Apoptosis levels in bovine Johne's disease ileal lesions and association with bacterial numbers.

Johne's disease (JD) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). While it is generally accepted that MAP employs immune subversion mechanisms, aspects of the host-pathogen relationship are not fully understood. We sampled 3 ileal tissue sections from 17 naturally infected cattle (n = 51 sections) to analyze differences in cell types, apoptosis, and phagocytic cells. Diffuse multibacillary (DM) was the most common lesion type (n = 17) followed by diffuse intermediate (DI; n = 15). DM lesions had significantly greater proportion of Treg cells (CD3+ FoxP3+) relative to all CD3+ T cells as compared to DI forms (P < .05). CD68+ individual cell size was significantly smaller in DM than in diffuse lymphocytic (DL) forms (P < .05). Area of caspase-3 positivity (apoptosis) was greater in DM lesions than DL (P < .05) and DI (P < .0001), and was linked to higher numbers of MAP within the macrophage.

Fasciola hepatica products can alter the response of bovine immune cells to Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. OBJECTIVES: Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). METHODS: This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. RESULTS: We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. CONCLUSIONS: Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Comparison of four commercially available ELISA kits for diagnosis of Fasciola hepatica in Irish cattle.

BACKGROUND: Fasciola hepatica is a liver parasite of mammals and it results in poor welfare outcomes and economic losses in ruminants. While faecal egg count is the test most commonly used for diagnosis, it does not indicate presence of migrating immature stages. Serological techniques increase sensitivity at all stages of the liver fluke infection. The aim of this study was to compare four commercially available ELISA tests for the diagnosis of F. hepatica. For this purpose, we tested three sample types; (i) known F. hepatica status sera from an experimental infection for the comparison of sensitivities and specificities, (ii) sera from pre- and post-flukicide-treated (albendazole, closantel, nitroxynil and triclabendazole) beef cattle to contrast the differences of seropositivity before and after treatment, and (iii) bulk tank milk samples from dairy herds sampled during high and low F. hepatica exposure periods for assessing seasonal variations with the four tests available. Samples were tested using ELISA kits supplied by four manufacturers (Ildana Biotech, IDEXX, Svanova, and Bio-X). Samples were analysed simultaneously and in duplicate. RESULTS: In the control population Ildana, IDEXX and Bio-X presented 100% sensitivity (Se) and specificity (Sp), Svanovir presented a Se of 59% and a Sp of 96%. In flukicide-treated beef cattle, kits highlighted decreasing antibody levels 90 days post-treatment in variable degrees. Finally, bulk milk showed a significant decrease in ELISA value between high and low fluke exposure periods with all tests studied. CONCLUSIONS: Se and Sp found in the present study, confirm that Ildana, IDEXX and Bio-X are accurate for the detection of F. hepatica exposure in Irish cattle. Svanovir Se and Sp in this population, indicate that a larger study is necessary to confirm this test characteristic in Irish herds. In post-treatment use, Bio-X showed a consistent and significant decrease of ELISA value in all groups treated, denoting to be a reliable tool for assessing treatment effect at 90 days post-treatment. Finally, all tests showed to be a reliable tool for the F. hepatica monitoring of high and low exposure seasons, using bulk tank milk samples.

Epidemiological investigation of a severe rumen fluke outbreak on an Irish dairy farm.

Although the rumen fluke, Calicophoron daubneyi is now very common and widespread throughout Western Europe, reports of clinical cases are still rare. This study explores the epidemiological background to a severe rumen fluke outbreak in 6-month-old heifers on a dairy farm in Ireland. Sequence analysis of the cytochrome oxidase subunit 1 (Cox1) gene of the rumen fluke metacercariae on pasture failed to identify predominant, possibly pathogenic subtypes. However, estimates of metacercarial load indicated that the animals were exposed to a daily dose of about 5334 C. daubneyi metacercariae for a period of 3 weeks resulting in the build-up of very large numbers of immature worms in the small intestine. It is hypothesized that specific environmental conditions may favour this parasite over its competitor, the liver fluke, Fasciola hepatica, possibly by allowing it to emerge earlier. The possibility that C. daubneyi may be better adapted to the Irish climate than F. hepatica together with the fact that selective treatment against F. hepatica effectively frees the niche for C. daubneyi, may result in the gradual replacement of F. hepatica by C. daubneyi.

