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Abstract Background Enzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit) for use with species for which it was not originally developed: elk (Cervus elaphus), bison (Bison bison) and caribou (Rangifer tarandus). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species. Results We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively. Conclusions Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were developed. In order to generate reliable test results, it is essential to evaluate the test and perform modifications if deemed necessary. Despite the challenges inherent to wildlife diagnostics, we have shown that several methods can be used to improve confidence in test results.

Assessing wildlife health in remote regions requires a multi-faceted approach, which commonly involves convenient samplings and the need of identifying and targeting relevant and informative indicators. We applied a novel wildlife health framework and critically assessed the value of different indicators for understanding the health status and trends of an endangered tundra caribou population. Samples and data from the Dolphin and Union caribou herd were obtained between 2015 and 2021, from community-based surveillance programs and from captured animals. We documented and categorized indicators into health determinants (infectious diseases and trace elements), processes (cortisol, pathology), and health outcomes (pregnancy and body condition). During a recent period of steep population decline, our results indicated a relatively good body condition and pregnancy rates, and decreasing levels of stress, along with a low adult cow survival. We detected multiple factors as potential contributors to the reduced survival, including Brucella suis biovar 4, Erysipelothrix rhusiopathiae and lower hair trace minerals. These results remark the need of targeted studies to improve detection and investigations on caribou mortalities. We also identified differences in health indicators between captured and hunter sampled caribou, highlighting the importance of accounting for sampling biases. This integrative approach that drew on multiple data sources has provided unprecedented knowledge on the health in this herd and highlights the value of documenting individual animal health to understand causes of wildlife declines., (© 2023. Springer Nature Limited.)

Competing Interests: Competing interests: None declared.

Wild animal trade for human consumption is a global issue, involving complex interactions between economics, culture, food security and conservation. Whilst being a biodiversity issue, it is also a major public health concern, with recent epidemics and pandemics of zoonotic pathogens linked to interactions with wildlife. At three time points, between March 2017 and June 2018, a longitudinal sero-survey of 150 market vendors from three wet markets in Laos (selling vegetables, domestic animal meat and/or wildlife meat) was conducted to determine if vendors had been differentially exposed to three endemic bacterial pathogens - Orientia tsutsugamushi, Rickettsia typhi , and Leptospira spp. A total of 367 serum samples were tested by IgG enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA, for scrub typhus group (STG) and typhus group (TG) only). Among vendors, 32.7% were IgG-positive for at least one pathogen, 13.3% sero-converted during the study. Multi-season occupancy modelling for STG indicated a significantly higher prevalence of STG IgG in vegetable vendors (27.3%) and wildlife vendors (28.4%) than in domestic animal meat vendors (6.9 %, p=0.05), and higher in Phonsavanh market (OR=9.6, p=0.03) compared to Lak Sao and Salavan markets. Estimated mean incidence was 57 cases per 10,000 per 7.5-month period. For TG, vendor age had a significant effect on prevalence (OR=1.04, p=0.006), estimated mean incidence was 64 cases per 10,000 per season (7.5-month period). Despite individuals selling domestic meat having a higher prevalence of Leptospira infections than those that did not (11.6% versus 4.5%), the difference was not significant. Whilst this study has a number of limitations, including vendors changing what food types they sold and no investigation of exposure outside of markets, the finding that the risk of exposure of vendors to zoonotic pathogens may be associated with types of food sold for human consumption warrants further investigation., Competing Interests: Declarations of interest: none

