Feasibility of meat juice as an alternative to serum on commercial ELISA for surveillance of exemplar viruses; Pestivirus and BoHV-1, in cattle and wild deer

doi:10.21203/rs.3.rs-4800925/v1

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Feasibility of meat juice as an alternative to serum on commercial ELISA for surveillance of exemplar viruses; Pestivirus and BoHV-1, in cattle and wild deer

https://doi.org/10.21203/rs.3.rs-4800925/v1

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Serology is a vital tool for disease surveillance but relies on obtaining blood samples, which can be difficult especially from wildlife species. Meat Juice Serology (MJS) presents an alternative but requires validation before being more confidently and widely applied. Here, MJS was performed on cattle and wild deer samples from Northern Ireland, to detect antibodies to the viruses that cause Bovine Viral Diarrhoea (BVD) and Infectious Bovine Rhinotracheitis (IBR) (Pestivirus A/B and Bovine Herpesvirus-1 (BoHV-1), respectively). Greater mean volumes of meat juice were obtained from cattle tissue frozen at -20⁰C than those at -80⁰C (p < 0.05). Antibodies to Pestivirus (unspeciated), were detected in three (2.6%) deer serum samples (n = 116), and one paired meat juice sample from deer neck muscle. No antibodies to IBR were detected in any deer (n = 116). In cattle, p80 ELISA performed at recommended dilution (1:9) (n = 40) and showed high correlation between serum and meat juice (r = 0.64, p < 0.05), but poor correlation at adjusted dilution (1:1) (n = 37, r = 0.18, p = 0.28). In deer, meat juice tested at adjusted dilution (1:1) (n = 94) showed moderate correlation with serum tested at recommended dilution (r = 0.49, p < 0.05). For IBR, cattle were tested on gE ELISA (n = 40). High correlation was found between serum and meat juice, tested to manufacturer’s recommended dilution (rho = 0.57, p < 0.05). Deer samples were tested on gB ELISA, and meat juice correlated poorly to serum (n = 113) at both recommended (1:1) (n = 113, r = 0.13, p = 0.25) and adjusted (9:1) dilution (n = 113, r = 0.08, p = 0.52).

Meat juice serology may represent an appropriate alternative to serum for Pestivirus and BoHV-1 in cattle, without the need to modify dilution. In deer, this study indicates that meat juice may be used to monitor for Pestivirus, but cannot be recommended for BoHV-1, without further evaluation and quality control.

Meat Juice

Serology

Cattle

Deer

Pestivirus

Bovine Viral Diarrhoea

Infectious Bovine Rhinotracheitis

BoHV-1

ELISA

Meat Juice Serology utilises the fluid released from muscle tissue post-mortem. This fluid, meat juice (MJ), is comprised of intercellular and extracellular fluid, blood, lymph and proteins, including antibodies (Wallander et al., 2015). Therefore, the meat juice matrix has the potential to be substituted for blood serum on commercially available enzyme-linked immunosorbent assay (ELISA) kits where serum is difficult or impossible to obtain, for example, in wildlife studies. Meat juice serology is also an attractive alternative to blood serum for passive surveillance in livestock as muscle tissue can easily be obtained at the abattoir by personnel without extra training, thus it is relatively easy and cost effective (Loreck et al., 2020). Legislation and protocol guidelines for meat juice sampling are already in place for several microorganisms and animal hosts in the European Union, for instance for Trichinella surveillance (Commission Implementing Regulation (EU) No. 2015/1375) and Salmonella surveillance in Germany and Denmark (Loreck et al., 2020; Mousing et al., 1997). Further, meat juice sampling also represents a lower safety and biosecurity risk than exsanguination or on-farm visits for venipuncture (Molina et al., 2008).

