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.

Bactericidal efficacies of food additive grade calcium hydroxide toward Legionella pneumophila.

Food additive grade calcium hydroxide (FdCa(OH)2) in the solution of 0.17% was evaluated for its bactericidal efficacies toward Legionella pneumophila with or without sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine, at room temperature (RT) (25°C ± 2°C) and 42°C, either with or without 5% fetal bovine serum (FBS). Besides, FdCa(OH)2 in different concentration solutions were prepared in field water samples (hot spring and bath tab water) and evaluated for their bactericidal efficacies at 42°C. FdCa(OH)2 (0.17%) inactivated the L. pneumophila to the undetectable level (<2.6 log CFU/ml) within 5 min and 3 min, respectively, at RT and 42°C, with 5% FBS. At RT and 42°C, NaOCl inactivated L. pneumophila to the undetectable level within 5 min, without 5% FBS, but with 5% FBS, it could only inactivate this bacterium effectively (≥3 log reductions). Conversely, at RT and 42°C, the mixture of 0.17% FdCa(OH)2 and 200 ppm NaOCl could inactivate L. pneumophila to the undetectable level, respectively, within 3 min and 1 min, even with 5% FBS, and it was elucidated that FdCa(OH)2 has a synergistic bactericidal effect together with NaOCl. FdCa(OH)2 0.05% solution prepared in hot spring water could inactivate L. pneumophila to the undetectable within 3 min at 42°C. So, FdCa(OH)2 alone could show nice bactericidal efficacy at 42°C, even with 5% FBS, as well as in field water samples.

Inhibitory effect of grapefruit seed extract (GSE) on avian pathogens.

The inhibitory activities of grapefruit seed extract (GSE) on avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis (SI) and Escherichia coli (EC) were evaluated. Original GSE contained 0.24% benzalkonium chloride (BZC), however, 0.0025% BZC solution could not inactivate bacteria. The activity of diluted GSE (×100, ×500 and ×1,000 with redistilled water) against selected viruses and bacteria was evaluated in this study. The GSE solutions were incubated with the pathogens over a period of time after which the remaining viruses were titrated and the bacterial colonies were counted. In the presence of organic material-5% fetal bovine serum (FBS), the test solutions were sprayed at 1 cm and 30 cm distances to test the efficacy of GSE in a spray form. Furthermore, the efficacy of GSE against bacteria on clothes was tested using non-woven cloth. GSE×100 reduced the viral titer of both AIV and NDV even in 5% FBS condition. IBDV showed high resistance to GSE. GSE×1,000 inactivated both SI and EC within 5 sec, even in the presence of 5% FBS. The disinfectant was able to maintain its efficacy in the spray form at 30 cm distance. GSE was also effective against SI and EC inoculated on fabric. GSE is a potential novel disinfectant against viruses and bacteria, effective even within a short contact time.

Bactericidal efficacy of a quaternary ammonium compound with food additive grade calcium hydroxide toward Salmonella Infantis and Escherichia coli on abiotic carriers.

The bactericidal efficacies of 0.2% food additive grade calcium hydroxide (FdCa(OH)2) solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500) and their mixture-Mix500 (FdCa(OH)2 powder added at final concentration 0.2% to QACx500)-were investigated at two different temperatures (room temperature (RT) (25 ± 2°C) and 2°C), using varying contact time, with or without presence of organic materials (5% fetal bovine serum: FBS), either in suspension or on abiotic carrier (steel, rubber and plastic). In the suspension test, QACx500 could inactivate Salmonella Infantis at effective level (≥3 log reductions), within 30 sec and 5 sec, respectively, with or without 5% FBS at RT; however, at 2°C it required 30 min and 1 min, respectively. Mix500 revealed the same efficacy as QACx500 at RT, but, at 2°C it required 1 min and 30 sec, respectively with or without FBS. Whereas, 0.2% FdCa(OH)2 solution alone could inactivate S. Infantis within 1 min and 3 min, respectively at RT and 2°C, even with 5% FBS. In the carrier test, single disinfectant required bit more (3 or 5 min) contact time to reduce bacterial load (S. Infantis or Escherichia coli) down to the effective level on rubber surface than that on steel and plastic surface. However, Mix500 could inactivate both bacteria on carrier surfaces within 1 min, even at 2°C. Thus, synergistic effects were observed in the suspension test and the carrier test at both temperatures toward both bacteria.

