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.

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.

Isolation, Identification, and Molecular Characterization of Newcastle Disease Virus from Field Outbreaks in Chickens in Afghanistan.

Newcastle disease viruses (NDVs) in Afghanistan were isolated from three chicken farms and identified using a hemagglutination test and reverse transcription-polymerase chain reaction assay. Three isolates from each farm were sequenced to characterize the part of their fusion protein gene around the cleavage site. The characteristics of the fusion protein genes of the three isolates shown by phylogenic analysis indicated that the isolates were velogenic, belonged to the class II subgenotype VII 1.1, and were closely related to an identified Chinese NDV isolate. To our knowledge, this is the first time that NDV isolates from Afghanistan have been partially sequenced.

Aislamiento, identificación y caracterización molecular del virus de la enfermedad de Newcastle de brotes de campo en pollos en Afganistán Se aislaron virus de la enfermedad de Newcastle (NDV) en Afganistán de tres granjas de pollos y se identificaron mediante una prueba de hemaglutinación y un ensayo de transcripción reversa y reacción en cadena de la polimerasa. Se secuenciaron tres aislados de cada granja para caracterizar la parte correspondiente al gene de la proteína de fusión alrededor del sitio de disociación. Las características de los genes de la proteína de fusión de los tres aislamientos mostrados por el análisis filogenético indicaron que los aislamientos eran velogénicos, pertenecían a la clase II subgenotipo VII 1.1 y estaban estrechamente relacionados con un aislado del virus de Newcastle identificado en China. Hasta donde se sabe, esta es la primera vez que los aislamientos del virus de Newcastle de Afganistán se han secuenciado parcialmente.

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.

Molecular analysis reveals expansion of Fasciola hepatica distribution from Afghanistan to China.

Recently, phosphoenol pyruvate carboxykinase (pepck) and DNA polymerase delta (pold) were established as reliable nuclear markers for species identification of Fasciola spp. in multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism-based assays, respectively. Currently, little is known about Fasciola species distribution in Central Asia. Therefore, the objective of the present study was to perform precise molecular species identification of liver flukes from Afghanistan and to reveal their dispersal route(s) via phylogenetic analysis based on mitochondrial nad1 haplotypes. Ninety-two Fasciolaflukes collected from sheep in Kabul, Afghanistan, were identified as F. hepatica based on pepck and pold screening. Although the pepck fragment pattern obtained via multiplex PCR analysis could not distinguish the species of the seven Fasciola flukes, the pepck nucleotide sequence data confirmed that they were F. hepatica.The 20 nad1 haplotypes detected among the Afghani liver flukes were closely related to those from China and Egypt, with the FSTvalue (-0.003, P = .41) between the F. hepatica populations from Afghanistan and China confirming a very close relationship. Nucleotide diversity was greater in the population from Afghanistan compared with that from China, indicating that the Afghani population was older, and that the dispersal direction of F. hepatica was from Afghanistan to China. The results of the present study contribute to our understanding of the dispersal of F. hepatica from its predicted origin, the Fertile Crescent.

Enhancement of bactericidal effects of sodium hypochlorite in chiller water with food additive grade calcium hydroxide.

An alkaline agent, namely food additive grade calcium hydroxide (FdCa(OH)2) in solution at 0.17%, was evaluated for its bactericidal efficacies in chiller water with sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine. Without organic material presence, NaOCl could inactivate Salmonella Infantis and Escherichia coli within 5 sec, but in the presence of fetal bovine serum (FBS) at 0.5%, the bactericidal effects of NaOCl were diminished completely. FdCa(OH)2 solution required 3 min to inactivate bacteria with or without 5% FBS. When NaOCl and FdCa(OH)2 were mixed at the final concentration of 200 ppm and 0.17%, respectively, the mixed solution could inactivate bacteria at acceptable level (103 reduction of bacterial titer) within 30 sec in the presence of 0.5% FBS. The mixed solution also inhibited cross-contamination with S. Infantis or E. coli on chicken meats. It was confirmed and elucidated that FdCa(OH)2 has a synergistic effect together with NaOCl for inactivating microorganisms.