BVD and IBR are economically important viral diseases of cattle, caused by Pestivirus A/ Pestivirus B and Bovine Herpesvirus-1 (BoHV-1), respectively (AHI, 2020; DAFM & AFBINI, 2023). BVD is currently subject to compulsory eradication programmes in Northern Ireland (NI) and the Republic of Ireland (ROI) and an IBR eradication scheme is due to commence in ROI in 2024. Abattoir surveillance using MJS could represent an important tool for post-eradication surveillance of Bovine Viral Diarrhoea (BVD) in cattle, similar to its use for Infectious Bovine Rhinotracheitis (IBR) antibody detection (Graham et al., 2021), which is approved under Animal Health Law Regulation (EU) 2020/689, annex III section 4 (European Commission, 2020). MJS could also be considered for wild deer surveillance, as deer have the potential to act as a maintenance host of these pathogens (Miller et al., 2013; Passler et al., 2010; Roug et al., 2012), which could be a potential source of re-introduction of the diseases to the domestic livestock population, undermining biosecurity measures and disease freedom status.

Several studies have reported suitable agreement between blood serum and meat juice (MJ) ELISA testing against a range of parasitic, bacterial and viral agents, although these range from mild to excellent (Abrantes et al., 2024; Casas et al., 2011; Charlier et al., 2009; Healy et al., 2024; Vico and Mainar-Jaime, 2011; Wallander et al., 2015).

While meat juice serology is currently in use in several animal species, for various pathogenic organisms (Abrantes et al., 2024; Berger-Schoch et al., 2011; European Commission, 2020; Healy et al., 2024; Loreck et al., 2020; Meemken et al., 2011; Meemken et al., 2014), several aspects of the matrix are still poorly evaluated, and harmonisation of protocols and nomenclature around this assay should be addressed. There are currently no guidelines in place for meat juice extraction protocols; such as freeze/thaw processes or optimum tissue type. Consensus on the requirement to alter kit-specified dilutions protocols (to account for the differences between meat juice and serum antibody titres (Loreck et al., 2020; Nielsen et al., 1998; Wallander et al., 2015)) is also lacking. Studies alternately refer to meat juice as ‘muscle extract’ (Lyashchenko et al., 2021), ‘muscle fluid’ (Mortensen et al., 2001), ‘exudate’ (Healy et al., 2024; Le Potier, 1998) or ‘transudate’ (Molina et al., 2008), and the test itself is also alternatively referred to as Meat Juice ELISA (MJE)(Snary et al., 2010).

Currently, there are few studies where meat juice serology has been described for deer tissue analysis (Abrantes et al., 2024; Witkowski et al., 2015). The aims of the present work, therefore, where to determine the utility of meat juice serology in the detection of these exemplar viruses in cattle and wild deer samples on commercially available ELISA kits by measuring agreement between paired meat juice and serum, and to examine whether the protocols recommended for sera can be similarly applied to the meat juice matrix.

Sampling methodology

Two studies were conducted. Study 1 was performed on cattle to establish protocols for meat juice extraction and identify factors which could potentially affect meat juice yield; initial freeze temperature and tissue type. Paired cattle serum and meat juice samples were also tested on commercial ELISA kits for Pestivirus p80-reacting antibody and BoHV-1 glycoprotein E-reacting antibody, to determine agreement between these matrices. Study 2 was subsequently performed on wild deer samples to detect Pestivirus p80-reacting antibody and BoHV-1 glycoprotein B-reacting antibody on paired serum/meat juice samples. Samples for each study were obtained as follows:

Cattle

Samples were harvested from 40 cattle (n = 190) submitted to the Agri-food & Biosciences Institute, Veterinary Sciences Division, Disease Surveillance & Investigation Branch (AFBINI, VSD, DSIB, Stormont, Belfast) between November 2020 and September 2021 for routine diagnostic post-mortem examination on site. Twenty-nine females and eleven males, aged five years or under were sampled (Table 1). A blood clot from the heart and muscle tissue measuring approximately 3 x 1 x 1 cm from the diaphragm, neck, masseter and tongue were placed in sterile containers. Blood was refrigerated at + 4°C for up to 24 hrs, then centrifuged at 3,000 RPM for 10 minutes (Thermo SL40R) to retrieve serum, which was subsequently frozen at -20⁰C before use. Muscle samples were immediately frozen at either − 80°C or -20°C until extraction.