Bactericidal Efficacy of Food Additive-Grade Calcium Hydroxide Against Salmonella Infantis on Eggshells.

The bactericidal efficacy of food additive-grade calcium hydroxide [FdCa(OH)2] was evaluated for inactivation of Salmonella Infantis and Salmonella Enteritidis in liquid and Salmonella Infantis on contaminated eggshells. The activity of FdCa(OH)2 was also compared with that of sodium hypochlorite (NaOCl) containing 150 ppm chlorine (150 ppm NaOCl). FdCa(OH)2 solutions (0.1% and 0.2%) in the presence or absence of organic materials (5% calf serum [CS]) at pH 12.6 were used to inactivate Salmonella Infantis and Salmonella Enteritidis in a reaction tube or on eggshells artificially contaminated with Salmonella Infantis. Both 0.1% and 0.2% FdCa(OH)2 were capable of inactivating Salmonella Infantis and Salmonella Enteritidis in liquid at >3 log10 colony-forming units (CFU)/ml within 3 and 1 min of contact time, respectively, even in the presence of 5% CS. Additionally, 0.1% and 0.2% FdCa(OH)2 reduced bacterial levels on contaminated eggshells to >3 log10 CFU/ml, within 3 and 1 min, respectively, in the presence of 5% CS. Without CS, 0.1% and 0.2% FdCa(OH)2 could reduce bacteria on eggshells to >3 log10 CFU/ml within 1 min and 30 sec, respectively. In contrast, 150 ppm NaOCl solution could not inactivate bacteria on eggshells down to >3 log10 CFU/ml within 3 min contact time, either with or without CS, and no bacterial reduction was observed in redistilled water. The findings of the present study indicate that FdCa(OH)2 solution has high efficacy against foodborne bacteria and may be a good candidate for enhancement of biosecurity at farms and egg processing plants.

Virucidal activity of a quaternary ammonium compound associated with calcium hydroxide on avian influenza virus, Newcastle disease virus and infectious bursal disease virus.

A quaternary ammonium compound (QAC) was evaluated for its virucidal efficacies with food additive grade calcium hydroxide (FdCa(OH)2). When the QAC was diluted 1:500 (QACx500) with redistilled water (dW2), it inactivated avian influenza virus (AIV) within 30 sec at 25°C, while at 2°C, it required 1 hr for inactivation. When FdCa(OH)2 powder was added to QACx500 at a final concentration of 0.17%, the mixture, namely Mix500, inactivated AIV within 3 min at 2°C. After contamination with 1% fetal bovine serum (FBS), Mix500 inactivated AIV within 2 hr at 2°C, but QACx500 did not. These results indicate synergistic effects of the QAC and FdCa(OH)2 solutions on virucidal activity.

Virucidal Efficacy of a Quaternary Ammonium Compound with Food Additive-Grade Calcium Hydroxide Toward Avian Influenza Virus and Newcastle Disease Virus on Abiotic Carriers.

The virucidal efficacies of a 0.2% food additive-grade calcium hydroxide [FdCa(OH)2] solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500), and their mixture [Mix500; FdCa(OH)2 powder added at a final concentration of 0.2% to QACx500] were investigated as fomites for avian influenza virus (AIV) and Newcastle disease virus (NDV) on abiotic carriers (steel, rubber, and plastic) at two different temperatures (room temperature [RT; 25 ± 2 C] and 2 C). These viruses were seeded on coupons (5 cm×5 cm) of rubber, steel, or plastic with 5% fetal bovine serum. After complete drying, the coupons were covered with the test solutions at RT or 2 C. After fixed incubation periods, viruses were recovered from the coupons and titrated. At RT, Mix500 required a short time (3 min) to inactivate AIV and NDV to effective levels (≥3 log virus reduction) on rubber, steel, and plastic carriers compared with QAC or FdCa(OH)2. At low temperature, QACx500 inactivated AIV on steel and plastic carriers to effective levels within 60 min, whereas Mix500 did so within 10 min. QACx500 and FdCa(OH)2 solutions could inactivate NDV on steel and plastic carriers within 20 and 10 min, respectively, and Mix500 could do so within 3 min. Viruses on the carriers required longer incubation periods for inactivation at 2 C than at 25 C. These results demonstrate desirable synergistic virucidal effects of Mix500 for important poultry viruses on abiotic carriers, while indicating high applicability within poultry farming.

