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 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.

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.

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.

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.

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.

Serological Cross-Reactivity Between Bovine alphaherpesvirus 2 and Bovine alphaherpesvirus 1 in a gB-ELISA: A Case Report in Italy.

In this study, we demonstrated for the first time in Italy, the serological cross-reactivity between Bovine alphaherpesvirus 2 (BoHV-2) and Bovine alphaherpesvirus 1 (BoHV-1). Five months after arriving at a performance test station in Central Italy, a 6-month-old calf, which was part of a group of 57 animals, tested positive for BoHV-1 in a commercial gB-ELISA test. It was immediately transferred to the quarantine unit and subjected to clinical observation and serological and virological investigations. During this period, the calf showed no clinical signs. The results from laboratory investigations demonstrated the presence of antibodies via competitive glycoprotein B (gB) ELISAs, indirect BoHV-1 ELISAs, and indirect BoHV-2 ELISAs. Furthermore, the plaque reduction assay provided evidence for the presence of antibodies only for BoHV-2, whereas the virus neutralization test showed negative results for both BoHV-1 and BoHV-5. These findings strongly suggest the occurrence of a serological cross-reactivity between BoHV-2 and BoHV-1. Interference of BoHV-2 antibodies in serological BoHV-1 diagnostics should be considered during routine IBR tests, especially when animals are kept in a performance test station.

Cross-Reactivity Antibody Response after Vaccination with Modified Live and Killed Bovine Viral Diarrhoea Virus (BVD) Vaccines.

Pestivirus A or bovine viral diarrhoea virus (BVDV) type 1 is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. The objective of the present study was to verify whether animals immunised with four commercial vaccines also developed a protective humoral immunity against other viral subgenotypes than those contained in each vaccine. Four groups of 25 bovines each were formed and vaccinated according to the manufacturer's instructions of the commercial vaccines. On sera collected 28 days after the last vaccination, virus neutralisation tests (VNT) were performed using homologous and heterologous viruses and enzyme-linked immunosorbent assay (ELISA) methods. Finally, the VNT results were comparatively evaluated through a statistical analysis. Serological results highlighted that, although with a different degree of efficiency, the four vaccines resulted in not developing a solid antibody-mediated cross-immunity against all the strains used.

Evaluation of Passive Immunity Induced by Immunisation Using Two Inactivated gE-deleted Marker Vaccines against Infectious Bovine Rhinotracheitis (IBR) in Calves.

Different types of vaccines against Infectious Bovine Rhinotracheitis (IBR) are commercially available. Among these, inactivated glycoprotein E (gE)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. Here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated gE-deleted marker vaccines to calves. We vaccinated 12 pregnant cattle devoid of neutralising antibodies against Bovine alphaherpesvirus 1 (BoHV-1) and divided them into two groups with 6 animals each. Both groups were injected with a different inactivated gE-deleted marker vaccine administrated via intranasal or intramuscular routes. An additional 6 pregnant cattle served as the unvaccinated control group. After calving, the number of animals in each group was increased by the newborn calves. In the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day 180 (PCD180). In addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until PCD180. The results indicated that inactivated glycoprotein E (gE)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and PCD180. Moreover, results showed that, in calf serum, passive immunity persists until PCD180.

Welfare Assessment in Shelter Dogs by Using Physiological and Immunological Parameters.

This study aimed to evaluate the state of welfare of a group of dogs during the first month after entering the shelter by using different stress parameters. Blood and fecal samples were collected from a group of 71 dogs at the time of admission to the shelter. In 46 of these dogs, sampling was repeated after four weeks. Well-recognized welfare biomarkers, such as fecal cortisol and leukocytes, as well as some innovative parameters (ß-endorphin and lysozyme) were determined. Uni- and multivariate statistical analyses were used to evaluate their interactions and changes over time. Neutrophils (p < 0.01), lysozyme (p < 0.05), and fecal cortisol (p < 0.05) decreased, while lymphocytes (p < 0.05) increased after four weeks compared to the first days of being in the shelter, suggesting an improvement in the dogs' welfare over time. A principal component analysis extracted three bipolar components (PCs), explaining 75% of the variance and indicating negative associations between neutrophil and lymphocyte (PC1), lysozyme and ß-endorphin (PC2), cortisol and lysozyme (PC3). The associations between these variables within each PC also confirmed the intricate relationships between the hypothalamic-pituitary-adrenal (HPA) axis and the immune system as well as the importance of a multiparametric approach in evaluating welfare.

Antibody Responses to Bovine Alphaherpesvirus 1 (BoHV-1) in Passively Immunized Calves.

To date, in countries where infectious bovine rhinotracheitis (IBR) is widespread, its control is associated with deleted marker vaccines. These products lack one or more genes responsible for the synthesis of glycoproteins or enzymes. In Europe, the most widely used marker vaccine is one in which glycoprotein E (gE-) is deleted, and it is marketed in a killed or modified-live form. Using this type of immunization, it is possible to differentiate vaccinated animals (gE-) from those infected or injected with non-deleted (gE+) products using diagnostic tests specific for gE. The disadvantage of using modified-live gE-products is that they may remain latent in immunized animals and be reactivated or excreted following an immunosuppressive stimulus. For this reason, in the last few years, a new marker vaccine became commercially available containing a double deletion related to genes coding for gE and the synthesis of the thymidine-kinase (tk) enzyme, the latter being associated with the reduction of the neurotropism, latency, and reactivation of the vaccine virus. Intramuscularly and intranasally administered marker products induce a humoral immune response; however, the mother-to-calf antibody kinetics after vaccination with marker vaccines is poorly understood. This review discusses several published articles on this topic.

Contrast-enhanced ultrasonography of maternal and fetal blood flows in pregnant bitches.

We evaluated the potential usefulness of CEUS to assess fetal-maternal circulation during pregnancy in dogs. Nine bitches were examined at 23, 30, and 45 days of gestation using an ultrasound machine (LOGIQ E9) and SonoVue® contrast media as echo-signal enhancer. Qualitative and quantitative evaluation of contrast enhancement patterns of uterine artery and utero/placental vessels were performed on recorded images. Independently of the gestational periods, the qualitative evaluation showed the initial wash-in phase from the first appearance of the uterine artery to the rapid distribution in embryonic vesicles or placenta to the progressive washout, whilst there was no enhancement of either embryos or fetuses in any bitch. Independent of gestational age, parameters derived from quantitative analysis of time intensity-curves of contrast enhancement (peak intensity, time to peak, rise time, washout) did not vary between proximal placenta, distal placenta, and uterine artery. With the progression of gestation, AUC values did not change in both proximal and distal placenta, but in the uterine artery it was lower (P ≤ 0.05) at day 30 than at day 23 (464.8 ±â€¯16.1 vs.596.4 ±â€¯28.1, respectively). In conclusion, CEUS appears to safely permit evaluation of the maternal and fetal vessels in the first two third of gestation, without any clinically relevant adverse effects. Further studies in a larger number of bitches in different stages of pregnancy are needed to establish standard parameters for normal pregnancies that can be used to detect abnormalities of pregnancy.