Wildlife and wildlife interfaces with people and livestock are essential surveillance targets to monitor emergent or endemic pathogens or new threats affecting wildlife, livestock, and human health. However, limitations of previous investments in scope and duration have resulted in a neglect of wildlife health surveillance (WHS) systems at national and global scales, particularly in lower and middle income countries (LMICs). Building on decades of wildlife health activities in LMICs, we demonstrate the implementation of a locally-driven multi-pronged One Health approach to establishing WHS in Cambodia, Lao PDR and Viet Nam under the WildHealthNet initiative. WildHealthNet utilizes existing local capacity in the animal, public health, and environmental sectors for event based or targeted surveillance and disease detection. To scale up surveillance systems to the national level, WildHealthNet relies on iterative field implementation and policy development, capacity bridging, improving data collection and management systems, and implementing context specific responses to wildlife health intelligence. National WHS systems piloted in Cambodia, Lao PDR, and Viet Nam engaged protected area rangers, wildlife rescue centers, community members, and livestock and human health sector staff and laboratories. Surveillance activities detected outbreaks of H5N1 highly pathogenic avian influenza in wild birds, African swine fever in wild boar (Sus scrofa), Lumpy skin disease in banteng (Bos javanicus), and other endemic zoonotic pathogens identified as surveillance priorities by local stakeholders. In Cambodia and Lao PDR, national plans for wildlife disease surveillance are being signed into legislation. Cross-sectoral and trans-disciplinary approaches are needed to implement effective WHS systems. Long-term commitment, and paralleled implementation and policy development are key to sustainable WHS networks. WildHealthNet offers a roadmap to aid in the development of locally-relevant and locally-led WHS systems that support the global objectives of the World Organization for Animal Health's Wildlife Health Framework and other international agendas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The role and impact of infectious diseases in wildlife population dynamics are increasingly recognized, yet disease information is variably incorporated into wildlife management frameworks. This discrepancy is particularly relevant for Rangifer tarandus (caribou or reindeer), a keystone circumarctic species experiencing widespread population declines. The primary objective of this review was to characterize the available peer-reviewed literature on infectious diseases of Rangifer by using a scoping review methodology. Three databases of peer-reviewed literature-Web of Science, BIOSIS previews, and Scopus-were searched and 695 articles met the criteria for initial review. After screening for relevance and language, 349 articles, published between 1967 and 2020, remained. More than half of the excluded articles (181/346; 52%) were left out because they were not published in English; the majority of these excluded articles (120) were in Russian. From the 349 included articles, 137 (39%) pertained to wild (as opposed to semidomesticated or captive) Rangifer populations. Articles on infectious disease in wild Rangifer were published in 40 different journals across various disciplines; the most common journals were disease and parasitology oriented, accounting for 55% of included articles. Most studies were descriptive (87%), followed by experimental (9%). Of the pathogen taxa investigated, helminths were the most common, comprising 35% of articles. Rangifer subspecies were not equally represented in the literature, with barren-ground caribou (R. t. groenlandicus; n=40) and woodland caribou (R. t. caribou; n=39) having the greatest abundance and diversity of infectious disease information available. Few studies explicitly examined individual or population-level impacts of disease, or related disease to vital population rates, and only 27 articles explicitly related results to management or conservation. Findings from this review highlight an unbalanced distribution of studies across Rangifer ecotypes, a preference for dissemination in disease-specialized publication venues, and an opportunity for investigating population-level impacts that may be more readily integrated into caribou conservation frameworks., (© Wildlife Disease Association 2022.)

We tested animals from wildlife trade sites in Laos for the presence of zoonotic pathogens. Leptospira spp. were the most frequently detected infectious agents, found in 20.1% of animals. Rickettsia typhi and R. felis were also detected. These findings suggest a substantial risk for exposure through handling and consumption of wild animal meat.

African Swine Fever (ASF) is a highly contagious and fatal viral disease affecting both domestic and wild suids. The virus was introduced to Southeast Asia in early 2019 and has since spread rapidly throughout the region. Although significant efforts have been made to track and diagnose the disease in domestic pigs, very little is known about ASF in free-ranging wild boar and their potential role in maintaining the disease within Southeast Asia. Through a collaboration between government and non-government actors in Laos, Viet Nam, and Cambodia, investigations were conducted to (a) characterize the interface between domestic pigs and wild boar, (b) document risk factors for likely ASF spillover into wild boar populations by way of this interface, and (c) determine whether ASF in wild boar could be detected in each country. An extensive overlap between wild boar habitat and domestic pig ranging areas was found around villages bordering forests in all three countries, creating a high-risk interface for viral spillover between domestic pig and wild boar populations. Fifteen and three wild boar carcasses were detected through passive reporting in Laos and Viet Nam, respectively, in 2019 and early 2020. Four of five carcasses screened in Laos and two of three in Viet Nam were confirmed positive for African swine fever virus using real-time PCR. There were no confirmed reports of wild boar carcasses in Cambodia. This is the first confirmation of ASF in wild boar in Southeast Asia, the result of a probable viral spillover from domestic pigs, which highlights the importance of early reporting and monitoring of ASF in wild boar to enable the implementation of appropriate biosecurity measures., (© 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

