Worms and welfare: Behavioural and physiological changes associated with gastrointestinal nematode parasitism in lambs.

Parasitism with gastrointestinal nematodes (GIN) is a worldwide issue impacting negatively on animal production, health, and welfare. Therefore, early diagnostic signs of parasitism are required to allow for timely interventions. The objective of this study was to evaluate the behavioural and physiological changes in lambs associated with GIN infection. We used 30, 8-month-old Romney-cross wethers, that were administered anthelmintics until faecal egg counts (FEC) were zero and housed in an indoor facility. The study lasted 9 weeks, which comprised a 3-week pre-treatment, and a 6-week treatment phase. Lambs were randomly assigned to one of two treatments (n = 15/treatment) trickle-dosed with: 1) 1500 infective third stage larvae (L3) three days/week for 6 weeks (27,000 total L3; challenged), or 2) water 3 days/week for 6 weeks (control). Within each pen there were 5 pairs of lambs (balanced for liveweight), with each pair comprising a challenged and control lamb. Blood, faecal, and saliva samples were collected 1 week pre-treatment and weekly for 6 weeks of treatment. Behaviour was observed (e.g., feeding, lying, standing) from video-camera recordings using scan sampling every 5 min for 8 h, 1 day pre-treatment and on the day immediately prior to physiological sampling across the 6-week treatment phase (7 days in total). Accelerometers were attached to each lamb to continuously monitor behaviour from 3 weeks pre-treatment and for the remainder of the study. Liveweight, body condition, faecal soiling and faecal consistency scoring were performed weekly as was lipidomic analysis of plasma samples. From week 2 of treatment, challenged lambs spent less time feeding and more time lying than control lambs until week 5 of treatment (P ≤ 0.01). At week 3 of treatment, elevated lipids (mainly triglycerides and phospholipids), loose faeces and faecal soiling around the anus were observed in challenged lambs compared with controls (P ≤ 0.05). From week 4 of treatment, FEC were elevated in the challenged compared to control lambs (P ≤ 0.05). There was also lower liveweight gain at 4 and 5 weeks of treatment in the challenged lambs compared with control lambs (P ≤ 0.05). These results show a clear timeline of changes in behaviour (e.g., feeding and lying), lipids such as triglycerides, and digestive function (e.g., faecal soiling) suggestive of GIN subclinical disease, which show promise for use in future studies on early identification of subclinical GIN parasitism in lambs.

Evaluation of the accuracy of the IDvet serological test for Mycoplasma bovis infection in cattle using latent class analysis of paired serum ELISA and quantitative real-time PCR on tonsillar swabs sampled at slaughter.

Mycoplasma bovis (Mbovis) was first detected in cattle in New Zealand (NZ) in July 2017. To prevent further spread, NZ launched a world-first National Eradication Programme in May 2018. Existing diagnostic tests for Mbovis have been applied in countries where Mbovis is endemic, for detecting infection following outbreaks of clinical disease. Diagnostic test evaluation (DTE) under NZ conditions was thus required to inform the Programme. We used Bayesian Latent Class Analysis on paired serum ELISA (ID Screen Mycoplasma bovis Indirect from IDvet) and tonsillar swabs (qPCR) for DTE in the absence of a gold standard. Tested samples were collected at slaughter between June 2018 and November 2019, from infected herds depopulated by the Programme. A first set of models evaluated the detection of active infection, i.e. the presence of Mbovis in the host. At a modified serology positivity threshold of SP%> = 90, estimates of animal-level ELISA sensitivity was 72.8% (95% credible interval 68.5%-77.4%), respectively 97.7% (95% credible interval 97.3%-98.1%) for specificity, while the qPCR sensitivity was 45.2% (95% credible interval 41.0%-49.8%), respectively 99.6% (95% credible interval 99.4%-99.8%) for specificity. In a second set of models, prior information about ELISA specificity was obtained from the National Beef Cattle Surveillance Programme, a population theoretically free-or very low prevalence-of Mbovis. These analyses aimed to evaluate the accuracy of the ELISA test targeting prior exposure to Mbovis, rather than active infection. The specificity of the ELISA for detecting exposure to Mbovis was 99.9% (95% credible interval 99.7%-100.0%), hence near perfect at the threshold SP%=90. This specificity estimate, considerably higher than in the first set of models, was equivalent to the manufacturer's estimate. The corresponding ELISA sensitivity estimate was 66.0% (95% credible interval 62.7%-70.7%). These results confirm that the IDvet ELISA test is an appropriate tool for determining exposure and infection status of herds, both to delimit and confirm the absence of Mbovis.

