High-Pressure Processing of Different Tissue Homogenates from Pigs Challenged with the African Swine Fever Virus.

African swine fever (ASF) is a disease that is a growing threat to the global swine industry. Regulations and restrictions are placed on swine movement to limit the spread of the virus. However, these are costly and time-consuming. Therefore, this study aimed to determine if high-pressure processing (HPP) sanitization techniques would be effective against the ASF virus. Here, it was hypothesized that HPP could inactivate or reduce ASF virus infectivity in tissue homogenates. To test this hypothesis, 30 aliquots of each homogenate (spleen, kidney, loin) were challenge-infected with the Turin/83 strain of ASF, at a 10 7.20 median hemadsorption dose (HAD)50/mL. Subsequently, eight aliquots of each homogenate were treated with 600 millipascal (600 MPa) HPP for 3, 5, and 7 min. Six untreated aliquots were used as the controls. Virological results showed a reduction in the viral titer of more than 7-log. These results support the validity of the study hypothesis since HPP treatment was effective in inactivating ASFV in artificially prepared samples. Overall, this study suggests the need for further investigation of other ASFV-contaminated meat products.

Assessment of BoAHV-1 Seronegative Latent Carrier by the Administration of Two Infectious Bovine Rhinotracheitis Live Marker Vaccines in Calves.

Seronegative latent carriers (SNLCs) are animals that carry the virus without detectable antibodies and pose a risk for disease transmission and diagnostic challenges, suggesting the importance of consideration of marker vaccines in managing them. Therefore, in this study, we evaluated two modified live infectious bovine rhinotracheitis (IBR) marker vaccines (single and double deletions) for their ability to generate SNLC calves. These vaccines were administered to four groups (n = 3 in each group) of three-month-old calves in the presence or absence of passive immunity. Three hundred days after the first vaccination and after confirming the IBR seronegativity of all animals, dexamethasone was administered intravenously for five consecutive days. Only animals immunized with the modified live IBR marker vaccine (single deletion) in the absence of passive immunity exhibited a more enduring immune response than those vaccinated in the presence of passive immunity. Moreover, the administration of a modified live IBR marker vaccine (double deletion) to calves with passive immunity generated SNLC. These findings underscore the potential of live IBR marker vaccine (double-deletions) to aid serological diagnostic tools and develop vaccination protocols in achieving the desired immune response, particularly in the context of latent carrier status, offering valuable insights into optimizing vaccination strategies for effective IBR control.

Biological Containment for African Swine Fever (ASF) Laboratories and Animal Facilities: The Italian Challenge in Bridging the Present Regulatory Gap and Enhancing Biosafety and Biosecurity Measures.

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures.

Genomic Characterization of a Wild-Type Bovine alphaherpesvirus 1 (BoAHV-1) Strain Isolated in an Outbreak in Central Italy.

Bovine alphaherpesvirus-1 (BoAHV-1) infection is common in cattle worldwide. However, information on the spread of BoAHV-1-circulating strains in Italy remains limited. In this study, we investigated an outbreak characterized by severe respiratory symptoms in a cattle herd (n = 30) located in Central Italy. BoAHV-1 was isolated from three cattle in a cell culture, which confirmed viral infection. Next, we characterized one (16453/07 TN) of the three isolates of BoAHV-1 using whole-genome sequencing. BLASTn and phylogenetic analysis revealed a nucleotide identity >99% with all BoAHV-1 strains belonging to subtype 1.1, highlighting the genetic stability of the virus. This study reports the first full genomic characterization of a BoAHV-1 isolate in Italy, enriching our understanding of the genetic characteristics of the circulating BoAHV-1 strain in Italy.

The Production of Recombinant African Swine Fever Virus Lv17/WB/Rie1 Strains and Their In Vitro and In Vivo Characterizations.

Lv17/WB/Rie1-Δ24 was produced via illegitimate recombination mediated by low-dilution serial passage in the Cos7 cell line and isolated on PAM cell culture. The virus contains a huge ~26.4 Kb deletion in the left end of its genome. Lv17/WB/Rie1-ΔCD-ΔGL was generated via homologous recombination, crossing two ASFV strains (Lv17/WB/Rie1-ΔCD and Lv17/WB/Rie1-ΔGL containing eGFP and mCherry markers) during PAM co-infection. The presence of unique parental markers in the Lv17/WB/Rie1-ΔCD-ΔGL genome indicates at least two recombination events during the crossing, suggesting that homologous recombination is a relatively frequent event in the ASFV genome during replication in PAM. Pigs infected with Lv17/WB/Rie1-Δ24 and Lv17/WB/Rie1/ΔCD-ΔGL strains have shown mild clinical signs despite that ASFV could not be detected in their sera until a challenge infection with the Armenia/07 ASFV strain. The two viruses were not able to induce protective immunity in pigs against a virulent Armenia/07 challenge.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune-pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig's immune system. A better understanding of these immune-pathological mechanisms would help to design suitable vaccines against this disease.

