Host immune response as well as virulence factors are key in disease susceptibility. There are no known association studies of HLA class I and II alleles with chikungunya (CHIKV) infection in Latin American population. We aim to identify Human Leukocyte Antigen (HLA) alleles present in patients with CHIKV infection when compared to healthy controls, as well as allele association with the clinical spectrum of the disease. A cross-sectional analysis nested in a community cohort was carried out. We included patients 18 years and older with serological confirmation of CHIKV infection. HLA typing of HLA-A, HLA-B and HLA-DRB1 alleles was performed. Two-by-two tables were used to establish associations between allele presence and clinical characteristics. Data from 65 patients with confirmed CHIKV infection were analyzed for HLA typing. CHIKV infection was associated with the presence of HLA-A*68, HLA-B*35, HLA-DRB*01, HLA-DRB1*04 and HLA-DRB1*13 alleles with statistical significance when compared to healthy subjects. A statistically significant relationship was found between the presence of rash in the face or the abdomen and the presence of HLA-DRB1*04. Our study demonstrated that in our cohort, HLA type I as well as type II alleles are associated with CHIKV infection, and specifically an HLA type II allele with dermatological symptoms. Further research is needed to set a path for future investigation on genes outside the HLA system to improve knowledge in the pathophysiology of CHIKV infection and its host-pathogen interaction.

Leishmaniasis is caused by protozoans of the Leishmania genus, which includes more than 20 species capable of infecting humans worldwide. In the Americas, the most widespread specie is L. braziliensis, present in 18 countries, including Bolivia. The taxonomic position of the L. braziliensis complex has been a subject of controversy, complicated further by the recent identification of a particular subpopulation named L. braziliensis atypical or outlier. The aim of this study was to carry out a systematic analysis of the L. braziliensis complex in Bolivia and to describe the associated clinical characteristics. Forty-one strains were analyzed by sequencing an amplified 1245 bp fragment of the hsp70 gene, which allowed its identification as: 24 (59%) L. braziliensis, 16 (39%) L. braziliensis outlier and one (2%) L. peruviana. In a dendrogram constructed, L. braziliensis and L. peruviana are grouped in the same cluster, whilst L. braziliensis outlier appears in a separate branch. Sequence alignment allowed the identification of five non-polymorphic nucleotide positions (288, 297, 642, 993 and 1213) that discriminate L. braziliensis and L. peruviana from L. braziliensis outlier. Moreover, nucleotide positions 51 and 561 enable L. peruviana to be discriminated from the other two taxa. A greater diversity, was observed in L . braziliensis outlier than in L. braziliensis- L. peruviana. The 41 strains came from 32 patients with tegumentary leishmaniasis, among which 22 patients (69%) presented cutaneous lesions (11 caused by L. braziliensis and 11 by L. braziliensis outlier) and ten patients (31%) mucocutaneous lesions (eight caused by L. braziliensis, one by L. braziliensis outlier and one by L. peruviana). Nine patients (28%) simultaneously provided two isolates, each from a separate lesion, and in each case the same genotype was identified in both. Treatment failure was observed in six patients infected with L. braziliensis and one patient with L. peruviana.

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated to the absence of vaccines and therapeutical tools. Although the exact transmission pathway of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free-living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free-ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free-living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae, and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated to high incidence of bovine besnoitiosis in the country. To date, this is the first report of a Besnoitia besnoiti-like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings.

