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African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig ReLA protein; a subunit of the nf-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV. African swine fever virus (ASFV) is a large DNA virus and sole member of the family Asfarviridae that causes a mostly lethal haemorrhagic disease, African swine fever (ASF), in domestic pigs and Eurasian wild boar. ASFV can genetically be separated into 24 genotypes that cause the same disease, but immunological cross-protection is limited and poorly understood 1. The introduction of ASF into a country results in trade restrictions and pig losses, thus the disease has a high socioeconomic consequence for both commercial and backyard farmers 2. Accordingly, the spread of this disease is a serious concern for the global pig industry. Following the incursion of a genotype II ASFV into the Caucasus in 2007 the virus has spread through Russia, entered the European Union in 2014 and, in 2018, was detected for the first time in China. Since then the Chinese pig population has declined by at least 20% and ASFV has further spread across many countries in South East Asia 3,4. Combating this global threat is hampered by the lack of a vaccine and is particularly difficult in production systems with poor biosecurity which are more vulnerable to virus introduction and contact with wild suids 1. ASFV infects all members of the family Suidae, which as well as domestic pigs (Sus scrofa domesticus), wild boar (Sus scrofa spp) and others also includes bushpigs (Potamochoerus spp.) and warthogs (Phacochoerus spp.), which are considered natural reservoir hosts 5. In domestic pigs and wild boar acute and subacute forms dominate,

Pancreas disease (PD) caused by the salmonid alphavirus (SAV) has been the most significant cause of mortalities in Irish farmed salmon Salmo salar L. over the past decade. SAV is a single-strand positive-sense RNA virus, originally thought to be unique to salmonids, but has recently been detected using real-time RT-PCR in a number of wild non-salmonid fish. In the present report, 610 wild flatfish (common dab Limanda limanda, plaice Pleuronectes platessa and megrim Lepidorhombus whiffiagonis) were caught from the Irish and Celtic Seas and screened for SAV using real-time RT-PCR and sequencing. In general, a very low prevalence was recorded in common dab and plaice, except for 1 haul in Dublin Bay where 25% of common dab were SAVpositive. SAV sequence analysis supported the fact that real-time RT-PCR detections were specific and further characterised the detected viruses within SAV Subtype I, the predominant subtype found in farmed salmon in Ireland. KEY WORDS: SAV · Pancreas disease · Wild fish · Horizontal transmission · Atlantic salmon · Aquaculture Resale or republication not permitted without written consent of the publisherDis Aquat Org 109: [1][2][3][4][5][6][7] 2014 MATERIALS AND METHODSFish were sampled onboard research vessels demersal trawling in the Irish and Celtic Seas, using a 2 to 3 metre beam trawl. Fish were caught from 4 trawls in the Irish Sea: 3 in Dublin Bay (October 2010 twice and February 2011) and 1 in Wexford Harbour (October 2010). A much broader annual groundfish survey was completed on the RV 'Celtic Voyager' in the Celtic Sea in December 2012, whereby the fish were caught from multiple hauls covering the south east to the north west of Ireland. In all cases, sampling was completed onboard within 1 h of capture. Either heart or gill tissue was taken and preserved by storage in RNAlater ® for testing in the laboratory. Pancreas and liver tissue were collected from Irish Sea flatfish into neutralbuffered formalin, and stained using haematoxylin and eosin according to standard methods for histopathological examination.Total RNA was extracted from 610 flatfish from either ~15 mg heart or gill tissue samples using a Qiagen TissueLyser and a standard protocol from the Qiagen RNeasy ® mini kit. Gill tissue was used in this study because SAV persists for a long time in this tissue (Graham et al. 2010). RNA was eluted in a 50 µl volume and a subsample treated with Qiagen RNase-Free DNase. Treated RNA was subsequently purified using a Qiagen RNeasy minElute kit and stored at -80°C. Real-time RT-PCR primers targeting the nsP1 gene (Hodneland & Endresen 2006) were used to test for the presence of SAV RNA using a 1-step real-time reverse transcriptionpolymerase chain reaction (RT-PCR) with Platinum ® qRT-PCR Thermoscript™ (Invitrogen) and an ABI 7500 thermal cycler (Applied Biosystems). A total real time RT-PCR reaction volume of 20 µl, containing 2 µl of sample RNA, 300 nM of each primer and 200 nM of probe was subjected to the following thermal profile: 50°C for 20 min and 95°C fo...

Infectious pancreatic necrosis is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality and disease-management costs. Significant outbreaks of the disease occurred in farmed Atlantic salmon in Ireland between 2003 and 2007, associated with imported ova and smolts. As the virus was known to occur in the country since the development of aquaculture in the 1980s, this study examined archived samples to determine whether these older isolates were associated with virulent forms. The study showed that two genotypes of IPNV were present in the 1990s, genotype 3 and genotype 5. A more virulent subtype of the virus first appeared in 2003 associated with clinical outbreaks of IPN, and this subtype is now the most prevalent form of IPNV found in the country. The data also indicated that IPNV in Ireland is more closely related to Scottish and continental European isolates than to Norwegian, Chilean and Australasian genogroup 5 isolates.

Validation of a novel quantitative real-time PCR using TaqMan® minor groove binder (MGB) chemistry is described for sensitive and rapid detection of Vibrio aestuarianus, an increasingly important pathogen of Pacific cupped oyster Crassostrea gigas aquaculture. Primers and TaqMan® MGB hydrolysis probe were designed to specifically amplify a 58bp DNA fragment of the V. aestuarianus dnaJ gene. Real-time PCR selectivity was empirically tested using DNA extracted from isolates of V. aestuarianus and a selection of different aquatic bacterial species, including other Vibrio spp. Theoretical selectivity was assessed through sequence comparison using the NCBI BLAST similarity tool. Quantitative PCR plasmid standards were generated to test assay linearity, amplification efficiency and the limit of quantitation (LOQ), according to International Organisation for Standardisation ISO 16140 validation recommendations. LOQ ranged between 5 and 10 PCR copies, although the detection range extended beyond this with reduced precision. Applied performance was tested using C. gigas samples taken from a selection of Irish aquaculture sites. Increasing levels of V. aestuarianus, accompanied by the development of tissue pathology in examined oysters, were found at 1 site that was sampled repeatedly in 2013. Rapid, sensitive and reproducible detections of V. aestuarianus from C. gigas tissue samples were attained during this validation study with a small sample size, and a practical application for disease management is described.

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The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.