The Detection of Vaccine Virus and Protection of a Modified Live, Intranasal, Trivalent Vaccine in Neonatal, Colostrum-Fed Calves with an Experimental Bovine Respiratory Syncytial Virus Challenge.

The efficacy of an intranasal (IN) bovine respiratory syncytial virus (BRSV) vaccine administered in the presence of passive immunity was assessed. Pooled colostrum was administered by intubation to 50 beef-dairy crossbred calves the day they were born. The calves were transported to a research facility and were blocked by age and sex, and randomly assigned into two groups: sham-vaccinated intranasally with a placebo (sterile water) or vaccinated with a trivalent (BRSV, bovine herpesvirus 1 and bovine parainfluenza 3) modified live viral (MLV) vaccine. The calves were 9 ± 2 days old when vaccinated (day 0). The calves were challenged by aerosolized BRSV on days 80 and 81 as a respiratory challenge. The study was terminated on day 88. Lung lesion scores (LLS) were significantly lower for calves vaccinated with trivalent MLV vaccine than those for calves that were sham-vaccinated. Serum neutralization (SN) antibody against BRSV in calves vaccinated with the trivalent MLV vaccine demonstrated an anamnestic response on day 88. After challenge, the calves sham-vaccinated with the placebo lost weight, while those vaccinated with the trivalent MLV vaccine gained weight. In this study, colostrum-derived antibodies did not interfere with the immune response or protection provided by one dose of the trivalent MLV vaccine.

Third-generation smallpox vaccines induce low-level cross-protecting neutralizing antibodies against Monkeypox virus in laboratory workers.

Due to the discontinuation of routine smallpox vaccination after its eradication in 1980, a large part of the human population remains naïve against smallpox and other members of the orthopoxvirus genus. As a part of biosafety personnel protection programs, laboratory workers receive prophylactic vaccinations against diverse infectious agents, including smallpox. Here, we studied the levels of cross-protecting neutralizing antibodies as well as total IgG induced by either first- or third-generation smallpox vaccines against Monkeypox virus, using a clinical isolate from the 2022 outbreak. Serum neutralization tests indicated better overall neutralization capacity after vaccination with first-generation smallpox vaccines, compared to an attenuated third-generation vaccine. Results obtained from total IgG ELISA, however, did not show higher induction of orthopoxvirus-specific IgGs in first-generation vaccine recipients. Taken together, our results indicate a lower level of cross-protecting neutralizing antibodies against Monkeypox virus in recipients of third-generation smallpox vaccine compared to first-generation vaccine recipients, although total IgG levels were comparable.

XBB.1.5 monovalent mRNA vaccine booster elicits robust neutralizing antibodies against XBB subvariants and JN.1.

COVID-19 vaccines have recently been updated to specifically encode or contain the spike protein of the SARS-CoV-2 XBB.1.5 subvariant, but their immunogenicity in humans has yet to be fully evaluated and reported, particularly against emergent viruses that are rapidly expanding. We now report that administration of an updated monovalent mRNA vaccine booster (XBB.1.5 MV) to previously uninfected individuals boosted serum virus-neutralizing antibodies significantly against not only XBB.1.5 (27.0-fold increase) and EG.5.1 (27.6-fold increase) but also key emerging viruses such as HV.1, HK.3, JD.1.1, and JN.1 (13.3- to 27.4-fold increase). Individuals previously infected by an Omicron subvariant had the highest overall serum neutralizing titers (ID50 1,504-22,978) against all viral variants tested. While immunological imprinting was still evident with the updated vaccines, it was not nearly as severe as observed with the previously authorized bivalent BA.5 vaccine. Our findings strongly support the official recommendation to widely apply the updated COVID-19 vaccines.

Deep immunological imprinting due to the ancestral spike in the current bivalent COVID-19 vaccine.