The COVID-19 pandemic has re-focused attention on mechanisms that lead to zoonotic disease spillover and spread. Commercial wildlife trade, and associated markets, are recognized mechanisms for zoonotic disease emergence, resulting in a growing global conversation around reducing human disease risks from spillover associated with hunting, trade, and consumption of wild animals. These discussions are especially relevant to people who rely on harvesting wildlife to meet nutritional, and cultural needs, including those in Arctic and boreal regions. Global policies around wildlife use and trade can impact food sovereignty and security, especially of Indigenous Peoples. We reviewed known zoonotic pathogens and current risks of transmission from wildlife (including fish) to humans in North American Arctic and boreal biomes, and evaluated the epidemic and pandemic potential of these zoonoses. We discuss future concerns, and consider monitoring and mitigation measures in these changing socio-ecological systems. While multiple zoonotic pathogens circulate in these systems, risks to humans are mostly limited to individual illness or local community outbreaks. These regions are relatively remote, subject to very cold temperatures, have relatively low wildlife, domestic animal, and pathogen diversity, and in many cases low density, including of humans. Hence, favorable conditions for emergence of novel diseases or major amplification of a spillover event are currently not present. The greatest risk to northern communities from pathogens of pandemic potential is via introduction with humans visiting from other areas. However, Arctic and boreal ecosystems are undergoing rapid changes through climate warming, habitat encroachment, and development; all of which can change host and pathogen relationships, thereby affecting the probability of the emergence of new (and re-emergence of old) zoonoses. Indigenous leadership and engagement in disease monitoring, prevention and response, is vital from the outset, and would increase the success of such efforts, as well as ensure the protection of Indigenous rights as outlined in the United Nations Declaration on the Rights of Indigenous Peoples. Partnering with northern communities and including Indigenous Knowledge Systems would improve the timeliness, and likelihood, of detecting emerging zoonotic risks, and contextualize risk assessments to the unique human-wildlife relationships present in northern biomes., Competing Interests: DJ was employed by the company Nyati Health Consulting, British Columbia, Canada. DL was employed by Nunavut Tunngavik Inc., Ottawa, Canada. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AP declared a past co-authorship with one of the authors SO to the handling Editor., (Copyright © 2021 Keatts, Robards, Olson, Hueffer, Insley, Joly, Kutz, Lee, Chetkiewicz, Lair, Preston, Pruvot, Ray, Reid, Sleeman, Stimmelmayr, Stephen and Walzer.)

This study investigates the occurrence of erythematous lip lesions in a captive sun bear population in Cambodia, including the progression of cheilitis to squamous cell carcinoma, and the presence of Ursid gammaherpesvirus 1. Visual assessment conducted in 2015 and 2016 recorded the prevalence and severity of lesions. Opportunistic sampling for disease testing was conducted on a subset of 39 sun bears, with histopathological examination of lip and tongue biopsies and PCR testing of oral swabs and tissue biopsies collected during health examinations. Lip lesions were similarly prevalent in 2015 (66.0%) and 2016 (68.3%). Degradation of lip lesion severity was seen between 2015 and 2016, and the odds of having lip lesions, having more severe lip lesions, and having lip lesion degradation over time, all increased with age. Cheilitis was found in all lip lesion biopsies, with histological confirmation of squamous cell carcinoma in 64.5% of cases. Single biopsies frequently showed progression from dysplasia to neoplasia. Eighteen of 31 sun bears (58.1%) had at least one sample positive for Ursid gammaherpesvirus 1. The virus was detected in sun bears with and without lip lesions, however due to case selection being strongly biased towards those showing lip lesions it was not possible to test for association between Ursid gammaherpesvirus 1 and lip squamous cell carcinoma. Given gammaherpesviruses can play a role in cancer development under certain conditions in other species, we believe further investigation into Ursid gammaherpesvirus 1 as one of a number of possible co-factors in the progression of lip lesions to squamous cell carcinoma is warranted. This study highlights the progressively neoplastic nature of this lip lesion syndrome in sun bears which has consequences for captive and re-release management. Similarly, the detection of Ursid gammaherpesvirus 1 should be considered in pre-release risk analyses, at least until data is available on the prevalence of the virus in wild sun bears., Competing Interests: The authors have declared that no competing interests exist.

