SARS-CoV-2 Seroprevalence Studies in Pets, Spain.

SARS-CoV-2 can infect domestic animals such as cats and dogs. The zoonotic origin of the disease requires surveillance on animals. Seroprevalence studies are useful tools for detecting previous exposure because the short period of virus shedding in animals makes detection of the virus difficult. We report on an extensive serosurvey on pets in Spain that covered 23 months. We included animals with exposure to SARS-CoV-2-infected persons, random animals, and stray animals in the study. We also evaluated epidemiologic variables such as human accumulated incidence and spatial location. We detected neutralizing antibodies in 3.59% of animals and showed a correlation between COVID-19 incidence in humans and positivity to antibody detection in pets. This study shows that more pets were infected with SARS-CoV-2 than in previous reports based on molecular research, and the findings highlight the need to establish preventive measures to avoid reverse zoonosis events.

Tuberculosis-Associated Death among Adult Wild Boars, Spain, 2009-2014.

We investigated adult Eurasian wild boar (Sus scrofa) survival and death in 2 tuberculosis-endemic populations with different harvest pressure in Spain. Overall, tuberculosis accounted for 30% of total deaths. Increased survival in protected areas has direct implications for wild boar management and tuberculosis control.

Spatiotemporal interactions between wild boar and cattle: implications for cross-species disease transmission.

Controlling infectious diseases at the wildlife/livestock interface is often difficult because the ecological processes driving transmission between wildlife reservoirs and sympatric livestock populations are poorly understood. Thus, assessing how animals use their environment and how this affects interspecific interactions is an important factor in determining the local risk for disease transmission and maintenance. We used data from concurrently monitored GPS-collared domestic cattle and wild boar (Sus scrofa) to assess spatiotemporal interactions and associated implications for bovine tuberculosis (TB) transmission in a complex ecological and epidemiological system, Doñana National Park (DNP, South Spain). We found that fine-scale spatial overlap of cattle and wild boar was seasonally high in some habitats. In general, spatial interactions between the two species were highest in the marsh-shrub ecotone and at permanent water sources, whereas shrub-woodlands and seasonal grass-marshlands were areas with lower predicted relative interactions. Wild boar and cattle generally used different resources during winter and spring in DNP. Conversely, limited differences in resource selection during summer and autumn, when food and water availability were limiting, resulted in negligible spatial segregation and thus probably high encounter rates. The spatial gradient in potential overlap between the two species across DNP corresponded well with the spatial variation in the observed incidence of TB in cattle and prevalence of TB in wild boar. We suggest that the marsh-shrub ecotone and permanent water sources act as important points of TB transmission in our system, particularly during summer and autumn. Targeted management actions are suggested to reduce potential interactions between cattle and wild boar in order to prevent disease transmission and design effective control strategies.

Tertiary lymphoid organs in wild boar exposed to a low-virulent isolate of African swine fever virus.

Despite the great interest in the development of a vaccine against African swine fever (ASF) in wild boar, the immunological mechanisms that induce animal protection are still unknown. For this purpose, tertiary lymphoid organs (TLOs) of wild boar were characterised and compared with mucosa-associated lymphoid tissues (MALTs) by histopathology, histomorphometry and immunohistochemistry (CD3, CD79, PAX5, LYVE1, fibronectin). In addition, real-time polymerase chain reaction (qPCR) and immunohistochemistry (p72) were used to evaluate the presence of ASF virus (ASFV) in blood and tissues samples, respectively. TLOs were observed in animals infected with a low-virulent ASFV isolate (LVI), animals co-infected with low and high-virulent ASFV isolates (LVI-HVI) and animals infected only with the high virulence isolate (HVI). TLOs in LVI and LVI-HVI groups were located adjacent to the mucosa and presented a similar structure to MALT. Immunoexpresion of p72 observed in the inflammatory cells adjacent to TLOs/MALTs confirmed its development and reactivity generated by ASF attenuated isolates. Immunohistochemical evaluation, based on cellular composition (T and B lymphocytes), and histomorphometrical study revealed a more pronounced maturation of TLOs/MALTs in the LVI-HVI group. It is currently unclear whether these formations play a protective role by contributing to local immunity in chronic inflammatory diseases. However, the structural similarities between TLOs and MALTs and the location of TLOs close to the mucosa suggest that they may perform a similar function, facilitating a local protective response. Nevertheless, further investigations are warranted to assess the cellular and humoral dynamics of these lymphoid organs induced by attenuated isolates.

