In vitro primary porcine alveolar macrophage cell toxicity and African swine fever virus inactivation using five commercially supply compound disinfectants under various condition.

Efficacy of African swine fever virus (ASFV) inactivation using five commercially supply compound disinfectants was evaluated under various condition. Virucidal efficacy demonstrated that products A and E could inactivate at 1:800 within 1 min for both temperatures, while products B, C and D inactivated at 1:400. However, product D could inactivate at 1:800 when the exposure time was extended to 30 min and effected only 20°C. In addition, the cytotoxicity demonstrated that products A, B, C, D and E did not significantly affect to cell at 1:51,200, 1:12,800, 1:12,800, 1:25,600 and 1:12,800 dilution, respectively. In conclusion, these disinfectants could inactivate ASFV, however, the application of these products should be performed under safety precautions to prevent cytotoxicity in humans and animals.

Accuracy and precision guidelines for optimal breeding time in bitches using in-house progesterone measurement compared with chemiluminescent microparticle immunoassay.

BACKGROUND AND AIM: The concentration of serum progesterone is commonly used to determine the optimal mating time in bitches, and to diagnose reproductive-related abnormalities. This study aims to compare the serum progesterone results obtained by rapid fluorescence immunochromatography assay (RFICA) with those obtained by chemiluminescent microparticle immunoassay (CMIA) from the same serum samples to develop a standard guideline for optimal breeding time. MATERIALS AND METHODS: Serum progesterone levels were measured in 124 bitches using RFICA and CMIA. Simple linear regression and correlation analyses were performed to analyze the data. The percentage difference between the maximum and minimum progesterone values in the same serum sample in the same assay was compared using Wilcoxon's rank-sum test. RESULTS: The present study showed a strong linear dependence of the results obtained by RFICA on those obtained by CMIA as R2=0.8976, with regression coefficient of 0.9474 and p<0.05, including the regression model was CMIA = (0.9483 × RFICA) - 0.761. Moreover, five critical measurement times during estrous in bitches showed statistically significant differences (p<0.05), except at the fertilizable period, which showed a non-significant difference (p>0.05). CONCLUSION: This study demonstrated that it is presumably acceptable to use the RFICA and CMIA methods interchangeably for quality progesterone measurements in serum samples from bitches. However, when considering the use of the RFICA method, it is advisable to carefully interpret the results and follow the interpretation guidelines. Finally, RFICA in the present study provides a reliable and convenient option for veterinarian practitioners to measure canine progesterone levels in-house.

In vitro cytotoxicity and virucidal efficacy of potassium hydrogen peroxymonosulfate compared to quaternary ammonium compound under various concentrations, exposure times and temperatures against African swine fever virus.

BACKGROUND AND AIM: The selection and proper application of disinfectants are crucial to the prevention of many diseases, so disinfectants must be evaluated before being used for the prevention of African swine fever (ASF). Three disinfectant products belonging to the group of potassium hydrogen peroxymonosulfates, product A and product B, and a quaternary ammonium compound called product C, were examined in vitro for host cell cytotoxicity and the efficacy of ASF virus inactivation. The study parameters included various concentrations, exposure times, temperatures, and degrees of cytotoxicity. MATERIALS AND METHODS: Three disinfectant products were evaluated for cytotoxicity using primary porcine alveolar macrophage (PAM) cells at dilutions from 1:200 to 1:51,200. Disinfectants in concentrations of 1:200, 1:400, and 1:800 were prepared, the pH and the virucidal activity were tested. An equal volume of each dilution was mixed with the ASF virus and incubated at room temperature (20°C) or on ice (4°C) for 1 min, 5 min, or 30 min. Hemadsorption (HAD) or rosette formation was observed using an inverted microscope for 5 days after inoculation, and the virus titer was calculated as HAD50/mL. Each treatment and virus control were tested in triplicate, and the titers were reported as means and standard deviations. The reduction factor was used to measure inactivation. RESULTS: Products A, B, and C at 1:400, 1:800, and 1:25,600 of dilution, respectively, did not show significant cytotoxic effects on PAM cells. Products A and B could inactivate ASF virus at 1:200 dilution within 5 min after exposure at 4°C. However, at 20°C, the exposure time had to be extended to 30 min to inactivate the virus. Product C could inactivate the virus at 1:400 dilution within 5 min under both temperature conditions, whereas at 1:800 dilution, the exposure time had to be extended to 30 min to completely inactivate the virus at 20°C. CONCLUSION: All disinfectants could inactivate ASF virus in various concentrations, under appropriate exposure times and reaction temperatures, and there was no evidence of host cell cytotoxicity. For the control of ASF in pig farms, the appropriate concentration, ambient temperature, and contact time of these disinfectants should be taken into account.