Local animal health services in rural communities are mainly provided by village animal health workers (VAHW), although the participation and contribution of VAWHs to disease prevention is uncertain. To address this, a desktop review of national VAWH data between 2011 - 2020 also conducted in December 2020, supporting a detailed survey on the involvement of VAHWs in disease prevention programs conducted between February to March 2014. The survey used guided group discussion with VAHWs (n = 198) from the two Cambodian provinces of Kampong Cham and Pursat. This study identified that VAHWs generated less than 22% of their annual household incomes from animal health services. Less than one-third had vaccinated livestock against FMD, with none having vaccinated cattle every six months during the study period, and nearly half of the VAHWs having never vaccinated their own cattle against FMD. As no privately-provided FMD vaccination services occurred in these communities, with all vaccines delivered through the government-subsidised program, the findings confirmed that VAHWs only vaccinated animals against FMD when vaccines were made available by the Government. The desktop review found that the number of VAHWs in 2020 declined by more than 24% since 2017 and the proportion of female VAHWs was consistently low, with a mean of 8.26 (± 1.019). These findings confirm there are considerable weaknesses in the VAHW system in Cambodia, particularly in contributing to FMD control. Cambodian animal health authorities require more effective policies to strengthen the current VAHW system, improving: their services delivery; their retention as ‘active’; their development of more sustainable roles with lower ‘dropout’ rates; and the prolonged gender inequity. With the limited availability of government-subsidised FMD vaccination currently, extension programs that engage VAHWs and farmers in seeking privately funded and delivered FMD vaccination that incorporates appropriate multivalent FMD serotype vaccines of high quality, delivered in small dose vials from a robust cold chain, is suggested. This strategy would assist VAHWs to contribute to the provision of private livestock vaccination services that are likely essential for sustainable FMD prevention and control in Cambodia.

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporter and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

RT-PCR is currently the standard diagnostic method to detect symptomatic and asymptomatic individuals infected with SARS-CoV-2. However, RT-PCR results are not immediate and may falsely be negative before an infected individual sheds viral particle in the upper airway where swabs are collected. Infected individuals emit volatile organic compounds (VOCs) in their breath and sweat that are detectable by trained dogs. Here we evaluate the diagnostic accuracy of dog detection against SARS-CoV-2 infection. Fifteen dogs previously trained at two centres in Australia were presented to axillary sweat specimens collected from known SARS-CoV-2 human cases and non-cases. The true infection status of the cases and non-cases were confirmed based on RT-PCR results as well as clinical presentation. Across dogs, the overall diagnostic sensitivity (DSe) was 95.6% (95%CI: 93.6%-97.6%) and diagnostic specificity (DSp) was 98.1% (95%CI: 96.3%-100.0%). The DSp decreased significantly with non-case specimens sourced from UAE ( P-value < 0.001). The location of evaluation did not impact the detection performances. The accuracy of detection varied across dogs and experienced dogs revealed a marginally better DSp ( P-value = 0.003). The potential and limitations of this alternative detection tool are discussed.

Porcine epidemic diarrhea virus (PEDV) produces infection in pigs characterized by vomiting and diarrhea. PEDV is transmitted via oral-fecal and a very low oral dose is enough to infect susceptible pigs, causing devastating consequences in production. A 10,000-sow farrow-to-wean farm located in northwest Mexico was infected with PEDV. After the observation of the first clinical signs, an outbreak investigation take into place to determine the most probably source of infection. A systematic collection of samples including rectal swabs, gestation and lactation feed, surface swabs from the interior or feed bins and many points of the feed truck delivering the implicated feed was performed. Samples were tested for PEDV polymerase chain reaction (PCR). Positive PCR results showed the evidence of PEDV RNA in lactating feed, the interior walls of the feed bins and in the interior of the auger boom of the feed truck. This, connected with the location of first clinical signs point that the most probably incursion of PEDV in to this breeding herd was contaminated feed. This paper shows how feed or feed transport can be a potential source of PEDV infection in farms and highlight the importance of stablishing biosecurity programs to mitigate the risk of PEDV infections.

African swine fever (ASF), is a serious global concern from an ecological and economic point of view. While it is well established that its main transmission routes comprise contact between infected and susceptible animals and transmission through contaminated carcasses, the specific mechanism leading to its long-term persistence is still not clear. Among others, a proposed mechanism involves the potential role of convalescent individuals, which would be able to shed the virus after the end of the acute infection. Using a spatially explicit, stochastic, individual-based model, we tested: 1) if ASF can persist when transmission occurs only through infected wild boars and infected carcasses; 2) if the animals that survive ASF can play a relevant role in increasing ASF persistence chances; 3) how hunting pressure can affect the ASF probability to persist. The scenario in which only direct and carcass-mediated transmission were contemplated had 52% probability of virus persistence 10 years after the initial outbreak. The inclusion of survivor-mediated transmission corresponded to slightly higher persistence probabilities (57%). ASF prevalence during the endemic phase was generally low, ranging 0.1-0.2%. The proportion of seropositive individuals gradually decreased with time and ranged 4.5 – 6.6%. Our results indicate that direct and carcass-mediated infection routes are sufficient to explain and justify the long-term persistence of ASF at low wild boar density and the ongoing geographic expansion of the disease front in the European continent. During the initial years of an ASF outbreak, hunting should be carefully evaluated as a management tool, in terms of potential benefits and negative side-effects, and combined with an intensive effort for the detection and removal of wild boar carcasses. During the endemic phase, further increasing hunting effort should not be considered as an effective strategy. Additional effort should be dedicated to finding and removing as many wild boar carcasses as possible.

