COVID-19 transmission between the community and meat processing plants in Ireland: A retrospective modelling study.

Outbreaks of COVID-19 in meat processing plants (MPPs) were recorded globally throughout the pandemic. There was speculation these outbreaks resulted in dissemination of COVID-19 throughout the surrounding county leading to high incidence rates. We aimed to investigate the dynamics of spread between MPPs and their surrounding counties. In this retrospective longitudinal study, data were collected on the number and size of outbreaks in 33 MPPs and county infections in Ireland between March 2020 and May 2021. These data were used to investigate the relationship between outbreaks in MPPs and county infection rates through statistical analysis, and the development of a novel SEIR model. We found an association between the number of MPPs present in a county and county incidence rates, however, incidence rates in the counties did not increase as a consequence of an outbreak in an MPP. The model results indicate that county incidence rates in the weeks prior to an MPP outbreak could reliably predict the size of that outbreak in a plant, r(49) = 0·62, p < 0·0001, RMSD = 5·6. In Ireland, outbreaks in MPPs were strongly correlated with high levels of infection in the surrounding county, rather than being a driver of infection in the county. The modified SEIR model described here can provide an explanation of the generative process required to cause outbreaks of the size and scale that occur in MPPs.

Inactivation and Recovery of High Quality RNA From Positive SARS-CoV-2 Rapid Antigen Tests Suitable for Whole Virus Genome Sequencing.

The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from "spent" RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with Ct values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.

Assessment of Environmental and Occupational Risk Factors for the Mitigation and Containment of a COVID-19 Outbreak in a Meat Processing Plant.

Throughout the COVID-19 pandemic, meat processing plants have been vulnerable to outbreaks of SARS-CoV-2 infection. Transmission of the virus is difficult to control in these settings because of a combination of factors including environmental conditions and the specific nature of the work. This paper describes a retrospective outbreak investigation in a meat processing plant, a description of the measures taken to prevent or contain further outbreaks, and insights on how those with specific knowledge of the working environment of these plants can collaborate with public health authorities to ensure optimal outbreak control. The plant experienced 111 confirmed positive asymptomatic cases in total with an estimated attack rate of 38% during a five-week period. 4 weeks after the first case, mass screening of all workers was conducted by the public health authorities. Thirty-two workers tested positive, of which 16 (50%) worked in one particular area of the plant, the boning hall (n = 60). The research team prepared and carried out semi-structured interviews with the plant personnel who were charged with COVID control within the plant. They carried out assessments of operational risk factors and also undertook air quality monitoring in the boning hall and abattoir. The air quality measurements in the boning hall showed a gradual build-up of carbon dioxide and aerosol particles over the course of a work shift, confirming that this poorly ventilated area of the plant had an environment that was highly favorable for aerosol transmission of SARS-CoV-2. Assessment of operational conditions incorporated visual surveys of the plant during the working day. Prior to and during the first 2 weeks of the outbreak, multiple measures were introduced into the plant by management, including physical distancing, provision of educational material to workers, visitor restrictions, and environmental monitoring. After the implementation of these measures and their progressive refinement by plant management, the factory had no further linked cases (clusters) or outbreaks for the following 198 days. The tailored approach to risk mitigation adopted in this meat processing plant shows that generic risk mitigation measures, as recommended by public health authorities, can be successfully adapted and optimized by designated plant emergency response teams.

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Pathogens, patterns of pneumonia, and epidemiologic risk factors associated with respiratory disease in recently weaned cattle in Ireland.

We examined the pathogens, morphologic patterns, and risk factors associated with bovine respiratory disease (BRD) in 136 recently weaned cattle ("weanlings"), 6-12 mo of age, that were submitted for postmortem examination to regional veterinary laboratories in Ireland. A standardized sampling protocol included routine microbiologic investigations as well as polymerase chain reaction and immunohistochemistry. Lungs with histologic lesions were categorized into 1 of 5 morphologic patterns of pneumonia. Fibrinosuppurative bronchopneumonia (49%) and interstitial pneumonia (48%) were the morphologic patterns recorded most frequently. The various morphologic patterns of pulmonary lesions suggest the involvement of variable combinations of initiating and compounding infectious agents that hindered any simple classification of the etiopathogenesis of the pneumonias. Dual infections were detected in 58% of lungs, with Mannheimia haemolytica and Histophilus somni most frequently recorded in concert. M. haemolytica (43%) was the most frequently detected respiratory pathogen; H. somni was also shown to be frequently implicated in pneumonia in this age group of cattle. Bovine parainfluenza virus 3 (BPIV-3) and Bovine respiratory syncytial virus (16% each) were the viral agents detected most frequently. Potential respiratory pathogens (particularly Pasteurella multocida, BPIV-3, and H. somni) were frequently detected (64%) in lungs that had neither gross nor histologic pulmonary lesions, raising questions regarding their role in the pathogenesis of BRD. The breadth of respiratory pathogens detected in bovine lungs by various detection methods highlights the diagnostic value of parallel analyses in respiratory disease postmortem investigation.