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

Effect of pre-supplementation with Pleurotus sajor-caju crude extracts on body weight and consequence responses of leukocytes and immune organs in fancy carp following inoculation with Aeromonas veronii.

AIM: The present study aimed at highlighting the effects of oyster mushroom (Pleurotus sajor-caju), as a dietary supplement on growth performance, differential leukocytes population, and histological changes of melanomacrophage centers (MMCs) in spleen and kidney of fancy carp on bacterial infection. MATERIALS AND METHODS: A total of 60 fancy carp were allocated into four groups according to feed formulations including; (1) basal diet with 2% crude extract of P. sajor-caju, (2) basal diet with 2% b-glucan, whereas Group 3, and Group 4 were positive and negative control, which were fed only basal diet. Diets were provided for 30 days, thereafter, fish of Group 1 to Group 3 were intraperitoneally injected with Aeromonas veronii (1.8×109 CFU), whereas Group 4 was injected with normal saline. At day 7 post-bacterial inoculation, all fish were weighed, whole blood was collected for differential white blood cell count, and two visceral organs, posterior kidney and spleen, were collected from euthanized fish to observe histological changes, particularly MMCs. RESULTS: No significant differences in body weight were found (p>0.05) at 1st week of the experiment; however, fish body weight was significantly increased from week 2 to week 4 of the experiment. Increased monocyte number was found in carp fish fed with the P. sajor-caju or b-glucan supplemented diets compared to the control groups (p<0.05). The proliferation of monocyte in fish was consistent with increased number and size of MMCs in hemotopoietic organs, posterior kidney and spleen, especially in fancy carp fed with of P. sajor-caju crude extract and commercially available b-glucan before bacterial inoculation in fish. CONCLUSION: These findings indicate that crude polysaccharide from P. sajor-caju can be potentially used as a feed additive that might promote innate immune function in fish.

Bactericidal efficacy of potassium peroxymonosulfate under various concentrations, organic material conditions, exposure timing and its application on various surface carriers.

Four concentrations of potassium peroxymonosulfate (PPMS) were evaluated for bactericidal activities and indicated that the concentration is less than the manufacturing-recommended concentrations, must extend the exposure time for bacterial inactivation. However, even with and without of organic material contamination, did not show marked inactivation difference. In addition, all concentrations were inactivated on all carrier surfaces within 30 sec, except on rubber where inactivation occurred within 1 min. However, quaternary ammonium compounds were inactivated on stainless steel and plastic within 1 min and 30 sec, respectively, but not inactivated within 5 min on rubber surfaces. Conclusion, PPMS inactivated bacteria under optimal concentration, organic material conditions, exposure timing and on carrier surfaces which can useful as an alternative disinfectant for biosecurity enhancement.

Bactericidal and virucidal efficacies of food additive grade calcium hydroxide under various concentrations, organic material conditions, exposure duration, and its stability.