The current COVID-19 pandemic highlights the need for zoonotic infectious disease surveillance. Avian influenza virus (AIV) poses a significant threat to animal and public health due to its pandemic potential. Virus-contaminated water has been suggested as an important AIV spread mechanism among multiple species. Nevertheless, few studies have characterized the global AIV subtype diversity and distribution in environmental water. Therefore, this study aims to provide an updated descriptive and phylogenetic analysis of AIVs isolated in water samples from high risk-sites for influenza outbreaks (i.e., live bird markets, poultry farms, and wild bird habitats) on a global scale. A total of 234 hemagglutinin (HA) gene sequences of 21 subtypes were reported from nine countries between 2003 – 2020. Fourteen AIV subtypes were solely reported from Asian countries. Most of the viral sequences were obtained in China and Bangladesh with 47.44% and 23.93%, respectively. Likewise, the greatest global AIV subtype diversity was observed in China with twelve subtypes. Live bird markets represented the main sampling site for AIV detection in water samples (64.10%), mostly from poultry cage water. Nevertheless, the highest subtype diversity was observed in water samples from wild bird habitats, especially from the Izumi plain and the Dongting Lake located in Japan and China, respectively. Water from drinking poultry troughs evidenced the greatest subtype diversity in live bird markets, meanwhile, environmental water used by ducks had the highest number of different subtypes in poultry farms. The maximum-likelihood phylogenetic tree showed that some HA sequences were closely related among different poultry/wild bird-related environments from different geographic origins. Therefore, the results suggest that even though the availability of HA gene sequences in public-access databases varies greatly among countries, environmental AIV surveillance represents a useful tool to elucidate potential viral diversity in wild and domestic bird populations.

Porcine circoviruses are important pathogens of production swine. Porcine circovirus type 1 (PCV1) is non-pathogenic, and discovered as a contaminant of a porcine kidney cell line, PK-15. The discovery of pathogenic variant, PCV2, occurred in the late 90’s in association with post-weaning multi-systemic wasting disease syndrome (PMWS), which is characterized by wasting, respiratory signs and lymphadenopathy in weanling pigs. A new PCV type, designated as PCV3, was discovered in 2016, in pigs manifesting porcine dermatitis and nephropathy syndrome (PDNS), respiratory distress and reproductive failure. Pathological manifestations of PCV3 Infections include systemic inflammation, vasculitis and myocarditis. A 4 th PCV type, PCV4, was identified in 2020 in pigs with PDNS, respiratory and enteric signs. All the pathogenic PCV types are detected in both healthy and morbid pigs. They cause chronic, systemic infections with various clinical manifestations. Dysregulation of the immune system homeostasis is a pivotal trigger for pathogenesis in porcine circoviral infections. While the study of PCV3 immunobiology is still in its infancy lessons learned from PCV2 and other circular replication-associated protein (Rep)-​encoding single stranded(ss) (CRESS) DNA viruses can inform the field of exploration for PCV3. Viral interactions with the innate immune system, interference with dendritic cell function coupled with the direct loss of lymphocytes compromises both innate and adaptive immunity in PCV2 infections. Dysregulated immune responses leading to the establishment of a pro-inflammatory state, immune complex associated hypersensitivity, and the necrosis of lymphocytes and immune cells are key features of PCV3 immunopathogenesis. A critical overview of the comparative immunopathology of PCV2 and PCV3/4, and directions for future research in the field are presented in this review.