Low transmission risk of African swine fever virus between wild boar infected by an attenuated isolate and susceptible domestic pigs.

African swine fever (ASF) is a lethal infectious disease that affects domestic and wild pigs. This complex virus has already affected five continents and more than 70 countries and is considered to be the main threat to the global swine industry. The disease can potentially be transmitted directly through contact with infectious animals, or indirectly by means of contaminated feed or environments. Nevertheless, the knowledge regarding the transmission patterns of different ASF virus isolates at the wildlife-livestock interface is still limited. We have, therefore, assessed the potential transmission of an attenuated ASF virus isolate between infectious wild boar and directly exposed domestic pig. We registered 3,369 interspecific interactions between animals, which were brief and mostly initiated by wild boar. The major patterns observed during the study were head-to-head contact owing to sniffing, thus suggesting a high probability of pathogen transmission. However, only one of the five domestic pigs had a short period of viremia and became serologically positive for ASF virus antibodies. It was additionally discovered that the wild boar did not transmit the virulent virus isolate to the domestic pigs, which suggests that the presence of attenuated ASF virus isolates in affected areas may control the spreading of other more virulent isolates. These outcomes may help make decisions related to large-scale targeted management actions against ASF in field conditions.

Intrapancreatic accessory spleens in African swine fever infection of wild boar (Sus scrofa).

Intrapancreatic accessory spleen (IPAS) is one of the most frequent congenital splenic anomalies in humans; however, studies in veterinary medicine are scarce. This study aimed to describe the macroscopic, histopathological and immunohistochemical features of 11 suspected cases of IPAS in wild boar piglets of 3-4 months old. Seven of the 11 animals were immunised with a low virulence isolate of African swine fever virus (ASFV) and subsequently challenged with a highly virulent ASFV isolate (LVI-HVI group). The remaining four animals were exclusively infected with a highly virulent isolate of ASFV (HVI group). Grossly, lesions comprised focal or multifocal reddish areas of variable shape, located on the surface of the pancreatic tail or within the parenchyma. Histological and immunohistochemical studies (anti-CD79 and CD3) confirmed the presence of IPAS in eight of the 11 cases. IPAS shared the same histological structure and alterations as those observed in the original spleen. The immunohistochemical study against ASFV revealed the presence of VP72+ cells in both the spleen and IPAS of seven of the eight piglets. The results of this study describe for the first time the presence of IPAS in ASFV infection of wild boar (Sus scrofa) regardless the isolate and suggest that the infection may induce the development of ectopic splenic tissue due to an increased demand for phagocytic cells from the reticuloendothelial system. However, further studies are needed to understand the immunological mechanisms that trigger the formation of these accessory organs.

Permeability of artificial barriers (fences) for wild boar (Sus scrofa) in Mediterranean mixed landscapes.