Environmental, Occupational, and Demographic Risk Factors for Clinical Scrub Typhus, Bhutan.

Underdiagnosis and underreporting of scrub typhus has increasingly affected public health in Bhutan since its initial detection in 2008. Identifying scrub typhus risk factors would support early diagnosis and treatment for this nonspecific febrile disease, reducing the incidence of potentially fatal complications. We conducted a hospital-based, case‒control study during October‒December 2015 in 11 scrub typhus‒prone districts. We identified harvesting cardamom as the major risk factor (odds ratio 1,519; p<0.001); other factors were traditional housing, largely caused by an outside toilet location, as well as owning a goat and frequently sitting on grass. Harvesting vegetables, herding cattle in the forest, and female sex were protective. Age had a nonlinear effect; children and the elderly were more likely to seek treatment for clinical scrub typhus. This study has informed public health policies and awareness programs for healthcare workers through development of National Guidelines for Prevention, Treatment and Control of Scrub Typhus in Bhutan.

What Are Sheep Doing? Tri-Axial Accelerometer Sensor Data Identify the Diel Activity Pattern of Ewe Lambs on Pasture.

Monitoring activity patterns of animals offers the opportunity to assess individual health and welfare in support of precision livestock farming. The purpose of this study was to use a triaxial accelerometer sensor to determine the diel activity of sheep on pasture. Six Perendale ewe lambs, each fitted with a neck collar mounting a triaxial accelerometer, were filmed during targeted periods of sheep activities: grazing, lying, walking, and standing. The corresponding acceleration data were fitted using a Random Forest algorithm to classify activity (=classifier). This classifier was then applied to accelerometer data from an additional 10 ewe lambs to determine their activity budgets. Each of these was fitted with a neck collar mounting an accelerometer as well as two additional accelerometers placed on a head halter and a body harness over the shoulders of the animal. These were monitored continuously for three days. A classification accuracy of 89.6% was achieved for the grazing, walking and resting activities (i.e., a new class combining lying and standing activity). Triaxial accelerometer data showed that sheep spent 64% (95% CI 55% to 74%) of daylight time grazing, with grazing at night reduced to 14% (95% CI 8% to 20%). Similar activity budgets were achieved from the halter mounted sensors, but not those on a body harness. These results are consistent with previous studies directly observing daily activity of pasture-based sheep and can be applied in a variety of contexts to investigate animal health and welfare metrics e.g., to better understand the impact that young sheep can suffer when carrying even modest burdens of parasitic nematodes.

Gastrointestinal nematode infection affects overall activity in young sheep monitored with tri-axial accelerometers.