Summary: This study evaluates through modeling the possible individual and combined effect of three populational parameters of pathogens (reproduction rate; rate of novelty emergence; and propagule size) on the colonization of new host species – putatively the most fundamental process leading to the emergence of new infectious diseases. The results are analyzed under the theoretical framework of the Stockholm Paradigm using IBM simulations to better understand the evolutionary dynamics of the pathogen population and the possible role of Ecological Fitting. The simulations suggest that all three parameters positively influence the success of colonization of new hosts by a novel parasite population but contrary to the prevailing belief, the rate of novelty emergence (e.g. mutations) is the least important factor. Maximization of all parameters result in a synergetic facilitation of the colonization and emulates the expected scenario for pathogenic microorganisms. The simulations also provide theoretical support for the retention of the capacity of fast-evolving lineages to retro-colonize their previous host species/lineage by ecological fitting. Capacity is, thus, much larger than we can anticipate. Hence, the results support the empirical observations that opportunity of encounter (i.e. the breakdown in mechanisms for ecological isolation) is a fundamental determinant to the emergence of new associations – especially Emergent Infectious Diseases - and the dynamics of host exploration, as observed in SARS-CoV-2. Insights on the dynamics of Emergent Infectious Diseases derived from the simulations and from the Stockholm Paradigm are discussed.

African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, “point-of-care” (POC) tests have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. Along these lines, the INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) promises virus antigen detection under field conditions within minutes. In the present study, we evaluated the performance of the assay with selected high-quality reference blood samples, and also with real field samples from wild boar carcasses in different stages of decay from the ongoing ASF outbreak in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct carcass testing without any modification resulted in a drastically reduced sensitivity of only 12.5%. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of carcass samples. A diagnostic specificity of 100% was observed. However, most of the German ASF cases in wild boar would have been missed using the lateral flow assay (LFA) alone. Therefore, the antigen-specific LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen.

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from 8 species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Recent transmission events between nearby farms and the park were not found based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Monogenean infection of the internal organs is extremely rare when compared to external infections. This study describes mass mortality of Nile tilapia (Oreochromis niloticus L.) originating from co-infection with Enterogyrus spp. and Aeromonas jandaei following transport stress. The first fish deaths occurred on day 1 post-transport, while cumulative mortality reached approximately 90% by day 10 post-stocking. An atypical amount of pale (whitish) faeces floating on the surface of the water as well as typical clinical signs of motile Aeromonas septicemia, were reported. Adult monogeneans and countless eggs of monogeneans were found in the stomachs and the intestines of both moribund and dead fish, respectively. Two strains of A. jandaei were isolated from the kidneys. Scanning electron microscope microphotographs of the stomach revealed the presence of numerous monogeneans penetrating deep into the gastric tissue, and diffuse lesions filled with bacilliform bacteria. This is the first report of co-infection by Enterogyrus spp. and A. jandaei in Nile tilapia and the first report of E. coronatus, E. foratus, and E. malbergi parasitizing tilapia in Brazil. These findings indicate that synergic co-infection by Monogenean stomach parasites (E. coronatus, E. foratus, and E. malbergi) and A. jandaei may induce high mortalities in tilapia following transport stress.

Ticks are involved in the transmission of various pathogens and some tick-borne diseases cause significant problems for the health of humans and livestock. Despite their obvious importance, the composition of viral communities in ticks, and their interactions with pathogens, is poorly understood, particularly in Eastern Europe that constitutes (via bird migrations for example) a major hub for animal-arthropod vectors exchanges. The aim of this study was first to describe the virome of Dermacentor sp., Rhipicephalus sp. and Haemaphysalis sp. ticks collected from poorly investigated regions of Romania (Iasi and Tulcea counties) located at the intersection of various biotopes, countries and routes of migrations. We then focused the study on viruses that could have potential relevance for human and animal health. More than 500 ticks were collected in 2019 from the environment and from small ruminants and analyzed by high-throughput transcriptome sequencing. Among the viral communities infecting Romanian ticks, viruses belonging to the Flaviviridae, Phenuiviridae and Nairoviridae families were identified and full genomes were derivedPhylogenetic analyses placed them in clades where mammalian isolates are found, suggesting that these viruses could constitute novel arboviruses. We also assessed the bacterial microbiome of the collected ticks. The characterization of these microbial communities increases the knowledge of the diversity of viruses in Eastern Europe and provide a basis for further studies on the relationship between ticks and tick-borne viruses.