BACKGROUND: Fences are one of the most widespread manmade features in nature, constituting an artificial limitation to the movement of wildlife. To date, their effects on wildlife behavior have been understudied but this knowledge is required to design effective management procedures. Using 21 GPS-monitored wild boar, we evaluated the permeability of different types of fences and described temporal patterns and spatial hotspots for crossing events. A fence's permeability was inferred by the crossing success, i.e., the number of times that animals crossed a barrier vs the number of times they did not cross. The vulnerability of fences at watercourses was explored by assessing whether the frequency of crossings was higher around watercourse intersections than expected by chance. RESULTS: Well-maintained big game proof fences were the most effective in reducing successful wild boar crossings; they were, on average, 30% more efficient than livestock type fences. Crossing success was higher for males than females and during the food shortage period than in the food abundance period. The frequency of crossings around watercourses was higher than expected by chance, especially in moderately and well-maintained big game proof type fences. CONCLUSION: While no fence type was 100% wild boar proof, well-maintained big game proof fences substantially constrained the movement of boar. However, they are vulnerable around watercourses. Managing the conflicts in which this species is involved, such as shared infections and agricultural damage, would require fences that are even more effective than the ones analyzed here, ideally in conjunction with other preventive actions. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of the clinical evolution and transmission of SARS-CoV-2 infection in cats by simulating natural routes of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current pandemic disease denominated as Coronavirus Disease 2019 (COVID-19). Several studies suggest that the original source of this virus was a spillover from an animal reservoir and its subsequent adaptation to humans. Of all the different animals affected, cats are one of the most susceptible species. Moreover, several cases of natural infection in domestic and stray cats have been reported in the last few months. Although experimental infection assays have demonstrated that cats are successfully infected and can transmit the virus to other cats by aerosol, the conditions used for these experiments have not been specified in terms of ventilation. We have, therefore, evaluated the susceptibility of cats using routes of infection similar to those expected under natural conditions (exposure to a sneeze, cough, or contaminated environment) by aerosol and oral infection. We have also evaluated the transmission capacity among infected and naïve cats using different air exchange levels. Despite being infected using natural routes and shed virus for a long period, the cats did not transmit the virus to contact cats when air renovation features were employed. The infected animals also developed gross and histological lesions in several organs. These outcomes confirm that cats are at risk of infection when exposed to infected people, but do not transmit the virus to other cats with high rates of air renovation.

Differences in wild boar spatial behaviour among land uses and management scenarios in Mediterranean ecosystems.

The ubiquitous wild boar is causing diverse and growing conflicts of socio-ecological and economic relevance worldwide. For that reason, knowledge of its spatial ecology is crucial to designing effective management programmes. But this knowledge is scarce in Mediterranean areas with mixed land uses. We describe the spatial ecology and habitat selection of 41 adult wild boar monitored using GPS collars and analyse the effects of sex and the period (food shortage period, hunting season and food abundance period) under different land uses (protected areas, mixed farms and fenced hunting estates). The spatial ecology of wild boar was characterised by marked temporality, mediated by sex and the land uses in the area. The activity (ACT), daily range (DR) and home range (HR) were higher for males than females, and in mixed farms versus fenced hunting estates, while the lowest values were obtained in protected areas. These effects were more marked for ACT and DR (movement) than HR. The selection of scrublands and avoidance of woodlands was observed where drive hunt events occur (mixed farms and fenced estates), but not in the protected areas. The differences in the requirements, reproductive behaviour and, interestingly, response to disturbance according to sex may explain this dissimilar behaviour. Disturbance originated higher movement rates and the selection of sheltering land cover as a refuge during the hunting season. This information is useful for designing species monitoring and management programmes; including both preventive and reactive actions in response to events such as outbreaks of African swine fever and agricultural damage produced by wild boar in Europe.

The Role of Interleukine-10 and Interferon-γ as Potential Markers of the Evolution of African Swine Fever Virus Infection in Wild Boar.

African swine fever virus (ASFv) is one of the most challenging pathogens to affect both domestic and wild pigs. The disease has now spread to Europe and Asia, causing great damage to the pig industry. Although no commercial vaccine with which to control the disease is, as yet, available, some potential vaccine candidates have shown good results in terms of protection. However, little is known about the host immune mechanisms underlying that protection, especially in wild boar, which is the main reservoir of the disease in Europe. Here, we study the role played by two cytokines (IL-10 and IFN-γ) in wild boar orally inoculated with the attenuated vaccine candidate Lv17/WB/Rie1 and challenged with a virulent ASFv genotype II isolate. A group of naïve wild boar challenged with the latter isolate was also established as a control group. Our results showed that both cytokines play a key role in protecting the host against the challenge virus. While high levels of IL-10 in serum may trigger an immune system malfunctioning in challenged animals, the provision of stable levels of this cytokine over time may help to control the disease. This, together with high and timely induction of IFN-γ by the vaccine candidate, could help protect animals from fatal outcomes. Further studies should be conducted in order to support these preliminary results and confirm the role of these two cytokines as potential markers of the evolution of ASFV infection.

A new method for sampling African swine fever virus genome and its inactivation in environmental samples.