AIM: This study aimed to evaluate the bactericidal and virucidal activity of food additive grade calcium hydroxide (FdCa(OH)2) under various concentrations, organic material conditions, and exposure duration including its stability. MATERIALS AND METHODS: The FdCa(OH)2 powder as well as the 0.17% and 3% solutions were evaluated for bacteria and virus inactivating efficacies against Salmonella infantis (SI), Escherichia coli, Newcastle disease virus (NDV), and avian influenza virus (AIV), in the absence or presence of organic materials. In addition, the stability of FdCa(OH)2, was also examined using wet-dry conditions and under sunlight. RESULTS: The FdCa(OH)2 powder could inactivate both NDV and AIV in the absence and presence of organic materials within a 3 min exposure period. The bactericidal efficacy using solution form revealed that 0.17% and 3% of FdCa(OH)2 could inactivate SI in the absence and presence of organic materials within 3 min of exposure. However, 3% of FdCa(OH)2 inactivated E. coli both with and without organic materials within 3min, while 0.17% required 5 min to be efficacious. The virucidal efficacy also showed that 0.17% FdCa(OH)2 could inactivate NDV in the absence and presence of organic materials within 10 min and 30 min, respectively. However, AIV inactivation was achieved within 30 sec under all conditions. In addition, under wet and dry conditions, FdCa(OH)2 powder demonstrated high efficacy when re-suspended at least 16 times for NDV and 7 times for AIV. Simultaneously, the FdCa(OH)2 powder retained its efficacy under the sunlight during up to 4 months for NDV and at least 6 months for AIV. CONCLUSION: The present study indicates that FdCa(OH)2 powder and solutions could inactivate SI, E. coli, NDV, and AIV while retaining good stability under challenging environmental conditions. Finally, the FdCa(OH)2 is safe for consumers because it is of food additive grade and can be useful as an alternative disinfectant, especially for biosecurity enhancement on and around poultry farms.

Stability and virucidal efficacies using powder and liquid forms of fresh charcoal ash and slaked lime against Newcastle disease virus and Avian influenza virus.

AIM: The present study was examined the virucidal activity comparison between fresh charcoal ash (FCA) and slaked lime (SL) against avian influenza virus (AIV) and Newcastle disease virus (NDV), using powder and liquid forms, either in the absence or presence of organic materials. In addition, both FCA and SL were evaluated for the persistence of virucidal activity in wet and dry conditions and stability of the solution. MATERIALS AND METHODS: Two hundred milligrams of FCA or SL powders were mixed with 100 µl of AIV or NDV in the absence of organic material or 33% of organic materials. In the same time, 400 µl of 1%, 5%, or 10% solution samples were mixed with 100 µl of each virus and then incubated at room temperature for an indicated time. After that, the mixed solution was stop activity of sample using 500 µl of 1M Tris-HCl pH 7.2. Each treatment was titrated onto Madin-Darby canine kidney cells or chicken embryo fibroblasts for AIV or NDV, respectively, for determining the efficacy of viral inactivation. In addition, the stability of powder under the wet-dry condition and solution stability under room temperature was examined. RESULTS: The results demonstrated that the FCA and SL in powder form could inactivate AIV and NDV even in the absence or presence of organic materials. In the liquid form, 5% and 10% of FCA could inactivate AIV and NDV either in the absence or presence of organic materials. Alongside, 1%, 5%, and 10% of SL could inactivate both viruses. 10% of FCA solution could inactivate virus at a shortest time when compared with other concentrations. In addition, the efficacy of wet-dry conditions of FCA was limited when compared with SL. On the other hand, it is demonstrated that the FCA solution was more stable and kept at room temperature longer than SL. CONCLUSION: The FCA may, hence, be used as an alternative virucide, while applying it to prevent spreading of poultry disease on commercial chicken farms and also backyard chickens, especially in developing countries, including in rural areas of Thailand.

The study of effect of didecyl dimethyl ammonium bromide on bacterial and viral decontamination for biosecurity in the animal farm.