Deer

Sampling packs containing sterile blood tubes, 30 mL universal containers and submission forms were supplied to participating deer stalkers. Approximately 10 mL of jugular blood was collected in a sterile blood tube and neck tissue sample measuring approximately 3 x 1 x 1 cm per animal, were harvested at the larder by trained and licensed stalkers and representatives of the Northern Ireland Forest Service during the legal hunting season between September 2022 and March 2023. All samples were refrigerated at + 4°C at the larder until collection, then transported on ice. Mean cull to collection turnaround time was 4 days. Blood samples were immediately centrifuged upon collection at 3,000 RPM for 10 minutes (Thermo SL40R) to retrieve serum, which was subsequently frozen at -20⁰C, and muscle samples were frozen at -20°C prior to the extraction process.

Meat Juice extraction

Tissue samples were thawed overnight (18-24hrs) under ambient conditions, over a polythene bag with an opening cut from the end to create a funnel, and fluid collected in a falcon tube (Fig. 1). Following extraction the meat juice was centrifuged at 3000 RPM for 10 minutes and the supernatant (meat juice) retrieved and frozen until required for testing. Freeze temperature analysis on Meat Juice yield was performed on a random subset of 31 cattle samples where tissue was initially frozen at either − 80⁰C (n = 45) or -20°C (n = 48).

BVD/BD p80 ELISA.

Applied Biosystems PrioCHECK™ Rum. BVD/BD p80 Ab Serum & Milk blocking ELISA Kit (ThermoFisher Scientific) (n = 116). Serum and controls were tested according to kit instructions, with the exception of the dilution trials, where meat juice was tested at a lower dilution (1:1), detailed below.

Antibody levels were expressed in terms of percentage inhibition (%INH) and interpreted as %INH < 50 negative, 50 ≤ %INH < 80 weak positive, %INH ≥ 80 positive. Manufacturer’s analytical sensitivity is 97% and specificity is 98% for BVDV and 96% and 100%, respectively, for BDV.

IBR gE ELISA

IDEXX IBR/BHV-1 gE Ab DIVA ELISA (IDEXX Laboratories Ltd.) (n = 105) was used according to the manufacturer’s instructions.

Antibody levels were expressed in terms of sample-negative ratio (S/N): <0.6 = positive; 0.61–0.7 = inconclusive; >0.7 = negative.

IBR gB Blocking ELISA.

IDEXX IBR gB X3 (IDEXX Switzerland GmbH, Switzerland) was used according to the kit instructions (n = 116), except in the dilution comparison study, where meat juice samples were also tested at a lower dilution factor (9:1).

Antibody levels were expressed as a blocking percentage and interpreted as Blocking % < 45 Negative, 45 ≤ Blocking % < 55 Suspect, Blocking % ≥ 55 Positive.

Statistical Analyses

All statistical analyses were performed in R Studio version 4.2.2 (RStudio, 2020), with statistical significance given as p = 0.05. Suitability of all statistical tests was determined by graphical analysis. Data were transformed, where appropriate, using Box-Cox or Tukey’s Ladder of Powers methods. Correlation between matrices was determined by Pearson’s correlation coefficient and Spearman’s correlation.

Animal demographic data (Table 1): Cattle samples included 29 females and 11 males aged from 3 months to 5 years (n = 40). Deer samples included 77 females, 34 males and 5 of unknown species. Deer age estimates were made by trained stalkers using dental analysis. Young deer estimated to be between 1–3 years made up the majority of samples (n = 91), followed by adults over 4 years old (n = 11) and immature animals under 1 year (n = 4). Ten animals were submitted without estimates of age. Fallow deer samples were submitted from a larder in Co. Armagh, managed by the Northern Ireland Forest Service. Fallow samples were culled in Moneyscalp, Donard Park and Tollymore, Co. Down (n = 49), Randalstown, Co. Antrim (n = 18) and Bangor, North Down (n = 2). Sika deer samples were submitted from a larder in Co. Fermanagh which services a 1000-acre private estate and around 7000 acres of surrounding area (n = 47).

