The use of binding-prediction models to identify M. bovis-specific antigenic peptides for screening assays in bovine tuberculosis

The identification of MHC class II-restricted antigenic peptides for inclusion into vaccines and/or as diagnostic test reagents for mycobacterial infections remains a high research priority. To expedite discovery of such peptides, numerous bioinformatic tools have been developed to predict whether a given peptide is likely to form a stable binding interaction with MHC class II molecules. However, no prediction tool dedicated to the identification of bovine MHC (BoLA) class II-restricted peptides is currently available. Using experimental immunogenicity data derived from the stimulation of whole blood of Mycobacterium bovis-infected cattle with 105 individual M. bovis-derived peptides, we have compared the ability of a novel BoLA DRB3 structure-based prediction method (Hepitom) with the human MHC class II binding predictor model ProPred in predicting peptides that induce bovine T-cell activation. When a stringent cut off for considering peptide antigenicity was applied, the sensitivities of Hepitom and ProPred in detecting immunogenic peptides were 62% and 77%, respectively. In contrast, the Hepitom model showed greater specificity, with values of 66% and 34% for Hepitom and ProPred, respectively. Using all peptides, seven out of eleven M. bovis proteins were identified as being highly immunogenic. All but one of these antigens were also identified when just the Hepitom predicted peptides were used, while only four of the seven were identified using the ProPred predicted peptides. In conclusion, we demonstrate that the Hepitom model is a useful pre-screening tool to select peptides for further immunogenicity studies in cattle without major impact on the identification of antigenic M. bovis proteins.     

Exercising upper respiratory videoendoscopic evaluation of 100 nonracing performance horses with abnormal respiratory noise and/or poor performance

Reasons for performing study: Although well documented in racehorses, there is paucity in the literature regarding the prevalence of dynamic upper airway abnormalities in nonracing performance horses. Objective: To describe upper airway function of nonracing performance horses with abnormal respiratory noise and/or poor performance via exercising upper airway videoendoscopy. Methods: Medical records of nonracing performance horses admitted for exercising evaluation with a chief complaint of abnormal respiratory noise and/or poor performance were reviewed. All horses had video recordings of resting and exercising upper airway endoscopy. Relationships between horse demographics, resting endoscopic findings, treadmill intensity and implementation of head and neck flexion during exercise with exercising endoscopic findings were examined. Results: Dynamic upper airway obstructions were observed in 72% of examinations. Head and neck flexion was necessary to obtain a diagnosis in 21 horses. Pharyngeal wall collapse was the most prevalent upper airway abnormality, observed in 31% of the examinations. Complex abnormalities were noted in 27% of the examinations. Resting laryngeal dysfunction was significantly associated with dynamic arytenoid collapse and the odds of detecting intermittent dorsal displacement of the soft palate (DDSP) during exercise in horses with resting DDSP was only 7.7%. Exercising endoscopic observations were different from the resting observations in 54% of examinations. Conclusions: Dynamic upper airway obstructions were common in nonracing performance horses with respiratory noise and/or poor performance. Resting endoscopy was only helpful in determining exercising abnormalities with recurrent laryngeal neuropathy. Potential relevance: This study emphasises the importance of exercising endoscopic evaluation in nonracing performance horses with abnormal respiratory noise and/or poor performance for accurate assessment of dynamic upper airway function.     

Locking compression plate osteosynthesis of sacral fractures in three heifers

Objective: To describe a technique used for reduction and internal fixation of sacral fractures in cattle. Study Design: Case series. Animals: Three heifers aged 19–32 months with deformation of the tail head and tail paralysis attributable to a closed fracture of S5 or S4 and S5. Methods: Fractures were reduced and repaired under general anesthesia. A narrow, 6–10‐hole, locking compression plate (LCP) was placed on the spinous processes of S5 and the first coccygeal (Co1) using 5.0 mm locking head screws. Follow‐up inquiries were made by telephone (3/3), and cows were examined clinically and radiographically 2 (1 cow) to 9 (1cow) months postoperatively. Results: No complications were encountered. The range of movement of the tail improved so that soiling of the tail during defecation was no longer a problem. In all heifers, the normal top line was restored and there was no narrowing of the pelvic canal. Unassisted calving occurred in all heifers, and follow‐up radiographs for 2 cows showed complete fracture healing. Conclusion: LCP‐osteosynthesis of the spinous processes provided sufficient stability for internal fixation of sacral fractures. Marked improvement in tail function occurred postoperatively without decompression by laminectomy.     

