One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

<p>Abstract</p> <p>Background</p> <p>Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.</p> <p>Results</p> <p>A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using <it>in vitro </it>transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).</p> <p>Conclusions</p> <p>The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 10²copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.</p

Teška Auzdyk infekcija u jednomjesečnih devinih mladunaca (Camelus dromedarius).

On 11 March 2002 two approximately one-month-old, one-humped camel calves (C. dromedarius) were presented to the University Veterinary Teaching Hospital, King Faisal University, Al-Hasa, Saudi Arabia (SA), suffering from severe lesions on the lips and hard palates. Samples were collected and virological, pathological and serological investigations were carried out. The disease was confirmed to be Auzdyk. The situation was discussed in relation to the severity of the disease, a condition seen for the first time in this country, in these very young camels,. The results confirmed that fairly young camel calves, 1-month old, can suffer a severe form of the disease.Na sveučilišnu veterinarsku kliniku King Faisal Sveučilišta u Saudijskog Arabiji (SA), dovedena su dva mladunca jednogrbih deva u dobi od približno mjesec dana s jako izraženim lezijama na usnama i tvrdom nepcu. Uzeti su uzorci kako bi se obavile virološka, patološka i serološka pretraga. Potvrđeno je da je riječ o Ausdyk infekciji. Razmatrana je jačina bolesti i njena pojava u mladih deva po prvi put u ovoj zemlji. Rezultati potvrđuju da i sasvim mlade deve, stare mjesec dana, mogu oboljeti s jako izraženim znakovima bolesti

A Novel Strategy for Determining Protective Antigens of the Parapoxvirus, Orf Virus

AbstractWe investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy

Population genomics of louping ill virus provide new insights into the evolution of tick-borne flaviviruses

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors

Cryptosporidiosis in calves

Cryptosporidiosis is a widespread zoonotic parasitic disease affecting livestock all over the world. Despite its prevalence, there is very little evidence about transmission routes to young calves, and how it could affect them long-term. Many commonly used disinfectants on farm are unable to inactivate Cryptosporidium oocysts, and some commercially available disinfectants, which claim to work, do not appear to have sufficient evidence available to the research and farming community. This work shows multiple commercial disinfectants which have been directly compared against each other for their efficacy against Cryptosporidium oocysts. The idea that transmission could occur from adult cattle via direct contamination of calf pens with faecal material has been disputed in scientific literature. Older research suggests that adult cattle are not infected with the same species that the calves have, however more recent research with new oocyst concentration techniques has found this not to be the case. It is essential therefore that the genotypes of Cryptosporidium are determined to see if adult cattle pose a risk to their calves. Genotyping using microsatellite analysis gives a more in-depth look at the type of C. parvum present. The aim was, therefore, to determine the risk that adult cattle pose to their calves with regard to C. parvum oocyst transmission on both a dairy and a beef farm in Scotland. Using these methods, it was discovered that adult dairy cattle are unlikely to play a major role in the transmission of C. parvum to their calves. Most of the adult cattle on the dairy farm were predominantly shedding C. parvum however calves on the same farm presented with different multilocus genotype. On the beef farm, however, many of the adult cattle did share the same multilocus genotype as their calves, and so pose more of a risk for oocyst transmission to their calves. The species and genotypes of Cryptosporidium present in Scottish wildlife has very few published studies, therefore the aim was to determine the prevalence of C. parvum in samples from rabbits and pheasants in Scotland. Rabbit faecal samples collected from 18 farms from across Scotland revealed C. parvum to be the most prevalent species; an unusual discovery as it was previosuly believed that C. cuniculus was the most prevalent species in wild rabbits. Despite this the DNA was very difficult to genotype which may indicate that the oocyst load in the faeces of rabbits was small, or that the PCR may have been affected by inhibition. If there is little DNA present, rabbits are unlikely to pose a major threat to calves with regard to C. parvum oocyst transmission. The pheasants also presented with C. parvum as the most prevalent species, although very few shared the same genotype that was present in the calves at the pheasant samples location. Very few oocysts are required to cause cryptosporidiosis in a calf, so even if co-located wildlife do not appear to be shedding high numbers of oocysts, there is still a small risk of transmission present. Young calves affected with cryptosporidiosis tend to make a full recovery under the right management, and the clinical signs clear up within a couple of weeks. It is not known whether or not there is a long-term effect on the calves ability to gain weight following infection with the parasite. Therefore the aim was to compare calves with different levels of clinical cryptosporidiosis to calves with no signs of clinical disease and weigh these animals periodically until they went to market at 6 months of age. It was found that calves with severe disease gained significantly less weight than those with no clinical disease and even animals with mild cryptosporidiosis suffered reduced weight gain over 6 months. This result demonstrates the economic cost that the parasite could have to the farming community on a long-term basis. Commonly used disinfectants are typically ineffective against Cryptosporidium oocysts, and those that are on the market have very little evidence to support their efficacy. Therefore, seven commercial disinfectants were tested for their efficacy to inactivate Cryptosporidium oocysts based on excystation rate and sporozoite to shell ratio. It was identified that hydrogen peroxide and hydrogen peroxide-based disinfectants are the most successful at inactivating oocysts, but only when the disinfectant is freshly prepared. Testing the efficacy of disinfectants once the disinfectant had been made up for 7 days showed that the best performing disinfectant with regard to having the least degradation over seven days was KENOTMCOX. As many farmers are unlikely to make disinfectant up fresh every time it is used, it is useful to know that despite the high efficacy of some products, time since the product was prepared significantly reduces this. It was also found that pens contaminated with faecal material are likely to reduce the efficacy of hydrogen peroxide-based disinfectants and so it is important to clean pens before disinfection. Therefore, this PhD has addressed the knowledge gaps in the literature regarding the role of adult cattle, rabbits and pheasants in the transmission of C. parvum to calves. Neither one poses a major risk due to the low oocyst output and mixed C. parvum genotypes present. It is more likely therefore that calves maintain infection through widespread environmental contamination caused by other infected calves. This work has shown how infection with C. parvum in the first few weeks of life has a significant effect on the weight gain achieved over a 6-month period and so cryptosporidiosis has a significant effect on livestock production and on the profitability of the farm business. The efficacy of commercial disinfectants has provided the advice that disinfectants should be made up fresh and used on an area that has already been cleaned of faecal material in order to inactivate as many of the oocysts as possible

