Mycobacterium microti Infection in Free-Ranging Wild Boar, Spain, 2017-2019.

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that causes pathology in many mammals. M. microti infections have been found in some countries in Europe. We report an outbreak of tuberculosis caused by M. microti in wild boars in Spain.

Characterization of biosecurity practices among cattle transport drivers in Spain.

Transmission of pathogens between farms via animal transport vehicles is a potential concern; however, the available information on driver routines and biosecurity measures implemented during transport is limited. Given the above, the aim of this study was to describe and characterize the prevailing practices and biosecurity measures adopted by cattle transport drivers in Spain. Eighty-two drivers were surveyed via face-to-face or remotely. The survey included questions on general characteristics of the drivers (type of journeys and vehicles) together with biosecurity practices implemented during cattle transport and vehicle hygiene practices. Results showed that several risky practices are performed quite frequently such as visiting different premises with different levels of risk (e.g., breeder and fattening farms); entering the farm premises to load/unload animals, passing by several farms to load and unload animals, or not always cleaning and disinfecting the vehicle between travels, among others. To explore similarities among the drivers and identify groups sharing specific practices, hierarchical clustering on principal components (HCPC) was computed on the results of multiple correspondence analysis (MCA). The first three MCA dimensions (out of 13) were retained in the agglomerative clustering and four different clusters were identified. Clusters 1 and Cluster 4 accounted for 39.5% and 29.6% of respondents, respectively. The clusters were mainly differentiated by practices in the loading/unloading of cattle, such as the frequency of contact with animals remaining on the farm, and the frequency of the vehicle's disinfection between farms. Cluster 2 and Cluster 3 were of similar size, about 15% of respondents each. Cluster 2 consisted of drivers who mainly made journeys to slaughterhouse, while drivers in Cluster 3 were characterised by the use of working clothes and boots. Based on these findings, it is advisable to increase awareness on the role that animal transport can have in the spread of pathogens between cattle farms and the importance of biosecurity in preventing such transmission. There is also a need to support animal transport professionals in such task, not only through the development of initiatives to increase awareness, but also through the investment in improving cleaning and disinfection facilities and to consider the economic cost associated with some practices to not compromise the economic viability of the sector.

Corrigendum: Examination of critical factors influencing ruminant disease dynamics in the Black Sea Basin.

[This corrects the article DOI: 10.3389/fvets.2023.1174560.].

Introduction of a PRRSV-1 strain of increased virulence in a pig production structure in Spain: virus evolution and impact on production.

BACKGROUND: A strain of Porcine reproductive and respiratory syndrome virus (PRRSV), showing characteristics of enhanced virulence, affected a pyramidal production system from Spain with 7600 sows in 4 genetic nuclei and 13,000 sows in multipliers. Different PRRSV strains circulating in this production system from December 2020 to October 2021 were detected and sequenced. The spread of each isolate was examined and their impact on health and production in three of the affected farms was evaluated. RESULTS: The newly emerged PRRSV isolate with enhanced virulence entered the system before the onset of the study (January 2020) and afterwards four significantly different clades were detected during the study period in different farms, probably because of independent introduction events. The diversification of the enhanced virulence strain was higher for those clades (substitution rates up to 1.1% nucleotides/year) compared to other PRRSV strains present in the production system (up to 0.17%), suggesting a faster spread and adaptation. The impact of the infection in the first affected farm was dramatic, with an average abortion rate above 27% during 17 weeks before returning to the baseline production. Fertile sow mortality reached 6.5% for 39 weeks. In two farms infected later by other clades of this enhanced virulence strain, the impact was less acute; despite the fact that for parameters such as the proportion of stillbirths or mummies, more than 10 months were needed to recover pre-outbreak values. In the examined nurseries, mortalities reached peaks between 28 and 50% and several months were needed to return to normality. CONCLUSION: Introduction of a PRRSV strain of enhanced virulence in a production system where several farms were previously positive for other PRRSV strains, resulted in a fast spread such as would be observed in naïve farms. The productive and health impact was very high taking several months to return to normality.

Government veterinarians' perceptions of routine biosecurity focused on dairy cattle farms in north-western and north-eastern Spain.