Trial design to estimate the effect of vaccination on tuberculosis incidence in badgers

The principal wildlife reservoir of Mycobacterium bovis in Ireland is the European badger. Studies in the Republic of Ireland (RoI) have shown that badgers culled in association with cattle herd tuberculosis breakdowns (focal culling) have a higher prevalence of infection than the badger population at large. This observation is one rationale for the medium term national strategy of focal badger culling. A vaccination strategy for the control of bovine tuberculosis (bTB) in badgers is a preferred long-term option. The Bacillus Calmette-Guérin (BCG) vaccine has been shown to decrease disease severity in captive badgers under controlled conditions. As the vaccine has been tested in a controlled environment with precise information on infection pressure, it cannot be assumed a priori that the effects of vaccination are similar in the wild, where other environmental and/or ecological factors prevail. For this reason we have designed a vaccine field trial to assess the impact of vaccination on the incidence of TB infection in a wild badger population. The selected study area for the vaccine trial (approximately 755 square kilometers) is divided into three zones each of which has similar characteristics in terms of size, number of main badger setts, cattle herds, cattle and land classification type. Three vaccination levels (100%, 50% and 0%) will be allocated to the three zones in a way that a gradient of vaccination coverage North to South is achieved. The middle zone (zone B) will be vaccinated at a 50% coverage but zone A and C will be randomly allocated with 100% or 0% vaccination coverage. Vaccination within zone B will be done randomly at individual badger level. The objective of this paper is to describe the design of a field tuberculosis vaccination trial for badgers, the epidemiological methods that were used to design the trial and the subsequent data analysis. The analysis will enable us to quantify the magnitude of the observed vaccination effect on M. bovis transmission in badgers under field conditions and to improve our knowledge of the biological effects of vaccination on susceptibility and infectiousness.     

Genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype I strains

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been based on ORF5/GP5 and ORF7/N protein variations. Complete viral genome studies are limited and focused on a single or a few set of strains. Moreover, there is a general tendency to extrapolate results obtained from a single isolate to the overall PRRSV population. In the present study, six genotype-I isolates of PRRSV were sequenced from ORF1a to ORF7. Phylogenetic comparisons and the variability degree of known linear B-epitopes were done considering other available full-length genotype-I sequences. Cytokine induction of all strains was also evaluated in different cellular systems. Non structural protein 2 (nsp2) was the most variable part of the virus with 2 out of 6 strains harboring a 74 aa deletion. Deletions were also found in ORF3 and ORF4. Phylogenetic analyses showed that isolates could be grouped differently depending on the ORF examined and the highest similarity with the full genome cluster was found for the nsp9. Interestingly, most of predicted linear B-epitopes in the literature, particularly in nsp2 and GP4 regions, were found deleted or varied in some of our isolates. Moreover, 4 strains, those with deletions in nsp2, induced TNF-α and 3 induced IL-10. These results underline the high genetic diversity of PRRSV mainly in nsp1, nsp2 and ORFs 3 and 4. This variability also affects most of the known linear B-epitopes of the virus. Accordingly, different PRRSV strains might have substantially different immunobiological properties. These data can contribute to the understanding of PRRSV complexity.     

Update on vaccination of cattle and wildlife populations against tuberculosis

A recombinant bovine herpesvirus 5 lacking thymidine kinase and glycoprotein E genes (BoHV-5gEΔTKΔ) was evaluated as a live experimental vaccine. In a first experiment, ten-months-old calves were vaccinated intramuscularly (n=9) or remained as controls (n=8) and 42 days later were challenged with BoHV-5 or BoHV-1 intranasally. The four control calves challenged with BoHV-5 developed severe depression and neurological signs and were euthanized in extremis at days 13 and 14 pos-infection (pi); the five vaccinated animals challenged with BoHV-5 remained healthy. The titers of virus shedding were reduced (p<0.01) from days 3 to 7 post-infection (pi) in vaccinated animals. Control and vaccinated calves challenged with BoHV-1 presented mild transient respiratory signs; yet the magnitude of virus shedding was reduced (p<0.05) in vaccinated animals (days 5, 9 and 11pi). In a second experiment, young calves (100-120 days-old) were vaccinated (n=15) or kept as controls (n=5) and subsequently challenged with a BoHV-1 isolate. Control calves developed moderate to severe rhinitis and respiratory distress; two were euthanized in extremis at days 5 and 9 pi, respectively. In contrast, vaccinated animals were protected from challenge and only a few developed mild and transient nasal signs. The duration and titers of virus shedding after challenge were reduced (p<0.05) in vaccinated animals comparing to controls. In both experiments, vaccinated animals developed antibodies to gE only after challenge. These results demonstrate homologous and heterologous protection and are promising towards the use of the recombinant BoHV-5gEΔTKΔ in vaccine formulations to control BoHV-5 and BoHV-1 infections.     

