Christopher Finnegan

Development of an improved real time PCR for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results

With the aim to erradicate Enzootic Bovine Leukosis from Poland, a more sensitive real-time polymerase chain reaction was required and developed to detect proviral Bovine leukaemia virus (BLV) DNA, the causative agent of Enzootic Bovine Leukosis (EBL). This new method proved more sensitive for our needs, than the current protocols available in the public domain. DNA was extracted from peripheral blood leukocytes of 51 cattle, which had given rise to doubtful serological test results by ELISA, and from mesenteric lymph nodes of six cattle that were slaughtered as EBL suspect cases. Additionally, fourteen DNA samples were obtained from animals with a strong BLV antibody response by ELISA. All real-time data were compared to results obtained from three different nested PCR methods. All 14 strongly positive ELISA samples were positive in all PCR tests. The real-time assay in comparison to the conventional PCR methods detected 7.8% (4/51) more specimens positive for BLV nucleic acid and showed a detection limit down to one copy. These observations represent the first report in the value of using a real-time method to help elucidate the disease status of animals when inconclusive ELISA results are obtained in the diagnostic laboratory. Thus, this method should be recommended for use in countries which have implemented an EBL-eradication programme, where a low level of BLV infection is evident.

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England.

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapox- and orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera.

Molluscum contagiosum in two donkeys

MOLLUSCUM contagiosum (MC) is a common skin infection in man and mainly seen in children, caused by the molluscum contagiosum virus (MCV). The typical pox virus particle morphology and genomic organisation of MCV led to its classification as a member of the family Poxviridae, subfamily Chordopoxvirinae, where it is the sole member of the genus Molluscipoxvirus (King and others 2011). MCV exists as two genetic subtypes, MCV1 and MCV2, each with several variants (Trama and others 2007) that have a similar clinical presentation in human beings. Lesions in human beings and animals are generally multifocal and are characterised by small, waxy, firm papules occurring principally on the face, trunk and in the genital region but can also be found in the oral cavity (Thompson and others 1998, Scott and Miller 2010). MC has been observed in other species including chickens, sparrows, pigeons, chimpanzees, kangaroos, dogs and horses (Ginn and others 2007). Equine MCV is thought to be identical to, or closely related to, human MCV. MCV has never been experimentally transmitted between animals (Mitchell 1953, Postlethwaite 1970); attempts to grow MCV in culture have failed. This property differentiates the virus from the orthopoxvirus of Uasin Gishu (UG) caused by Uasin Gishu disease virus (UGDV). UGDV produces histologically and clinically similar lesions to MCV but the virus can be grown in culture (Scott and Miller 2010). UGDV is antigenically similar to cowpox and vaccinia viruses (Scott and Miller 2010). Very little is known about its exact modality of transmission, with fomites and direct prolonged contact often quoted (Scott and Miller 2010). There is also very limited evidence of transmission between horses and man. Similarly, there is only speculation about the transmission between horses and man. In horses, MC is a self-limiting cutaneous infection with multiple small papules arising …

Confirmation of myxomatosis in a European brown hare in Great Britain.

MYXOMATOSIS is a disease of wild European and domestic rabbits ( Oryctolagus cuniculus ) caused by infection with myxoma virus, which is found naturally in some Sylvilagus rabbit species of South America and California. Sylvilagus species show few clinical signs. However, myxomatosis was introduced into Australia (1950), continental Europe (1952), Great Britain (1953) and Ireland (1954). This was as a control method for wild European rabbits. The initial mortality in Great Britain was 99 per cent. Myxomatosis is normally transmitted between host rabbits when virus particles adhere to the piercing mouthparts of a biting insect vector. In Great Britain the rabbit flea ( Spilopsyllus cuniculi ) is the most important vector of the disease. Other blood-sucking insects may play a minor role as vectors in some circumstances. Myxomatosis has rarely been reported in the European brown hare ( Lepus europaeus ). Initial experimental inoculation in 1937 of wild-caught brown hares in Australia using virulent myxoma virus from rabbits failed to elicit disease …

Use of Rapid Cycle Real-Time PCR for the Detection of Rabies Virus

Classical rabies virus (RV) is a member of the Lyssavirus genus within the Rhabdoviridae family, and is a causative agent of rabies. The virus has a wide host range, most probably including all mammals.

Colostrum replacer and bovine leukaemia virus sero-positivity

Bovine Leukemia Virus (BLV), a Deltaretrovirus in the Retroviridae family, is the causative agent of Enzootic Bovine Leucosis (EBL). Whilst EBL is still endemic in the Americas and Eastern Europe, most Western European countries are disease free in accordance with European Union legislation e.g. Great Britain has held EBL-free status since 1999. BLV infection is life-long and the presence of antibody and integrated proviral DNA are indicators of exposure to the virus.Screening for antibodies is the primary means of diagnosis. Here we present three case reports of animals which tested BLV antibody positive. As there was no evidence that the animals or their in-contacts were infected with BLV the result was somewhat confusing. Investigation revealed that the animals had been fed a colostrum replacer that was produced outside of Great Britain. As BLV is endemic in some countries, it was considered that BLV antibodies may have been present in the colostrum replacer and thus passively acquired resulting in BLV sero-positivity. The hypothesis was supported by the declining serological antibody titre seen on serial blood sampling. We present data on the serological and molecular testing conducted on the animals and product in support of the hypothesis. The above mentioned cases were all linked by the same brand of colostrum replacer; in addition we provide data derived from the investigation of colostrum replacers from other manufacturers. The policy and International Trade implications will also be discussed.

Detection of Bovine Leukaemia Virus Antibodies and Proviral DNA in Colostrum Replacers

Great Britain has been bovine leukaemia virus (BLV) disease free since 1999. We recently reported three separate incidents of BLV seropositivity on farms with home-reared cattle due to the use of colostrum replacer rather than infection with BLV (Emerg. Infect. Dis., 19, 2013, 1027). These cases were all linked via the use of the same brand of colostrum replacer. Here, we investigate further by examining multiple brands of colostrum replacer for proviral DNA and BLV antibodies. BLV antibodies were detected in 7 of the colostrum replacers tested, with PCR concurring in two cases. Thus, the use of these BLV antibody-positive colostrum replacers may also lead to false-positive serological diagnostics.