Meat Juice extraction on cattle tissue

Overall mean yield from − 80⁰C was M = 1.03mL (n = 45, SD = 1.16, 95% CI = 0.68 to 1.38), while from − 20⁰C mean yield was M = 1.81mL (n = 48, SD = 1.32, 95% CI = 1.43 to 2.19). Box-Cox transformation was applied. Two-way ANOVA showed freezing temperature had a statistically significant effect on tissue yield (F_{1, 86}= 9.17, p < 0.05), with non-significant overall effect for tissue (F_{2, 86}= 0.18, p = 0.83). On yield by temperature t-test, the difference was determined to be due to between temperature yield from masseter (t₃₀ = 2.94, p < 0.05, 95% CI = 0.32 to 1.84) (Figure. 2).

Cattle Pestivirus p80 ELISA

At kit recommended 1:9 dilution, one-way ANOVA showed no statistically significant differences in %INH between tissue types (F₃ = 1.21, p = 0.31). %INH in serum and meat juice when tested at this dilution were significantly correlated (Pearson’s r(₂₉) = 0.64, p < 0.05, 95% CI = 0.36 to 0.81)(Fig. 3A).

Cattle Pestivirus p80 ELISA dilution factor analysis

Due to the consensus that meat juice contains around 10x fewer antibodies than serum (Loreck et al., 2020; Nielsen et al., 1998; Wallander et al., 2015), the manufacturer’s recommended 1:9 dilution was tested against modified 1:1 dilution for paired samples on eighteen animals, 37 tissue samples per dilution. Pearson’s correlation coefficient for serum samples tested at 1:9 dilution against meat juice samples tested at modified 1:1 showed poor positive correlation (r₃₅ = 0.18, p = 0.28, 95% CI= -0.15 to 0.48) (Fig. 3B).

Cattle IBR gE

Thirty cases were analysed on IBR gE DIVA-marker ELISA at kit recommended dilution (n = 105). Six cases (20.0%) contained at least one positive tissue sample. Data could not be transformed. Kruskal-Wallis analysis showed good agreement between S/N values between all sample matrices (χ² ₁₁₉ = 2.27, p = 0.52) (Fig. 4A) with high correlation between serum and meat juice (Spearman’s rho = 0.57, p < 0.05) (Fig. 4B).

Deer Meat Juice extraction

All samples were thawed from − 20°C, based on the higher yield results from this temperature in study 1. Of 116 animals, 94 yielded meat juice (81.0%). Yield was rounded to the nearest 500µL. Mean yield from these samples, approximately 3 x 1 x1 cm in size, was M = 578.72µL (n = 94, SD = 616.16, 95% CI = 452.52 to 704.92). Species. Mean yield from fallow was M = 457.84µL (n = 51, SD = 448.82, 95% CI = 331.61 to 584.07), from sika M = 742.68µL (n = 41, SD = 760.80, 95% CI = 502.54 to 982.82), from unknown species M = 300µL (n = 2, SD = 282.84, 95% CI= -2241.22 to 2841.22). Sex. Mean yield for males was M = 587.04 µL (n = 27, SD = 450.98, 95% CI = 408.64 to 765.44), females M = 583.85µL (n = 65, SD = 682.14, 95% CI = 414.82 to 752.87), and from unknown sex M = 300µL (n = 2, SD = 282.84, 95% CI= -2241.22 to 2841.22) (Fig. 5A).

Data were transformed by Tukey’s Ladder of Powers and ANOVA showed no statistically significant differences in yield by species or sex (F₂ = 2.94, p = 0.06 and F₂ = 0.56, p = 0.57, respectively) (Fig. 5B). Tukey HSD post-hoc analysis showed Fallow-Sika differences in meat juice yield accounted for the borderline significance in species (diff = 0.01, p = 0.06, 95% CI = 0.0004 to 0.03).

Deer Pestivirus p80 ELISA dilution factor analysis

Sera were tested to manufacturer’s recommended 1:9 dilution and corresponding meat juice, were tested at modified 1:1 dilution. Paired t-test showed statistically significant differences between meat juice and serum (t₉₃= -3.83, p < 0.05, 95% CI= -12.00 to -3.81). Moderate correlation was demonstrated between serum and meat juice (Pearson’s r(₉₂) = 0.49, p < 0.05, 95% CI = 0.32 to 0.63)(Fig. 6).

