Bovine Herpesvirus Research Articles

Development of a Synthesized Gene Unique to Lumpy Skin Disease Virus and Its Application in Serological Differentiation of Naturally Infected from Vaccinated Cattle with Attenuated Goat Pox Vaccine

Lumpy skin disease (LSD) is an important infectious disease caused by lumpy skin disease virus (LSDV) in bovine. LSDV, sheep pox virus (SPPV), and goat pox virus (GTPV) from the same genus Capripoxvirus (CaPV) of the Poxviridae family exhibit a nucleotide sequence similarity of up to 97%. Therefore, attenuated vaccines of GTPV and SPPV are often used to vaccinate cattle against LSD. However, available serological testing methods cannot accurately differentiate cattle vaccinated with GTPV from those infected with LSDV, posing a significant risk for disease spread. In this study, we developed a synthesized gene unique to LSDV as a differential antigen to detect serum antibodies specific to LSDV and differentiate naturally infected from vaccinated animals (DIVA). We used it for an in‐house indirect enzyme‐linked immunosorbent assay (iELISA), and no cross‐reaction with positive sera for bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Mycobacterium bovis (M. Tb), Pasteurella multocida (P. multocida), and Mycoplasma bovis (M. bovis). The cut‐off value (S/P%) was 30% for in‐house iELISA. The corresponding diagnostic specificity was 100% (95% CI: 88.43–100), and the diagnostic sensitivity was 93.3% (95% CI: 77.93–99.18). The intra‐assay coefficient of variation (CV) ranged from 1.08% to 4.11%, and the interassay CV was 0.00%–8.90%. Furthermore, 200 clinical serum samples were examined, in the vaccinated herd, there were no positive samples (0/141) indicating the strong differentiation ability of this method. On the other hand, in the infected herds, the overall positivity was 33.90% (20/59) (95% CI: 22.08–47.39). In summary, a valuable synthesized protein unique to LSDV was developed and showed a promising application in an iELISA with high specificity and sensitivity in differentiating cattle infected with LSDV from those vaccinated with GTPV.

Haematological and serum biochemical profile of bovines infected with infectious bovine rhinotracheitis

Haematological and serum biochemical profile of bovines infected with infectious bovine rhinotracheitis

Virus as Teratogenic Agents.

Viral infectious diseases are important causes of reproductive disorders, as abortion, fetal mummification, embryonic mortality, stillbirth, and congenital abnormalities in animals and in humans. In this chapter, we provide an overview of some virus, as important agents in teratology.We begin by describing the Zika virus, whose infection in humans had a very significant impact in recent years and has been associated with major health problems worldwide. This virus is a teratogenic agent in humans and has been classified as a public health emergency of international concern (PHEIC).Then, some viruses associated with reproductive abnormalities on animals, which have a significant economic impact on livestock, are described, as bovine herpesvirus, bovine viral diarrhea virus, Schmallenberg virus, Akabane virus, and Aino virus.For all viruses mentioned in this chapter, the teratogenic effects and the congenital malformations associated with fetus and newborn are described, according to the most recent scientific publications.

Reactivity Induced by Inactivated Infectious Bovine Rhinotracheitis Vaccination and Its Related Factors in Holstein Calves Following A Modified Live Vaccine Schedule

Reactivity Induced by Inactivated Infectious Bovine Rhinotracheitis Vaccination and Its Related Factors in Holstein Calves Following A Modified Live Vaccine Schedule

Genetic characterisation of Indian bovine herpesvirus-1 with other ruminant herpesviruses in relation to glycoproteins C

Genetic characterisation of Indian bovine herpesvirus-1 with other ruminant herpesviruses in relation to glycoproteins C

Resposta anamnéstica contra o vírus da diarreia viral bovina e herpesvírus bovino tipo 1 em novilhas Holandesas jovens vacinadas com quatro diferentes formulações comerciais