Durability of alkaline agents' bactericidal efficacies in litter under field conditions.

Alkaline agents are well-known for their disinfection capacities against pathogens even at the presence of organic materials, but the durability of their bactericidal efficacies under field conditions is unknown. Therefore, within the present study, two alkaline agents, namely bioceramic (BCX) derived from chicken feces and food additive grade Ca(OH)2 (FdCa(OH)2) derived from natural lime stone, were evaluated for the persistence of their bactericidal efficacies in litter, under simulated field conditions. BCX powder mixed at 50% concentration in litter or FdCa(OH)2 powder at 20% concentration in litter lost their bactericidal efficacies at 3 days post exposure of chicks, and thereafter, both mentioned alkaline agents could not inactivate bacteria down to the acceptable level (≥3 log10 CFU/ml reduction).

Accuracy of the evaluation method for alkaline agents' bactericidal efficacies in solid, and the required time of bacterial inactivation.

An alkaline agent, namely food additive grade calcium hydroxide (FdCa (OH)2) in the powder form, was evaluated for its bactericidal efficacies in chicken feces at pH 13. The point for this evaluation was neutralization of the alkaline agent's pH at the time of bacterial recovery, since otherwise the results are substantially misleading. Without neutralization of the FdCa (OH)2 pH, the spiked bacteria were killed within min at the time of recovery in aqueous phase, but not in the solid form in feces, hence, it has been demonstrated that when bacteria were in solid, it took longer time than in liquid for the alkaline agent to inactivate them down to the acceptable level (≥3 log10 CFU/ml).

Virucidal Properties of Bioceramic Derived from Chicken Feces pH 13 and its Stability in Harsh Environments.

Bioceramic derived from chicken feces (BCX) is a material produced by a sintering process for the purpose of use in animal farms to control livestock infectious diseases. In the present study, BCX at pH 13 was evaluated for the durability of its virucidal activity in simulated field conditions. First it was shown that BCX had activity toward Newcastle disease virus, infectious bursal disease virus, and goose parvovirus within 3 min and toward avian influenza virus (AIV) within 1 hr. BCX was further tested by keeping it under simulated harsh environmental conditions with sunlight for several weeks as well as by repeatedly soaking it with water and drying under sunlight many times. After sampling every 2 consecutive weeks and every 2 (of 9) consecutive resuspensions, BCX was evaluated for its efficacy against AIV. Evaluation under the harsh conditions illustrated that BCX could retain its satisfactory efficacy toward AIV throughout 7 wk and through 9 resuspensions. It is hence concluded that BCX is an excellent material for applying in livestock farming as a trapping disinfectant, due to its efficacy to inactivate various viruses, and that this efficacy is prolonged even under harsh environmental conditions.

Inactivation of bacteria on surfaces by sprayed slightly acidic hypochlorous acid water: in vitro experiments.

The capacity of slightly acidic hypochlorous acid water (SAHW), in both liquid and spray form, to inactivate bacteria was evaluated as a potential candidate for biosecurity enhancement in poultry production. SAHW (containing 50 or 100 ppm chlorine, pH 6) was able to inactivate Escherichia coli and Salmonella Infantis in liquid to below detectable levels (≤2.6 log10 CFU/ml) within 5 sec of exposure. In addition, SAHW antibacterial capacity was evaluated by spraying it using a nebulizer into a box containing these bacteria, which were present on the surfaces of glass plates and rayon sheets. SAHW was able to inactivate both bacterial species on the glass plates (dry condition) and rayon sheets within 5 min spraying and 5 min contact times, with the exception of 50 ppm SAHW on the rayon sheets. Furthermore, a corrosivity test determined that SAHW does not corrode metallic objects, even at the longest exposure times (83 days). Our findings demonstrate that SAHW is a good candidate for biosecurity enhancement in the poultry industry. Spraying it on the surfaces of objects, eggshells, egg incubators and transport cages could reduce the chances of contamination and disease transmission. These results augment previous findings demonstrating the competence of SAHW as an anti-viral disinfectant.