The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes

The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.     

Lead Isotopic and Metallic Pollution Record in Tree Rings from the Copperbelt Mining�CSmelting Area, Zambia

The composition of tree rings and soils was studied at several locations affected by smelting and transportation in the vicinity of Kitwe (Copperbelt, Zambia). The contents of cobalt (Co), copper (Cu), manganese (Mn), and lead (Pb) and the ²⁰⁶Pb/²⁰⁷Pb isotope ratios in the tree rings were interpreted in relation to potential sources of contamination such as smelter production, acidification of the environment, soil composition, raw material processing, and atmospheric suspended particulate matter (SPM). The highest Co contents in the tree rings correspond to maximum ore production in the mid-1970s. Acidification through SO₂ emissions is documented in the increased Mn contents from the mid-1980s. The isotopic composition of the tree rings of the studied tree species varies in the interval 1.16?C1.34 and the youngest parts of all the studied trees exhibit a low ²⁰⁶Pb/²⁰⁷Pb ratio (<1.17). The soil isotope composition varies in the range ²⁰⁶Pb/²⁰⁷Pb?=?1.18?C1.35. The Pb isotope composition in the soils and tree rings was formed by a combination of lithogenic Pb (²⁰⁶Pb/²⁰⁷Pb????1.3), Pb in processed ores (²⁰⁶Pb/²⁰⁷Pb????1.2), and SPM (automobile) Pb (²⁰⁶Pb/²⁰⁷Pb??1.1). As the soils in the distant region have high ²⁰⁶Pb/²⁰⁷Pb ratios (>1.3) in the whole profile and simultaneously the youngest parts of the tree rings of tree species growing in this soil have a low ²⁰⁶Pb/²⁰⁷Pb ratio (<1.17), it can be assumed that the Pb in the youngest parts of the tree species is derived from absorption of SPM Pb through the bark rather than root uptake. The absence of Pb with a low ²⁰⁶Pb/²⁰⁷Pb ratio in soils in the distant area is probably affected by fires in the herbaceous and bush undergrowth and plant litter, which prevents Pb from biomass from entering the soil and mobilize it back into the atmosphere.     

Mercury (Hg) Transport in a High Arctic River in Northeast Greenland

In a warming climate, mercury (Hg) pathways in the Arctic can be expected to be affected. The Hg transport from the high arctic Zackenberg River Basin was assessed in 2009 in order to describe and estimate the mercury transported from land to the marine environment. A total of 95 water samples were acquired and filtered (0.4 ??m pore size), and Hg concentrations were determined in both the filtered water and in the sediment. A range of other elements were also measured in the water samples. Hg concentrations in the filtered water were in general highest in the beginning of the season when the water came mainly from melted snow. THg concentrations in the sediment were in general relatively constant or slightly decreasing until mid-August, where after the concentrations increased. A principal component analysis separated the samples into spring, summer and autumn samples indicating seasonal characteristics of the patterns of element concentrations. The total amount of Hg in the sediment transported was estimated to 2.6 kg. Approximately 60% of the sediment-transported Hg occurred during a 24-h flood in the beginning of August caused by a glacial lake outburst flood. The total amount of transported dissolved Hg was estimated to 46 g, and 13% of this transport occurred during the 24-h flood. If it is assumed that the Hg transport by Zackenberg River is representative for the general glacial rivers in East Greenland, the total Hg transport into the North Atlantic from Greenland alone is approximately 4.6 tons year^?1 with an estimated annual freshwater discharge of ??440 km³.