The implementation of biosecurity measures in livestock production systems can be affected by the psychosocial factors of its stakeholders, which can be observed through their knowledge, attitudes and perceptions/practices. In Spain, there are no regulations per se to promote biosecurity. Of all stakeholders, farmers and veterinarians have been addressed in previous biosecurity studies, but not veterinarians belonging specifically to the government services. This study explores this particular group's perceptions of routine biosecurity in livestock production systems in north-western and north-eastern Spain, an understanding of which could help to improve the implementation of biosecurity measures on farms. Eleven interviews were conducted with veterinarians from different levels of the government services in Galicia and Catalonia, and were analyzed through content analysis. Dairy cattle farms were considered as the reference livestock production systems. The respondents stress the limited availability of staff and time resources for biosecurity. The advisory role of government veterinarians is not well recognized among farmers, who feel that their services prioritize their sanctioning role. In fact, government veterinarians consider that farmers only implement biosecurity measures to avoid being sanctioned, and not because they are aware of the importance of biosecurity. Meanwhile, the participants comment that biosecurity regulations should be flexible and need to consider the contexts of the farms where biosecurity measures are implemented. Finally, government veterinarians are willing to attend biosecurity meetings together with all farm stakeholders, at which the government services could be informed about biosecurity issues on farms. The person who could take on the biosecurity advisory role should be defined, along with further discussion of such matters as the responsibilities of each stakeholder. Government veterinary services need to be considered in studies of biosecurity operations in order to improve their implementation. It is therefore concluded that government veterinarians are seeking to balance their own institutional perspective with that of farmers and veterinarians in the routine implementation of biosecurity measures.

Improving the communication between farmers and veterinarians to enhance the acceptability of bovine tuberculosis eradication programmes.

France and Spain have been fighting against bovine tuberculosis (bTB) for years, even though new outbreaks continue to appear on both sides of the border, generating misconceptions about the disease and social distrust in the eradication programme and competent authorities. The perceived disease risk and the commitment of all interested parties are key factors for the successful implementation of control programmes, as they might influence the acceptability of recommended measures. Effective communication can contribute to increasing knowledge, trust and stakeholders' engagement, thus ensuring the acceptability of the eradication programme. This study was conducted in Catalonia (Spain) and Pyrenees-Atlantiques (France) in the frame of the INNOTUB project (https://innotub.eu/) to characterise the communication on bTB in the trans-Pyrenees region and provide recommendations to improve it. The communication on bTB was characterised by analysing 153 (Spain) and 66 (France) online freely available texts, published between 2018 and 2020, through Content Analysis and Critical Metaphor Analysis. Moreover, six farmers and four veterinarians were in-depth interviewed in each area to gather information about the communication on bTB. Interviews were made in original languages and analysed using a qualitative thematic approach. A pilot participatory intervention inspired by the Systematic Tool for Behavioural Assumption Validation and Exploration (STAVE) method was used to develop a list of proposals to improve communication and to promote the creation of territorial networks/committees on bTB prevention and control. It included three focus groups with farmers and veterinarians, a meeting with representatives of the regional veterinary services, and a final deliberative workshop. Results highlight the existence of a controversial debate on bTB and a heterogeneous understanding between stakeholders. Institutional and scientific communication mainly focus on bTB detection and control while other aspects are left in the background. On the contrary, farmers extend their communication to a greater variety of topics. The metaphorical framing strongly differed among actors, while veterinary services and researchers "fight" against bTB and "progress" toward the eradication, farmers place themselves in a framework of "sacrifice" and, particularly in Spain, they play a passive role. The proposals developed by the participants to improve the current communication on bTB included: (i) create participatory meeting spaces to share opinions and information; (ii) improve data accessibility (on epidemiological situations); (iii) develop clearer and written protocols and informative visual material; (iv) redesign the training courses (v) increase the stakeholders' participation in the design of protocols.

Evaluation Survey on Agreement with Existing Definitions of Biosecurity with a Focus on Livestock.

Disease prevention, including biosecurity, surveillance, and traceability are key aspects to minimize the risk of animal diseases causing harm to society. Diseases for which biosecurity are needed depend on species of interest, e.g., African swine fever, avian influenza, or foot-and-mouth disease. However, several definitions of biosecurity co-exist in the literature. Under the new COST Action "Biosecurity Enhanced Through Training Evaluation and Raising Awareness" (BETTER) CA20103, we launched an initial survey on the agreement with eight existing definitions of (livestock) biosecurity, to rank keywords to consider before attempting a more consolidated definition, and to select the desirable qualities of a definition of livestock biosecurity. Respondents (N = 316) had a male-female gender ratio close to one, were mostly between 25 and 54 years old, and had animal health as the main field of expertise (30% were government officials). The significant most popular biosecurity definition was the one that conceptualized the rules of 5B's (bio-exclusion, bio-containment, bio-compartmentation, bio-prevention, and bio-preservation). The top two keywords to consider for the consolidation of the biosecurity definition were "prevention" and "measures". The optimal biosecurity definition needs to be operational and related to animal health but also comprehensible, simple, and related to public health. The survey results highlight the need for the integration of more aspects in the existing definitions of livestock biosecurity (prevention of zoonoses and preservation of the environment and diversity).

