Magnitude and kinetics of multifunctional CD4+ and CD8β+ T cells in pigs infected with swine influenza A virus

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4⁺ and CD8β⁺ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ⁺TNF-α⁺IL-2⁺ multifunctional CD4⁺ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4⁺ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4⁺ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4⁺ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4⁺ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67⁺perforin⁺ CD8β⁺ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β⁺ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0182-3) contains supplementary material, which is available to authorized users.     

Behaviour of equine influenza virus in a naïve population: a practitioner's perspective

We describe the behaviour of equine influenza (EI) virus infection in a na?ve population as observed by equine veterinary practitioners. The clinical signs displayed by infected horses and the highly contagious nature of the disease are discussed, as well as the treatment and management of infected horses.     

In vivo evaluation of vaccine efficacy against challenge with a contemporary field isolate from the α cluster of H1N1 swine influenza virus

Influenza A virus vaccines currently contain a mixture of isolates that reflect the genetic and antigenic characteristics of the currently circulating strains. This study was conducted to evaluate the efficacy of a trivalent inactivated swine influenza virus vaccine (Flusure XP) in pigs challenged with a contemporary α-cluster H1N1 field isolate of Canadian swine origin. Pigs were allocated to vaccinated, placebo, and negative-control groups and monitored for respiratory disease for 5 d after challenge. On the challenge day and 5 d after challenge the serum of the vaccinated pigs had reciprocal hemagglutination inhibition antibody titers 40 for all the vaccine viruses but ≤ 20 for the challenge virus. Gross lesions were present in the lungs of all pigs that had been inoculated with the challenge virus, but the proportion of lung tissue consolidated did not differ significantly between the placebo and vaccinated pigs. However, the amount of virus was significantly reduced in the nasal secretions, lungs, and bronchoalveolar lavage fluid in the vaccinated pigs compared with the placebo pigs. These results indicate that swine vaccinated with Flusure XP were partially protected against experimental challenge with a swine α-cluster H1N1 virus that is genetically similar to viruses currently circulating in Canadian swine.     

The measurement of three cytokine transcripts in naïve and sensitized ovine peripheral blood mononuclear cells following in vitro stimulation with bluetongue virus serotype-23

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of na?ve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from na?ve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to na?ve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens.     

Induction of antibody and interferon-γ production in mice immunized with virus-like particles of swine hepatitis E virus

Virus-like particles (VLPs) composed of the truncated capsid protein of swine hepatitis E virus (HEV) were developed and immune responses of mice immunized with the VLPs were evaluated. IgG titers specific for the capsid protein of swine HEV were significantly higher for all groups of mice immunized with the VLPs than those of the negative control mice. Splenocytes from mice immunized with the VLPs also produced significantly greater quantities of interferon (IFN)-γ than interleukin (IL)-4 and IL-10. These newly developed swine HEV VLPs have the capacity to induce antigen-specific antibody and IFN-γ production in immunized mice.     

Clinical outcomes and virology of equine influenza in a naïve population and in horses infected soon after receiving one dose of vaccine

As part of the control measures of the equine influenza (EI) outbreak, in addition to the strategic use of vaccination to provide buffer zones around infected populations, approval was obtained to vaccinate Thoroughbred racing horses. We review the clinical expression of the disease and virus excretion in a population of racehorses that were exposed to EI approximately 7 days after administration of a single dose of the canarypox-vectored recombinant compared with a similar unvaccinated population of horses at a nearby racetrack. Although this study was undertaken opportunistically and under the difficult field conditions that prevailed during the outbreak, our observations demonstrate that an appropriate vaccine can be effectively used as a disease control measure, even in the face of an outbreak, and therefore should be rapidly implemented as soon as there is evidence of infection in a na?ve population.     

Enhanced inactivation of avian influenza virus at ?20°C by disinfectants supplemented with calcium chloride or other antifreeze agents

Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl₂)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at −20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl₂ inactivated 6 log₁₀ AIV within 5 min at −20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl₂ solution alone inactivated 5 log₁₀ AIV within 10 min. The results suggested that CaCl₂ is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons.     

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-na?ve regulatory T cells to proliferate

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)⁺ and N-protein⁻ moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein⁺ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein⁻ moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3⁺ regulatory T cells present within CD4⁺ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4⁺ T cells showed differences in regard to proliferation and frequency of Foxp3⁺ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.     