Endogenous and viral microRNAs in nasal secretions of water buffaloes (Bubalus bubalis) after Bubaline alphaherpesvirus 1 (BuHV-1) challenge infection.

Bubaline alphaherpesvirus 1 (BuHV-1) is a pathogen of water buffaloes responsible for economic loss worldwide. MicroRNAs (miRNAs) regulate gene expression produced by alphaherpesviruses and hosts. This study aimed at (a) unravelling the ability of BuHV-1 to produce miRNAs, including hv1-miR-B6, hv1-miR-B8, hv1-miR-B9; (b) measuring the host immune-related miRNAs associated to herpesvirus infection, including miR-210-3p, miR-490-3p, miR-17-5p, miR-148a-3p, miR-338-3p, miR-370-3p, by RT-qPCR; (c) identifying candidate markers of infection by receiver-operating characteristic (ROC) curves; (d) exploiting the biological functions by pathway enrichment analyses. Five water buffaloes BuHV-1 and Bovine alphaherpesvirus 1 (BoHV-1) free were immunized against Infectious Bovine Rhinotracheitis (IBR). Five additional water buffaloes served as negative controls. All animals were challenged with a virulent wild-type (wt) BuHV-1 via the intranasal route 120 days after the first vaccination. Nasal swabs were obtained at days (d) 0, 2, 4, 7, 10, 15, 30, and 63 post-challenge (pc). The animals of both groups shed wt BuHV-1 up to d7 pc. Results demonstrated that (a) miRNAs produced by the host and BuHV-1 could be efficiently quantified in the nasal secretion up to d63 and d15 pc, respectively; b) the levels of host and BuHV-1 miRNAs are different between vaccinated and control buffaloes; c) miR-370-3p discriminated vaccinated and control animals; d) host immune-related miRNAs may modulate genes involved in the cell adhesion pathway of the neuronal and immune system. Overall, the present study provides evidence that miRNAs can be detected in nasal secretions of water buffaloes and that their expression is modulated by BuHV-1.

Evaluation of an Immunization Protocol Using Bovine Alphaherpesvirus 1 gE-Deleted Marker Vaccines against Bubaline Alphaherpesvirus 1 in Water Buffaloes.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.

The Cell-Mediated Immune Response against Bovine alphaherpesvirus 1 (BoHV-1) Infection and Vaccination.

Bovine Alphaherpesvirus 1 (BoHV-1) is one of the major respiratory pathogens in cattle worldwide. Infection often leads to a compromised host immune response that contributes to the development of the polymicrobial disease known as "bovine respiratory disease". After an initial transient phase of immunosuppression, cattle recover from the disease. This is due to the development of both innate and adaptive immune responses. With respect to adaptive immunity, both humoral and cell-mediated immunity are required to control infection. Thus, several BoHV-1 vaccines are designed to trigger both branches of the adaptive immune system. In this review, we summarize the current knowledge on cell-mediated immune responses directed against BoHV-1 infection and vaccination.

Involvement of the MGF 110-11L Gene in the African Swine Fever Replication and Virulence.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease that causes extensive economic and animal welfare losses in the Eurasian pig (Sus scrofa) population. To date, no effective and safe vaccines have been marketed against ASF. A starting point for vaccine development is using naturally occurring attenuated strains as a vaccine base. Here, we aimed to remove the multigene family (MGF) 110 gene of unknown function from the Lv17/WB/Rie1 genome to improve the usability of the virus as a live-attenuated vaccine, reducing unwanted side effects. The MGF 110-11L gene was deleted using the CRISPR/Cas9 method, and the safety and efficacy of the virus were tested in pigs after isolation. The vaccine candidates administered at high doses showed reduced pathogenicity compared to the parental strain and induced immunity in vaccinated animals, although several mild clinical signs were observed. Although Lv17/WB/Rie1/d110-11L cannot be used as a vaccine in its current form, it was encouraging that the undesirable side effects of Lv17/WB/Rie1 at high doses can be reduced by additional mutations without a significant reduction in its protective capacity.

Assessment of Different Infectious Bovine Rhinotracheitis Marker Vaccines in Calves.

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

Disinfectants against African Swine Fever: An Updated Review.