Migration of Fasciola hepatica newly excysted juveniles is inhibited by high-mannose and oligomannose-type N-glycan-binding lectins.

Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-type N-glycans on the NEJ tegument may have potential for disrupting infection.

Development of a versatile in vitro method for understanding the migration of Fasciola hepatica newly excysted juveniles.

Fasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

Rumen fluke in Irish sheep: prevalence, risk factors and molecular identification of two paramphistome species.

BACKGROUND: Rumen flukes are trematode parasites found globally; in tropical and sub-tropical climates, infection can result in paramphistomosis, which can have a deleterious impact on livestock. In Europe, rumen fluke is not regarded as a clinically significant parasite, recently however, the prevalence of rumen fluke has sharply increased and several outbreaks of clinical paramphistomosis have been reported. Gaining a better understanding of rumen fluke transmission and identification of risk factors is crucial to improve the control of this parasitic disease. In this regard, a national prevalence study of rumen fluke infection and an investigation of associated risk factors were conducted in Irish sheep flocks between November 2014 and January 2015. In addition, a molecular identification of the rumen fluke species present in Ireland was carried out using an isolation method of individual eggs from faecal material coupled with a PCR. After the DNA extraction of 54 individual eggs, the nuclear fragment ITS-2 was amplified and sequenced using the same primers. RESULTS: An apparent herd prevalence of 77.3 % was determined. Several risk factors were identified including type of pasture grazed, regional variation, and sharing of the paddocks with other livestock species. A novel relationship between the Suffolk breed and higher FEC was reported for the first time. The predominant rumen fluke species found was C. daubneyi. Nevertheless, P. leydeni was unexpectedly identified infecting sheep in Ireland for the first time. CONCLUSIONS: An exceptionally high prevalence of rumen fluke among Irish sheep flocks has been highlighted in this study and a more thorough investigation is necessary to analyse its economic impact. The isolation of individual eggs coupled with the PCR technique used here has proven a reliable tool for discrimination of Paramphistomum spp. This technique may facilitate forthcoming studies of the effects of paramphistomosis on livestock production. The most noteworthy finding was the identification of P. leydeni affecting sheep in Ireland, however further studies are required to clarify its implications. Also, a significant relationship between Suffolk breed and a heavier infection was found, which can be used as a starting point for future research on control strategies of rumen fluke infection.

Comparison of diagnostic techniques for the detection of Cryptosporidium oocysts in animal samples.

While a large number of laboratory methods for the detection of Cryptosporidium oocysts in faecal samples are now available, their efficacy for identifying asymptomatic cases of cryptosporidiosis is poorly understood. This study was carried out to determine a reliable screening test for epidemiological studies in livestock. In addition, three molecular tests were compared to identify Cryptosporidium species responsible for the infection in cattle, sheep and horses. A variety of diagnostic tests including microscopic (Kinyoun's staining), immunological (Direct Fluorescence Antibody tests or DFAT), enzyme-linked immunosorbent assay (ELISA), and molecular methods (nested PCR) were compared to assess their ability to detect Cryptosporidium in cattle, horse and sheep faecal samples. The results indicate that the sensitivity and specificity of each test is highly dependent on the input samples; while Kinyoun's and DFAT proved to be reliable screening tools for cattle samples, DFAT and PCR analysis (targeted at the 18S rRNA gene fragment) were more sensitive for screening sheep and horse samples. Finally different PCR primer sets targetedat the same region resulted in the preferential amplification of certain Cryptosporidium species when multiple species were present in the sample. Therefore, for identification of Cryptosporidium spp. in the event of asymptomatic cryptosporidiosis, the combination of different 18S rRNA nested PCR primer sets is recommended for further epidemiological applications and also tracking the sources of infection.

Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA.

BACKGROUND: Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected. RESULTS: A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %-75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving. CONCLUSION: This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.

Identity of rumen fluke in deer.

As evidence is growing that in many temperate areas paramphistome infections are becoming more common and widespread, this study was undertaken to determine the role of deer as reservoirs for rumen fluke infections in livestock. A total of 144 deer faecal samples (88 from fallow deer, 32 from red deer and 24 samples from sika, sika/red deer hybrids) were screened for the presence of fluke eggs. Based on the ITS-2 rDNA locus plus flanking 5.8S and 28S sequences (ITS-2+), fluke eggs were identified to species level. Our results indicate that, of the 3 deer species, fallow deer had the highest fluke infection rates. Two rumen fluke species, Calicophoron daubneyi and Paramphistomum leydeni, with morphologically distinct eggs, were identified. Concurrent infections of the two paramphistome species and liver fluke, Fasciola hepatica, were common. Considering the comparatively low egg burdens observed in this study, it is unlikely that deer represent a significant source of infection for Irish livestock.

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.

Molecular species determination of cyathostomins from horses in Ireland.

Cyathostomins are globally important equine parasites, responsible for both chronic and acute pathogenic effects. The occurrence of mixed infections with numerous cyathostomin species hinders our understanding of parasite epidemiology, host-parasite dynamics, and species pathogenicity. There have been few studies of cyathostomin species occurring in horses in Ireland, where temperate climatic conditions with year-round rainfall provide suitable conditions for infection of grazing animals with bursate nematodes. Here, we amplified and sequenced the ITS-2 region of adult worms harvested at post-mortem from eleven adult horses between August 2018 and June 2020, and recorded species prevalence and abundance of worms recovered from the caecum, right ventral colon and left dorsal colon, using both BLAST and IDTAXA for taxonomic attribution. Phylogenetic relationships and community composition were also recorded and compared with other relevant studies, including a global meta-analysis. Overall, our results agree with previous studies that there does not seem to be a major difference in cyathostomin species occurrence in equids in different geographical regions. We confirmed the results of other workers in relation to the difficulties in discriminating between Cylicostephanus calicatus and Coronocyclus coronatus on the basis of ITS-2 sequences.

Validation of a serum ELISA test for cyathostomin infection in equines.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Panmictic structure of the Cryptosporidium parvum population in Irish calves: influence of prevalence and host movement.

In total, 245 Cryptosporidium parvum specimens obtained from calves in 205 Irish herds between 2003 and 2005 were subtyped by sequencing the glycoprotein gene gp60 and performing multilocus analysis of seven markers. The transmission dynamics of C. parvum and the influence of temporal, spatial, parasitic, and host-related factors on the parasite (sub)populations were studied. The relationship of those factors to the risk of cryptosporidiosis was also investigated using results from 1,368 fecal specimens submitted to the veterinary laboratories for routine diagnosis during 2005. The prevalence was greatest in the northwest and midwest of the country and on farms that bought in calves. The panmixia (random mating) detected in the C. parvum population may relate to its high prevalence, the cattle density, and the frequent movement of cattle. However, local variations in these factors were reflected in the C. parvum subpopulations. This study demonstrated the importance of biosecurity in the control of bovine cryptosporidiosis (e.g., isolation and testing of calves before introduction into a herd). Furthermore, the zoonotic risk of C. parvum was confirmed, as most specimens possessed GP60 and MS1 subtypes previously described in humans.

Investigating the role of wild carnivores in the epidemiology of bovine neosporosis.