Effects of single or trickle Haemonchus contortus experimental infection on digestibility and host responses of naïve Creole kids reared indoor

The objective of this study was to compare the effects of the type of Haemonchus contortus experimental infection (trickle infection, TI versus single infection, SI) on feed intake, nutrients digestibility, parasitological and haematological measures, and plasma leptin in Creole kids. The animals were infected over 2 periods (challenge 1 and challenge 2) of 6 weeks each, corresponding respectively to the primary and the secondary infection. Periods prior infection (1 week each) were considered as controls. The primary infection was realized with 35 Creole kids (18.40 ± 3.76 kg BW) housed in individual boxes and fed a hay-based diet. The secondary infection continued with 29 kids (21.90 ± 3.40 kg BW) from the initial 35. A total of 6 kids and 8 kids were slaughtered for measuring nematode burden at the end of the primary and the secondary infection, respectively. Measurements of nutrients digestibility were made at 0, 3 and 5 weeks post-infection for both challenges. Faecal egg count (FEC), blood eosinophilia and packed cell volume (PCV) were monitored weekly. Feed intake (dry matter intake, DMI) and nutrients digestibility were negatively affected by H. contortus infection only during the primary infection. Plasma leptin changed significantly over time (P = 0.0002) but was not affected by the infection type. Effect of infection type was observed only on crude protein digestibility during the primary infection, which was lower in the TI group (P < 0.01). The overall level of blood eosinophilia was significantly higher in the TI group (P < 0.0001) during both challenges. The overall FEC mean was significantly higher in the SI compared with the TI groups, during both challenges (P < 0.02). These results were related to the mean female length significantly higher in the SI group compared with the TI group during challenge 1 (P = 0.004), and the number of adult nematode significantly lower in the TI group compared with the SI group during the challenge 2 (P = 0.05). The results showed that the response of Creole kids to H. contortus experimental infection was in part dependent on the type of experimental infection. Our data suggest that plasma leptin would not be involved in the response of Creole kids against H. contortus infection, as no relationship between its plasma level and the transient reduction in voluntary feed intake observed in both groups during the primary infection was observed.     

Effect of Saccharomyces cerevisiae var. Boulardii and β-galactomannan oligosaccharide on porcine intestinal epithelial and dendritic cells challenged in vitro with Escherichia coli F4 (K88)

ABSTRACT: Probiotic and prebiotics, often called "immune-enhancing" feed additives, are believed to deal with pathogens, preventing the need of an immune response and reducing tissue damage. In this study, we investigated if a recently developed β-galactomannan (βGM) had a similar protective role compared to Saccharomyces cerevisiae var. Boulardii (Scb), a proven probiotic, in the context of enterotoxigenic Escherichia coli (ETEC) infection. ETEC causes inflammation, diarrhea and intestinal damage in piglets, resulting in large economic loses worldwide. We observed that Scb and βGM products inhibited in vitro adhesion of ETEC on cell surface of porcine intestinal IPI-2I cells. Our data showed that Scb and βGM decreased the mRNA ETEC-induced gene expression of pro-inflammatory cytokines TNF-α, IL-6, GM-CSF and chemokines CCL2, CCL20 and CXCL8 on intestinal IPI-2I. Furthermore, we investigated the putative immunomodulatory role of Scb and βGM on porcine monocyte-derived dendritic cells (DCs) per se and under infection conditions. We observed a slight up-regulation of mRNA for TNF-α and CCR7 receptor after co-incubation of DC with Scb and βGM. However, no differences were found in DC activation upon ETEC infection and Scb or βGM co-culture. Therefore, our results indicate that, similar to probiotic Scb, prebiotic βGM may protect intestinal epithelial cells against intestinal pathogens. Finally, although these products may modulate DC activation, their effect under ETEC challenge conditions remains to be elucidated.     

Do animal exhibitors support and follow recommendations to prevent transmission of variant influenza at agricultural fairs? A survey of animal exhibitor households after a variant influenza virus outbreak in Michigan

Influenza A viruses circulate in swine and can spread rapidly among swine when housed in close proximity, such as at agricultural fairs. Youth who have close and prolonged contact with influenza‐infected swine at agricultural fairs may be at increased risk of acquiring influenza virus infection from swine. Animal and human health officials have issued written measures to minimize influenza transmission at agricultural exhibitions; however, there is little information on the knowledge, attitudes, and practice (KAP) of these measures among animal exhibitors. After an August 2016 outbreak of influenza A(H3N2) variant (“H3N2v”) virus infections (i.e., humans infected with swine influenza viruses) in Michigan, we surveyed households of animal exhibitors at eight fairs (including one with known H3N2v infections) to assess their KAP related to variant virus infections and their support for prevention measures. Among 170 households interviewed, most (90%, 151/167) perceived their risk of acquiring influenza from swine to be low or very low. Animal exhibitor households reported high levels of behaviours that put them at increased risk of variant influenza virus infections, including eating or drinking in swine barns (43%, 66/154) and hugging, kissing or snuggling with swine at agricultural fairs (31%, 48/157). Among several recommendations, including limiting the duration of swine exhibits and restricting eating and drinking in the animal barns, the only recommendation supported by a majority of households was the presence of prominent hand‐washing stations with a person to monitor hand‐washing behaviour (76%, 129/170). This is a unique study of KAP among animal exhibitors and highlights that animal exhibitor households engage in behaviours that could increase their risk of variant virus infections and have low support for currently recommended measures to minimize infection transmission. Further efforts are needed to understand the lack of support for recommended measures and to encourage healthy behaviours at fairs.     

