Evaluation of chlorine dioxide in liquid state and in gaseous state as virucidal agent against avian influenza virus and infectious bronchitis virus.

The antiviral activity of chlorine dioxide (ClO2) in liquid (ClO2 gas dissolved liquid) and gaseous state against avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated. To evaluate the effect of ClO2 in liquid state, suspension tests (10 ppm) and carrier tests in dropping / wiping techniques (100 ppm) were performed. In the suspension test, virus titers were reduced below the detection limit within 15 sec after treatment, in spite of the presence of an accompanying organic matter. In the carrier test by dropping technique, AIV and IBV were reduced to below the detection limit in 1 and 3 min, respectively. Following wiping technique, no virus was detected in the wiping sheets after 30 sec of reaction. Both viruses adhering to the carriers were also reduced by 3 logs, thereby indicating that they were effectively inactivated. In addition, the effect of ClO2 gas against IBV in aerosols was evaluated. After the exposure of sprayed IBV to ClO2 gas for a few seconds, 94.2% reduction of the virus titer was observed, as compared to the pre-treatment control. Altogether, hence, ClO2 has an evident potential to be an effective disinfectant for the prevention and control of AIV and IBV infections on poultry farms.

Evaluation of antimicrobial efficacies of chlorine dioxide gas released into the air towards pathogens present on the surfaces of inanimate objects.

The efficacy of ClO2 gas, as surface disinfectant at around 1,000 ppb against avian orthoavulaviruses type 1 (AOAV-1), infectious bronchitis virus (IBV), Escherichia coli (EC), and Salmonella Enteritidis (SE) was evaluated at the required level (≥99.9% reduction) on various surfaces. Exposing the surfaces to ClO2 gas for 1 hr reduced AOAV-1, except for rayon sheets which required 3 hr. However, 1 hr of exposure did not effectively reduced IBV titer. In the case of EC, glass plates and plastic carriers needed 1 hr of exposure, while rayon sheets required 2 hr. SE on rayon sheets required 1 hr exposure, but on the other tested surfaces showed inadequate reduction. Overall, ClO2 gas is an effective disinfectant for poultry farms.

Complete genome constellations of two bovine rotavirus A strains isolated in Japan reveal a unique T9 NSP3 genotype.

Full genome sequencing of two bovine rotavirus A (RVA) strains isolated in Japan in 2019 revealed two genotype constellations; one had a constellation of G8-P[1]-I2-R2-C2-M2-A3-N2-T9-E2-H3. Thereupon, genotype T9 carried by RVA/Bovine-tc/JPN/AH1041/2022/G8P[1], constitutes a rare NSP3 genotype, and only two unusual Japanese bovine RVA strains have thus far been reported to carry this genotype. The other RVA/Bovine-tc/JPN/AH1207/2022/G6P[5] strain possessed a constellation of G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Phylogenetic analyses indicate that the majority of gene segments were most closely related to Japanese bovine RVAs, suggesting that both strains might have derived through multiple reassortment events from RVA strains circulating within Japanese cattle. The emergence of RVA strains in Japan and their reassortment with locally circulating atypical RVAs could have implications for current vaccination strategies.

Comparison of antigenicity between recently isolated bovine rotaviruses A and vaccine strains by cross-neutralizing antibody tests and their significance for immunization.

Three bovine rotaviruses A (RVAs) isolated from a cattle farm in Japan were serotyped by serum neutralization assay, as compared with the RVA strains contained in a vaccine used on the same farm. Antisera were prepared against the three isolates and the vaccine strains of bovine RVA. The results of cross-neutralization tests revealed that the RVA isolates from this farm differed somewhat in serotype. Collected plasma from calves for 6 weeks after colostrum ingestion showed that maternal antibodies acquired against all isolates gradually decreased, but antibodies toward one isolate increased by 6 weeks after the mentioned decreasing. These results suggest that rotavirus vaccines administered to cows should include all serotypes commonly found in calves with diarrhea.

Isolation, molecular characterization, and disinfectants susceptibility of swine-carried mammalian orthoreoviruses in Japan in 2020-2022.