ABSTRACT: This study evaluated the vaccine-induced serological response after administering four commercial formulations containing Bovine Viral Diarrhea Virus (BVDV) type-1, BVDV-2, and Bovine Herpesvirus type 1 (BoHV-1) to young heifers with circulating maternal antibody titers. The study also determined the anamnestic response to vaccinations after the complete metabolization of maternal antibodies when the calves reached six months. Eighty-seven Holstein heifers were selected and randomly distributed into four experimental groups based on the vaccine administered. The four vaccine-based experimental groups were as follows: vaccine A (n = 22), vaccine B (n = 22), vaccine C (n = 24), and vaccine D (n = 19), given on the 60th, 90th, and 180th day of life, respectively. Virus-neutralization (VN) tests were performed at baseline (day 60) and 30 days after administering the second (day 120) and third doses (day 210). We analyzed the effect of vaccine group (P ≤ 0.001), day of vaccination (P ≤ 0.001), and group versus vaccine day interaction (P ≤ 0.001) for antibody titers produced against BVDV-1, BVDV-2, and BoHV-1 using the PROC MIXED method (Statistical Analysis System -SAS 9.4). Antibody titers against BVDV-1, BVDV-2, and BoHV-1 were similar at baseline and on day 60 of life. The mean antibody titers were constant and persisted against BVDV-1 in heifers immunized with vaccines A and C. Heifers immunized with vaccine A alone had a similar effect against BVDV-2. Regarding BoHV-1, the antibody titers decreased between days 60 and 210 in groups B, C, and D. The antibody titer for heifers in group A also decreased between days 60 and day 120, and an intense increase in titers was observed on day 210. After being immunized with formulations B, C, and D, the frequency of animals with titers above protective levels for BVDV-1, BVDV-2, and BoHV-1 was very low or null. Indicators of anamnestic response were observed in heifers vaccinated with formulation A only. Therefore, it can be concluded that passive immunity negatively interferes with antibody production after vaccination.

WhatsApp como ferramenta para identificação de doenças em ruminantes e equinos não diagnosticadas e subnotificadas em Mato Grosso do Sul

ABSTRACT: This study explores the use of WhatsApp as a tool for identifying previously undiagnosed and underreported diseases in ruminants and horses in Mato Grosso do Sul, Brazil. The research, carried out from January 2022 to December 2023, involved a WhatsApp group of 302 public service and private sector veterinarians. The contacts were categorized as those that resulted in a necropsy by the veterinary diagnostic laboratory, the sending of a sample for histopathological analysis, or the non-sending of material. The results demonstrate the efficiency of using WhatsApp for diagnosing underreported diseases such as anaplasmosis, haemonchosis, rabies, blackleg disease, Mannheimia haemolytica pneumonia, bovine herpesvirus encephalitis, multiple mineral deficiency in cattle and copper poisoning in sheep, thus increasing diagnostic efficiency for veterinary laboratories. The tool also facilitated communication, resulting in an increase in conclusive diagnoses, the identification of previously undiagnosed diseases such as Chlamydia pecorum encephalomyelitis in buffalo, peripheral neuropathy in cattle, phosphorus deficiency in cattle, vitamin A deficiency, and perinatal pneumonia caused by Salmonella enterica subsp. arizonae in cattle. In addition, it was possible to identify underreported occurrences, such as cases of abortion and neonatal diarrhea and to implement the correction of diagnostic approaches in real-time via WhatsApp. The study concludes that WhatsApp improves communication between field veterinarians and diagnostic laboratories, providing valuable information on disease occurrence and diagnostic challenges.

Investigation of the immunomodulatory effect of inactive parapoxvirus (iPPVO) on infectious bovine rhinotracheitis (IBR) vaccine in cattle

In the study, alterations in antibody titers, proinflammatory and antiinflammatory cytokine levels were determined in serum samples collected at various periods before and after administration of inactive IBR and iPPVO to cattle. It was aimed to investigate the immunomodulatory effects of inactivated parapoxvirus ovis (iPPVO) in cattle vaccinated with inactivated-IBR vaccine. In the study, 40 unvaccinated, clinically healthy cattle of different breeds aged older then 3 months were used. Three groups of cattle were formed as control group 1 (n=10), control group 2 (n=10) and experimental group (n=20). iPPVO was applied to the cattle in the control group 1 and experimental groups on the 0, 2nd, and 4th days. Blood samples were colleted from all the animals after 6th hours of the injections applied on 0th and 4th days of the study. Commercially available ELISA kits were used to determine serum levels of of IL-2, IL-6, IL-12 and IFN-γ. Furthermore, virus neutralization test was also performed to detect virus neutralizing antibody titres. In the present study, serum levels of IL-2, IL-6 IL-12, and IFN-γ levels were found to be significantly higher in the experimental group compared to that of the control group 1 and control group 2 (p<0,05). The differences between control group 1 and control group 2 groups were not statistically significant. In conclusion, iPPVO increased the levels of cytokines in IBR vaccinated cattle due to its immunomodulatory effects. In addition, virüs neutralizing antibody titers were found to be significantly higher in cattle that received vaccine and iPPVO.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

An intrinsic network of polar interactions is responsible for binding of UL49.5 C-degron by the CRL2KLHDC3 ubiquitin ligase.