Animals suffering from parasitism typically display altered grazing behaviour and a voluntary reduction in feed intake. These changes are potentially important as indicators of disease. Recent advances in sensor technologies provide the opportunity to objectively measure animal activity while on pasture. Tri-axial accelerometers measure body movement in terms of acceleration, which can then be used to estimate physical activity over time. This study investigated if tri-axial measures of overall activity can be used to assess the impact of gastrointestinal nematode (GIN) infection in young sheep. To address this, the overall activity, faecal nematode egg count (FEC) and body weight of two treatment groups of Romney X Suffolk ram lambs were compared. Animals were monitored for four days using tri-axial accelerometer sensors mounted on a ram mating harness after 42-days grazing on contaminated pasture. On Day 0, all lambs were given anthelmintics. Subsequently, a Suppressive Treatment Group (n = 12) was treated with anthelmintics every two weeks. An Untreated Group (n = 12) did not receive further anthelmintics. Overall activity levels were monitored from Day 42 - 46. Activity level was calculated as vectorial dynamic body acceleration (VeDBA). Anthelmintic treatment had a significant effect on FEC but there was no evidence found for a treatment effect on body weight growth over the 42-day period. An effect of treatment and lamb starting weight on overall activity was found (beta = -0.74, 95 % CI -1.17 to -0.30, p =  0.002), identifying a negative impact of parasitism on activity in heavier animals. These results highlight the usefulness of this approach in assessing the effect of GIN parasitism on sheep monitored remotely. If a threshold value of activity could be determined, it could provide a useful tool for farmers and managers that serves as an early indicator of parasitism in sheep.

Evaluation of post-exposure prophylaxis practices to improve the cost-effectiveness of rabies control in human cases potentially exposed to rabies in southern Bhutan.

BACKGROUND: Rabies is endemic in southern Bhutan, associated with 1-2 human deaths and high post exposure prophylaxis (PEP) costs annually. Evaluation of clinicians' management of human cases potentially exposed to rabies could contribute to improving PEP prescribing practices to both reduce unnecessary costs associated with PEP and reach the target of zero human deaths due to rabies by 2023. METHODS: A cross-sectional survey of 50 clinicians' management of human cases potentially exposed to rabies was conducted in 13 health centers in high-rabies-risk areas of Bhutan during February-March 2016. RESULTS: Data were collected on clinicians' management of 273 human cases potentially exposed to rabies. The 50 clinicians comprised health assistants or clinical officers (55%) and medical doctors (45%) with a respective median of 19, 21 and 2 years' experience. There was poor agreement between clinicians' rabies risk assessment compared with an independent assessment for each case based on criteria in the National Rabies Management Guidelines (NRMG). Of the 194 cases for which clinicians recorded a rabies risk category, only 53% were correctly classified when compared with the NRMG. Clinicians were more likely to underestimate the risk of exposure to rabies and appeared to prescribe PEP independently of their risk classification.. Male health assistants performed the most accurate risk assessments while female health assistants performed the least accurate. Clinicians in Basic Health Units performed less accurate risk assessments compared with those in hospitals. CONCLUSIONS: This study highlights important discrepancies between clinicians' management of human cases potentially exposed to rabies and recommendations in the NRMG. In particular, clinicians were not accurately assessing rabies risk in potentially exposed cases and were not basing their PEP treatment on the basis of their risk assessment. This has significant implications for achieving the national goal of eliminating dog-mediated human rabies by 2030 and may result in unnecessary costs associated with PEP. Recommendations to improve clinician's management of human cases potentially exposed to rabies include: reviewing and updating the NRMG, providing clinicians with regular and appropriately targeted training about rabies risk assessment and PEP prescription, and regularly reviewing clinicians' practices.

Clinical and Epidemiological Patterns of Scrub Typhus, an Emerging Disease in Bhutan.

Scrub typhus (ST) is a vector-borne rickettsial infection causing acute febrile illness. The re-emergence of ST in the Asia-Pacific region represents a serious public health threat. ST was first detected in Bhutan in 2008. However, the disease is likely to be under-diagnosed and under-reported, and the true impact is difficult to estimate. At the end of 2014, the SD Bioline Tsutsugamushi TestTM rapid diagnostic test (RDT) kits became available in all hospitals to assist clinicians in diagnosing ST. We conducted a retrospective descriptive study, reviewing records from all hospitals of Bhutan to identify all RDT-positive clinical cases of ST in Bhutan in 2015. The aim was to evaluate the burden of ST in Bhutan, describe the demographic, spatial and temporal patterns of disease, and identify the typical clinical presentations. The annual incidence of RDT-positive cases of ST reporting to Bhutanese hospitals in 2015 was estimated to be 62 per 100,000 population at risk. The incidence of disease was highest in the southern districts with a subtropical climate and a high level of agricultural production. The highest proportion of cases (87%) was rural residents, with farmers being the main occupational category. The disease was strongly seasonal, with 97% of cases occurring between June and November, coinciding with the monsoon and agricultural production seasons. Common ST symptoms were not specific, and an eschar was noted by clinicians in only 7.4% of cases, which is likely to contribute to an under-diagnosis of ST. ST represents an important and neglected burden, especially in rural communities in Bhutan. The outcomes of this study will inform public health measures such as timely-awareness programmes for clinicians and the public in high-risk areas, to improve the diagnosis, treatment and clinical outcomes of this disease.

