Detection of non-primate hepaciviruses in UK dogs.

Non-primate hepacivirus (NPHV) has been identified in dogs, horses, bats and wild rodents. The presence of NPHV in dogs outside of the USA however is yet to be established. Here we describe for the first time the detection of NPHV in the UK dog population (described throughout the manuscript as CnNPHV). We examined tissues collected from dogs housed in a rehoming kennel where respiratory disease was endemic. CnNPHV RNA was detected in the tracheal tissues of 48/210 dogs by RT-PCR, and in the liver, lung and/or tracheal tissues of 12/20 dogs. The presence of CnNPHV RNA, and its tropism was confirmed by in situ hybridisation. Histopathological examination demonstrated a trend toward higher histopathological scores in CnNPHV RNA positive respiratory tissues, although, this was not statistically significant. Our findings broaden the geographic distribution and our understanding of CnNPHV. Further evidence of CnNPHV replication in canids warrants investigation.

New and emerging pathogens in canine infectious respiratory disease.

Canine infectious respiratory disease is a common, worldwide disease syndrome of multifactorial etiology. This review presents a summary of 6 viruses (canine respiratory coronavirus, canine pneumovirus, canine influenza virus, pantropic canine coronavirus, canine bocavirus, and canine hepacivirus) and 2 bacteria (Streptococcus zooepidemicus and Mycoplasma cynos) that have been associated with respiratory disease in dogs. For some pathogens a causal role is clear, whereas for others, ongoing research aims to uncover their pathogenesis and contribution to this complex syndrome. Etiology, clinical disease, pathogenesis, and epidemiology are described for each pathogen, with an emphasis on recent discoveries or novel findings.

Monitoring the bulk milk antibody response to BVDV: the effects of vaccination and herd infection status.

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus in the flaviviridae family which affects cattle worldwide. Bulk milk (BM) antibody testing is frequently used as a relatively quick method of assessing herd BVDV exposure; however, an understanding of the effects of vaccination and historic infection is essential for test interpretation. This study investigated the trends exhibited by monthly BM antibody analysis in 14 herds split into three categories. Category 1 herds (vaccinating/no persistently infected (PI) animals) began the study with mid-positive BM antibody titres and experienced an estimated increase of 0.007 optical density (OD) units per month (equating to a rise of 0.35 OD units in 50 months). Category 2 herds (not vaccinating/no PI animals) began the study with mid-positive BM antibody titres and experienced an estimated decrease of 0.005 OD units per month with antibody levels in one category 2 herd taking 1290 days to decrease from mid-positive to negative. Category 3 herds (vaccinating/PI animals present) began the study with high BM antibody titres which plateaued within this range throughout the 50-month observation period. Vaccination was observed to cause transient increases in BM antibody in a number of herds in categories 1 and 3.

Establishing a pilot bovine viral diarrhoea virus eradication scheme in Somerset.

Beginning in April 2006, 41 farms were recruited onto a pilot Bovine viral diarrhoea virus (BVDV) eradication programme across the south of England with the majority of study herds concentrated in Somerset. Each herd was assessed and where relevant cleared of persistently infected (PI) animals. Seven farms dropped out before whole herd screening could be performed. Of the remaining 34 farms, 20 (59 per cent) were classified as infected although two of these were initially misclassified as BVDV-free. Over the course of three years, 61 PIs were identified across 16 of the 20 infected farms. 72 per cent of PIs indentified on the first herd test were below two years of age. PI prevalence ranged from 0.2 to 3.1 per cent of infected herds and was highest in herds that did not vaccinate. By the end of 2009, 24/34 (71 per cent) of study farms were BVDV-free while 10 (29 per cent) remained infected.

Functional test of the influence of Wolbachia genes on cytoplasmic incompatibility expression in Drosophila melanogaster.

Wolbachia are inherited intracellular bacteria that infect a broad range of invertebrate hosts. They commonly manipulate host reproduction in a variety of ways and thereby favour their invasion into host populations. While the biology of Wolbachia has been extensively studied at the ecological and phenotypic level, little is known about the molecular mechanisms underlying the interaction between Wolbachia and their hosts. Recent comparative genomics studies of Wolbachia strains have revealed putative candidate genes involved in the expression of cytoplasmic incompatibility (CI) in insects. However the functional testing of these genes is hindered by the lack of available genetic tools in Wolbachia. To circumvent this problem we generated transgenic Drosophila lines expressing various Wolbachia CI candidate genes under the control of the GAL4/UAS system in order to evaluate their possible role in Wolbachia-related phenotypes in Drosophila. The expression of a number of these genes in Drosophila melanogaster failed to mimic or alter CI phenotypes across a range of Wolbachia backgrounds or in the absence of Wolbachia.

A histopathologic and immunohistochemical review of archived UK caprine scrapie cases.

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.

Epidemiological patterns of foot-and-mouth disease worldwide.