Porcine Sapovirus (SaV) was first identified by electron microscopy in the United States in 1980 and has since been reported from both asymptomatic and diarrheic pigs usually in mixed infection with other enteric pathogens. SaV as the sole etiological agent of diarrhea in naturally infected pigs has not previously been reported in the United States. Here, we used four independent lines of evidence including metagenomics analysis, real-time RT-PCR (rRT-PCR), histopathology, and in situ hybridization to confirm porcine SaV genogroup III (GIII) as the sole cause of enteritis and diarrhea in pigs. A highly sensitive and specific rRT-PCR was established to detect porcine SaV GIII. Examination of 184 fecal samples from the outbreak farm showed that pigs with clinical diarrhea had significantly lower Ct values (15.9 ± 0.59) compared to clinically unaffected pigs (35.8 ± 0.71). Further survey of 336 fecal samples from different states in the United States demonstrated that samples from pigs with clinical diarrhea had a comparable positive rate (45.3%) with those from non-clinical pigs (43.1%). However, the SaV-positive pigs with clinical diarrhea had significantly higher viral loads (Ct = 26.0 ± 0.5) than those positive but clinically healthy pigs (Ct = 33.2 ± 0.9). Phylogenetic analysis of 20 field SaVs revealed that all belonged to SaV GIII and recombination analysis indicated that intra-genogroup recombination occurred within the field isolates of SaV GIII. These results suggest that porcine SaV GIII plays an important etiologic role in swine enteritis and diarrhea and rRT-PCR is a reliable method to detect porcine SaV. Our findings provide significant insights to better understand the epidemiology and pathogenicity of porcine SaV.

Porcine astroviruses (PoAstVs) have been reported globally and are divided into at least five distinct lineages (PoAstV1-PoAsV5). The primary objective of this study was to summarize the scientific literature about the frequency of detection, associated clinical presentations, and type of samples and diagnostic tools used for the detection of porcine astroviruses. The secondary objective was to summarize the body of knowledge about the causal role in disease of PoAstVs using the Bradford Hill framework. A search was conducted using Centre for Biosciences and Agriculture International (CABI), MEDLINE, American Association of Swine Veterinarians (AASV) Swine Information Library (SIL) abstracts, swine conferences including American College of Veterinary Pathologists (ACVP), and American Association of Veterinary Laboratory Diagnosticians (AAVLD). From 168 studies identified by the search, 29 studies were eligible. Results indicated that 69% (20/29) of the literature on PoAstVs has been published between 2011 and 2018. Of 29 papers, 52% were detection studies (15 of 29) and 48% (14 of 29) were case-control studies. Seventy-two percent (21 of 29) reported differential diagnosis and 10% (3 of 29) reported histologic lesions, out of which 67% (2 of 3) associated the detection of PoAstV3 with development of polioencephalomyelitis. PCR-based assays were the most common diagnostic tools. Keywords: Swine, Astrovirus, Scoping review, Bradford Hill, PoAstV detection

The hypothesis that feed ingredients could serve as vehicles for the transport and transmission of viral pathogens was first validated under laboratory conditions. To bridge the gap from the laboratory to the field, this current project tested whether three significant viruses of swine could survive in feed ingredients during long-distance commercial transport across the continental US. One-metric ton totes of soybean meal (organic and conventional) and complete feed were spiked with a 10 mL mixture of PRRSV 174, PEDV, and SVA and transported for 23 days in a commercial semi-trailer truck, crossing 29 states, and 10,183 km. Samples were tested for the presence of viral RNA by PCR, and for viable virus in soy-based samples by swine bioassay and in complete feed samples by natural feeding. Viable PRRSV, PEDV, and SVA were detected in both soy products and viable PEDV and SVA in complete feed. These results provide the first evidence that viral pathogens of pigs can survive in representative volumes of feed and feed ingredients during long-distance commercial transport across the continental US.