Wet markets are a critical part of South-East Asian culture and economy. However, their role in circulation and transmission of both endemic and emerging disease is a source of concern in a region considered a hotspot of disease emergence. In the Lao People's Democratic Republic (Lao PDR, Laos), live and dead wild animals are frequently found in wet markets, despite legislation against the bushmeat trade. This is generally considered to increase the risk of disease transmission and emergence, although whether or not wildlife vendors themselves have indeed increased incidence of zoonotic disease has rarely been assessed. In preparation for a future longitudinal study of market vendors investigating vendors' exposure to zoonotic pathogens, we conducted a pilot survey of Lao market vendors of wildlife meat, livestock meat and vegetables, to identify demographic characteristics and potential control groups within markets. We also investigated baseline risk perception for infectious diseases among market vendors and assessed the association between risk perception and risk mitigation behaviours. The surveys conducted with 177 vendors revealed similar age, sex, ethnic background and geographical origin between vendor types, but differences in professional background and work history for livestock meat vendors. The perception of disease risk was very low across all vendors, as was the reported use of personal protective equipment, and the two appeared unrelated. Personal risk discounting and assumptions about transmission routes may explain this lack of association. This information will help inform the development of future research, risk communication and risk mitigation policy, especially in the light of the COVID-19 pandemic., (© 2020 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (SARS-CoV-2) that emerged in China highlight the importance of this viral family as a zoonotic public health threat. To gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in Viet Nam 2013-2014, we used consensus Polymerase Chain Reactions to detect coronavirus sequences. In comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. Most notably among field rats, the odds of coronavirus RNA detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). Coronaviruses were also detected in rodents on the majority of wildlife farms sampled (60.7%, 17/28). These coronaviruses were found in the Malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are raised on wildlife farms for human consumption as food. We identified six known coronaviruses in bats and rodents, clustered in three Coronaviridae genera, including the Alpha-, Beta-, and Gammacoronaviruses. Our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces on wildlife farms. The mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people., Competing Interests: The authors have declared that no competing interests exist.

Growing evidence suggests that multiple wildlife species can be infected with peste des petits ruminants virus (PPRV), with important consequences for the potential maintenance of PPRV in communities of susceptible hosts, and the threat that PPRV may pose to the conservation of wildlife populations and resilience of ecosystems. Significant knowledge gaps in the epidemiology of PPRV across the ruminant community (wildlife and domestic), and the understanding of infection in wildlife and other atypical host species groups (e.g., camelidae, suidae, and bovinae) hinder our ability to apply necessary integrated disease control and management interventions at the wildlife-livestock interface. Similarly, knowledge gaps limit the inclusion of wildlife in the FAO/OIE Global Strategy for the Control and Eradication of PPR, and the framework of activities in the PPR Global Eradication Programme that lays the foundation for eradicating PPR through national and regional efforts. This article reports on the first international meeting on, "Controlling PPR at the livestock-wildlife interface," held in Rome, Italy, March 27-29, 2019. A large group representing national and international institutions discussed recent advances in our understanding of PPRV in wildlife, identified knowledge gaps and research priorities, and formulated recommendations. The need for a better understanding of PPRV epidemiology at the wildlife-livestock interface to support the integration of wildlife into PPR eradication efforts was highlighted by meeting participants along with the reminder that PPR eradication and wildlife conservation need not be viewed as competing priorities, but instead constitute two requisites of healthy socio-ecological systems., (Copyright © 2020 Fine, Pruvot, Benfield, Caron, Cattoli, Chardonnet, Dioli, Dulu, Gilbert, Kock, Lubroth, Mariner, Ostrowski, Parida, Fereidouni, Shiilegdamba, Sleeman, Schulz, Soula, Van der Stede, Tekola, Walzer, Zuther, Njeumi and Meeting Participants.)

Overlap of cattle and wild elk ranges in southwestern Alberta foothills is an opportunity for inter-species interactions. To assess the spatio-temporal patterns of disease transmission risk between cattle and elk, several risk indexes were defined to represent different transmission routes. Risk indexes were estimated by combining elk telemetry data obtained from 168 GPS-collared elk, and cattle management information obtained by interviews conducted in 16 cow-calf operations overlapping the elk home range. We assessed the bias resulting from ignoring cattle movement related to seasonnal grazing practices, and the impact of the assessment of spatio-temporal patterns of risk. Direct transmission risk indexes peaked during winter months, due to aggregation at higher densities of both elk and cattle on winter ranges and winter pastures, respectively. However, a summer peak also was observed when risk indexes were not adjusted for pasture area, due to larger cattle summer pastures overlapping a higher number of elk telemetry locations. We identified periods when the proximity of elk to specific features (such as mineral blocks, hay land, winter-feeding areas, or water sources) may increase the risk of inter-species transmission. Indirect transmission risk indexes increased with the survival of pathogens in the environment, as the temporal constraint for cattle and elk overlap decreased. Finally, integrating pasture management information substantially influenced the magnitude and temporal patterns of transmission risk indexes, highlighting the importance of collecting detailed livestock management data in the context of assessing the risk of inter-species disease transmission., (Copyright © 2019. Published by Elsevier B.V.)