The impact of infection with Schmallenberg virus on weaning rate in Irish sheep flocks.

Schmallenberg virus (SBV) disease emerged in Europe in 2011, with the virus initially identified in Germany, and the first confirmed case of SBV infection in Ireland diagnosed in a dairy calf in October 2012. SBV was subsequently confirmed by RT-PCR in 49 cattle herds and 39 sheep flocks. While these studies provide a good representation of the spatial distribution of SBV in Ireland, they do not quantify the impact of SBV on productivity. The objectives of this study were to assess the impact of SBV on weaning rate in Irish sheep flocks, based on data reported by Irish sheep farmers, and to evaluate weaning rate in sheep flocks as an indicator to be used in emerging disease surveillance systems. A questionnaire on productivity and management practices in sheep flocks was developed to gather data from sheep farmers. Valid responses from 267 sheep farmers were received. Negative binomial regression indicated that flocks with a confirmed SBV diagnosis had a weaning rate 0.9 times that of flocks free of SBV. The 10% reduction in weaning rates as a result of SBV is a justifiable concern for farmers and should be considered in formulating flock breeding policy. This study shows the value of a production database as an indicator of an emerging disease and the economic impact of that disease in Irish sheep flocks.

Seroprevalence of Hepatitis E virus infection in the Irish pig population.

BACKGROUND: Hepatitis E is an acute viral disease of humans, occurring in explosive outbreaks in the developing world and as sporadic cases in returning travellers. Increasing recognition of indigenous transmission in Western countries suggests a zoonotic source of infection, most likely pigs. To determine if hepatitis E virus is present in Irish pigs, sera from 330 animals were examined for antibodies using a commercially available ELISA. FINDINGS: Antibodies were detected in 89 pigs (27%) in 13 herds (81%). CONCLUSIONS: Hepatitis E virus is present in most Irish pig herds and in many animals within these herds.

Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes.

Toxoplasma gondii and Chlamydophila abortus are the 2 most common infectious causes of ovine abortion worldwide. These obligate intracellular pathogens are associated with severe placentitis leading to abortion or stillbirth in pregnant ewes, and resulting in significant economic losses. The objectives of the current study were the development, validation, and application of a duplex real-time polymerase chain reaction (PCR) assay capable of quantifying the burden of infection by T. gondii and C. abortus in material submitted for diagnostic purposes. The validation was carried out using samples from ewes experimentally infected with these organisms. Based on the numbers of genome copies detected, an arbitrary cutoff level was established to correlate with significant pathological changes sufficient to give rise to abortion. When the PCR assay was applied to samples from 66 Irish farms with naturally occurring outbreaks of ovine abortion, toxoplasmosis and enzootic abortion of ewes (EAE) accounted for 14% and 20% of the farms, respectively, while on 6% of the farms, there was evidence of dual infection. When standard diagnostic techniques including histopathological examination, serological analysis, chlamydial antigen detection, and bacteriological culture, were used on samples from the same farms, toxoplasmosis was diagnosed in 17% of farms, and EAE in 12%; dual infection was diagnosed on 3% of the farms. In general, good agreement was found between the PCR and the standard methods. The duplex real-time PCR assay developed in this study has proved to be a very sensitive and rapid tool that might provide a valuable addition to the methods currently available for routine diagnosis of ovine abortions.

Seroprevalence of chlamydial infection in cattle in Ireland.