Two serum samples tested positive for Pestivirus antibody (p80%INH = 84.1, female sika and p80%INH 92.8, sika, unknown sex) and one tested low positive (p80%INH = 76.9, male sika). Pestivirus species could not be differentiated on gold-standard Virus Neutralisation Test (M. Lyons et al., unpublished at this time). All three positive samples were culled in County Fermanagh. Of the positive serum samples, only the p80%INH = 84.1 (female) and p80%INH = 76.9 (male) samples had sufficient corresponding meat juice for testing. The p80%INH = 84.1 serum sample corresponding meat juice tested low positive for BVDv antibody (p80%INH = 78.7), while the serum low positive p80%INH = 76.9 corresponding meat juice sample tested negative (p80%INH = 15.3).

Deer IBR gB Blocking ELISA dilution factor analysis

Sera were tested at recommended 1:1 dilution, while paired meat juice was tested at both kit recommended dilution and a modified dilution of 9:1. Box-Cox transformation was applied to paired samples. One-way ANOVA testing showed statistically significant differences between all matrices and dilutions (F₂ = 86.51, p < 0.05)(Fig. 7). Post-hoc Tukey HSD test for multiple comparison showed statistically significant differences between serum and meat juice at all dilution factors; Meat Juice 9:1-Meat Juice 1:1 p < 0.05 (t.ratio₁₇₀= -7.97, SE = 5969, 95% CI = 32139.64 to 64547.30), Serum 1:1-Meat Juice 1:1 p < 0.05 (t.ratio₁₇₀ = 6.93, SE = 5066, 95% CI= -48910.65 to -21642.63), Serum 1:1-Meat Juice 9:1 p < 0.05 (t.ratio₁₇₀ = 14.54, SE = 5686, 95% CI= -98698.03 to -68542.19).

Pearsons’s correlation for serum against meat juice at recommended dilution (1:1) was very weak (r₇₅ = 0.13, p = 0.24, 95% CI= -0.09 to 0.35), as was serum (1:1) against modified meat juice (9:1) (r₅₄ = 0.08, p = 0.52, 95% CI= -0.18 to 0.34). Meat juice (1:1) against modified meat juice (9:1) showed good positive correlation (r₅₅ = 0.62, p < 0.05, 95% CI = 0.43 to 0.76). All samples were negative for IBR.

Meat juice is often extracted from tissue that is easily accessible and of little commercial value. Diaphragmatic muscle is most frequently analysed as it is accessible and already used in Trichinella surveillance (Wallander et al., 2015). However, testing of meat juice from skeletal muscle such as foreleg and hindleg (Molina et al., 2008), forequarter and hindquarter (Kaden et al., 2009) and diaphragmatic muscle (Felin et al., 2017; Kaden et al., 2009; Wallander et al., 2015) as well as organ tissue, such as liver (Navarro et al., 2020), heart (Wallander et al., 2015; Yonemitsu et al., 2019) and tongue (Wallander et al., 2015), have been described. One study comparing tissue types found the highest agreement between serum and heart derived meat juice, with diaphragmatic tissue placed third (Wallander et al., 2015), while others found heart and sternomastoideus muscle to produce meat juice more reliably or at greater volumes than diaphragm (Nielsen et al., 1998; Yonemitsu et al., 2019). The present study found suitable agreement between cattle diaphragm, masseter and neck yields, thus, coupled with ease of access and low commercial value, neck was requested for the subsequent deer study.

A specialised funnel can be purchased for meat juice extraction (Kabe Labortechnik GmbH, Nuembrecht, Germany), however, more rudimentary methods have been described in other studies, such as dripping the thawing meat juice through a plastic bag tied with an elastic band or a clamp (Loreck et al., 2020; Meemken et al., 2014). Here, meat juice was obtained by thawing samples in an upturned 30 mL universal container over a polythene bag, cut to create a funnel. Meat juice was collected in a was 50 mL falcon tube.