To combat the evolving SARS-CoV-2 Omicron variants, bivalent COVID-19 mRNA vaccines, encoding both ancestral and Omicron BA.5 spikes, have replaced monovalent vaccines in numerous countries. However, fourth doses of either vaccine result in similar neutralizing antibody titers against Omicron subvariants, raising the possibility of immunological imprinting. To address this, we investigate antibody responses in 72 participants given three doses of a monovalent mRNA vaccine, followed by a bivalent or monovalent booster, or those with breakthrough infections with BA.5 or BQ. Bivalent boosters do not show notably higher binding or virus-neutralizing titers against various SARS-CoV-2 variants compared to monovalent ones. However, breakthrough infections lead to significantly better neutralization of Omicron subvariants. Multiple analyses, including antigenic mapping, suggest that the ancestral spike in bivalent vaccines is causing deep immunological imprinting, preventing broadening of antibodies to the BA.5 component, thereby defeating its intended goal. Its removal from future vaccine compositions is therefore strongly recommended.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Targeting Rabies-Specific IgM and IgG in Human Sera.

Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may influence RVNA isotype kinetics, potentially affecting the peak, and longevity, of RVNA immunoglobulin (IgG) levels. We developed rapid and reliable assays for quantifying the anti-rabies IgM/IgG class switch in human serum based on an indirect ELISA technique. The immune response was tracked in ten individuals naïve to the rabies vaccine by quantifying serum titers weekly, from day seven to day 42 post-immunization, using a serum neutralization assay and the ELISA IgM/IgG assays. The average RVNA IU/mL levels were at D0 ≤ 0.1, D7 0.24, D14 8.36, D21 12.84, D28 25.74 and D42 28.68. Levels of specific IgM antibodies to rabies glycoprotein (EU/mL) were higher, on average, at D7, 1.37, and from D14, 5.49, to D21, 6.59. In contrast, average IgG antibodies (EU/mL) predominated from D28, 10.03, to D42, 14.45. We conclude that levels of anti-rabies IgM/IgG at D28 characterize the isotype class switch. These assays, combined with serum neutralization assays, distinguished the RVNA levels in terms of the IgM/IgG responses and are expected to add to the diagnostic repertoire, provide additional information in establishing rabies vaccine regimens, both post- and pre-exposure prophylaxis, and contribute to research efforts.

Canine Distemper Virus in Tigers (Panthera tigris) and Leopards (P. pardus) in Nepal.

From wild dogs (Lycaon pictus) in the Serengeti to tigers (Panthera tigris altaica) in the Russian Far East, canine distemper virus (CDV) has been repeatedly identified as a threat to wild carnivores. Between 2020 and 2022, six Indian leopards (P. pardus fusca) presented to Nepali authorities with fatal neurological disease, consistent with CDV. Here, we report the findings of a serosurvey of wild felids from Nepal. A total of 48 serum samples were tested, comprising 28 Bengal tigers (P. t. tigris) and 20 Indian leopards. Neutralizing antibodies were identified in three tigers and six leopards, equating to seroprevalences of 11% (CI: 2.8-29.3%, n = 28) and 30% (CI: 12.8-54.3%, n = 20), respectively. More than one-third of seropositive animals were symptomatic, and three died within a week of being sampled. The predation of domestic dogs (Canis lupus familiaris) has been posited as a potential route of infection. A comparison of existing diet studies revealed that while leopards in Nepal frequently predate on dogs, tigers do not, potentially supporting this hypothesis. However, further work, including molecular analyses, would be needed to confirm this.

HoBi-like Pestivirus Is Highly Prevalent in Cattle Herds in the Amazon Region (Northern Brazil).

Pestiviruses are globally distributed and cause substantial economic losses to the cattle industry. In Brazil, the country with the world's largest cattle population, pestivirus infections are well described in some regions, such as in the south, where a high frequency of BVDV-2 is described and contrasts with the high prevalence of HoBi-like pestivirus (HoBiPeV) in the northeast. However, there is a lack of information about pestiviruses in the Amazon Region, in northern Brazil, with a cattle population estimated at 55.7 million head, which has a significant impact on the international livestock market. Therefore, this study investigated the seroprevalence and genetic variability of ruminant pestiviruses in 944 bovine serum samples from four states in northern Brazil: Pará (PA), Amapá (AP), Roraima (RR), and Amazonas (AM). Our results showed that 45.4% of the samples were seropositive (19.8% for BVDV-1, 14.1% for BVDV-2, and 20.9% for HoBiPeV). All samples were tested by RT-qPCR, and three were positive and classified as HoBiPeV in a phylogenetic analysis. These serological and molecular results contrast with those from other regions of the world, suggesting that the northern Brazilian states have a high prevalence of all bovine pestiviruses including HoBiPeV.

