Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus.

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Spatial and temporal analysis of sheep scab notifications in Scotland, 2014-2019.

BACKGROUND: Sheep scab is considered an endemic disease of great welfare and economic significance in the UK. METHOD: This paper provides an up-to-date assessment of the impact of Sheep Scab (Scotland) Order 2010 on sheep scab notifications in Scotland between 1 January 2014 and 31 December 2019, using data collected by the APHA. RESULTS: In total, 564 sheep scab notifications were reported from 503 unique holdings, of which 44 holdings (8.7%) reported more than one incident. The number of notifications did not differ between years, with 81, 84, 93, 101, 109 and 97 notifications recorded in 2014, 2015, 2016, 2017, 2018 and 2019, respectively: representing an average annual notification prevalence of 0.63% (1/159 flocks/year). A total of 413/564 records documented how notifications were resolved, with macrocyclic lactone and organophosphate treatments accounting for 79.6% and 20.4% of resolutions, respectively. CONCLUSION: Our results suggest that the Order has facilitated the notification of sheep scab in Scotland (including trends and preferred methods of resolution), allowed industry and government to identify previously unidentified potentially free areas as well as recurrent incidents on sheep farms, and start to understand better the geographical and temporal nature of scab outbreaks. However, concerns remain about a potential lack of engagement, evidenced by the low notification prevalence and stagnant annual notification rates.

Exploiting Scanning Surveillance Data to Inform Future Strategies for the Control of Endemic Diseases: The Example of Sheep Scab.

Scanning surveillance facilitates the monitoring of many endemic diseases of livestock in Great Britain, including sheep scab, an ectoparasitic disease of major welfare and economic burden. There is, however, a drive to improve the cost-effectiveness of animal health surveillance, for example by thoroughly exploiting existing data sources. By analysing the Veterinary Investigation Diagnosis Analysis (VIDA) database, this study aimed to enhance the use of existing scanning surveillance data for sheep scab to identify current trends, highlighting geographical "hotspots" for targeted disease control measures, and identifying a denominator to aid the interpretation of the diagnostic count data. Furthermore, this study collated and assessed the impact of past targeted disease control initiatives using a temporal aberration detection algorithm, the Farrington algorithm, to provide an evidence base towards developing cost-effective disease control strategies. A total of 2,401 positive skin scrapes were recorded from 2003 to 2018. A statistically significant decline in the number of positive skin scrapes diagnosed (p < 0.001) occurred across the study period, and significant clustering was observed in Wales, with a maximum of 47 positive scrapes in Ceredigion in 2007. Scheduled ectoparasite tests was also identified as a potential denominator for the interpretation of positive scrapes by stakeholders. Across the study period, 11 national disease control initiatives occurred: four in Wales, three in England, and four in Scotland. The majority (n = 8) offered free diagnostic testing while the remainder involved knowledge transfer either combined with free testing or skills training and the introduction of the Sheep Scab (Scotland) Order 2010. The Farrington algorithm raised 20 alarms of which 11 occurred within a period of free testing in Wales and one following the introduction of the Sheep Scab (Scotland) Order 2010. In summary, our analysis of the VIDA database has greatly enhanced our knowledge of sheep scab in Great Britain, firstly by identifying areas for targeted action and secondly by offering a framework to measure the impact of future disease control initiatives. Importantly this framework could be applied to inform future strategies for the control of other endemic diseases.

Kinetics of IgA and eosinophils following a low-dose, predominantly Haemonchus contortus infection of Boer goats.