Calcinated egg shell as a candidate of biosecurity enhancement material.

Calcinated egg shell (Egg-CaO), of which the main component is calcium oxide, was evaluated in the forms of powder and aqueous solutions for their efficacies as disinfectants against avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis and Escherichia coli. Egg-CaO powder inactivated these viruses within 3 min in the presence of 33% of fetal bovine serum (FBS). In Egg-CaO solutions, except AIV, all pathogens were inactivated within 1 hr, even in the presence of 5% of FBS. Without FBS, all pathogens, except AIV, were inactivated within 3 min, and AIV within 1 hr. In addition, persistence of virucidal activity against AIV and NDV of Egg-CaO powder was confirmed after exposure to sunlight for 2 weeks or resuspension with water for 7 times, simulating field harsh environments. Chick growth test was conducted to ensure the safety of the use of Egg-CaO powder in chicken cages and showed that it is safe to add Egg-CaO in litter or feed. In conclusion, Egg-CaO can be useful for the enhancement of biosecurity at farms.

Aerosol Disinfection Capacity of Slightly Acidic Hypochlorous Acid Water Towards Newcastle Disease Virus in the Air: An In Vivo Experiment.

Existence of bioaerosol contaminants in farms and outbreaks of some infectious organisms with the ability of transmission by air increase the need for enhancement of biosecurity, especially for the application of aerosol disinfectants. Here we selected slightly acidic hypochlorous acid water (SAHW) as a candidate and evaluated its virucidal efficacy toward a virus in the air. Three-day-old conventional chicks were challenged with 25 doses of Newcastle disease live vaccine (B1 strain) by spray with nebulizer (particle size <3 µm in diameter), while at the same time reverse osmosis water as the control and SAHW containing 50 or 100 parts per million (ppm) free available chlorine in pH 6 were sprayed on the treated chicks with other nebulizers. Exposed chicks were kept in separated cages in an isolator and observed for clinical signs. Oropharyngeal swab samples were collected from 2 to 5 days postexposure from each chick, and then the samples were titrated with primary chicken kidney cells to detect the virus. Cytopathic effects were observed, and a hemagglutination test was performed to confirm the result at 5 days postinoculation. Clinical signs (sneezing) were recorded, and the virus was isolated from the control and 50 ppm treatment groups, while no clinical signs were observed in and no virus was isolated from the 100 ppm treatment group. The virulent Newcastle disease virus (NDV) strain Sato, too, was immediately inactivated by SAHW containing 50 ppm chlorine in the aqueous phase. These data suggest that SAHW containing 100 ppm chlorine can be used for aerosol disinfection of NDV in farms.

Efficacy of scallop shell powders and slaked lime for inactivating avian influenza virus under harsh conditions.

The efficacy and stability of scallop shell powder (SSP) were investigated, in terms of its capacity to inactivate avian influenza virus (AIV), and compared with slaked lime (SL). An environmental simulation was conducted by emulating sunlight and wet-dry conditions. The powders were collected at consecutive 2-week intervals under sunlight and upon every resuspension. These materials were tested by mixing them with AIV and incubating the mixture for 3 min or 20 h, followed by AIV titration. At the same time, a pH buffering test was conducted by neutralization with Tris-HCl. The results revealed that SSP and SL have high alkalinity and excellent ability to inactivate AIV. In a simulated harsh environment, SSP and SL retained a satisfactory ability to inactivate AIV within 20 h throughout the experimental procedure. However, SSP was able to inactivate AIV during a short contact period (3 min), even under harsh conditions, and it was more resistant than SL to neutralization.