African swine fever (ASF) is currently the most dangerous disease for the global pig industry, causing huge economic losses, due to the lack of effective vaccine or treatment. Only the early detection of ASF virus (ASFV) and proper biosecurity measures are effective to reduce the viral expansion. One of the most widely recognized risks as regards the introduction ASFV into a country is infected animals and contaminated livestock vehicles. In order to improve ASF surveillance, we have assessed the capacity for the detection and inactivation of ASFV genome by using Dry-Sponges (3 M) pre-hydrated with a new surfactant liquid. We sampled different surfaces in ASFV-contaminated facilities, including animal skins, and the results were compared to those obtained using a traditional sampling method. The surfactant liquid successfully inactivated the virus, while ASFV DNA was well preserved for the detection. This is an effective method to systematically recover ASFV DNA from different surfaces and skin, which has a key applied relevance in surveillance of vehicles transporting live animals and greatly improves animal welfare. This method provides an important basis for the detection of ASFV genome that can be assessed without the biosafety requirements of a BSL-3 laboratory at least in ASF-affected countries, which may substantially speed up the early detection of the pathogen.

The spatial ecology of red deer under different land use and management scenarios: Protected areas, mixed farms and fenced hunting estates.

The knowledge regarding the spatial ecology of red deer (Cervus elaphus) in different environments is crucial if effective management actions are to be designed. However, this knowledge continues to be scarce in the complex contexts of mixed land use and management circumstances. This study describes the spatial ecology of red deer monitored using GPS collars in Mediterranean ecosystems of South-Central Spain, considering the effect of individual and seasonal (food shortage period, rut, hunting season and food abundance period) factors on different land use and management scenarios, namely protected areas, mixed farms and fenced hunting estates. Our results showed less activity (ACT), a shorter daily range (DR) and a smaller home range (HR) during the food shortage period: ACT: 0.38 ± (SD) 0.12; DR: 3010.9 ± 727.3 m; and weekly HR: 122.2 ± 59.6 ha. With regard to land use, individuals were less ACT and had a smaller DR on fenced hunting estates (ACT: 0.24 ± 0.12; DR: 1946.3 ± 706.7 m) than in protected areas (ACT: 0.59 ± 0.12; DR: 4071.4 ± 1068.2 m) or on mixed farms (ACT: 0.57 ± 0.29; DR: 5431.1 ± 1939.5 m) in all the periods studied. Red deer selected land cover with forage and shelter when foraging and resting, respectively. When drive hunt events occurred (mixed farms and fenced hunting estates), the deer were more prone to select safer habitats (scrublands) and avoid open areas (crops or grasslands) than were their counterparts in protected areas. The patterns observed can be explained by sexual and seasonal differences as regards requirements, the response to disturbances and, interestingly, population management. Our results provide useful information with which to design scientifically-based species adaptive management in response to relevant and timely situations in Europe, such as the potential transmission of shared infections, vehicle collisions, and damage to crops and ecosystems.

Detection of Hepatitis E Virus in Hyalomma lusitanicum Ticks Feeding on Wild Boars.

Little is known about the role of ticks in maintaining highly prevalent zoonotic viruses in wildlife, such as hepatitis E virus (HEV), which do not require ticks for transmission between animals and humans. In this cross-sectional study, adult female ticks were collected from Eurasian wild boar (Sus scrofa) in autumn 2015 in Spain. HEV RNA in both ticks and wild boar was evaluated by RT-qPCR. Twenty-nine adult Hyalomma lusitanicum ticks were collected from 29 wild boars. HEV RNA was detected in a total of 10 tick (34.4%) and 11 wild boar serum samples (37.9%). In two cases, detectable HEV RNA was found in a wild boar but not in the tick collected from them. In contrast, one HEV-positive tick was collected from an HEV-negative wild boar. All viral sequences were consistent with genotype 3f. We describe for the first time the presence of HEV RNA in adult Hyalomma lusitanicum ticks.

High Doses of Inactivated African Swine Fever Virus Are Safe, but Do Not Confer Protection against a Virulent Challenge.