Although few studies have investigated the prevalence of chlamydial infections in cattle, reported prevalence rates vary hugely. In order to assess the prevalence of this infection in cattle in Ireland, serum samples (100 herds, 20 samples/herd) collected for statutory screening for brucellosis were examined by soluble chlamydial antigen indirect ELISA. The assay detects antibodies to the two most common Chlamydiaceae spp. affecting cattle, namely Chlamydia abortus and Chlamydia pecorum. A total of 95 samples from 57 herds were seropositive, representing an observed prevalence rate of 4.75%. The parametric bootstrap estimate of the mean disease prevalence in the population was 6.04% (95%, CI 4.70-7.50). The results suggest the prevalence of chlamydial infection is low in cattle in Ireland.

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus.

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

Monitoring clinical outcomes, pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection.

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral-nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.

Neuropathologic features of Aleutian disease in farmed mink in Ireland and molecular characterization of Aleutian mink disease virus detected in brain tissues.

A neuropathologic survey was conducted on mink brains from the 5 licensed mink farms in Ireland. The survey was part of a transmissible spongiform encephalopathy surveillance study. Aleutian disease (AD) was present on 4 of the 5 farms (80%). Neuropathologic features of nonsuppurative meningoencephalitis were common in mink from the 4 affected farms but were absent in the mink from the fifth farm, which was free of AD. The meningoencephalitis was characterized by infiltrates of lymphocytes and plasma cells, which were present in meninges, perivascular spaces, and the brain parenchyma. Fibrinoid necrotizing arteritis was seen in 11 mink brains, all of which were obtained from a single farm. Aleutian mink disease virus (AMDV) sequences for the capsid protein VP2 were obtained from brain samples from all affected farms. Although containing previously unreported amino acid residues, similarities with European and North American isolates were observed in the hypervariable regions within VP2, suggesting Irish AMDV is related to those isolates. The predicted amino acid residues, suspected of conferring pathogenicity at certain positions of the VP2 sequence, were present in the viral nucleic acid sequences.

Use of continuous results to compare ELISAs for the detection of antibodies to non-structural proteins of foot-and-mouth disease virus.

Six tests for detection of antibodies against the non-structural proteins of foot-and-mouth disease virus (FMDV) were compared at an international workshop in Brescia, Italy in 2004 on the basis of dichotomous test results. However, as results from all of these assays were also available on a continuous scale, validation was extended by calculating and subsequently analysing the receiver-operator characteristic (ROC) curves and likelihood ratios (LR) for each test method. For the purposes of these analyses, test results for a total of 1337 sera were selected from the Brescia workshop dataset, 237 sera that had been obtained from cattle exposed to FMDV and 1100 sera obtained from cattle that were not exposed to the virus; sera from "exposed" cattle were considered to be "true positives" and sera from "non-exposed" cattle were considered to be "true negatives". Analysis of ROC curves showed that at specificities of both 99 and 99.5%, the IZS-Brescia and the Ceditest ELISA had significantly better detection rates in exposed cattle than the other ELISAs. The ROC analysis confirms the previous finding that the IZS-Brescia and the Ceditest ELISAs have both better detection rates in exposed cattle combined with a high specificity. The analysis of likelihood ratios provides information that may be very useful in the interpretation of test results, and a working example is presented to show how these likelihood ratios might be used in an objective approach to deciding the true infection status of surveyed populations.

Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle.

There has been much debate about the use of the so-called "vaccinate-to-live" policy for the control of foot-and-mouth disease (FMD) in Europe, according to which, spread of the FMD virus (FMDV) from future outbreaks could be controlled by a short period of "emergency" vaccination of surrounding herds, reducing the need for large-scale preemptive culling of at-risk animals. Since vaccinated animals may become subclinically infected with FMDV following challenge exposure, it is necessary to either remove all vaccinates (vaccinate-to-kill) or to detect and remove vaccinates in which virus is circulating or has established persistent infections (vaccinate-to-live), in order to rapidly regain the most favoured trading status of FMD-free without vaccination. The latter approach can be supported by testing vaccinated animals for the presence of antibodies to certain non-structural proteins (NSP) of FMDV, which are induced by infection with the virus, but not by vaccination with purified FMD vaccines. Using test sensitivity and specificity data established at a recent workshop on NSP assays [Brocchi E, Bergmann I, Dekker A, Paton DJ, Sammin DJ, Greiner M, et al. Comparative performance of six ELISAs for antibodies to the non-structural proteins of foot-and-mouth disease. Vaccine, in press], this paper examines the ways in which serological testing with NSP ELISAs can be used and interpreted and the effect that this will have on the confidence with which freedom from infection can be demonstrated within guidelines specified by the World Animal Health Organisation and the European Commission.