased tendency to bleed observed after minor trauma or surgical procedures in the breed. The findings may also be attributed to blood viscosity or to the concentration of citrate in the sample (ie, Greyhounds have higher hematocrit and less plasma per unit volume).     

Laryngeal rhinosporidiosis in a Belgian warmblood horse

In Belgium and even in northern Europe Rhinosporidium seeberi has not been reported in autochtonous people or animals. In this paper, the authors report the first observation of laryngeal masses, caused by Rhinosporidium seeberi, in a Belgian Warmblood horse. Moreover, laryngeal rhinosporidiosis is extremely rare since this localisation is only described in four human cases.     

First outbreak of Trypanosoma evansi in camels in metropolitan France

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.     

Leukemia inhibitory factor protein and receptors are expressed in the bovine adrenal cortex and increase cortisol and decrease adrenal androgen release

The release of adrenal steroids during acute stress is primarily regulated by adrenocorticotropic hormone (ACTH). In contrast, during chronic inflammatory stress additional factors are involved in regulating adrenal function. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that increases ACTH release from the pituitary. In addition, LIF and LIF receptors (LIFR) are expressed in the human adrenal cortex and the human adrenocortical tumor cell line H295R. Furthermore, LIF increases basal and ACTH-stimulated cortisol release from H295R cells. However, the expression of LIF and LIFR in non-human adrenal glands and the effects of LIF on the release of cortisol from adrenal cells of non-human species have not been determined. Furthermore, the effects of LIF on adrenal androgen release from all species are unknown. In this study, immunohistochemistry, Western blots, RT-PCR, and nucleotide sequencing was utilized to demonstrate that LIF and its receptor are expressed throughout the bovine adrenal cortex. Although LIF did not modify basal cortisol release from dispersed cells isolated from the bovine adrenal zona fasciculate, this cytokine increased ACTH-stimulated release of cortisol from these cells in a manner dependent on the LIF concentration and exposure interval. In contrast, LIF in a concentration-dependent and time-dependent manner decreased basal and ACTH-stimulated adrenal androgen release from dispersed cells isolated from the bovine adrenal zona reticularis. Because LIF release increases during inflammatory stress and this cytokine stimulates adrenal cortisol release and inhibits adrenal androgen release, this cytokine may play an important role in regulating the release of adrenal steroids during inflammatory stress.