The present study found that pre-extraction freeze temperature had a significant impact on meat juice yield and volume. Freezing of muscle samples causes the formation of ice crystals, a process known as nucleation, which influences the release of intracellular fluid and proteins into the intracellular spaces (Añón and Calvelo, 1980; Leonard et al., 2022). Fast freezing at -80⁰C causes the formation of small ice crystals, while a slower freeze rate (-20⁰C), causes larger crystals to form. Larger crystals disrupt the muscular structure to a higher extent, resulting in a greater volume of fluid release (Cook et al., 1926; Leonard et al., 2022; Petrović et al., 1993; Rahelić et al., 1985). Freeze temperature analysis in the cattle study found statistically significant greater overall meat juice yield when extracted from the higher freeze temperature (-20⁰C). Therefore, all deer samples for study 2 were frozen at -20⁰C. Adequate volumes of meat juice was extracted from 81.0% of these samples.

The concentration of antibodies in meat juice is known to be approximately 10x lower than in serum; prompting several studies to recommend meat juice should be analysed at more concentrated volumes than serum (Glor, 2013; Loreck et al., 2020). However, in the present study, cattle IBR gE values for meat juice showed good agreement with serum when tested at the manufacturer’s recommended dilution (modified meat juice analysis was not performed). European legislation for IBR testing in cattle (Animal Health Law Regulation (EU) No 2016/689, section 4 annex II) provides for the use of meat juice on ELISA as an alternative to serum, without the caveat of modifying the dilution of the meat juice. This is supported by the results obtained here, which indicate that meat juice would be a suitable serum substitution for IBR gE surveillance in cattle.

Dilution analyses of Pestivirus p80 values for cattle, comparing serum and meat juice tested at recommended 1:9 against meat juice also tested at modified dilution of 1:1 was performed here. Results indicated that meat juice correlated best with serum when tested at the recommended 1:9 dilution (R = 0.64) compared to the modified lower dilution (R = 0.18). There was a high correlation between meat juice at recommended and modified dilutions (R = 0.64); as dilution decreased, %INH increased, as would be expected. For deer samples, due to the discontinuation of the PrioCHECK™ Ruminant BVD p80 ELISA kit during the course of the study, only modified meat juice dilution could be assessed. A moderate correlation between the matrices was determined (R = 0.49). This indicates that meat juice, at this dilution, may a suitable alternative for serum on this ELISA in deer.

IBR glycoprotein B (gB) based tests have previously been determined as highest sensitivity and specificity than gE assays (Kramps et al., 2004) and, being competitive assays, are less sensitive to species-specificity as they don’t rely on anti-species conjugate. Therefore, this assay was used in the deer study. Here, meat juice was poorly correlated to serum at both recommended and modified dilutions and in all tissue, therefore, appears to be an inappropriate alternative to serum for deer on this assay.

Future recommendations

Dilution analysis on deer meat juice for Pestivirus would ideally have been performed here, however, the PrioCHECK BVD/BD p80 kit was discontinued during the course of this study. Replication of this study, using currently available Pestivirus ELISA would be strongly recommended.

The unavoidable use of non species-specific diagnostic tests may have impacted test sensitivity and specificity for both serum and meat juice in the deer study. However, it should be acknowledged that this is an intrinsic limitation to most wildlife studies. True validation of diagnostic assays for wildlife species are required in order to effectively survey them for pathogens of interest.

Further works to investigate the impact of repeated freeze-thaw cycles and storage conditions on meat juice samples post-extraction should be assessed, as these are factors known to affect serum.

As meat juice is a recommended matrix for IBR surveillance in cattle, guidelines should be adapted to include standardised tissue selection protocols.

Results here indicate that meat juice is a suitable substitute for serum for the detection of Pestivirus and IBR in cattle, without the requirement of dilution modification, on these serological assays. Meat juice did not correlate well to serum at either dilution for deer on IBR gB ELISA, thus, based on these results, cannot be recommended as an alternative matrix. Meat juice correlated modestly with serum for deer on Pestivirus p80 ELISA, at modified, lower dilution, and antibodies to Pestivirus (unspeciated) were detected in a deer meat juice sample with corresponding positive serum. However, assay sensitivity may be reduced for low positive viral titres, as evidenced by the negative Pestivirus result in a deer meat juice sample with a low positive serum result, which warrants further investigation. Where serum cannot be obtained, or as a secondary matrix, meat juice could represent a viable alternative in wild deer pestivirus surveillance, and may point to the potential greater application of MJS in the field of animal disease surveillance.

