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.

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.

A retrospective study of SARS-CoV-2 seroprevalence in dogs and cats in the Community of Madrid, Spain.

To date, susceptibility to SARS-CoV-2 infection in domestic animals including cats and dogs has been described. However, it is important to carry out passive surveillance of these animals to be aware of any changes in the outcomes of the disease in these species that may occur. In this study, we have performed a retrospective study in which we analyzed sera (n = 1,640) from random animals: dogs (n = 1,381) and cats (n = 259) belonging to both homes (n = 1,533) and animal protection centers (n = 107) in the Community of Madrid, Spain. Neutralizing antibodies were evaluated between November 2021 and May 2022 using a surrogate ELISA kit to determine the seroprevalence. Based on the results obtained, a few animals (both cats and dogs) presented neutralizing antibodies to SARS-CoV-2 (2.3%), all of them from private owners. However, the seroprevalence in cats (4.6%) resulted to be almost twice as much as in dogs (1.9%) which reinforces that cats' susceptibility to the infection seems higher than in the case of dogs, maybe due to the lower ACE2 expression of the dogs in the respiratory tract. These findings also confirm that the probability of infection is considerably higher in domestic animals in close contact with infected owners, compared to animals living in animal shelters whose contact with humans is markedly lower.

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.

Importance of genomic surveillance of SARS-CoV-2 in cats during reverse zoonosis events: potential viral evolution may occur.

The apparition of new variants of severe acute respiratory syndrome coronavirus 2 and lineages is constantly happening because of the high viral mutation rate. Since numerous reverse zoonosis events have been reported so far, genomic surveillance should be conducted in susceptible species to evaluate potential adaptations that may trigger the apparition of new variants. Here, we evaluate the evolution of the infection in a cat naturally infected in parallel with its owner, performing a comparative phylogenetic analysis. Sequencing analysis showed that both were infected with the Omicron BA.5/BF.1 lineage and revealed the presence of nucleotide substitution in the viral genome recovered from the cat with respect to the viral genome from the human sample. This nucleotide substitution (C11897A) produced the amino acid change Orf1a: Q3878K. Therefore, genomic surveillance in the case of reverse zoonosis events is still necessary in order to control possible adaptations of the virus to other susceptible species. IMPORTANCE Genomic surveillance of pets for severe acute respiratory syndrome coronavirus 2 is important to monitor the emergence of new variants of the virus associated with these animals. Pets can serve as a potential reservoir for the virus, and their close contact with humans increases the risk of transmission. By conducting genomic surveillance in pets, it is possible to detect and track new variants early on, allowing for more effective control measures to be put in place. This can help prevent the spread of these variants to human populations and potentially mitigate the impact of the pandemic. Furthermore, it may also provide insight into the evolution and spread of the virus within the animal population.

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.

The Omicron (B.1.1.529) SARS-CoV-2 variant of concern also affects companion animals.

The emergence of the Omicron variant (B.1. 1.529) has brought with it an increase in the incidence of SARS-CoV-2 disease. However, there is hardly any data on its incidence in companion animals. We have detected the presence of this new variant in domestic animals (dogs and cats) living with infected owners in Spain. None of the RT-qPCR positive animals (10.13%) presented any clinical signs and the viral loads detected were low. In addition, the shedding of viral RNA lasted a short period of time in the positive animals. Infection with this variant of concern (VOC) was confirmed by RT-qPCR and sequencing. These outcomes suggest a lower virulence of this variant in infected cats and dogs. They also demonstrate the transmission from infected humans to domestic animals and highlight the importance of active surveillance as well as genomic research to detect the presence of VOCs or mutations associated with animal hosts.

A subunit vaccine candidate based on the Spike protein of SARS-CoV-2 prevents infectious virus shedding in cats.

Of the numerous animal species affected by the SARS-CoV-2 virus, cats are one of the most susceptible, and cat-to-cat transmission has been described. Although cat-to-human infection has not, as yet, been demonstrated, preventive measures should be taken in order to avoid both viral infection in cats and transmission among them. In this respect, the application of an effective vaccine to at-risk populations would be a useful tool for controlling the disease in this species. Here, we test a new vaccine prototype based on the Spike protein of the virus in order to prevent infection and infectious virus shedding in cats. The vaccine employed in experimentation, and which is easily produced, triggered a strong neutralizing antibody response in vaccinated animals. In contrast to that which occurred with control animals, no infectious virus was detected in the oropharyngeal or rectal swabs of vaccinated cats submitted to a SARS-CoV-2 challenge. These results are of great interest as regards future considerations related to implementing vaccination programs in pets. The value of cats as vaccination trial models is also described herein.

First Detection of SARS-CoV-2 B.1.617.2 (Delta) Variant of Concern in a Symptomatic Cat in Spain.

Natural and experimental SARS-CoV-2 infection in pets has been widely evidenced since the beginning of the COVID-19 pandemic. Among the numerous affected animals, cats are one of the most susceptible species. However, little is known about viral pathogenicity and transmissibility in the case of variants of concern (VOCs) in animal hosts, such as the B.1.617.2 (Delta) variant first detected in India. Here, we have identified the B.1.617.2 (Delta) VOC in a cat living with a COVID-19 positive owner. The animal presented mild symptoms (sneezing) and a high viral load was detected in the oropharyngeal swab, suggesting that an active infection was occurring in the upper respiratory tract of the cat. Transmission from the owner to the cat occurred despite the human being fully vaccinated against SARS-CoV-2. This study documents the first detection of B.1.165.2 VOC in a cat in Spain and emphasizes the importance of performing active surveillance and genomic investigation on infected animals.