Biosecurity enhancement contributes to the reduction of various microbial pathogens. Mammalian orthoreoviruses (MRVs) which are increasingly recognized as potentially serious problems on swine industry were used as indicators of biosecurity enhancement on two pig farms. Twelve MRVs were detected and isolated from fecal specimens of healthy pigs collected from one of the two farms in Japan. By sequencing based on the partial S1 gene, MRV isolates were classified as MRV1 and MRV2. Additionally, the virucidal activities of disinfectants toward the isolated MRV1 were evaluated using quaternary ammonium compound (QAC) diluted 500 times with water (QAC-500), 0.17% food additive glade calcium hydroxide (FdCa(OH)2) solution, QAC diluted with 0.17% FdCa(OH)2 solution (Mix-500), sodium hypochlorite at 100 or 1,000 parts per million (ppm) of total chlorine (NaClO-100 or NaClO-1000, respectively). To efficiently inactivate MRV1 (≥3 log10 reductions), 0.17% FdCa(OH)2, Mix-500 and NaClO-1000 required 5 min, whereas it took 30 min for QAC-500. The number of MRV detections has decreased over time, after using Mix-500 for disinfection on the positive farm. These results suggest that different serotypes of MRVs are circulating among pigs, and that the occurrence of MRVs in the farms decreased consequent to more effective disinfection.

Evaluation of Virucidal Quantitative Carrier Test towards Bovine Viruses for Surface Disinfectants While Simulating Practical Usage on Livestock Farms.

Livestock farming is affected by the occurrence of infectious diseases, but outbreaks can be prevented by effective cleaning and disinfection along with proper farm management. In the present study, bovine coronavirus (BCoV) and bovine rotavirus A (RVA) were inactivated using food additive-grade calcium hydroxide (FdCa(OH)2) solution, quaternary ammonium compound (QAC) and their mixture through suspension tests as the primary screening, and afterward via carrier tests using dropping or dipping techniques as the secondary screenings. Viruses in the aqueous phase can be easily inactivated in the suspension tests, but once attached to the materials, they can become resistant to disinfectants, and require longer times to be inactivated. This highlights the importance of thorough cleaning with detergent before disinfection, and keeping elevated contact durations of proper disinfectants to reduce viral contamination and decrease infectious diseases incidence in farms. It was also reaffirmed that the suspension and carrier tests are necessary to evaluate disinfectants and thus determine their actual use. Particularly, the mixture of QAC and FdCa(OH)2 was found to exhibit synergistic and broad-spectrum effects compared to their use alone, and is now recommended for use on livestock farms.

G and P genotype profiles of rotavirus a field strains circulating in a vaccinated bovine farm as parameters for assessing biosecurity level.

After improvement of hygiene protocols on boots in a bovine operation (farm A) in Ibaraki, Japan in September 2017, mortality of calves and the detection of 4 viral pathogen indicators, including bovine rotavirus A (RVA), became significantly low for one year. Subsequently, in the present study, these indicators and mortality were monitored and confirmed all were still low, except for the detection rate of bovine RVA in calves less than 3 weeks old. The present study aimed to investigate G and P genotypic profiles of RVAs in farm A from 2018 to 2020. Molecular analysis using semi-nested multiplex RT-PCR of positive RVAs (n=122) and sequencing of selected samples revealed the presence of G6, G8, G10, P[1], P[5] and P[11] genotypes and the prevalence of G and/or P combination and mixed infections. The most common combination of G and P types was G10P[11] (41.8%), followed by mixed infection with G6+G10P[5] (11.5%). Phylogenetic analysis of RVAs showed clustering with bovine and other animal-derived RVA strains, suggesting the possibility of multiple reassortant events with strains of bovine and others animal origins. Noteworthy as well is that vaccinated cattle might fail to provide their offspring with maternal immunity against RVA infections, due to insufficient colostrum feeding. Our findings further highlight the importance of RVA surveillance in bovine populations, which may be useful to improving effective routine vaccination and hygiene practices on bovine farms.

Isolation and Molecular Characterization of Fowl Adenovirus and Avian Reovirus from Breeder Chickens in Japan in 2019-2021.

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.

Rapid in vitro virucidal activity of slightly acidic hypochlorous acid water toward aerosolized coronavirus in simulated human-dispersed droplets.