African Swine Fever (ASF), a hemorrhagic disease with a high mortality rate in suids, is transmitted via direct and indirect contact with infectious animals and contaminated fomites, respectively. ASF reached Europe in 2014, affecting 14 of the 27 EU countries including, recently, the Italian peninsula. The fast and unprecedented spread of ASF in the EU has highlighted gaps in knowledge regarding transmission mechanisms. Fomites, such as contaminated clothing and footwear, farming tools, equipment and vehicles have been widely reported in the spread of ASF. The absence of available vaccines renders biosecurity measures, cleaning and disinfection procedures an essential control tool, to a greater degree than the others, for the prevention of primary and secondary introductions of ASF in pig farms. In this review, available data on the virucidal activity of chemical compounds as disinfectants against the ASF virus (ASFV) are summarized together with laboratory methods adopted to assess the virucidal activity.

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet's agreement coefficient of 0.99 (95% confidence interval (CI): 0.96−1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3−100%) and 97.5% (95% CI: 91.3−99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen's κ: 0.96, 95% CI: 0.78−1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Herpesvirus Vaccines.

The Special Issue titled "Herpesvirus Vaccines" contains different articles and a review regarding veterinary and human herpesviruses [...].

Corrigendum: Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy.

[This corrects the article DOI: 10.3389/fvets.2021.665607.].

Enzootic Bovine Leukosis in Italy: Epidemiological Issues after Free Status Recognition and Measures Applied to Tackle the Last Persistent Clusters.

Enzootic Bovine Leukosis (EBL), caused by the bovine leukemia virus (BLV), has been eradicated in over 20 countries, most of which are in Western Europe. The European Commission, in 2017, declared Italy to be an officially EBL-free country by means of Commission Implementing Decision (EU) 2017/1910, despite the presence of some infection clusters located in four regions of Central-Southern Italy. As a consequence of persisting infection, the Italian Ministry of Health established specific eradication measures in these areas. In collaboration with the National Reference Laboratory for the Study of Ruminant Retroviral Infectious Diseases, the Ministry of Health employed data from the veterinary information system digital platform, combined with a gap analysis exercise, to monitor and verify the progress of control activities within infection clusters during the period 2018-2021. Our aim was to identify any remaining gaps and, consequently, specific measures to eliminate the factors favouring EBL persistence, on the basis of a description and analysis of the current data regarding epidemiological trends in Italian clusters. The final goal is to achieve the implementation of a less expensive surveillance plan in these areas, as well. The results of comprehensive analysis showed that the eradication activities had been effectively implemented by official local veterinary services, resulting in a drastic reduction of EBL outbreaks in most territories during the period 2018-2021.

Global Distribution and Genetic Heterogeneity of Border Disease Virus.

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae. Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy

The cattle industry is a major driving force for the Italian agricultural sector totalling about 5. 6 million heads for dairy and meat production together. It is particularly developed in the northern part of the country, where 70% of the whole Italian cattle population is reared. The cattle industry development in the rest of the country is hampered by the hard orography of the territories and a variety of socioeconomic features leading to the persistence of the traditional rural farming systems. The differences in the farming systems (industrial vs. traditional) also affect the health status of the farms. Whereas, Enzootic Bovine Leukosis (EBL) is almost eradicated across the whole country, in Southern Italy where Bovine Tuberculosis and Brucellosis are still present and Bluetongue is endemic due to the presence of the competent vector (Culicoides imicola), less investments are aimed at controlling diseases with economic impact or at improving farm biosecurity. On the other hand, with the eradication of these diseases in most part of the country, the need has emerged for reducing the economic burden of non-regulated endemic disease and control programs (CPs) for specific diseases have been implemented at regional level, based on the needs of each territory (for instance common grazing or trading with neighboring countries). This explains the coexistence of different types of programs in force throughout the country. Nowadays in Italy, among cattle diseases with little or no EU regulations only three are regulated by a national CP: Enzootic Bovine Leukosis, Bluetongue and Paratuberculosis, while Bovine Genital Campylobacteriosis and Trichomonosis are nationwide controlled only in breeding bulls. For some of the remaining diseases (Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea, Streptococcus agalactiae) specific CPs have been implemented by the regional Authorities, but for most of them a CP does not exist at all. However, there is a growing awareness among farmers and public health authorities that animal diseases have a major impact not only on the farm profitability but also on animal welfare and on the use of antibiotics in livestock. It is probable that in the near future other CPs will be implemented.

Evaluation of Safety and Efficacy of an Inactivated Marker Vaccine against Bovine alphaherpesvirus 1 (BoHV-1) in Water Buffalo (Bubalus bubalis).

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.