African swine fever (ASF) is currently the major concern of the global swine industry, as a consequence of which a reconsideration of the containment and prevention measures taken to date is urgently required. A great interest in developing an effective and safe vaccine against ASF virus (ASFV) infection has, therefore, recently appeared. The objective of the present study is to test an inactivated ASFV preparation under a vaccination strategy that has not previously been tested in order to improve its protective effect. The following have been considered: (i) virus inactivation by using a low binary ethyleneimine (BEI) concentration at a low temperature, (ii) the use of new and strong adjuvants; (iii) the use of very high doses (6 × 109 haemadsorption in 50% of infected cultures (HAD50)), and (iv) simultaneous double inoculation by two different routes of administration: intradermal and intramuscular. Five groups of pigs were, therefore, inoculated with BEI- Pol16/DP/OUT21 in different adjuvant formulations, twice with a 4-week interval. Six weeks later, all groups were intramuscularly challenged with 10 HAD50 of the virulent Pol16/DP/OUT21 ASFV isolate. All the animals had clinical signs and pathological findings consistent with ASF. This lack of effectiveness supports the claim that an inactivated virus strategy may not be a viable vaccine option with which to fight ASF.

Safety of African Swine Fever Vaccine Candidate Lv17/WB/Rie1 in Wild Boar: Overdose and Repeated Doses.

African swine fever (ASF) is a highly lethal infectious disease that affects domestic pigs and wild boar. Outbreaks of ASF have grown considerably in the last decade causing important economic consequences for the swine industry. Its control is hampered by the lack of an effective treatment or vaccine. In Europe, the wild boar is a key wild reservoir for ASF. The results of the oral vaccination trial of wild boar with Lv17/WB/Rie1 are hope for this problem. However, this vaccine candidate has certain safety concerns, since it is a naturally attenuated vaccine. Therefore, the current study aims to evaluate the safety of this vaccine candidate in terms of overdose (high dose) and repeated doses (revaccination) in wild boar. Low-dose orally vaccinated animals developed only a slight transient fever after vaccination and revaccination. This was also the case for most of the high-dose vaccinated wild boar, except for one of them which succumbed after revaccination. Although this fatality was related to hierarchical fights between animals, we consider that further studies are required for clarification. Considering these new results and the current epidemiological situation of ASF in wild boar, this vaccine prototype is a promising tool for the control of the disease in these wild populations, although further studies are needed.

Distinct African Swine Fever Virus Shedding in Wild Boar Infected with Virulent and Attenuated Isolates.

Since the reappearance of African swine fever virus (ASFV), the disease has spread in an unprecedented animal pandemic in Eurasia. ASF currently constitutes the greatest global problem for the swine industry. The wild boar (Sus scrofa) in which the pathogen has established wild self-sustaining cycles, is a key reservoir for ASFV, signifying that there is an urgent need to develop an effective vaccine against this virus. Current scientific debate addresses whether live attenuated vaccines (LAVs), which have shown promising results in cross-protection of susceptible hosts, may be feasible for vaccinations carried out owing to safety concerns. The objective of this study was, therefore, to compare the ASFV shedding in wild boar infected with virulent and attenuated (LAV) isolates. Different shedding routes (oral fluid and feces) and viremia rates were characterized in wild boar inoculated with Lv17/WB/Rie1 isolate (n = 12) when compared to those inoculated with the virulent Armenia07 isolate (n = 17). In general, fewer animals infected with the Lv17/WB/Rie1 isolate tested positive for ASFV in blood, oral fluid, and feces in comparison to animals infected with the virulent Armenia07 isolate. The shedding patterns were characterized in order to understand the transmission dynamics. This knowledge will help evaluate the shedding of new LAV candidates in wild boar populations, including the comparison with gene deletion mutant LAVs, whose current results are promising.

Detection of Antibodies against Mycobacterium bovis in Oral Fluid from Eurasian Wild Boar.

The presence of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) is a main concern in wildlife populations such as the Eurasian wild boar (Sus scrofa). Tests detecting antibodies against the MTC are valuable for tuberculosis (TB) monitoring and control and particularly useful in suids. The development of accurate, efficient, and non-invasive new tools to detect exposure to MTC would be highly beneficial for improving disease surveillance. This study aimed to determine if antibodies against MTC could be detected in oral fluid (OF) samples by a new ELISA test (IgG detection) from naturally TB-infected wild boar. For this, individual, paired serum and OF samples were collected from 148 live wild boar in two TB-status areas from Spain and quantitatively used to validate the new ELISA test. Antibodies against MTC were widely detected in OF samples, for which a significant positive correlation (r = 0.83) was found with the validated serology test. OF ELISA sensitivity and specificity were 67.3% and 100%, respectively. The results of this work suggest that OF samples have the potential to be used for MTC diagnosis as a further step in TB surveillance and control in suid populations. Based on our results, further research is warranted and could be performed using non-invasive new tools directly in field conditions to detect exposure to MTC.