The 2016-2017 introduction of peste des petits ruminants virus (PPRV) into livestock in Mongolia was followed by mass mortality of the critically endangered Mongolian saiga antelope and other rare wild ungulates. To assess the nature and population effects of this outbreak among wild ungulates, we collected clinical, histopathologic, epidemiologic, and ecological evidence. Molecular characterization confirmed that the causative agent was PPRV lineage IV. The spatiotemporal patterns of cases among wildlife were similar to those among livestock affected by the PPRV outbreak, suggesting spillover of virus from livestock at multiple locations and time points and subsequent spread among wild ungulates. Estimates of saiga abundance suggested a population decline of 80%, raising substantial concerns for the species' survival. Consideration of the entire ungulate community (wild and domestic) is essential for elucidating the epidemiology of PPRV in Mongolia, addressing the threats to wild ungulate conservation, and achieving global PPRV eradication.

Trade of bushmeat and other wildlife for human consumption presents a unique set of challenges to policy-makers who are confronted with multiple trade-offs between conservation, food security, food safety, culture and tradition. In the face of these complex issues, risk assessments supported by quantitative information would facilitate evidence-based decision making. We propose a conceptual model for disease transmission risk analysis, inclusive of these multiple other facets. To quantify several processes included in this conceptual model we conducted questionnaire surveys with wildlife consumers and vendors in semi-urban centers in Lao People's Democratic Republic (Lao PDR, Laos) and direct observations of consumer behaviors. Direct observation of market stalls indicated an estimated average of 10 kg bushmeat biomass per stall per hour. The socio-demographic data suggested that consumption of bushmeat in urban areas was not for subsistence but rather driven by dietary preference and tradition. Consumer behavioral observations indicated that each animal receives an average of 7 contacts per hour. We provide other key parameters to estimate the risk of disease transmission from bushmeat consumption and illustrate their use in assessing the total public health and socio-economic impact of bushmeat consumption. Pursuing integrative approaches to the study of bushmeat consumption is essential to develop effective and balanced policies that support conservation, public health, and rural development goals., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

A mass mortality event involving Chaerephon plicatus and Taphozous theobaldi bats occurred during a heat wave in April 2016 in Cambodia. This was investigated to clarify the causes of the die-off and assess the risk to public health. Field evidences, clinical signs, and gross pathology findings were consistent with a heat stress hypothesis. However, the detection of a novel bat paramyxovirus raises questions about its role as a contributing factor or a coincidental finding. Systematic documentation of bat die-offs related to extreme weather events is necessary to improve understanding of the effect of changing weather patterns on bat populations and the ecosystem services they provide., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Springer-Verlag GmbH Germany, part of Springer Nature 2019.)

Southern River terrapins ( Batagur affinis) are among the most critically endangered turtles in the world. To augment the Cambodia population, a head-start program was established for the endemic subspecies Batagur affinis edwardmolli in 2006, and in 2015, prerelease health assessments were performed on 70 subadults (hatch years, 2006-2011). Combined choanal/cloacal swab samples ( n = 70) were collected and screened by polymerase chain reaction (PCR) for Mycoplasma, herpesvirus, and ranavirus. Cloacal samples ( n = 50) were also collected and cultured for Salmonella sp. Of 70 tested samples, six (8.6%) were positive for Mycoplasma, and all other PCR and culture test results were negative. Phylogenetic analysis of the 16S ribosomal RNA gene placed the Mycoplasma sp. from B. affinis edwardmolli in the chelonian Mycoplasma cluster that groups within the Mycoplasma pulmonis clade. This mollicute was not associated with clinical disease (defined as observable clinical abnormalities, such as depression, lethargy, respiratory signs, and anorexia) and is likely part of the endemic microbial flora of these terrapins.