Bovine herpesvirus type 1 (BoHV-1) is a pathogen of cattle responsible for infectious bovine rhinotracheitis. The BoHV-1 UL49.5 is a transmembrane protein that binds to the transporter associated with antigen processing (TAP) and downregulates cell surface expression of the antigenic peptide complexes with the major histocompatibility complex class I (MHC-I). KLHDC3 is a kelch domain-containing protein 3 and a substrate receptor of a cullin2-RING (CRL2) E3 ubiquitin ligase. Recently, it has been identified that CRL2KLHDC3 is responsible for UL49.5-triggered TAP degradation via a C-degron pathway and the presence of the degron sequence does not lead to the degradation of UL49.5 itself. The molecular modeling of KLHDC3 in complexes with four UL49.5 C-terminal decapeptides (one native protein and three mutants) revealed their activity to be closely correlated with the conformation which they adopt in KLHDC3 binding cleft. To analyze the interaction between UL49.5 and KLHDC3 in detail, in this work a total of 3.6 μs long molecular dynamics simulations have been performed. The complete UL49.5-KLHDC3 complexes were embedded into the fully hydrated all-atom lipid membrane model with explicit water molecules. The network of polar interactions has been proposed to be responsible for the recognition and binding of the degron in KLHDC3. The interaction network within the binding pocket appeared to be very similar between two CRL2 substrate receptors: KLHDC3 and KLHDC2.

GARP and EARP are required for efficient BoHV-1 replication as identified by a genome wide CRISPR knockout screen.

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle.

Development of a recombinant protein-based ELISA for detection of antibodies against bovine herpesvirus 6 (BoHV6).

Bovine herpesvirus 6 (BoHV6) belongs to the Herpesviridae family, Gammaherpesvirinae subfamily and Macavirus genus. It is common in cattle, but was also detected in American bison (Bison bison) and water buffalo (Bubalus bubalis). The aim of the experiment was to develop an ELISA for serological examination of cattle sera for the presence of anti-BoHV6 specific antibodies. Viral DNA from a BoHV6-positive cow was amplified by qPCR and the resulting fragments of the gB and gH genes encoding glycoproteins B and H (gB and gH) were cloned into the pLATE52 vector to express recombinant gB (rgB) and gH (rgH) in Rosetta (DE3) E. coli. The expressed recombinant proteins were used as antigens in the developed ELISA. The proteins expressed had the expected molecular weight. A total of 143 sera were examined, and 141 of them were positive, according to the chosen cut-off values of 9% and 10% for the sample-to-positive ratios of the rgB and rgH antigens, respectively. The rgB and rgH recombinant antigens of BoHV6 were successfully expressed in E. coli and successfully used in a newly developed ELISA.

Carnitine palmitoyl-transferase 1A is potentially involved in bovine herpesvirus 1 productive infection

Bovine herpesvirus 1(BoHV-1) is an important bovine pathogen that causes great economic loss to cattle farms worldwide. The virus-productive infection in bovine kidney (MDBK) cells results in ATP depletion. The mechanisms are not well understood. Mitochondrial fatty acid β-oxidation (FAO) is an important energy source in many tissues with high energy demand. Since carnitine palmitoyl-transferase 1 A (CPT1A) is the rate-limiting enzyme of FAO, we investigated the interactions between virus-productive infection and CPT1A signaling. Here, we found that virus-productive infection at the later stage significantly decreased CPT1A protein levels in all the detected cells, including MDBK, A549, and Neuro-2A cells, differentially altered the accumulation of CPT1A proteins in the nucleus and cytosol, and re-localized the protein in the nucleus. Etomoxir (ETO), an irreversible inhibitor of CPT1A, inhibited viral replication and partially interfered with the ability of BoHV-1 to alter CPT1A accumulation in the nucleus but not in the cytosol. Furthermore, ETO consistently reduced RNA levels of two viral regulatory proteins (bICP0 and bICP22) and protein expression of virion-associated proteins during productive infection, further supporting the important roles of CPT1A signaling in BoHV-1 productive infection. These data, for the first time, suggest that CPT1A is potentially involved in BoHV-1 productive infection.