The virucidal activities were evaluated by spraying slightly acidic hypochlorous acid waters (SAHWs) containing various concentrations of free available chlorine - 100, 200, 300 and 500 ppm (SAHW-100, -200, -300 and -500, respectively) - toward aerosol of an avian coronavirus (infectious bronchitis virus: IBV). The viral solution was supplemented with 0.5% fetal bovine serum (FBS) to simulate normal human droplets generated by sneezing or coughing in a real-life scenario. The virus containing 0.5% FBS was sprayed and exposed to SAHWs for a few seconds in a closed chamber, before reaching the air sampler. The results showed that IBV exposed to SAHW-100 and -200 for a few seconds decreased by 0.21 log10 and 0.80 log10, respectively, compared to the pre-exposed samples to SAHWs as controls. On the other hand, reductions of 1.16 log10 and 1.67 log10 were achieved following the exposure to SAHW-300 and -500, respectively, within a few seconds. These results suggest that SAHWs have rapid in vitro virucidal activity toward aerosolized IBV. The findings obtained for IBV might basically be applicable in relation to SARS-CoV-2, given the resemblance between the two viruses. To prevent human-to-human transmissions by aerosols, the inactivation of viruses in the air by exposure to SAHWs for a few seconds seems to be an effective way.

Synergistic effects of quaternary ammonium compounds and food additive grade calcium hydroxide on microbicidal activities at low temperatures.

The microbicidal activities of mixtures of quaternary ammonium compounds (QACs) and food additive grade calcium hydroxide (FdCa(OH)2) were evaluated in a suspension test at -20°C using an anti-freeze agent (AFA) containing methanol, or at 1°C, with varying contact time, toward avian influenza virus (AIV), Newcastle disease virus (NDV), fowl adenovirus (FAdV), avian reovirus (ARV), Salmonella Infantis (SI) and Escherichia coli (EC). At -20°C, the mixtures could inactivate AIV and NDV within 30 min, FAdV and ARV within 5 sec, and SI and EC within 3 min, respectively. AFA did not inactivate viruses and bacteria within 30 min and 10 min, respectively. At 1°C, the mixtures inactivated FAdV and ARV within 30 sec, AIV within 10 min, and NDV within 30 min. A mixture of slaked lime (SL) and QAC could inactivate FAdV and ARV within 30 sec, but could not inactivate AIV and NDV even after 60 min at 1°C. SL could not substitute FdCa(OH)2 in order to exert the synergistic effects with QAC. Thus, QACs microbicidal activities were maintained or enhanced by adding FdCa(OH)2. It is hence recommended to use QACs with FdCa(OH)2, especially in the winter season.

Virucidal activity of slightly acidic hypochlorous acid water toward influenza virus and coronavirus with tests simulating practical usage.

Slightly acidic hypochlorous acid waters (SAHWs) with pH of 5.2-5.8 containing different concentrations of free available chlorine - 62, 119, 220, 300, and 540 ppm (SAHW-62, -119, -220, -300, and -540, respectively) - were evaluated for their virucidal activity toward a low pathogenic H7N1 avian influenza virus (AIV) and an infectious bronchitis virus (IBV) in suspension, abiotic carrier, and direct spray tests, with the presence of organic materials. In the carrier test, the dropping and wiping techniques were performed toward viruses on carriers. In the suspension test, SAHW-62 could decrease the viral titer of both AIV and IBV by more than 1000 times within 30 s. With the dropping technique, IBV on carriers showed high resistance to SAHW, while AIV on plastic carrier was inactivated to an effective level (≧3 log virus reduction) within 1 min. With the wiping technique, SAHW-62 could inactivate both AIV and IBV on wiped plastic carriers to an effective level within 30 s. However, SAHW-220 could not inactivate IBV in the wiping rayon sheet to an effective level. In the direct spray test, sprayed SAHW-300 within 10 min, and SAHW-540 within 20 min, inactivated AIV and IBV on the rayon sheets to undetectable level, respectively. Our study indicates that the usage of wipes with SAHW could remove viruses from plastic carriers, while viruses remained in the wipes. Besides, a small volume of sprayed SAHW was effective against the viruses on the rayon sheets for daily cleaning in the application area. The findings we obtained concerning IBV might basically be applicable in relation to SARS-CoV-2, given the resemblance between the two viruses.