The meat juice extraction technique adopted for this study was adequate for both cattle and deer samples, with yield results best when extracted from − 20°C, therefore this protocol would be recommended. However, it is worth noting that meat juice extraction is time consuming and yields are variable and unpredictable. Since deer Pestivirus prevalence is low and no IBR was detected, the cost benefit of routine surveillance in deer, by either serum or meat juice, should be considered.

To the best of the author’s knowledge, no other studies have attempted to assess the use of meat juice for Pestivirus analysis in either cattle or deer. Correlation studies for serum against meat juice for IBR are similarly unavailable for either species.

Ethics approval 

The manuscript does not contain clinical studies, patient data, or individual data. Samples were harvested from deer culled by trained and licenced stalkers in Northern Ireland, in accordance with legislation and within the designated open season.

Consent for publication 

All authors consent for publication. 

Availability of data and materials 

The datasets supporting the conclusions of this article are included within the article will be made available on request.

Competing interests 

The authors declare no competing interests. 

Funding 

This work was supported by DAERA’s annual postgraduate studentship, in contribution to the author’s PhD thesis (M Lyons).

Authors' contributions 

M Lyons – Conceptualisation, Methodology, Project administration, Data curation, Formal Analysis, Investigation, Writing – original draft, Writing – review & editing, A Lahuerta-Marin – Supervision, Writing – review & editing, J. Clarke – Methodology,

J. McConville – Methodology, S. Porter – Writing – review & editing, M. Guelbenzu – Methodology, E. Campbell – Conceptualisation, Methodology,

P. J. Collins – Methodology, ER Morgan –  Supervision, Investigation, Project administration, Writing – review & editing. 

Acknowledgements 

The authors would like to acknowledge all stalkers, estate managers, the Forest Service Northern Ireland, especially Donald Whiteside, for the kind provision of sample materials and AFBINI for their support and resources. We would also like to thank Dr David Graham, of Animal Health Ireland, for his advice and consultation.

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Table 1

Breakdown of demographics relating to the animals used in this study, including: a) sex, b) age class* and c) location data.* Age Class: Study 1-Cattle: Class1 = < 6 months, Class 2 = 6–11 months, Class 3 = 12–23 months, Class 4 = > 24 months. Phases 3; Study 2-Deer: Class1 = Immature (calves, fawns, kids), Class 2 = Young (yearling-3 year old), Class 3 = Adult ( = > 4), Class 4 = Age unknown.
Sex
Female		Male	Unknown
Study 1	29	11	0
Study 2	77	34	5
Age Class*
Class 1		Class 2	Class 3	Class 4
Study 1	2	4	12	22
Study 2	4	91	11	10
Location
	North Down	Randalstown	Co. Down	Fermanagh	NA
Study 1	0	0	0	0	40
Study 2	2	18	49	47	0

No competing interests reported.

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Editorial decision: Revision requested
06 Aug, 2024
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06 Aug, 2024
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Feasibility of meat juice as an alternative to serum on commercial ELISA for surveillance of exemplar viruses; Pestivirus and BoHV-1, in cattle and wild deer

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Abstract

Figures

Introduction

Methods

Sampling methodology

Cattle

Deer

Meat Juice extraction

Statistical Analyses

Results

Meat Juice extraction on cattle tissue

Cattle Pestivirus p80 ELISA

Cattle Pestivirus p80 ELISA dilution factor analysis

Cattle IBR gE

Deer Meat Juice extraction

Deer Pestivirus p80 ELISA dilution factor analysis

Deer IBR gB Blocking ELISA dilution factor analysis

Discussion

Conclusions

Declarations

References

Tables

Additional Declarations

Status:

Version 1