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

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 p0.05). 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

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% β-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 β-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 β-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

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. Conclusion: 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

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

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&ndash;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

The porcine socs and their involvement in the response to two strains of influenza a virus

International audienceThe Suppressors Of Cytokine Signaling (SOCS) and the Cytokine-Inducible SH2 domain containing protein (CISH) are a family of eight proteins which have emerged as key regulators of the immune system. Little is known about tissue expression of SOCS and data in pigs are still scarce. In order to go further in the study of porcine SOCS, additional data are needed. CISH and SOCS transcript expressions along with the stability of some reference genes were assessed in various tissues. Then, we characterized the immune response of Newborn Pig Trachea cells (NPTr) and Porcine Alveolar Macrophages (PAMs) to a German strain (H3N2) and a Canadian strain (H1N1) of swine influenza and the potential role of SOCS proteins in this response. Among SOCS, only SOCS1 appears to be over-expressed. Using Precision Cut Lung Slices (PCLS) we characterized the interaction of H3N2 and H1N1 with the respiratory mucosa in the first hours of infection using different multiplicity of infection. By immunofluorescence the presence of the virus in the epithelial cells was confirmed and the establishment of a type I interferon response along with the subsequent mRNA expression of antiviral genes such as Mx1, Mx2, OAS1, RNAseL and PKR were observed in PAMs and NPTr cells. Furthermore, the influence of H3N2 on cellular signalisation was analysed at early points of infection in NPTr cells. We observed a precocious activation of JAK/STAT and MAPK pathways such as p38 and ERK 1-2. Then, kinome and RTqPCR analyses of the impact of H1N1 infection of NPTr cells in the presence of an inhibitor of SOCS1/SOCS3 (pJAK2) is in progress. Together, our data show the establishment of a broad antiviral response in NPTr cells, PAMs, and PCLS against H3N2 and H1N1, and strongly suggest a regulation by SOCS1 requiring further studies