AIM: The aim of this study was to determine the effectiveness of the fourth-generation quaternary ammonium compounds, didecyl dimethyl ammonium bromide (DDAB), on the efficacy of bacterial and viral decontamination against pathogens commonly found in livestock industry including Salmonella infantis (SI), Escherichia coli, and avian influenza virus (AIV). MATERIALS AND METHODS: The DDAB was prepared at 500, 250, and 125 parts per million (ppm) for absent and present organic material. Meanwhile, 5% of fetal bovine serum in DDAB solution sample was used to mimic the presence of organic material contamination. 400 µl of each DDAB concentration was mixed with 100 µl of each pathogen (SI, E. coli, and AIV) and then incubated at room temperature or 4°C at various time points (5 s, 30 s, 1 min, 5 min, 10 min, 15 min, and 30 min). The activity of DDAB treatment was stopped using 500 µl of FBS. Each treatment sample was titrated on either deoxycholate hydrogen sulfide lactose agar plates or Madin-Darby canine kidney cells for bacteria and AIV, respectively. Each treatment was conducted in triplicates, and the pathogen inactivation was considered effective when the reduction factor was ≥3 log10. RESULTS: Our current study revealed that the DDAB inactivated SI, E. coli, and AIV under the various concentrations of DDAB, organic material conditions, exposure temperature, and exposure timing. In addition, the comparison of bactericidal and virucidal efficacy indicated that bacteria were more susceptible to be inactivated by DDAB as compared to viruses. However, DDAB showed marked inactivated differences in the absence or presence of organic materials. CONCLUSIONS: The DDAB may be a potential disinfectant for inactivating bacteria and viruses, especially enveloped viruses, in livestock farms. It can be useful as a disinfectant for biosecurity enhancement on and around animal farm.

Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells.

We analyzed the adaptation of influenza viruses to growth in differentiated airway epithelial cells of a new host by passaging an avian H9N2 virus three times in porcine precision-cut lung slices (PCLS). Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins, and two in the HA protein. In this study, we characterized the PB2 mutation G685R by generating recombinant H9N2 viruses containing the PB2 single mutation alone or in combination with one of the HA mutations (A190V or T212I). When analyzed in porcine cells - a tracheal cell line (NPTr) or PCLS - the PB2-685 mutant did not provide a growth advantage and had no effect on the ciliary activity which is a virulence marker of swine influenza viruses. Pathogenicity for mice was also not increased by the single PB2 mutation. However, both double mutants (HA-190+PB2-685 and HA-212+PB2-685) showed significantly increased virulence in mice. Therefore, the mutations in the HA and PB2 proteins may confer early adaptation of an avian H9N2 virus to a mammalian host. In conclusion, we expect that a broader ensemble of mutations will be required to render an H9N2 virus virulent for pigs.

Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses.

Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices.

Viral respiratory diseases remain of major importance in swine breeding units. Swine influenza virus (SIV) is one of the main known contributors to infectious respiratory diseases. The innate immune response to swine influenza viruses has been assessed in many previous studies. However most of these studies were carried out in a single-cell population or directly in the live animal, in all its complexity. In the current study we report the use of a trachea epithelial cell line (newborn pig trachea cells - NPTr) in comparison with alveolar macrophages and lung slices for the characterization of innate immune response to an infection by a European SIV of the H3N2 subtype. The expression pattern of transcripts involved in the recognition of the virus, interferon type I and III responses, and the host-response regulation were assessed by quantitative PCR in response to infection. Some significant differences were observed between the three systems, notably in the expression of type III interferon mRNA. Then, results show a clear induction of JAK/STAT and MAPK signaling pathways in infected NPTr cells. Conversely, PI3K/Akt signaling pathways was not activated. The inhibition of the JAK/STAT pathway clearly reduced interferon type I and III responses and the induction of SOCS1 at the transcript level in infected NPTr cells. Similarly, the inhibition of MAPK pathway reduced viral replication and interferon response. All together, these results contribute to an increased understanding of the innate immune response to H3N2 SIV and may help identify strategies to effectively control SIV infection.

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses.

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses.

BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.