Examination of critical factors influencing ruminant disease dynamics in the Black Sea Basin.

Introduction: Ruminant production in the Black Sea basin (BSB) is critical for national economies and the subsistence of rural populations. Yet, zoonoses and transboundary animal diseases (TADs) are limiting and threatening the sector. To gain a more comprehensive understanding, this study characterizes key aspects of the ruminant sector in nine countries of the BSB, including Armenia, Azerbaijan, Belarus, Bulgaria, Georgia, Moldova, Romania, Türkiye, and Ukraine. Methods: We selected six priority ruminant diseases (anthrax, brucellosis, Crimean Congo haemorrhagic fever (CCHF), foot-and-mouth disease (FMD), lumpy skin disease (LSD), and peste des petits ruminants (PPR)) that are present or threaten to emerge in the region. Standardized questionnaires were completed by a network of focal points and supplemented with external sources. We examined country and ruminant-specific data such as demographics, economic importance, and value chains in each country. For disease-specific data, we analysed the sanitary status, management strategies, and temporal trends of the selected diseases. Results and discussion: The shift from a centrally planned to a market economy, following the collapse of the Soviet Union, restructured the ruminant sector. This sector played a critical role in rural livelihoods within the BSB. Yet, it faced significant challenges such as the low sustainability of pastoralism, technological limitations, and unregistered farms. Additionally, ruminant health was hindered by informal animal trade as a result of economic factors, insufficient support for the development of formal trade, and socio-cultural drivers. In the Caucasus and Türkiye, where diseases were present, improvements to ruminant health were driven by access to trading opportunities. Conversely, European countries, mostly disease-free, prioritized preventing disease incursion to avoid a high economic burden. While international initiatives for disease management are underway in the BSB, there is still a need for more effective local resource allocation and international partnerships to strengthen veterinary health capacity, protect animal health and improve ruminant production.

Targeting the search of African swine fever-infected wild boar carcasses: A tool for early detection.

This study analyses the temporal and spatial distribution of found dead African swine fever (ASF)-positive wild boar carcasses from 2017 to January 2021 in affected European countries: Bulgaria, Estonia, Germany, Hungary, Latvia, Lithuania, Romania, Poland, Serbia and Slovakia. During this period, a total of 21,785 cases were confirmed in 19,071 unique locations. The temporal analysis of aggregated cases per month evidenced that most countries located in southern latitudes showed a higher number of cases between January and April, whereas in northern latitudes there was no clear temporal pattern. The space-time K-function evidenced a space-time clustering in the ASF-positive wild boar carcasses, which was most prominent within distances of 2 km and within 1 week. A Bayesian hierarchical spatial model was calibrated to evaluate the association between the probability of finding ASF-positive wild boar carcasses and landscape factors (i.e. the presence of a path and paved road), land use and wild boar abundance. Results showed the highest likelihood of finding ASF-positive wild boar carcasses in areas of transition between woodland and shrub, green urban areas and mixed forests. The presence of a path and a higher abundance of wild boar also increased slightly the odds of finding an ASF-positive dead wild boar. In summary, this paper aims to provide recommendations to design a search strategy to find ASF-infected wild boar carcasses, which is a crucial activity in the management of the disease, not just for surveillance purposes (i.e. the early detection of an introduction and the regular monitoring to understand the epidemiology and dynamics), but also for control, namely the disposal of infected carcasses as a virus source.

Description of a New Clade within Subtype 1 of Betaarterivirus suid 1 Causing Severe Outbreaks in Spain.

This report describes 28 genome sequences from a new clade within subtype 1 of Betaarterivirus suid 1, formerly known as porcine reproductive and respiratory syndrome virus 1. All share a potential recombinant pattern, with a highly pathogenic Italian strain as the putative major parental sequence and three other possible parents.

Animal diseases caused by orbiviruses, Algeria.

Antibodies against bluetongue virus were detected in cattle, sheep, goats, and camels in Algeria in 2008. Antibodies against epizootic hemorrhagic disease virus were detected in cattle, but antibodies against African horse sickness virus were not detected in horses and mules. Epizootic hemorrhagic disease in northern Africa poses a major risk for the European Union.

Quantitative assessment of the probability of bluetongue virus overwintering by horizontal transmission: application to Germany.