What counts? A review of in vitro methods for the enumeration of Mycobacterium avium subsp. paratuberculosis.

Enumeration of Mycobacterium avium subsp. paratuberculosis (MAP) from clinical samples or from inoculum suspensions for challenge studies is an essential pre-requisite for paratuberculosis research. However, MAP organisms are slow and challenging to grow in vitro, with a strong tendency to clump. Standard bacteriological methods are not well suited to enumerate MAP. Enumeration can be achieved by culture-based methods, direct microscopic counts, turbidimetry, pelleted weights and quantitative PCR. There is an unresolvable discrepancy between culture-based methods, enumerating cultivable MAP organisms, and other methods enumerating total MAP organisms irrespective of viability, hence these methods are not directly comparable. However, they are complementary and should be used in parallel when accuracy is required. We review currently available methods, their principles, limitations and equivalence between methods.

A synthesis of the patho-physiology of Mycobacterium avium subspecies paratuberculosis infection in sheep to inform mathematical modelling of ovine paratuberculosis.

This literature review of exposure to Mycobacterium avium subsp. paratuberculosis (MAP) in sheep enabled a synthesis of the patho-physiology of ovine paratuberculosis (PTB). These results could be used to inform subsequent modelling of ovine PTB. We reviewed studies of both experimental and natural exposure. They were generally comparable. Possible outcomes following exposure were latent infection, i.e. mere colonization without lesions; active infection, with inflammatory histopathology in the intestinal tissues resulting in mild disease and low faecal shedding; and affection, with severe intestinal pathology, reduced production, clinical signs and high faecal shedding. Latent infection was an uninformative outcome for modelling. By contrast, histological lesions and their grade appeared to be a good marker of active infection and progression stages to clinical disease. The two possible pathways following infection are non-progression leading to recovery and progression to clinical disease, causing death. These pathways are mediated by different immune mechanisms. This synthesis suggested that host-related characteristics such as age at exposure and breed, combined with pathogen-related factors such as MAP dose, strain and inoculum type for experimental infection, have a strong influence on the outcome of exposure. The material reviewed consisted of disparate studies often with low numbers of sheep and study-level confounders. Hence comparisons between and across studies was difficult and this precluded quantitative model parameter estimation. Nevertheless, it allowed a robust synthesis of the current understanding of patho-physiology of ovine PTB, which can inform mathematical modelling of this disease.

Using social network analysis to inform disease control interventions.

Contact patterns between individuals are an important determinant for the spread of infectious diseases in populations. Social network analysis (SNA) describes contact patterns and thus indicates how infectious pathogens may be transmitted. Here we explore network characteristics that may inform the development of disease control programes. This study applies SNA methods to describe a livestock movement network of 180 farms in New Zealand from 2006 to 2010. We found that the number of contacts was overall consistent from year to year, while the choice of trading partners tended to vary. This livestock movement network illustrated how a small number of farms central to the network could play a potentially dominant role for the spread of infection in this population. However, fragmentation of the network could easily be achieved by "removing" a small proportion of farms serving as bridges between otherwise isolated clusters, thus decreasing the probability of large epidemics. This is the first example of a comprehensive analysis of pastoral livestock movements in New Zealand. We conclude that, for our system, recording and exploiting livestock movements can contribute towards risk-based control strategies to prevent and monitor the introduction and the spread of infectious diseases in animal populations.