Eficacia virucida de un compuesto de amonio cuaternario con hidróxido de calcio con grado aditivo alimentario contra el virus de la influenza aviar y el virus de la enfermedad de Newcastle en vehículo abióticos. Las eficacias virucidas de una solución de hidróxido de calcio [FdCa(OH)2] con grado aditivo alimentario al 0.2%, de un compuesto de amonio cuaternario (QAC) diluido a 1:500 (QACx500) y su mezcla [Mix500; FdCa(OH)2 en polvo agregado en una concentración final de 0.2% en QACx500] se investigaron en forma de fómites para el virus de la influenza aviar (AIV) y para el virus de la enfermedad de Newcastle (NDV) en vehículos abióticos (acero, hule y plástico) a dos temperaturas diferentes (temperatura ambiente [RT; 25 ± 2 C] y 2 C). Estos virus se sembraron en placas (5 cm x 5 cm) de hule, acero o plástico con suero bovino fetal al 5%. Después del secado completo, los cupones se cubrieron con las soluciones bajo ensayo a temperatura ambiente o 2 C. Después de los períodos de incubación fijos, los virus se recuperaron de las placas y se titularon. A temperatura ambiente, la mezcla Mix500 requirió un corto tiempo (tres minutos) para desactivar a los virus de influenza y de Newcastle a niveles efectivos (reducción igual o mayor de tres logaritmos en el título viral) en las placa de hule, acero y plástico en comparación con el tratamiento QAC o FdCa(OH)2. A baja temperatura, el tratamiento QACx500 inactivó al virus de la influenza en placas de acero y plástico a niveles efectivos dentro de 60 minutos, mientras que el tratamiento Mix500 lo hizo en 10 minutos. Las soluciones QACx500 y FdCa(OH)2 pudieron inactivar al virus de Newcastle en las placas de acero y plástico dentro de 20 y 10 minutos, respectivamente, y el tratamiento Mix500 pudo hacerlo dentro de tres minutos. Los virus en los vehículos requirieron períodos de incubación más prolongados para la inactivación a 2 C que a 25 C. Estos resultados demuestran los efectos virucidas sinérgicos deseables del tratamiento Mix500 para virus aviares importantes en vehículos abióticos, además de que indican una alta aplicabilidad dentro de la avicultura.

Virucidal efficacy of food additive grade calcium hydroxide against surrogate of human norovirus.

An alkaline agent, namely, food additive grade calcium hydroxide (FdCa(OH)2) in the solution, powder and suspension forms was evaluated as a virucidal agent, using a murine norovirus (MNV) as the surrogate for human norovirus. The main constituent of FdCa(OH)2 is Ca(OH)2, which has pH 13 in 0.17% solution. The results showed that 0.17% FdCa(OH)2 solution could inactivate MNV within 30s even in the presence of organic materials (5% fetal bovine serum (FBS)). In a contaminated surface experiment, MNV with 5% FBS was inoculated on rayon sheets, and the result showed FdCa(OH)2 solution could markedly reduce virus titer within 1min. When mouse feces were spiked with MNV and FdCa(OH)2 powder as 10% and 20% w/w was added to the feces, these concentrations could inactivate the virus within 30min and 15min, respectively. Whereas, FdCa(OH)2 suspension at 2.5% and 5% could inactivate the virus within 30min and at 1% within 45min. These and additional results obtained here indicate that FdCa(OH)2 is an effective virucidal agent against MNV, and can serve as a useful alternative disinfectant for inactivation and prevention of human norovirus in house and hospital.

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.

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

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).

TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus.

Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.