Migratory movements and alteration of host communities through livestock production are examples of ecological processes that may have consequences on wildlife pathogens. We studied the effect of co-grazing of cattle and wild elk, and of elk migratory behaviour on the occurrence of the giant liver fluke, Fascioloides magna, in elk. Migratory elk and elk herds with a higher proportion of migratory individuals were significantly less likely to be infected with F. magna. This may indicate a decreased risk of infection for migratory individuals, known as the "migratory escape" hypothesis. Elk herds overlapping with higher cattle densities also had a lower prevalence of this parasite, even after adjustment for landscape and climate variables known to influence its life cycle. Serological evidence suggests that even in low-prevalence areas, F. magna is circulating in both elk and cattle. Cattle are "dead-end" hosts for F. magna, and this may, therefore, indicate a dilution effect where cattle and elk are co-grazing. Migratory behaviour and host community composition have significant effects on the dynamics of this wildlife parasite; emphasizing the potential impacts of decisions regarding the management of migratory corridors and livestock-wildlife interface.

Following an outbreak of Johne's disease on an elk farm in northern Alberta, Canada, fecal culture, fecal polymerase chain reaction (PCR), and serum enzyme-linked immunosorbent assay (ELISA) tests were performed on individual animals. The magnitude of the outbreak is described and the challenges associated with poor test agreement, as well as herd management options, are discussed.

Neospora caninum is an important abortive agent of domestic ruminants, but few diagnostic tools are available to reliably assess the exposure of wild cervid species such as elk (Cervus elaphus) to this pathogen, which limits our ability to understand their role in the life cycle of this parasite. In the absence of a gold standard test or panels of samples from individuals of known infection status, classical laboratory-based validation methods are not applicable. However, there are a number of statistical methods that can help in selecting an appropriate cut-off value and estimating the resulting diagnostic test performances. In this paper, the performance of a commercial competitive enzyme-linked immunosorbent assay (cELISA) on elk serum samples was evaluated with two statistical approaches: a mixture distribution model fitted to the cELISA results, and a Bayesian latent class analysis combining results from the cELISA and an indirect immuno-fluorescence antibody test. Both methods indicated that the commercial kit could be used on elk serum with the specifications recommended by the manufacturer. In particular, the optimal combination of sensitivity and specificity were obtained for a percentage of inhibition cutoff of 30%. The 95% probability interval of the proportion of elk exposed to N. caninum, adjusting for the sensitivity and specificity of this test in elk, was estimated between 1.3 and 7.4%. There was no association between the serological status of female elk and their pregnancy status. These results point out to the involvement of elk in a sylvatic cycle of N. caninum in this area.

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

For several years, the demand for pork has been on the rise in Nepal. To assess the importance of pork as a carrier of zoonotic agents, we performed a cross-sectional study in the Kathmandu Valley of Nepal, in which we serologically determined the infection status of slaughtered pigs with regard to three of the most important parasites transmitted through pork consumption: Trichinella spp., Taenia solium cysticerci, and Toxoplasma gondii. From 2007 to 2010, 742 pigs were sampled at slaughter, of which 0.1% (95% confidence interval [CI] 0.0-0.7%) were found positive for Trichinella infection, 13.8% (95% credibility interval [CrI] 0.8-28.5%) for T. solium cysticercosis, and 11.7% (95% CI 5.2-17.5%) for Toxoplasma infection. Further monitoring of the related animal and human disease burden and strengthening of food safety protocols throughout the pork production chain are strongly recommended.

Trypanosoma evansi is responsible for the most largely distributed animal trypanosomosis, affecting a wide range of wild and domestic animals. Its surveillance requires the implementation of standardized and reliable diagnostic tools. Although the development of polymerase chain reaction (PCR) tools has greatly improved our diagnostic capacity, factors affecting their sensitivity need to be acknowledged and accounted for in the interpretation of results. The targeted gene and the primer design have already been shown to greatly affect the sensitivity of a PCR, and the best-performing sets of primers have been previously identified. However, the sensitivity of the PCR is also largely influenced by the DNA extraction or sample preparation method. In this paper, we selected 6 DNA extraction or blood sample preparation methods representative of what would be used in a budget-constrained setting: phenol-chloroform, Chelex(®), Flexigen (Qiagen(®)) kit, Genekam(®) kit and two original protocols using sodium hydroxide. We studied the effects of the preparation method on the detection limit of the subsequent PCR. Our results show that the extraction method strongly affects the PCR sensitivity. The classical phenol-chloroform extraction method allowed for the PCR with the lowest detection limit. Some combinations of extraction method and primer set had detection limits that were not compatible with their use as a reliable diagnostic test, and would severely reduce the performance of a surveillance program. Therefore, we encourage laboratories to carefully select their sample preparation and PCR protocols, depending on the aimed sensitivity, cost, safety, time requirement and objectives., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