Diagnostics for Viral Pathogens in Veterinary Diagnostic Laboratories.

Laboratory testing is one part of clinical diagnosis, and quick and reliable testing results provide important data to support treatment decision and develop control strategies. Clinical viral testing has been shifting from traditional virus isolation and electron microscopy to molecular polymerase chain reaction and point-of-care antigen tests. This shift in diagnostic methodology also means change from looking for infectious virions or viral particles to hunting viral antigens and genomes. With technological development, it is predicted that metagenomic sequencing will be commonly used in veterinary clinical diagnosis for unveiling the whole picture of microbes involved in diseases in the future.

Serology in Bovine Infectious Disease Diagnosis.

Serologic diagnosis is used to identify evidence of infection or vaccination by specific agents, or for population surveillance. The enzyme-linked immunosorbent assay and the serum (virus) neutralizing tests are most used for bovine serologic diagnosis. Although infectious agent-specific antibodies may include immunoglobulin M, immunoglobulin G, and immunoglobulin A, the antibody class is rarely specifically identified in diagnostic laboratory testing. When interpreting the results of serology, consider whether the antibodies are due to an agent that causes life-long infection, transient infection with no history of vaccination, or transient infection with a history of vaccination. Paired serology is necessary to confirm recent infection in cattle with a history of vaccination.

Investigation of the effect of mutual vaccination with pest des petits ruminants and polyvalent foot and mouth disease vaccines on the immune response of sheep.

Background: Pest des petits ruminants (PPRs) and foot and mouth disease (FMD) are two viral infectious diseases affecting sheep dramatically causing great economic losses. Therefore, attention should be directed toward their control, especially through the application of well-designed vaccination schedules with specific potent vaccines. Aim: Determination of the possibility of sheep vaccination with PPR and FMD vaccines in a mutual schedule. Methods: Different groups of sheep have vaccinated with live attenuated PPR vaccine and inactivated polyvalent FMD vaccine in a mutual manner (one before the other at weekly intervals or simultaneously) followed by monitoring of the induced immunity to both vaccines using serum neutralization test (SNT) and enzyme linked immune sorbent assay (ELISA). Results: SNT and ELISA revealed that there was no antagonizing effect of any vaccine on the immune response to the mutual vaccination of sheep to the other where the obtained antibody titers in single vaccinated sheep groups were similar to those in the simultaneous vaccinated group. Conclusion: Simultaneous vaccination of sheep with PPR and polyvalent FMD vaccine is of applicable benefit saving time, effort, and stress factors on the animals.

Development of ELISA for the detection of antibodies against VP2 protein of bluetongue virus serotype-1.

Serotype-specific diagnosis of bluetongue virus (BTV) is necessary for sero-surveillance and taking effective control measures. The VP2 is the major serotype determining protein and BTV-1 is the most predominant serotype in India. In the present study, an indirect ELISA (i-ELISA) was optimized for the detection of serotype-specific antibody against BTV-1 serotype. The VP2 protein of BTV-1 was expressed in a prokaryotic expression system and used to optimize i-ELISA to detect VP2 antibodies of BTV-1 in serum samples of both small and large ruminants. Serum samples (n = 363) classified as positive and negative for antibodies to BTV-1 by serum neutralization test (SNT) and also positive and negative for BTV antibodies by c-ELSIA kit (VMRD, USA) were used to determine the cut-off value, diagnostic sensitivity (DSn), and diagnostic specificity (D-Sp) using receiver operating characteristic (ROC) analysis. The percent positivity (PP) value >30.10% was accepted as the cut-off for i-ELISA at which DSn of 99.52% and D-Sp of 99.35% was observed with a 95% confidence interval. Further, there was no cross-reactivity with other available BTV serotypes in the country. The study indicated serotype-specific i-ELISA is sensitive, specific and suitable alternative to tedious SNT method for determining serotype. The assay will also help in the serotype-specific epidemiological studies and implementation of future control strategies including vaccination and selection of suitable serotype as a vaccine candidate.