AIMS: Most breeds of goat are more susceptible to nematode infection than sheep, and this appears to be a consequence of less effective immune responses. Several papers have considered the effectiveness of eosinophils and immunoglobulin A (IgA) in goats but differences in the induction of responses have not been studied in the same detail. The aim of this study was to look at the induction of eosinophil and IgA responses in Boer goats reared indoors under intensive conditions. METHODS AND RESULTS: The goats were experimentally infected with a low dose of 2400 Haemonchus contortus, Trichostrongylus spp. and Oesophagostomum spp. at a 6:1:1 ratio. Faecal egg counts (FEC), packed cell volume (PCV), IgA activity against third-stage larvae and peripheral eosinophilia were measured twice a week for eight weeks. The infection generated an IgA response but did not significantly increase peripheral eosinophilia in the 25 infected kids compared with the 4 control animals. FEC was not associated with IgA activity or eosinophilia. CONCLUSION: A detailed analysis of IgA and eosinophil responses to deliberate nematode infection in Boer goats showed that there was an increase in nematode-specific IgA activity but no detectable eosinophil response. In addition, there was no association between increased IgA activity or eosinophilia with egg counts and worm burdens. These suggest that IgA and eosinophils do not act to control nematode infection in goats.

The dynamics of ovine gastrointestinal nematode infections within ewe and lamb cohorts on three Scottish sheep farms.

Gastrointestinal nematodes (GIN) are a serious concern for sheep producers worldwide. However, there is a paucity of evidence describing the epidemiology of GIN on modern UK sheep farms. The aim of this paper was to understand whether expected seasonal variations of infection are still found in ewes and lambs under varying management strategies in temperate climates. Faecal egg counts (FEC) were conducted on freshly voided samples collected from groups of ewes and lambs every third week for twelve months on three farms in southeast Scotland. The patterns of egg output have been described here in relation to management practices undertaken on the farms. Despite changes in farming practice and climatic conditions, the findings complement historical studies detailing the epidemiology of GIN. Findings include a periparturient rise in ewe FEC on two of the farms, while lambing time treatment appeared to suppress this on the third farm. On the same two farms lamb FEC increased during the summer, reaching a peak in the autumn. The work also highlights how the ad hoc use of anthelmintics does little to impact these patterns.

Evaluation of lameness detection using radar sensing in ruminants.

BACKGROUND: Lameness is a major health, welfare and production-limiting condition for the livestock industries. The current 'gold-standard' method of assessing lameness by visual locomotion scoring is subjective and time consuming, whereas recent technological advancements have enabled the development of alternative and more objective methods for its detection. METHODS: This study evaluated a novel lameness detection method using micro-Doppler radar signatures to categorise animals as lame or non-lame. Animals were visually scored by veterinarian and radar data were collected for the same animals. RESULTS: A machine learning algorithm was developed to interpret the radar signatures and provide automatic classification of the animals. Using veterinary scoring as a standard method, the classification by radar signature provided 85 per cent sensitivity and 81 per cent specificity for cattle and 96 per cent sensitivity and 94 per cent specificity for sheep. CONCLUSION: This radar sensing method shows promise for the development of a highly functional, rapid and reliable recognition tool of lame animals, which could be integrated into automatic, on-farm systems for sheep and cattle.

Performance of the Psoroptes ovis antibody enzyme-linked immunosorbent assay in the face of low-level mite infestation.

Psoroptes ovis mites, the causative agent of sheep scab, can severely compromise sheep welfare and production. However, in subclinical infections, mite detection is difficult increasing the risk of spread. A recent serodiagnostic test, based on detecting host antibodies to the P ovis allergen, Pso o 2, has made the detection of subclinical infection possible. The use of this test was demonstrated in subclinical situations, through an opportunistic observational study on an extensive hill farm and a lowland flock with recently introduced, quarantined livestock. Twelve animals were tested from each group. Breeding ewes and lambs on the hill farm had seroprevalences of 16 per cent (12.5-17.8 per cent) and 8.3 per cent (4.8-10.1 per cent), respectively. Quarantined store lambs had a seroprevalence of 16.7 per cent (13.2-18.5 per cent); no evidence of P ovis was found in quarantined replacement ewes. By detecting subclinical infection, this serological test could be a powerful tool in sheep scab control, for quarantine procedures, accreditation programmes, and possibly regional or national eradication protocols.

Boer goats appear to lack a functional IgA and eosinophil response against natural nematode infection.