Establishment and utilization of an evaluation system for virucidal activity of disinfectants against a coronavirus with apparent applicability to SARS-CoV-2.

Decontamination of pathogens on surfaces of substances is very important for controlling infectious diseases. In the present experiments, we tested various disinfectants in aqueous phase as well as on plastic surface carrying a viral inoculum, through dropping and wiping decontamination techniques, comparatively, so as to evaluate virucidal efficacies of those disinfectants toward an avian coronavirus (infectious bronchitis virus: IBV). We regard this evaluation system applicable to SARS-CoV-2. The disinfectants evaluated were 0.17% food additive glade calcium hydroxide (FdCa(OH)2) solution, sodium hypochlorite at 500 or 1,000 ppm of total chlorine (NaClO-500 or NaClO-1,000, respectively), NaClO at 500 ppm of total chlorine in 0.17% FdCa(OH)2 (Mix-500) and quaternary ammonium compound (QAC) diluted 500-fold in water (QAC-500). In the suspension test, all solutions inactivated IBV inoculum that contained 5% fetal bovine serum (FBS) under detectable level within 30 sec. In the carrier test, all solutions, except NaClO-500, could inactivate IBV with 0.5% FBS on a carrier to undetectable level in the wiping-sheets and wiped-carriers. We thus conclude that suspension and carrier tests should be introduced to evaluate disinfectants for the field usage, and that this evaluation system is important and workable for resultful selection of the tested disinfectants against avian coronavirus and SARS-CoV-2 on surfaces, particularly on plastic fomite.

Regression of viral pathogen indicators due to improvement of hygiene protocols on boots in a bovine farm.

Rotavirus A (RVA), bovine torovirus (BToV), bovine enterovirus (BEV) and bovine coronavirus (BCV) at a bovine farm in Ibaraki prefecture were monitored by one-step multiplex reverse transcription polymerase chain reaction (RT-PCR), with the aim of confirming the reduction of "viral pathogen indicators". A total of 960 bovine fecal samples were collected from calves less than 2 month-old within the period from October 2016 to October 2018 every 2 months at the bovine farm. In each sampling, 40 samples were taken from calves 3 week-old or less, and 40 samples from calves over 3 week-old, in principle. At the end of September 2017, the farm introduced improvement of hygiene protocols on boots by exchanging boots and appropriate usage of a footbath at the entrance of calf sheds. In the comparison of the virus detection by RT-PCR, prevalence of all 4 viruses was significantly reduced (P<0.01) in calves 3 week-old or less after the improvement. The mortality of calves less than 2 month-old was also significantly reduced after the improvement of hygiene protocols. These data suggest that the proper control of boots at calf sheds is important, perhaps even vital, for rearing hygiene measures at bovine farms so as to attain substantial decrease in the prevalence of pathogens.

Longitudinal and cross-sectional detection of four bovine enteric viruses by multiplex- reverse transcription polymerase chain reaction: Identification of possible indicator viruses to assess biosecurity level at bovine farms.

It can be judged that if the detection frequency of prevalent pathogenic viruses decreases, biosecurity has been enhanced. To monitor bovine farm biosecurity levels, one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of group A rotavirus (RVA), bovine torovirus (BToV), bovine enterovirus (BEV), and bovine coronavirus (BCV) was designed, with the aim of configuring candidates for "viral pathogen indicators". A total of 322 bovine fecal samples were collected from calves aged less than three months at 48 bovine farms in Ibaraki and Chiba prefectures. At farm A, 20 calves were selected and sampled weekly for 12 weeks (184 samples); at farm B, 10 calves were selected and sampled for five weeks (50 samples); and at the rest of the 46 farms, 88 calves were sampled once. The screening on the 358 field samples proved positive for 27 RVA, 4 BToV, 55 BEV, and 52 BCV. In the successive sampling, RVA was detected once but not continuously, whereas BEV and BCV were detected in succession for up to five weeks. The results revealed that RVA was the primary agent among the positive samples obtained from calves aged three weeks or less, while BEV was the primary among those from the older than three weeks old. They can be employed as useful viral pathogen indicators for soundly evaluating biosecurity at bovine farms.