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.

Large-scale study on virological and serological prevalence of SARS-CoV-2 in cats and dogs in Spain.

The disease produced by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is currently one of the primary concerns worldwide. Knowing the zoonotic origin of the disease and that several animal species, including dogs and cats, are susceptible to viral infection, it is critical to assess the relevance of pets in this pandemic. Here, we performed a large-scale study on SARS-CoV-2 serological and viral prevalence in cats and dogs in Spain in order to elucidate their role and susceptibility. Samples from animals in contact with COVID-19 positive people and/or compatible symptoms (n = 492), as well as from random animals (n = 1024), were taken. Despite the large number of animals analyzed, only 12 animals (eight dogs and four cats), which represents 0.79% of the total analyzed animals (n = 1516), were positive for viral SARS-CoV-2 RNA detection by reverse transcription quantitative PCR (RT-qPCR) in which viral isolation was possible in four animals. We detected neutralizing antibodies in 34 animals, four of them were also positive for PCR. This study evidences that pets are susceptible to SARS-CoV-2 infection in natural conditions but at a low level, as evidenced by the low percentage of positive animals detected, being infected humans the main source of infection. However, the inclusion of animals in the surveillance of COVID-19 is still recommended.

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.

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.

First Detection of SARS-CoV-2 B.1.1.7 Variant of Concern in an Asymptomatic Dog in Spain.

Natural SARS-CoV-2 infection in pets has been widely documented during the last year. Although the majority of reports suggested that dogs' susceptibility to the infection is low, little is known about viral pathogenicity and transmissibility in the case of variants of concern, such as B.1.1.7 in this species. Here, as part of a large-scale study on SARS-CoV-2 prevalence in pets in Spain, we have detected the B.1.1.7 variant of concern (VOC) in a dog whose owners were infected with SARS-CoV-2. The animal did not present any symptoms, but viral loads were high in the nasal and rectal swabs. In addition, viral isolation was possible from both swabs, demonstrating that the dog was shedding infectious virus. Seroconversion occurred 23 days after the first sampling. This study documents the first detection of B.1.1.7 VOC in a dog in Spain and emphasizes the importance of performing active surveillance and genomic investigation on infected animals.

Environment, vector, or host? Using machine learning to untangle the mechanisms driving arbovirus outbreaks.

Climatic, landscape, and host features are critical components in shaping outbreaks of vector-borne diseases. However, the relationship between the outbreaks of vector-borne pathogens and their environmental drivers is typically complicated, nonlinear, and may vary by taxonomic units below the species level (e.g., strain or serotype). Here, we aim to untangle how these complex forces shape the risk of outbreaks of Bluetongue virus (BTV); a vector-borne pathogen that is continuously emerging and re-emerging across Europe, with severe economic implications. We tested if the ecological predictors of BTV outbreak risk were serotype-specific by examining the most prevalent serotypes recorded in Europe (1, 4, and 8). We used a robust machine learning (ML) pipeline and 23 relevant environmental features to fit predictive models to 24,245 outbreaks reported in 25 European countries between 2000 and 2019. Our ML models demonstrated high predictive performance for all BTV serotypes (accuracies > 0.87) and revealed strong nonlinear relationships between BTV outbreak risk and environmental and host features. Serotype-specific analysis suggests, however, that each of the major serotypes (1, 4, and 8) had a unique outbreak risk profile. For example, temperature and midge abundance were as the most important characteristics shaping serotype 1, whereas for serotype 4 goat density and temperature were more important. We were also able to identify strong interactive effects between environmental and host characteristics that were also serotype specific. Our ML pipeline was able to reveal more in-depth insights into the complex epidemiology of BTVs and can guide policymakers in intervention strategies to help reduce the economic implications and social cost of this important pathogen.

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.

Natural SARS-CoV-2 Infection in Kept Ferrets, Spain.

We found severe acute respiratory syndrome coronavirus 2 RNA in 6 (8.4%) of 71 ferrets in central Spain and isolated and sequenced virus from 1 oral and 1 rectal swab specimen. Natural infection occurs in kept ferrets when virus circulation among humans is high. However, small ferret collections probably cannot maintain virus circulation.

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.

Global emergence and evolutionary dynamics of bluetongue virus.

Bluetongue virus (BTV) epidemics are responsible for worldwide economic losses of up to US$ 3 billion. Understanding the global evolutionary epidemiology of BTV is critical in designing intervention programs. Here we employed phylodynamic models to quantify the evolutionary characteristics, spatiotemporal origins, and multi-host transmission dynamics of BTV across the globe. We inferred that goats are the ancestral hosts for BTV but are less likely to be important for cross-species transmission, sheep and cattle continue to be important for the transmission and maintenance of infection between other species. Our models pointed to China and India, countries with the highest population of goats, as the likely ancestral country for BTV emergence and dispersal worldwide over 1000 years ago. However, the increased diversification and dispersal of BTV coincided with the initiation of transcontinental livestock trade after the 1850s. Our analysis uncovered important epidemiological aspects of BTV that may guide future molecular surveillance of BTV.