Application of multiblock analysis to identify key areas and risk factors for dairy cow persistence

The present study analysed the importance of individual variables and different thematic blocks of production areas, management, and herd infectious disease status on cow persistence, characterised by herd on-farm mortality rate (MR), culling rate (CR), and mean age of culled cows (MAofCC) applying multiblock partial least squares (mbPLS) analysis. This study included 120 free-stall dairy herds with ≥ 100 cows. Data on the previous year’s predominant cow housing system and management practices were collected, and on-farm measurements and cow scoring were performed. Bulk tank milk (BTM) and heifer blood samples (10 samples per herd) were collected and analysed for antibodies against the selected pathogens. In total, 172 variables were aggregated into 14 thematic blocks. The annual CR, MR, and MAofCC values were calculated for each herd. Thematic blocks with significant impact on cow persistence (included herd MR, CR and MAofCC) were ‘infectious diseases’ (block importance index out of all blocks = 13.6%, 95% CI 10.3; 20.5), ‘fertility management’ (16.3%, 95% CI 6.8; 26.9), ‘lactating cow management’ (11.5%, 95% CI 6.4; 17.8), ‘milking’ (11.3%, 95% CI 3.2; 17.1), ‘herd characteristics’ (10.1%, 95% CI 6.3; 14.2), ‘close-up period management’ (9.7%, 95% CI 2.7; 15.7), ‘calving management’ (7.9%, 95% CI 3.1; 11.4) and ‘disease management’ (7.3%, 95% CI 0.2; 12.0). Variable categories with the highest importance in explaining composite outcome including herd MR, CR and MAofCC were rear-end and udder lesions in ≥ 20% of the cows, BTM and heifers seropositive to bovine respiratory syncytial virus, vaccination against bovine herpesvirus 1, twice daily milking and herd location in Northwest region. Larger herd size, higher levels of milk yield, and rearing predominantly Holstein breed cattle were herd factors associated with poorer cow persistency. Grazing cows and having semi-insulated barns were associated with lower CR and MR, respectively. Heat detection and farm pregnancy testing strategies were significant factors in the fertility block. Using disposable dry papers for teat cleaning and not using any wet teat-cleaning tools were risk factors for high MR. A robotic milking system was protective for increased herd MR and CR. A high pre-calving body condition score and poor rear body cleanliness of ≥ 30% of cows were associated with inferior herd persistency outcomes. Calving in group pens with deep litter bedding was associated with a lower CR. Multiblock PLS model is innovative tool that helped to identify most influential farming areas but also single risk factors associated with cow persistency described by multiple parameters.

Comprehensive diagnosis of abortion in cattle

Abstract. Abortion is one of the most common factors that has an extremely negative eff ect on the reproductive capacity of cattle, resulting in signifi cant economic losses. Abortions can be caused by infectious and non-infectious agents. Diagnosis of abortion is a diffi cult task, since only about 40% of abortions are diagnosed as infectious, and the remaining 60% - as unknown etiology. The goal was to establish the infectious etiology of abortion using complex diagnostics. The research was carried out in the laboratories of "Center of Veterinary Diagnostics" LLC (Kyiv). The selection of samples for research was carried out in the section hall of the LLC "Center of Veterinary Diagnostics" during the autopsy. The material for the study was a fetus and a vaginal smear from an aborted cow. Abortion occurred at 4 months of pregnancy. To establish the etiology, the following research methods were used: patho-anatomical, bacteriological, histological and molecular genetic (PCR-RT). Autolysis, light gray foci ≤ 2 mm in diameter in the lungs, and multiple light gray foci ≤ 1 mm in diameter in the liver were the main pathological fi ndings. The main pathohistological changes detected were focal myocarditis, intranuclear inclusion bodies in hepatocytes, multifocal coagulation necrosis in the lungs and liver, and diff use interstitial nephritis. Bacteriological culture was negative. DNA of infectious bovine rhinotracheitis virus (IBRV) and DNA of bovine herpesvirus type 4 (BHV-4) was isolated from the examined samples of fetal tissues and vaginal smear by PCR-RT. Using a comprehensive study, infectious agents and changes characteristic of the infectious process were identifi ed. On the basis of the obtained results, the etiology of the abortion was established as co-infection of IBRV and BHV-4. Complex diagnostics provides an opportunity to establish a cause-and-eff ect relationship between the identifi ed infectious agent and pathological changes in the fetus and the etiology of abortion. The results of a set of studies provide information on the manifestation of herpesvirus co-infection and complement the existing knowledge that can be used in the diagnosis of abortions in cows. Key words: abortion, cattle, IBRV, BHV-4, coinfection, PCR-RT, pathohistology, diagnosis.

Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

Molecular detection of bovine alphaherpesvirus-1 in cases of reproductive disorders among cattle and buffaloes in Gujarat.

Bovine alphaherpesvirus-1 (BoAHV-1) is an important viral pathogen that causes significant economic losses to the dairy industry. The present study aimed to determine the prevalence of BoAHV-1 in cases of bovine reproductive disorder. Clinical samples were collected from various villages in Gujarat using specialized FTA® cards and were tested using real-time PCR assay targeting the gB gene of BoAHV-1. Out of 401 animals, 18.20% (95% CI: 14.74-22.28%) tested positive for BoAHV-1 DNA. The percentage positivity of BoAHV-1 was 20.37% in abortion cases and 19.55% in retention of fetal membrane cases, while only one out of nine metritis cases screened in the study was positive for BoAHV-1 DNA. A higher percentage positivity in buffaloes (22.14%) compared to cattle (16.30%) was recorded, but this difference was not statistically significant (p = 0.169). The frequency of BoAHV-1 detection was higher among crossbreeds (16.76%) and exotics (19.61%) than among indigenous cattle (8.82%), although this difference was not statistically significant (p = 0.400). There was also no significant difference in frequency distribution among animals of varying parity, ranging from 15.20 to 33.33% (p = 0.540). This study confirms the widespread circulation of BoAHV-1 and highlights the need for its control and prevention.

Infectious Bovine Rhinotracheitis Post-Eradication Program in the Autonomous Province of Bolzano, Italy: A Retrospective Study on Potential Bovine Herpesvirus Type 2 Cross-Reactivity.

Bovine alphaherpesviruses, BoAHV, can cause respiratory, genital and neurological disorders. In particular, bovine alphaherpesvirus type 1 (BoAHV1) is one of the most significant ruminant pathogens worldwide and it can heavily damage the livestock industry. BoAHV1 can cause infectious bovine rhinotracheitis (IBR) along with fertility disorders. Bovine alphaherpesvirus type 2 (BoAHV2) can cause two different conditions as well: pseudo-lumpy skin disease (PSLD) and bovine herpetic mammillitis (BHM). The autonomous province of Bolzano (Italy) has adopted several strategies to control and eradicate IBR, and it was declared in 2000 to be IBR-free by the European Commission. Since 2001, a post-eradication monitoring program has overseen the serological analysis of bulk milk and, in the presence of a positive result, a follow-up examination is performed on the individual blood serum of all bovines older than 24 months that belong to bulk milk-positive herds. Despite the detection of positives in both bulk milk and serum samples, South Tyrol has been declared IBR-free, as these positives have never been confirmed through seroneutralization. Between 2014 and 2022, approximately 41,000 bulk milk (averaging 4300 samples/year) and 3229 serum samples were tested for BoAHV1. The aim of this study was to evaluate the post-eradication program for IBR with a particular focus on the potential cross-reactivity with BoAHV2; for this reason, serum samples were also tested for BoAHV2 antibodies. This study could be of great importance for those countries that submit herds to an IBR monitoring and eradication program; performing further analyses to confirm and explain false positive outcomes would increase the reliability of the obtained results.

Development and evaluation of a monoclonal antibody-based blocking ELISA to detect antibodies against the E2 protein of bovine viral diarrhea virus-1

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future.

The Safety and Protective Efficacy Evaluation of an Attenuated M. bovis-BoHV-1 Bivalent Vaccine in Rabbits.

Bovine respiratory disease (BRD) is a global prevalent multifactorial infection primarily caused by viral and bacterial coinfections. In China, Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the predominant pathogens associated with BRD. Our previous study involved the development of attenuated M. bovis HB150 and BoHV-1 gG-/tk- vaccine strains, which were thoroughly assessed for their safety profiles and protective efficacy in cattle. In this study, we applied a combination of vaccines in varying ratios and used a rabbit model to determine the safety and protective efficacy. We used PCR/RT-PCR to detect the postimmunization and challenge shedding of M. bovis and BoHV-1. Additionally, we measured antibody titers and the expression of IFN-β and TNF-α to evaluate the humoral and cellular immune responses, respectively. Furthermore, we performed a histopathological analysis to assess lung damage. Our study provides evidence of the safety and effectiveness of the bivalent M. bovis-BoHV-1 vaccine in rabbits, particularly when applying a combination of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain. The bivalent vaccine significantly enhanced both the long-term antibody immune response and cellular protection against the M. bovis and BoHV-1 challenge. These findings provide a valuable model for the potential application in cattle.