Dynamic Network of Interactions in the Wildlife-Livestock Interface in Mediterranean Spain: An Epidemiological Point of View.

The correct management of diseases that are transmitted between wildlife and livestock requires a reliable estimate of the pathogen transmission rate. The calculation of this parameter is a challenge for epidemiologists, since transmission can occur through multiple pathways. The social network analysis is a widely used tool in epidemiology due to its capacity to identify individuals and communities with relevant roles for pathogen transmission. In the present work, we studied the dynamic network of interactions in a complex epidemiological scenario using information from different methodologies. In 2015, nine red deer, seven fallow deer, six wild boar and nine cattle were simultaneously monitored using GPS-GSM-Proximity collars in Doñana National Park. In addition, 16 proximity loggers were set in aggregation points. Using the social network analysis, we studied the dynamic network of interactions, including direct and indirect interactions, between individuals of different species and the potential transmission of pathogens within this network. The results show a high connection between species through indirect interactions, with a marked seasonality in the conformation of new interactions. Within the network, we differentiated four communities that included individuals of all the species. Regarding the transmission of pathogens, we observed the important role that fallow deer could be playing in the maintenance and transmission of pathogens to livestock. The present work shows the need to consider different types of methodologies in order to understand the complete functioning of the network of interactions at the wildlife/livestock interface. It also provides a methodological approach applicable to the management of shared diseases.

Clinical Course and Gross Pathological Findings in Wild Boar Infected with a Highly Virulent Strain of African Swine Fever Virus Genotype II.

African swine fever (ASF) is a notifiable disease that in recent years has spread remarkably in Europe and Asia. Eurasian wild boar (Sus scrofa) plays a key role in the maintenance and spread of the pathogen. Here we examined gross pathology of infection in wild boar with a highly virulent, hemadsorbing genotype II ASF virus (ASFV) strain. To this end, six wild boars were intramuscularly inoculated with the 10 HAD50 Arm07 ASFV strain, and 11 wild boars were allowed to come into direct contact with the inoculated animals. No animals survived the infection. Clinical course, gross pathological findings and viral genome quantification by PCR in tissues did not differ between intramuscularly inoculated or contact-infected animals. Postmortem analysis showed enlargement of liver and spleen; serosanguinous effusion in body cavities; and multiple hemorrhages in lungs, endocardium, brain, kidneys, urinary bladder, pancreas, and alimentary system. These results provide detailed insights into the gross pathology of wild boar infected with a highly virulent genotype II ASFV strain. From a didactic point of view, this detailed clinical course and macroscopic description may be essential for early postmortem detection of outbreaks in wild boar in the field and contribute to disease surveillance and prevention efforts.

Adenovirus-vectored African Swine Fever Virus Antigens Cocktail Is Not Protective against Virulent Arm07 Isolate in Eurasian Wild Boar.

: African swine fever (ASF) is a viral disease of domestic and wild suids for which there is currently no vaccine or treatment available. The recent spread of ASF virus (ASFV) through Europe and Asia is causing enormous economic and animal losses. Unfortunately, the measures taken so far are insufficient and an effective vaccine against ASFV needs to be urgently developed. We hypothesized that immunization with a cocktail of thirty-five rationally selected antigens would improve the protective efficacy of subunit vaccine prototypes given that the combination of fewer immunogenic antigens (between 2 and 22) has failed to elicit protective efficacy. To this end, immunogenicity and efficacy of thirty-five adenovirus-vectored ASFV antigens were evaluated in wild boar. The treated animals were divided into different groups to test the use of BioMize adjuvant and different inoculation strategies. Forty-eight days after priming, the nine treated and two control wild boar were challenged with the virulent ASFV Arm07 isolate. All animals showed clinical signs and pathological findings consistent with ASF. This lack of protection is in line with other studies with subunit vaccine prototypes, demonstrating that there is still much room for improvement to obtain an effective subunit ASFV vaccine.