Comparative immune response and pathogenicity of the H9N2 avian influenza virus after administration of Immulant®, based on Echinacea and Nigella sativa,in stressed chickens

Avian influenza vaccines are commonly used in the poultry industry, and some medicinal plants can increase the efficacy of such vaccines. The objective of this study was to evaluate the effect of Immulant^® (IMU) (a commercial product based on Echinacea and Nigella sativa) on stress induced by dexamethasone (DEX) in chickens vaccinated (VAC) against the H9N2 avian influenza virus (AIV-H9N2). Seven experimental groups were included: the negative control, VAC, DEX, VAC + DEX, VAC + DEX + IMU, VAC + IMU and IMU groups. The vaccinated chickens (at 10 days of age) were injected daily with DEX for three days pre-vaccination and for three days pre-challenge and orally administered 1% IMU for 6 weeks post-vaccination (PV). The chickens were then challenged intranasally with AIV-H9N2 at 28 days PV. Serum, blood, tracheal and cloacal swabs and tissue samples were collected in the 1^st and 4^th weeks PV and at different time points post-challenge. The results showed significant changes (P ≤ 0.05) in oxidative stress and antioxidant biomarkers (malondialdehyde, nitric oxide and reduced glutathione), haematological and immunological parameters, final live weights, relative organ weights and histopathological lesions between the VAC+DEX group and the VAC group. Moreover, IMU significantly increased protection rates post-challenge, HI antibody titers and heterophil phagocytic activity and decreased DEX-induced stress and virus shedding titers. In conclusion, oral administration of 1% IMU for six weeks can enhance the immune response after AI-H9N2 vaccination and reduce the pathogenicity of infection in stressed chickens.     

Oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 enhances the alleviation of clinical signs caused by respiratory infection with avian influenza virus H9N2

The combined use of cytokines has shown synergistic and/or additive effects in controlling several viral infections of livestock animals. However, little is known concerning the practical use of chicken cytokine combinations to control avian diseases. Here, we investigated the antiviral efficacy of oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) using attenuated Salmonella enterica serovar Typhimurium in chickens infected with avian influenza virus (AIV) H9N2. Our results demonstrate that oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 produced a greater alleviation of clinical signs caused by respiratory infection with AIV H9N2 in chickens, when compared to administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18 alone. Mortality, clinical symptom severity, and feed and water intake were used to access treatment effectiveness. This enhancement of antiviral immunity was further confirmed by evidence of reduced rectal shedding and decreased replication of AIV H9N2 in several different tissues of challenged chickens including trachea, lung, cecal tonsil, and brain. Furthermore, oral co-administration of chIFN-α and chIL-18 more efficiently modulated the immune responses of chickens against AIV H9N2 by enhancing both humoral and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results suggest that the combined administration of two chicken cytokines, chIFN-α and chIL-18, using attenuated S. enterica serovar Typhimurium as an oral carrier, provides an effective means for controlling respiratory disease caused by AIV H9N2 infection.     

Limited efficacy of Fever Tag? temperature sensing ear tags in calves with naturally occurring bovine respiratory disease or induced bovine viral diarrhea virus infection

Temperature sensing ear tags were tested in 1) auction-derived calves with 50% incidence of bovine respiratory disease, and 2) specific pathogen-free calves infected with bovine virus diarrhea virus. There were no false positives, but tag placement, probe displacement, and a high threshold for activation all contributed to failure to reliably detect sick calves.     

Piloting the effectiveness of pig health education in combination with oxfendazole treatment on prevention and/or control of porcine cysticercosis,gastrointestinal parasites,African swine fever and ectoparasites in Angónia District,Mozambique

A community-based intervention combining health education (HE) and treatment of pigs for control of porcine cysticercosis (PC), gastrointestinal (GI) helminths, African swine fever (ASF) and external parasites was tested involving six villages of resource-poor smallholder pig farmers. Farmers and pigs of six rural villages were randomly allocated into group 1 (HE), which served as controls, and group 2 (HE?+?OFZ) pigs received a single oral dose of 30 mg/kg OFZ. Farmers were trained in pig health, housing and feeding. The proportion of farmers with confined pigs, the adoption rate of the introduced pig pen, the sero-prevalence of PC and ASF, the prevalence and intensity of GI nematodes and the prevalence of ectoparasites were measured at 9, 15 and 24 months after initiation and compared to a baseline survey to seek the effectiveness of the interventions. There was no clear effect of the intervention on the sero-prevalence of PC, but analysis of the rate of change in prevalence between the two groups showed significant effect with the rate of change to lower prevalence in the HE?+?OFZ group compared to the HE group. Although HE managed to improve the farmer’s knowledge in the control and prevention of ASF and ectoparasites, there was no significant reduction in the sero-prevalence of ASF and the prevalence of ectoparasites throughout the two-year period. The reported ineffectiveness of the intervention in this study suggested that more research is needed to develop more effective methods for controlling PC, ASF and pig parasites.