Antigenic characterization of the SARS-CoV-2 Omicron subvariant BA.2.75.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2.75 emerged recently and appears to be spreading. It has nine mutations in spike compared with the currently circulating BA.2, raising concerns that it may further evade vaccine-elicited and therapeutic antibodies. We found BA.2.75 to be moderately more neutralization resistant to sera from vaccinated/boosted individuals than BA.2 (1.8-fold), similar to BA.2.12.1 (1.1-fold), but more neutralization sensitive than BA.4/5 (0.6-fold). Relative to BA.2, BA.2.75 showed heightened resistance to class 1 and class 3 monoclonal antibodies targeting the spike-receptor-binding domain while gaining sensitivity to class 2 antibodies. Resistance was largely conferred by G446S and R460K mutations. BA.2.75 was slightly resistant (3.7-fold) to bebtelovimab, a therapeutic antibody with potent activity against all Omicron subvariants. BA.2.75 also exhibited a higher binding affinity to host receptor ACE2 than other Omicron subvariants. BA.2.75 provides further insight into SARS-CoV-2 evolution as it gains transmissibility while incrementally evading antibody neutralization.

Seroprevalence and Associated Risk Factors of Lumpy Skin Disease of Cattle in Selected Districts of Afar Region, Ethiopia.

Background: Lumpy skin disease (LSD) is one of Ethiopia's most economically significant transboundary livestock illnesses. The disease has a significant economic impact on pastoral household livestock owners, who rely significantly on their cattle as a source of income. Methods: A cross-sectional study was undertaken in selected districts of Afar region from November 2018 to May 2019 primarily intended to estimate the prevalence of lumpy skin disease serologically in local Afar cattle as well as identify potential associated factors. A multistage sampling method was employed to select study districts, peasant association, herd size and study units. A total of 384 sera were processed using serum neutralization test (SNT) method to detect antibodies against lumpy skin disease virus. Relevant data were refined and further analyzed using stata version 14. Results: In the study districts, the overall animal level seroprevalence was found to be 7.6% (N = 29/384; 95% confidence interval: 4.90-10.20) and the overall herd level prevalence was found to be 20.8% (n = 15/72; 95% confidence interval: 11.42-30.18). Only district was shown to be statistically significant (P = 0.004) in terms of LSD occurrence among the relevant factors studied. Cattle in Chifra district were 20.18 times more likely to contract LSD infection than cattle in Dubti district, when Asayita district was used as the reference group. Conclusion: The present study finding confirmed the presence of the disease in the study districts of afar region and coordinated intervention set to be in place.

Preparation and the assessed efficacy of oral baits for the vaccination of free-roaming dogs against rabies.

Background and Aim: Rabies is considered a highly fatal zoonotic disease and many deaths in humans have been associated with dog bites. This study was designed to prepare an oral anti-rabies vaccine in the form of baits to eliminate the disease in free-roaming dogs and subsequently protect humans from dog bites. Materials and Methods: The Evelyn Rokintniki Abelseth (ERA) rabies virus strain was propagated in baby hamster kidney cell cultures and adjusted to the recommended dose for application. Four forms of oral baits were employed with the rabies vaccine, which was evaluated for safety, acceptability, and potency in different dog groups. Enzyme-Linked Immunosorbent Assay (ELISA) and the serum neutralization test (SNT) were used to determine the protective rabies antibody titer in the sera of vaccinated dogs. Results: According to the results, a dose of 3 mL of the ERA strain, containing a viral titer of 107.6 TCID50/mL, induced a mean antibody titer of 25.6 by SNT, and the PI% was 75.7 by Block ELISA, providing a protective level of the rabies antibody in 100% of vaccinated dogs. All used baits were found to be safe, inducing no abnormal general post-vaccination signs (the signs are limited to mild fever, mild loss of appetite, and mild-to-moderate loss of energy for 24-36 h after vaccination). Conclusion: It was found that most of the accepted and highly potent bait types consisted of a mixture of wheat flour, vegetable oil, sodium alginate, corn starch, meat meal, cellulose gum, and water. This dog meal was covered with bran and edible wax to seal the bait cavity after inserting the vaccine sachet. This bait was able to induce a protective level of rabies antibodies in 100% of vaccinated dogs after receiving one bait/dog. Hence, such a bait could be recommended for use in the protection of free-roaming dogs and the elimination of the disease.