Even though bluetongue virus (BTV) transmission is apparently interrupted during winter, bluetongue outbreaks often reappear in the next season (overwintering). Several mechanisms for BTV overwintering have been proposed, but to date, their relative importance remain unclear. In order to assess the probability of BTV overwintering by persistence in adult vectors, ruminants (through prolonged viraemia) or a combination of both, a quantitative risk assessment model was developed. Furthermore, the model allowed the role played by the residual number of vectors present during winter to be examined, and the effect of a proportion of Culicoides living inside buildings (endophilic behaviour) to be explored. The model was then applied to a real scenario: overwintering in Germany between 2006 and 2007. The results showed that the limited number of vectors active during winter seemed to allow the transmission of BTV during this period, and that while transmission was favoured by the endophilic behaviour of some Culicoides, its effect was limited. Even though transmission was possible, the likelihood of BTV overwintering by the mechanisms studied seemed too low to explain the observed re-emergence of the disease. Therefore, other overwintering mechanisms not considered in the model are likely to have played a significant role in BTV overwintering in Germany between 2006 and 2007.

Biosecurity in pig farms: a review.

The perception of the importance of animal health and its relationship with biosecurity has increased in recent years with the emergence and re-emergence of several diseases difficult to control. This is particularly evident in the case of pig farming as shown by the recent episodes of African swine fever or porcine epidemic diarrhoea. Moreover, a better biosecurity may help to improve productivity and may contribute to reducing the use of antibiotics. Biosecurity can be defined as the application of measures aimed to reduce the probability of the introduction (external biosecurity) and further spread of pathogens within the farm (internal biosecurity). Thus, the key idea is to avoid transmission, either between farms or within the farm. This implies knowledge of the epidemiology of the diseases to be avoided that is not always available, but since ways of transmission of pathogens are limited to a few, it is possible to implement effective actions even with some gaps in our knowledge on a given disease. For the effective design of a biosecurity program, veterinarians must know how diseases are transmitted, the risks and their importance, which mitigation measures are thought to be more effective and how to evaluate the biosecurity and its improvements. This review provides a source of information on external and internal biosecurity measures that reduce risks in swine production and the relationship between these measures and the epidemiology of the main diseases, as well as a description of some systems available for risk analysis and the assessment of biosecurity. Also, it reviews the factors affecting the successful application of a biosecurity plan in a pig farm.

Most likely causes of infection and risk factors for tuberculosis in Spanish cattle herds.

INTRODUCTION: This study aims to assess the most likely causes of Bovine tuberculosis (bTB) breakdowns in Spanish cattle herds and to identify the main risk factors at farm-level. METHODS: Causes of bTB breakdowns were assessed through a qualitative risk-assessment based on decision-trees by analysing surveillance data from 3819 bTB breakdowns detected during 2014-2016. Results were compared to veterinary officers' (VO) opinions. Risk factors were identified through a case-control study with data from 196 bTB cases and 160 controls collected during 2014-2018. RESULTS: The decision tree analysis identified residual infections and interactions with wildlife as the most frequent causes of breakdowns (36% each), followed by purchasing infected cattle (14%). These results were not supported by VOs' opinions. According to the regression models, the risk of bTB increased by sharing pastures (odds ratios [OR] = 2.7; 95% confidence interval [CI] = 1.6-4.4) and by increasing inwards cattle movements. The presence of wildlife reservoirs represented a significant risk for extensively-managed farms if other cattle farms are situated within a one-kilometre radius (OR = 2.3; 95% CI = 1.1-5.1). DISCUSSION: To prevent bTB breakdowns, efforts should be devoted to decrease the likelihood of residual infections and improve farm biosecurity. The adoption of biosecurity measures might be influenced by farmers' perceptions, which should be carefully evaluated to ensure the effectiveness of such strategies.

Extended-Spectrum ß-Lactam Resistant Klebsiella pneumoniae and Escherichia coli in Wild European Hedgehogs (Erinaceus europeus) Living in Populated Areas.

Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of ß-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of ß-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.

Factors Associated with the Introduction of Mycobacterium avium spp. Paratuberculosis (MAP) into Dairy Herds in Galicia (North-West Spain): The Perception of Experts.

This study aimed at quantifying expert opinions on the risk factors involved in Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds. For this purpose, potential risk factors associated with the introduction of MAP into dairies were chosen based on a literature review and discussions with researchers and veterinarians. For each factor, a decision tree was developed, and key questions were included in each. Answers to these key questions led to different events within each decision tree. An expert opinion workshop was organized (following the recommendations of the OIE), and ordinal values ranging from 0 to 9 (i.e., a null to very high likelihood of infection) were assigned to each event. The potential risk factors were also incorporated into a structured questionnaire that was responded to by 93 farms where the sanitary status against MAP was known. Thereby, based on the values given by the experts and the information collected in the questionnaires, each farm was assigned a score based on their MAP entry risk. From these scores (contrast variable) and using a ROC curve, the cut-off that best discriminated MAP-positive and -negative farms was estimated. The most important risk factors for the introduction of MAP, according to expert opinions, involved purchase and grazing practices related to animals under six months of age. The scores obtained for each farm, also based on the expert opinions, allowed MAP positive/MAP negative farms to be discriminated with 68.8% sensitivity and 68.7% specificity. These data should be useful for focusing future training initiatives and improving risk-reduction strategies in the dairy industry.