Development of a framework for genotyping bovine-derived Cryptosporidium parvum, using a multilocus fragment typing tool

Background: There is a need for an integrated genotyping approach for C. parvum; no sufficiently discriminatory scheme to date has been fully validated or widely adopted by veterinary or public health researchers. Multilocus fragment typing (MLFT) can provide good differentiation and is relatively quick and cheap to perform. A MLFT tool was assessed in terms of its typeability, specificity, precision (repeatability and reproducibility), accuracy and ability to genotypically discriminate bovine-derived Cryptosporidium parvum. Methods: With the aim of working towards a consensus, six markers were selected for inclusion based on their successful application in previous studies: MM5, MM18, MM19, TP14, MS1 and MS9. Alleles were assigned according to the fragment sizes of repeat regions amplified, as determined by capillary electrophoresis. In addition, a region of the GP60 gene was amplified and sequenced to determine gp60 subtype and this was added to the allelic profiles of the 6 markers to determine the multilocus genotype (MLG). The MLFT tool was applied to 140 C. parvum samples collected in two cross-sectional studies of UK calves, conducted in Cheshire in 2004 (principally dairy animals) and Aberdeenshire/Caithness in 2011 (beef animals). Results: Typeability was 84 %. The primers did not amplify tested non-parvum species frequently detected in cattle. In terms of repeatability, within- and between-run fragment sizes showed little variability. Between laboratories, fragment sizes differed but allele calling was reproducible. The MLFT had good discriminatory ability (Simpson’s Index of Diversity, SID, was 0.92), compared to gp60 sequencing alone (SID 0.44). Some markers were more informative than others, with MS1 and MS9 proving monoallelic in tested samples. Conclusions: Further inter-laboratory trials are now warranted with the inclusion of human-derived C. parvum samples, allowing progress towards an integrated, standardised typing scheme to enable source attribution and to determine the role of livestock in future outbreaks of human C. parvum

Suitability of loci for multiple-locus variable-number of tandem-repeats analysis of Cryptosporidium parvum for inter-laboratory surveillance and outbreak investigations

Cryptosporidium parvum is the major cause of livestock and zoonotically-acquired human cryptosporidiosis. The ability to track sources of contamination and routes of transmission by further differentiation of isolates would assist risk assessment and outbreak investigations. Multiple-locus variable-number of tandem-repeats (VNTR) analysis provides a means for rapid characterization by fragment sizing and estimation of copy numbers, but structured, harmonized development has been lacking for Cryptosporidium spp. To investigate potential for application in C. parvum surveillance and outbreak investigations, we studied nine commonly used VNTR loci (MSA, MSD, MSF, MM5, MM18, MM19, MS9-Mallon, GP60 and TP14) for chromosome distribution, repeat unit length and heterogeneity, and flanking region proximity and conservation. To investigate performance in vitro, we compared these loci in 14 C. parvum samples by capillary electrophoresis in three laboratories. We found that many loci did not contain simple repeat units but were more complex, hindering calculations of repeat unit copy number for standardized reporting nomenclature. However, sequenced reference DNA enabled reproducible fragment sizing and inter-laboratory allele assignation based on size normalized to that of the sequenced fragments by both single round and nested polymerase chain reactions. Additional Cryptosporidium loci need to be identified and validated for robust inter-laboratory surveillance and outbreak investigations