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay.

The reverse genetics system is a valuable tool in the virological study of RNA viruses. With the availability of reverse genetics, the porcine reproductive and respiratory syndrome virus (PRRSV) has been utilized as a viral vector for the expression of foreign genes of interest. Here, we constructed a full-length cDNA clone of a highly pathogenic PRRSV (HP-PRRSV) TA-12 strain. Using this cDNA clone, we generated a reporter virus expressing a gaussia luciferase (Gluc) via an additional subgenomic RNA between ORF7 and 3'UTR. This reporter virus exhibited similar growth kinetics to the wild-type (WT) virus and remained genetically stable for at least ten passages in MARC-145 cells. In cells infected with this reporter virus, the correlation between the expression levels of Gluc in culture media and the virus titers suggested that Gluc is a good indicator of the reporter virus infection. With this reporter virus, we further established the Gluc readout-based assays for antiviral drug screening and serum neutralizing antibody detection that exhibited comparable performance to the classical assays. Taken together, we established a reverse genetics system of HP-PRRSV and generated a novel reporter virus that could serve as a valuable tool for antiviral drug screening and serum neutralizing antibody detection.

Serum Neutralization of SARS-CoV-2 Omicron BA.1 and BA.2 after BNT162b2 Booster Vaccination.

The SARS-CoV-2 Omicron variant BA.2 sublineage is rapidly replacing earlier Omicron lineages, suggesting BA.2 has increased vaccine evasion properties. We measured neutralization titers of authentic BA.1 and BA.2 isolates in serum samples from persons who received the BNT162b2 booster vaccine. All samples neutralized BA.1 and BA.2 at equal median values.

Experimental veterinary SARS-CoV-2 vaccine cross neutralization of the Delta (B.1.617.2) variant virus in cats.

SARS-CoV-2 has exhibited varying pathogenesis in a variety of Mammalia family's including Canidae, Mustelidae, Hominidae, Cervidae, Hyaenidae, and Felidae. Novel SARS-CoV-2 variants characterized by spike protein mutations have recently resulted in clinical and epidemiological concerns, as they potentially have increased infectious rates, increased transmission, or reduced neutralization by antibodies produced via vaccination. Many variants have been identified at this time, but the variant of continuing concern has been the Delta variant (B.1.617.2), due to its increased transmissibility and infectious rate. Felines vaccinated using an experimental SARS-CoV-2 spike protein-based veterinary vaccine mounted a robust immune response to the SARS-CoV-2 spike protein. Using a reporter virus particle system and feline serum, we have verified that vaccinated felines produce antibodies that neutralize the SARS-CoV-2 Wuhan strain and variant B.1.617.2 at comparable levels.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay.

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.

Equine Herpesvirus Type 1 Myeloencephalitis in the Brazilian Amazon.

An investigative and epidemiological study was carried out for equine herpesvirus type 1 (HVE-1) in 10 outbreaks of neurological disease from different farms in the state of Pará, Brazil. 25 horses were studied: six male and 19 females, aged between one and 13 years. A necropsy of six horses was performed, and the others recovered either with or without treatment (T1-vitamin B1 + dexamentasone; T2-vitamin B1 + flunixim meglumine). Animals that received treatment recovered after eight days. The main clinical signs observed were motor incoordination, progressive paresis, thoracic and/or pelvic limbs abducted after induction of clinical examination, knuckling of the hind fetlocks, sagging and swaying of the hindquarters while standing or walking and paresis. All animals were positive: 88% (22/25) in nested PCR and 72% (18/25) in serum neutralization (including three negatives in serology). Focal brownish areas compatible with hemorrhage were found in the white and gray matter of the spinal cord of two animals. On histological analysis, there were perivasculitis and neutrophilic vasculitis in the gray matter of the spinal cord and brain. Based on the evidence, this work proves the circulation of HVE-1 in the Amazon biome, mainly in the state of Pará, Brazil.

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens.

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.