Foot-and-Mouth Disease (FMD) is a clinical syndrome in animals due to FMD virus that exists in seven serotypes, whereby recovery from one sero-type does not confer immunity against the other six. So when considering intervention strategies in endemic settings, it is important to take account of the characteristics of the different serotypes in different ecological systems. FMD serotypes are not uniformly distributed in the regions of the world where the disease still occurs. For example, the cumulative incidence of FMD serotypes show that six of the seven serotypes of FMD (O, A, C, SAT-1, SAT-2, SAT-3) have occurred in Africa, while Asia contends with four sero-types (O, A, C, Asia-1), and South America with only three (O, A, C). Periodically there have been incursions of Types SAT-1 and SAT-2 from Africa into the Middle East. This paper describes the global dynamics for the seven sero-types and attempts to define FMD epidemiological clusters in the different regions of the world. These have been described on a continent by continent basis. The review has reaffirmed that the movement of infected animals is the most important factor in the spread of FMD within the endemically infected regions. It also shows that the eco-system based approach for defining the epidemiological patterns of FMD in endemic, which was originally described in South America, can apply readily to other parts of the world. It is proposed that any coordinated regional or global strategy for FMD control should be based on a sound epidemiological assessment of the incidence and distribution of FMD, identifying risk sources as either primary or secondary endemic eco-systems.

Planning for the progressive control of foot-and-mouth disease worldwide.

In the wake of on-going successful programmes for global eradication of rinderpest and the current effort to contain the spread of avian influenza, the progressive world-wide control of FMD must be regarded as a major contribution to the international public good. FMD is the single most animal disease constraint to international trade in animal products. Its control is relevant, on the one hand, to protecting the livestock industries of industrialised countries and, on the other, to the livelihoods and income generation of developing countries, where, as a general rule, FMD continues to be endemic. The strategy that is advocated in this paper is one that is based on progressive risk reduction of FMD in the context of progressive market access of livestock commodities from developing countries. It is suggested that FMD control should be linked to improvement in livelihoods of livestock dependent communities in the FMD endemic settings. It is expected that this in turn will lead to increasing demand for effective national veterinary services and disease surveillance. This strategy has also taken lessons from the global rinderpest eradication programme and regional FMD control programmes in Europe and South America. The strategy that is advocated for the progressive control of FMD in the endemic settings is based on a seven stage process within a horizon of about 30 years, namely: (1) Assessing and defining national FMD status; (2) instituting vaccination and movement control; (3) suppressing virus transmission to achieve absence of clinical disease; (4) achieving freedom from FMD with vaccination in accordance with the OIE standards; (5) achieving freedom from FMD without vaccination in accordance with the OIE standards; (6) extending FMD free zones; and (7) maintaining FMD Freedom. Concomitant with progressive FMD control, there needs be the encouragement of such risk reduction measures as in-country commodity processing in order to encourage regulated trade in livestock commodities without unduly increasing the risk of disease spread. Finally, the progressive control of FMD should also be seen as part of reducing the overall, world-wide threat of infectious diseases to human health and economic development.

Foot-and-mouth disease virus infection in fetal lambs: tissue tropism and cytokine response.

Foot-and-mouth disease virus (FMDV) can cause transplacental infection and death in fetal lambs. This study investigates the pathogenesis of FMDV infection in ovine fetuses using in-situ hybridization (ISH) to detect viral transcripts in tissue and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays to quantify the fetal cytokine response to infection. FMDV ribonucleic acid (RNA) was localized mainly to the heart and skeletal muscles of fetuses and was only occasionally expressed in the lingual epithelium, demonstrating that FMDV has a different tissue tropism in the fetus compared with that in adult sheep. There was early expression of genes encoding anti-viral cytokines (IFN-alpha and IFN-beta) in fetuses at 2 and 4 days post-infection (dpi), followed by a marked rise in the transcription of pro-inflammatory cytokine genes (IFN-gamma, TNF-alpha and IL-1alpha) from 7 to 18 dpi, particularly in the heart. The degree of cytokine mRNA expression correlated with fetal infection and was likely to be a factor in fetal death. In contrast, cytokine gene expression in infected neonatal lambs was much less and mainly occurred between 2 and 4 dpi. This study identifies two key factors in the pathogenicity of FMDV in fetal lambs: viral tropism for cardiac and skeletal muscles, and a marked cytokine response following infection.

Foot-and-mouth disease virus crosses the placenta and causes death in fetal lambs.