To face the worldwide threat of Surra caused by Trypanosoma evansi, international organizations have stressed the need to evaluate and standardize diagnostic tools. PCR detection of T. evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared. In this work, we compared the performances of six primer pairs-TBR1/2 (Masiga et al., 1992), ESAG6/7 (Holland et al., 2001a, b), TEPAN1/2 (Panyim et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples. TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitaemia below one parasite per ml in rat blood. They presented the highest sensitivity in cattle samples as well as a high specificity, without non-specific products nor false positive reactions out of 84 negative cattle samples tested. ESAG6/7 showed equivalent results with purified DNA and rat samples but presented non-specific products with Thai dairy cattle samples, leading to non interpretable results. TEPAN1/2 was not able to detect less than 0.1 pg of purified DNA or 50 trypanosomes/ml in rat blood. In cattle, TEPAN1/2 primers detected only 36% of the positives detected by TBR1/2. Given the parasitemic level detected, pMUTEC F/R, TRYP1 R/S and TRYP4 R/S were not more sensitive than classical microscopic examination of the buffy coat. TBR1/2, TEPAN1/2, pMUTEC F/R and TRYP4 R/S did not cross-reacted with Babesia sp., Trypanosoma theileri and Anaplasma marginale. TBR1/2 was the most sensitive primer set to detect T. evansi in purified DNA, rodent blood and cattle blood, and did not show cross reaction with the other pathogens tested: it should be therefore preferred for epidemiological surveys. These results confirmed that TBR1/2 primers remain the reference for the detection of Trypanozoon DNA and should therefore be included in subsequent evaluations of new diagnosis tools based on DNA detection., (Copyright 2010 Elsevier B.V. All rights reserved.)

Trypanosoma evansi is generally considered a mild pathogen in bovines. However, in Asia, acute and chronic signs have been observed in cattle, with high levels of parasitaemia, abortion and death. Investigations in Asian cattle are needed to better understand this epidemiological situation. To generate comparable data at a regional level, development and standardization of an antibody-enzyme linked immunosorbent assay for T. evansi (ELISA/T. evansi) was initiated and applied in an epidemiological survey carried out in dairy cattle in Thailand. A batch of 1979 samples was collected from dairy farms located throughout the country's four regions. Soluble T. evansi antigens initially produced in France were also produced in Thailand for comparison and technology transfer. Screening of 500 samples allowed us to identify reference samples and to determine the cut-off value of the ELISA. Seropositive animals - some of them confirmed by PCR - were found in the four regions, in 12 out of 13 provinces, in 22 out of 31 districts, in 56 farms out of 222 (25%, 95%CI+/-6%) and in 163 animals out of 1979 (8.2, 95%CI+/-1.2%). Estimated seroprevalence in 35 farms ranged between 1% and 30%, and in 21 farms it was >30%. Approximately 25% of survey cattle were exposed to the infection, in various situations. A sub-sample of 160 sera was tested on both antigens. Wilcoxon's (Z=1.24; p=0.22) and McNemars's tests (CHI2=3.55; p=0.09) did not show any significant differences, showing that the locally produced antigen is suitable for further evaluation in the surrounding countries. Use of this standardized serological method will broaden knowledge of the prevalence and impact of the disease at the regional level in South-East Asia. Further validation of this ELISA will be necessary in other host species such as buffalo, horse and pig.

A learning capacity for feeding is described in many insect species including vectors of diseases, but has never been reported in tsetse flies (Diptera, Glossinidae), the cyclic vectors of human (sleeping sickness) and animal trypanosomoses in Africa. Repeated feeding on the same host species by a disease vector is likely to increase the within-species disease-transmission risk, but to decrease it between species. An experiment with cattle and reptiles in a stable provides evidence that the species of host selected for the second blood meal in tsetse flies depends on the host encountered for the first blood meal when the between-meal interval is 2 days. This preference disappears when the between-meal interval is extended to 3 days. The energetic advantages of this acquired preference and its importance in trypanosomoses epidemiology are discussed.