Neospora caninum is a protozoan parasite, primarily associated with bovine abortion. The only definitive hosts discovered to date are carnivores. This study aimed to identify the role of mammalian carnivores in the epidemiology of bovine neosporosis. A sample bank of serum, fecal and brain samples was established: American mink (Mustela vison), red foxes (Vulpes vulpes), pine martens (Martes martes), badgers (Meles meles), stoats (Mustela erminea), otters (Lutra lutra) and feral ferrets (Mustela putorius). Approximately 1% of mink and 1% of fox samples were positive by IFAT. According to PCR analysis of DNA extracted from brain tissue, 3% of the mink, 4% of the otters and 6% of the foxes examined were infected with N. caninum. All fecal samples tested negative for N. caninum DNA (n = 311), suggesting that the species that tested positive were intermediate not definitive hosts. This is the first time that tissues from mustelids have tested positive for N. caninum. The need to test 2 relatively large (~200 mg) targeted parts of the brain to avoid false negatives was also identified. The relatively low prevalence of N. caninum in Irish carnivores suggests that the local ecology of a species has an important influence on its epidemiological role.

A coprological survey of parasites of wild carnivores in Ireland.

The increasing movement of people to wilderness areas, shrinking of wildlife habitats and the resulting urbanisation of wildlife has led to growing concerns about the transfer of parasitic diseases, particularly from contaminated faeces. Faecal samples from wild carnivores in Ireland were examined for the presence of protozoan and nematode parasites. Red fox (Vulpes vulpes) samples (n = 91) were positive for Uncinaria stenocephala (38%), Eucoleus aerophilus (26%), Toxocara canis (20%), Trichuris vulpis (4%) and Isospora-like oocysts (9%). Badger (Meles meles) samples (n = 50) were positive for Uncinaria criniformis (40%), E. aerophilus (6%) and Isospora-like oocysts (16%). No parasites were observed in pine marten (n = 48; Martes martes) faeces. Approximately 5% of American mink (Mustela vison) samples were positive for Cryptosporidium by polymerase chain reaction (identified as Cryptosporidium andersoni (n = 3) and 'mink' genotype (n = 1)). The results suggest that wild carnivores in Ireland have a range of parasites, although it is unclear from the present study to what extent these infections are associated with morbidity. While it can be expected that, via their faeces, wild carnivores contribute to the spread of these parasites, they are unlikely the primary source of environmental contamination. Therefore, they should not always be the principal target of control measures.

A qualitative study of perceived barriers and facilitators to sustainable parasite control on thoroughbred studs in Ireland.

In view of the rising threat of anthelmintic resistance in parasite populations and the planned change of classification of anthelmintics in Ireland for food animals to prescription-only medicines, there is a need for increased focus on parasite control strategies for horses. Effective parasite control programs (PCPs) are complex, necessitating the application of risk-assessment based on host immune status, infection pressure, type of parasite and seasonality to determine the requirement for anthelmintic administration and an understanding of parasite biology to employ effective, non- therapeutic, parasite control measures. The aim of this study was to explore horse breeders' attitudes and behaviours towards general parasite control practices and anthelmintic use on thoroughbred studs in Ireland using qualitative research methods, in order to help identify barriers to the implementation of sustainable equine PCPs supported by veterinary involvement. One-to-one, qualitative, semi-structured interviews were conducted with 16 breeders, using an interview topic guide that allowed for an open questioning style. The topic guide facilitated discussion around the following areas: (i) parasite control measures (general approach, (ii) veterinary involvement, (iii) use of anthelmintics, (iv) use of diagnostics, pasture management), (v) recording of anthelmintic use and (vi) anthelmintic resistance. Convenience, purposive (subjective selection) sampling was used to select a small group of participants that reflected the characteristics of current thoroughbred breeders in Ireland taking into account farm type, farm size, geographical location. The interviews were transcribed, and a method of inductive thematic analysis (a data driven identification and analysis of themes) was performed. Assessment of current behaviours by these participants found that PCPs were primarily based on the prophylactic use of anthelmintics without a strategic rationale. A key driver of behaviour regarding parasite prevention practices was localised routine, dictated by tradition, that gave breeders a sense of confidence and protection regarding parasite control. The perceptions of the benefits of parasitology diagnostics were varied and their application to control was poorly understood. Anthelmintic resistance was recognised as an industry threat but not an issue for individual farms. By using a qualitative approach, this study offers insights into the possible barriers to the adoption of sustainable PCPs on Irish thoroughbred farms and highlights the need for end-user involvement in the preparation of future guidelines.