Gastrointestinal nematode infection is one of the major diseases affecting small ruminants. Although some breeds of goats are quite resistant, many breeds of goats are relatively susceptible. This study used a combined parasitological, immunological, bioinformatic and statistical approach to examine the role of goat IgA and eosinophils in protection against Teladorsagia circumcincta. Molecular modelling suggested that the transmembrane domain of the high affinity IgA receptor was dysfunctional in goats. Statistical analyses failed to find any association in naturally infected goats between high IgA or eosinophil responses and low faecal egg counts. Together these results indicate that IgA and eosinophil responses against T. circumcincta are less effective in goats than sheep.

Black-pigmented anaerobic bacteria associated with ovine periodontitis.

Periodontitis is a polymicrobial infectious disease that causes occlusion change, tooth loss, difficulty in rumination, and premature culling of animals. This study aimed to detect species of the genera Porphyromonas and Prevotella present in the periodontal pocket of sheep with lesions deeper than 5mm (n=14) and in the gingival sulcus of animals considered periodontally healthy (n=20). The presence of microorganisms was evaluated by polymerase chain reaction (PCR) using specific primers for Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gulae, Prevotella buccae, Prevotella intermedia, Prevotella loescheii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, and Prevotella tannerae. Prevalence and risk analysis were performed using Student's t-test and Spearman's correlation. Among the Prevotella and Porphyromonas species detected in the periodontal lesions of sheep, P. melaninogenica (85.7%), P. buccae (64.3%), P. gingivalis (50%), and P. endodontalis (50%) were most prevalent. P. gingivalis (15%) and P. oralis (10%) prevailed in the gingival sulcus. P. gulae and P. tannerae were not detected in the 34 samples studied. Data evaluation by t-test verified that occurrence of P. asaccharolytica, P. endodontalis, P. gingivalis, P. buccae, P. intermedia, P. melalinogenica, and P. nigrescens correlated with sheep periodontitis. The findings of this study will be an important contribution to research on pathogenesis of sheep periodontitis and development of its control measures.

Opportunities and challenges for the application of microfluidic technologies in point-of-care veterinary diagnostics.

There is a growing need for low-cost, rapid and reliable diagnostic results in veterinary medicine. Point-of-care (POC) tests have tremendous advantages over existing laboratory-based tests, due to their intrinsic low-cost and rapidity. A considerable number of POC tests are presently available, mostly in dipstick or lateral flow formats, allowing cost-effective and decentralised diagnosis of a wide range of infectious diseases and public health related threats. Although, extremely useful, these tests come with some limitations. Recent advances in the field of microfluidics have brought about new and exciting opportunities for human health diagnostics, and there is now great potential for these new technologies to be applied in the field of veterinary diagnostics. This review appraises currently available POC tests in veterinary medicine, taking into consideration their usefulness and limitations, whilst exploring possible applications for new and emerging technologies, in order to widen and improve the range of POC tests available.

Investigation and treatment of ovine psoroptic otoacariasis.

BACKGROUND: Psoroptic otoacariasis has been described worldwide and is caused by a mite morphologically indistinguishable from the sheep scab mite Psoroptes ovis. A single treatment of affected sheep with 200 µg/kg of injectable ivermectin is reported to be curative. CASE REPORT: Psoroptes mites were isolated following treatment with ivermectin, but treatment with moxidectin at 1 mg/kg caused complete cessation of clinical signs. Affected animals were seropositive to Pso o 2 antigen enzyme-linked immunosorbent assay (ELISA) and had serum haptoglobin concentrations that overlapped with those described for field infections of classical sheep scab. CONCLUSIONS AND CLINICAL IMPORTANCE: Psoroptic otoacariasis is not controlled by single treatments of injectable ivermectin but resolves after a single treatment with injectable moxidectin. Pso o 2 ELISA can detect infection with Psoroptes spp. mites but cannot distinguish between sheep scab and psoroptic otoacariasis.