Whole-Genome SNP Analysis Identifies Putative Mycobacterium bovis Transmission Clusters in Livestock and Wildlife in Catalonia, Spain.

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting.

Influence on the implementation of biosecurity measures in dairy cattle farms: Communication between veterinarians and dairy farmers.

This study was carried out in two regions in Spain (Catalonia and Galicia) through eight focus groups; four for dairy farmers and four for veterinarians. The results showed that dairy farmers and veterinarians attributed responsibility to one another for not following biosecurity practices. The study brings to light contradictions among veterinarians and certain individual veterinary practices that participated in the study, which lead to doubt and confusion on the part of dairy farmers. Distinct perceptions were also identified of the role that government authorities should play in relation both to training and sanctions as a means of improving biosecurity on dairy farms. Additionally, the participants expressed varying opinions as to whether biosecurity measures ought to be made mandatory or remain voluntary. Results from this study highlight the need to promote initiatives through which distinct stakeholders such as veterinarians, government authorities, and dairy farmers can develop consensus-based messages on the implementation of biosecurity practices.

Phylogenetic relationships investigation of Mycobacterium caprae strains from sympatric wild boar and goats based on whole genome sequencing.

Tuberculosis (TB) in wildlife challenges epidemiological surveillance and disease control. An outbreak of TB was detected in a free-ranging wild boar population of a Natural Park in Catalonia (Spain) and the outbreak investigation was conducted in the area. During the study period (2015-2020), 278 wild boars were analysed by gross pathology, histopathology, mycobacterial culture and DVR-spoligotyping. In addition, all cattle (49) and goat (47) herds of the area were tested with tuberculin skin test. TB compatible lesions were detected in 21 wild boars, and Mycobacterium caprae was isolated in 17 of them with two different spoligotypes: SB0415 (13) and SB1908 (4). Only two goat herds showed TB positive animals that were subsequently slaughtered. M. caprae with the spoligotypes SB0416 and SB0415 were isolated from these animals. To investigate the phylogenetic relationships and the transmission chain of the outbreak, nine strains isolated from six wild boars and three goats of the study area were analysed by whole genome sequencing (WGS) followed by single nucleotide polymorphism (SNP) analysis by maximum likelihood and median-joining network inference methods. Results indicated that infected wild boars maintained M. caprae strains circulation in their own population and have likely transmitted the infection to goats, thus acting as TB reservoirs, compromising the success of livestock TB eradication campaigns and posing a risk for public health. The results also highlighted the usefulness of WGS followed by SNP analysis in providing relevant epidemiological information when detailed contact data are missing.

Persistence of highly pathogenic avian influenza virus (H7N1) in infected chickens: feather as a suitable sample for diagnosis.

Selection of an ideal sample is a vital element in early detection of influenza infection. Rapid identification of infectious individuals or animals is crucial not only for avian influenza virus (AIV) surveillance programmes, but also for treatment and containment strategies. This study used a combination of quantitative real-time RT-PCR with an internal positive control and a cell-titration system to examine the presence of virus in different samples during active experimental AIV infection and its persistence in the infected carcasses. Oropharyngeal/cloacal swabs as well as feather pulp and blood samples were collected from 15-day-old chicks infected with H7N1 highly pathogenic AIV (HPAIV) and the kinetics of virus shedding during active infection were evaluated. Additionally, several samples (muscle, skin, brain, feather pulp and oropharyngeal and cloacal swabs) were examined to assess the persistence of virus in the HPAIV-infected carcasses. Based on the results, feather pulp was found to be the best sample to detect and isolate HPAIV from infected chicks from 24 h after inoculation onwards. Kinetic studies on the persistence of virus in infected carcasses revealed that tissues such as muscle could potentially transmit infectious virus for 3 days post-mortem (p.m.), whilst other tissues such as skin, feather pulp and brain retained their infectivity for as long as 5-6 days p.m. at environmental temperature (22-23 degrees C). These results strongly favour feather as a useful sample for HPAIV diagnosis in infected chickens as well as in carcasses.