Inhibition of infectious bursal disease virus transmission using bioceramic derived from chicken feces.

Bioceramic powder (BCX), at pH 13.0, derived from chicken feces, was evaluated for its efficacy to inactivate virus and inhibit virus horizontal transmission by fecal-oral route, using infectious bursal disease virus (IBDV) vaccine strain D78 as a challenge virus. Three 1-week-old SPF chicks were vaccinated per os and used as seeder birds. Six hours later, 3 sentinel 1-week-old SPF chicks were introduced into the same cage. Results revealed that BCX had excellent efficacy to inactivate IBDV within 3 min. Treating IBDV contaminated litter in the cage with BCX could prevent transmission of IBDV to new sensitive chicks completely. Further, transmission of IBDV to the sentinel chicks was significantly inhibited by adding BCX to litter and chicken feed. These data suggest that BCX at pH 13, derived from chicken feces, has excellent efficacy to inactivate IBDV, which can be applied in bedding materials for preventing viral transmission during production round. It is a good material that can effectively be used for enhancing biosecurity system in poultry farms.

Evaluation of sprayed hypochlorous acid solutions for their virucidal activity against avian influenza virus through in vitro experiments.

Hypochlorous acid (HOCl) solutions were evaluated for their virucidal ability against a low pathogenic avian influenza virus (AIV), H7N1. HOCl solutions containing 50, 100 and 200 ppm chlorine (pH 6) or their sprayed solutions (harvested in dishes placed at 1 or 30 cm distance between the spray nozzle and dish) were mixed with the virus with or without organic materials (5% fetal bovine serum: FBS). Under plain diluent conditions (without FBS), harvested solutions of HOCl after spraying could decrease the AIV titer by more than 1,000 times, to an undetectable level (< 2.5 log10TCID50/ml) within 5 sec, with the exception of the 50 ppm solution harvested after spraying at the distance of 30 cm. Under the dirty conditions (in the presence of 5% FBS), they lost their virucidal activity. When HOCl solutions were sprayed directly on the virus on rayon sheets for 10 sec, the solutions of 100 and 200 ppm could inactivate AIV immediately after spraying, while 50 ppm solution required at least 3 min of contact time. In the indirect spray form, after 10 sec of spraying, the lids of the dishes were opened to expose the virus on rayon sheets to HOCl. In this form, the 200 ppm solution inactivated AIV within 10 min of contact, while 50 and 100 ppm could not inactivate it. These data suggest that HOCl can be used in spray form to inactivate AIV at the farm level.

Inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder.

Scallop shell powder produced by calcination process - the average diameter of the powder particles being 20 µm (SSP) - was further ground into nano-sized particles, with average diameter of 500 nm, here designated CaO-Nano. Solution of CaO-Nano could inactivate avian influenza virus within 5 sec, whereas the solution of SSP could not even after 1 hr incubation. CaO-Nano solution could also inactivate Newcastle disease virus and goose parvovirus within 5 sec and 30 sec, respectively. The virus-inactivating capacity (neutralizing index: NI>3) of the solution was not reduced by the presence of 20% fetal bovine serum. CaO-Nano solution seems to be a good candidate of materials for enhancement of biosecurity in farms.

Inactivation of goose parvovirus, avian influenza virus and phage by photocatalyst on polyethylen terephthalate film under light emitting diode (LED).

The inactivation effect of a novel photocatalyst on polyethylene terephthalate film on goose parvovirus (GPV), avian influenza virus (AIV) and Qß phage was evaluated. Under a light emitting diode (LED) light (range 410-750 nm), GPV was inactivated by irradiation at 1,000 lux for 6 hr, while AIV and Qß phage were inactivated by irradiation at 150 lux for 2 hr. These data suggest that this new photocatalyst can potentially be used as one of the materials to inactivate viruses in the indoor environment and help us to prevent viral infectious diseases through indirect contact.