Eighteen pregnant sheep, six at 45 days gestation and twelve at 75 days gestation, were infected with foot-and-mouth disease virus (FMDV) type O UKG 34/2001. Two sheep from each gestational group were killed at 2, 4, and 7 days post-inoculation (dpi). Three sheep, pregnant for 75 days at infection, were killed at 17 and 18 dpi. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and virus isolation (VI) were used to detect viral RNA and infectious virus, respectively, in fetal tissues taken post mortem. Eleven fetuses were obtained from the six sheep inoculated at day 45 of gestation. Of these, two of three fetuses at 2 dpi had viral RNA detected by RT-PCR and virus was detected in one by VI. Viral RNA was detected in two of four fetuses at 4 dpi, while viral RNA and virus were detected in all four fetuses at 7 dpi. No gross abnormalities were evident in these fetuses. In the group inoculated at day 75 of gestation, viral RNA was detected in three of four fetuses at 4 dpi. Virus and viral RNA were detected in three of four fetuses at 7 dpi. Of the seven fetuses examined at 17 and 18 dpi, viral RNA was detected in five, and four of these had died in utero. Gross abnormalities including haemorrhage and oedema in a number of tissues were evident in many of the fetuses in this group, but no vesicular lesions were found. Viral RNA and virus were detected in the amniotic fluid associated with infected fetuses. This study is the first to demonstrate that FMDV may cause transplacental infection and fetal death.

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

The control of bovine viral diarrhoea virus in Europe: today and in the future.

This paper summarises the views of a European group of scientists involved in the control of bovine viral diarrhoea virus (BVDV), as part of a European Union Thematic Network. The group concludes that the technical tools and the knowledge needed to eradicate BVDV are at hand, as proven by successful national control schemes in several European countries. A generic model for BVDV control is presented, which includes biosecurity, elimination of persistently infected animals and surveillance as central elements. These elements are termed 'systematic', in contrast to control efforts without clear goals and surveillance to evaluate progress. The network concludes that a systematic approach is needed to reach a sustainable reduction in the incidence and prevalence of BVDV in Europe. The role of vaccines in systematic control programmes is considered as an additional biosecurity measure, the effect of which should be evaluated against cost, safety and efficacy. It is also concluded that active participation by farmers' organisations is a strong facilitator in the process that leads up to the initiation of control, and that public funding to support the initiation of organised BVD control programmes can be justified on the basis of expected wider societal benefits, such as animal welfare and reduction in the use of antibiotics. If applied successfully, the focus on biosecurity in systematic BVD control programmes would also reduce the risk of the introduction and spread of other epizootic and zoonotic agents, thereby improving both cattle health and welfare in general, as well as increasing the competitiveness of the cattle industry.

Identification of novel non-autonomous CemaT transposable elements and evidence of their mobility within the C. elegans genome.

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations.

Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

The Caenorhabditis briggsae genome contains active CbmaT1 and Tcb1 transposons.

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.

CemaT1 is an active transposon within the Caenorhabditis elegans genome.

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike Tc transposons in mutator strains of C. elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control Tc activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome.

Analysis of variation of bovine viral diarrhoea virus E2 sequence following transplacental infection of cattle.

The genetic and antigenic diversity observed in field isolates of bovine viral diarrhoea virus (BVDV) is thought to occur during acute infection because of the genetic stability observed in BVDV throughout the lifetime of persistently infected (PI) cattle. In this study, 15 cows in early pregnancy were inoculated with identical challenge doses obtained from a single infectious inoculum of the virologically cloned isolate Pe515nc. In order to examine the diversity that may develop in utero in the PI foetus, the variable E2 sequence of the virus isolated directly from the serum of each PI calf was compared. A high degree of sequence similarity was demonstrated, with 0-4 nucleotide differences out of 608 bases compared. Thus, the virus showed relatively few genomic changes in any of the PI calves, although we observed that the in utero environment did provide some opportunity for genetic variation to become established.

The role of the defective interfering particle DI9c in mucosal disease in cattle.

Mucosal disease occurs in cattle persistently infected with a noncytopathogenic strain of bovine viral diarrhoea virus (BVDVnc) following in utero infection. The disease can be initiated by superinfection with a cytopathogenic biotype (BVDVc) of the virus with antigenic "homology" to the persisting virus. A BVDVc isolated from a clinical case of mucosal disease has been discovered to consist of a defective interfering particle, DI9, and an associated BVDVnc helper virus. A defective virus corresponding to DI9 was recently recovered from an infectious cDNA clone and was named DI9c. To evaluate the role of DI9 in the pathogenesis of mucosal disease a two-part experimental study was carried out which included clinical, haematological, pathological and virological investigations. Eight of nine calves persistently infected with BVDVnc were experimentally inoculated with DI9c. The defective virus was propagated in cells preinfected with the same strain of virus used to persistently infect the calves in utero. The calves were euthanased on days 4, 7, 14, 21, 28, 40, 40 or 87 post inoculation. None of the inoculated animals developed classical mucosal disease, neither clinically nor pathologically. DI9c was not found in serum, nasal swab or tissue samples from the calves by observing cytopathogenic effect and/or using a polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. DI9c did not replicate to a detectable extent in these assays, and its participation in the pathogenesis of mucosal disease could not be proven.