Survey of the knowledge and perceptions of horse owners in Ireland of common clinical conditions and their impact.

BACKGROUND: As the primary decision-maker for their horse's health and welfare, owners' knowledge of clinical conditions may impact their horse's health. Anecdotal evidence suggests that the emotional impact of equine illness on the owner can be severe but research is lacking. OBJECTIVES: To evaluate horse owners' self-declared knowledge of eight common equine health conditions and perceptions of the quality of information available; to ascertain respondents' perceptions of the severity of impact on their horse of the conditions and potential emotional impact on themselves and to establish the factors of greatest concern to owners when their horse has a health condition and influential factors on end-of-life decisions. STUDY DESIGN: Online survey of horse owners. METHODS: An online survey comprising three sections was created: respondent demographics; vignettes of clinical conditions; concerns and decision-making regarding illness and euthanasia. Vignettes focused on arthritis; Pars Pituitary Intermedia Dysfunction; Equine Metabolic Syndrome (EMS); gastric ulcers; high intestinal worm burden; dermatophilosis ('mud rash'); quidding and head tossing; and equine asthma. Respondents were self-selecting. The survey was circulated for completion by horse owners residing in Ireland and Northern Ireland obtaining 491 valid responses. RESULTS: Respondents declared that they had the highest knowledge of mud rash, with 84.1% rating their knowledge as very good compared to 42% of respondents for EMS. There was positive correlation between the perceived impact on their horse and emotional impact on the owner for all conditions ranging from weak (arthritis 0.36, P < 0.001) to strong (EMS 0.62, P < 0.001). Pain, discomfort, and quality of life were reported as the most common concerns when their horse has a medical condition or when considering euthanasia. MAIN LIMITATIONS: Potential bias among respondents towards computer literate owners, with good literacy and interest in equine health conditions. CONCLUSIONS: Knowledge of equine health conditions, perceived impact of conditions on the horse and emotional impact on their owners varied significantly between conditions.

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.

Age-stratified Bayesian analysis to estimate sensitivity and specificity of four diagnostic tests for detection of Cryptosporidium oocysts in neonatal calves.

There is no gold standard diagnostic test for the detection of bovine cryptosporidiosis. Infection is usually highest in 2-week-old calves, and these calves also excrete high numbers of oocysts. These factors may give rise to variations in the sensitivity and specificity of the various diagnostic tests used to detect infection in calves of various ages. An age-stratified Bayesian analysis was carried out to determine the optimum diagnostic test to identify asymptomatic and clinical Cryptosporidium sp. infection in neonatal calves. Fecal samples collected from 82 calves at 1 week, 2 weeks, 3 weeks, and 4 weeks of age were subjected to the following tests: microscopic examination of smears stained with either phenol-auramine O or fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium monoclonal antibody, nested-PCR, and quantitative real-time PCR. The results confirmed a high prevalence of Cryptosporidium sp. infection, as well as a high level of oocyst excretion, in 2-week-old calves. The sensitivities of all the tests varied with the age of the calves. Quantitative real-time PCR proved to be the most sensitive and specific test for detecting infection irrespective of the age of the calf. The microscopic techniques were the least sensitive and exhibited only moderate efficiency with 2-week-old calves excreting large numbers of oocysts, the majority of which were diarrheic. It was concluded that, when interpreting the results of routine tests for bovine cryptosporidiosis, cognizance should be taken of the sensitivity of the tests in relation to the age of the calves and stage of infection.