Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Prolonged Detection of PCV2 and Anti‐PCV2 Antibody in Oral Fluids Following Experimental Inoculation

The onset, level and duration of PCV2 and anti‐PCV2 antibody in oral fluid were evaluated using samples collected from experimentally inoculated pigs for 98 days post‐inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (i) negative control group; (ii) inoculated with PCV2a (strain ISU 40895) on DPI 0; (iii) inoculated with PCV2a (strain ISU‐40895) on DPI 0 and re‐challenged at DPIs 35 and 70; (iv) inoculated with PCV2a (ISU‐40895), PCV2b (PVG4072) and PCV2a (ISU‐4838) on DPIs 0, 35 and 70, respectively. Serum was collected from each animal, and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by quantitative polymerase chain reaction (qPCR) and anti‐PCV2 IgG, IgA and IgM antibody isotypes by enzyme‐linked immunosorbant assay (ELISA). Serum was assayed for anti‐PCV2 IgG by ELISA. Anti‐PCV2 antibodies (IgG, IgM and IgA) were detected in oral fluid from experimentally inoculated pigs from DPI 14 with IgG and IgA clearly present at 98 DPI. PCV2 was detected in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout 98 DPI. Overall, the data indicated that PCV2 infection in swine is prolonged, persists in the face of an active antibody response and can be efficiently monitored using oral fluid specimens.     

Bioaerosol and surface sampling for the surveillance of influenza A virus in swine

Influenza A virus in swine is of significant importance to human and veterinary public health. Environmental sampling techniques that prove practical would enhance surveillance for influenza viruses in swine. The primary objective of this study was to demonstrate the feasibility of bioaerosol and surface sampling for the detection of influenza virus in swine barns with a secondary objective of piloting a mobile application for data collection. Sampling was conducted at a large swine operation between July 2016 and August 2017. Swine oral fluids and surface swabs were collected from multiple rooms. Room‐level air samples were collected using four bioaerosol samplers: a low volume polytetrafluoroethylene (PTFE) filter sampler, the National Institute for Occupational Safety and Health's low volume cyclone sampler, a 2‐stage Andersen impactor and/or one high volume cyclonic sampler. Samples were analysed using quantitative RT‐PCR. Data and results were reported using a mobile data application. Eighty‐nine composite oral fluid samples, 70 surface swabs and 122 bioaerosol samples were analysed. Detection rates for influenza virus RNA in swine barn samples were 71.1% for oral fluids, 70.8% for surface swabs and 71.1% for the PTFE sampler. Analysis revealed a statistically significant relationship between the results of the PTFE sampler and the surface swabs with oral fluid results (p < 0.001 and p < 0.01 respectively). In addition, both the PTFE sampler (p < 0.01) and surface swabs (p = 0.03) significantly correlated with, and predicted oral fluid results. Bioaerosol sampling using PTFE samplers is an effective hands‐off approach for detecting influenza virus activity among swine. Further study is required for the implementation of this approach for surveillance and risk assessment of circulating influenza viruses of swine origin. In addition, mobile data collection stands to be an invaluable tool in the field by allowing secure, real‐time reporting of sample collection and results.     

Infection Dynamics of Pandemic 2009 H1N1 Influenza Virus in a Two‐Site Swine Herd

Influenza A viruses are common causes of respiratory disease in pigs and can be transmitted among multiple host species, including humans. The current lack of published information on infection dynamics of influenza viruses within swine herds hinders the ability to make informed animal health, biosecurity and surveillance programme decisions. The objectives of this serial cross‐sectional study were to describe the infection dynamics of influenza virus in a two‐site swine system by estimating the prevalence of influenza virus in animal subpopulations at the swine breeding herd and describing the temporal pattern of infection in a selected cohort of growing pigs weaned from the breeding herd. Nasal swab and blood samples were collected at approximately 30‐day intervals from the swine breeding herd (Site 1) known to be infected with pandemic 2009 H1N1 influenza virus. Sows, gilts and neonatal pigs were sampled at each sampling event, and samples were tested for influenza virus genome using matrix gene RRT‐PCR. Influenza virus was detected in neonatal pigs, but was not detected in sow or gilt populations via RRT‐PCR. A virus genetically similar to that detected in the neonatal pig population at Site 1 was also detected at the wean‐to‐finish site (Site 2), presumably following transportation of infected weaned pigs. Longitudinal sampling of nasal swabs and oral fluids revealed that influenza virus persisted in the growing pigs at Site 2 for at least 69 days. The occurrence of influenza virus in neonatal pigs, but not breeding females, at Site 1 emphasizes the potential for virus maintenance in this dynamic subpopulation, the importance of including this subpopulation in surveillance programmes and the potential transport of influenza virus between sites via the movement of weaned pigs.     

Susceptibility of Pigs to Zoonotic Hepatitis E Virus Genotype 3 Isolated from a Wild Boar

In Europe, zoonotic hepatitis E virus (HEV) genotype 3 strains mainly circulate in humans, swine and wild boar. The aim of this study was to investigate the potential transmission of a wild boar originating HEV strain (WbHEV) to swine by intravenous or oral inoculation and to study the consequences of infection of a WbHEV strain, a WbHEV strain previously passaged in a pig and a swine HEV strain after oral inoculation. Firstly, an intravenous infection was performed for which five piglets were divided into two groups with three pigs inoculated with a WbHEV field strain and two pigs inoculated with a HEV‐negative swine liver homogenate. All pigs were necropsied 8, 9 and 10 days post‐inoculation. Secondly, an oral infection of 56 days was performed on 12 piglets divided into four groups inoculated with a WbHEV strain, a WbHEV strain previously passaged in swine, a swine HEV strain or a HEV‐negative swine liver homogenate. After intravenous inoculation, HEV RNA was detected in serum, bile, liver, spleen, duodenum, jejunum, colon, lung, gastro‐hepatic lymph nodes and faeces in all infected piglets. After oral inoculation, HEV RNA was detected in serum, bile, liver, gastro‐hepatic lymph nodes and faeces. Most of HEV‐inoculated pigs became seropositive at day 15. This study provides experimental evidence of early viral spread throughout the organism after intravenous infection with a WbHEV strain and supports the notion that such a zoonotic strain could be transmitted via the natural faecal–oral route of infection between wild boar and pigs but also between pigs.     

Quantification of Lumpy Skin Disease Virus Following Experimental Infection in Cattle

Lumpy skin disease along with sheep pox and goatpox are the most serious poxvirus diseases of livestock, and are caused by viruses that belong to the genus Capripoxvirus within the subfamily Chordopoxvirinae, family Poxviridae. To facilitate the study of lumpy skin disease pathogenesis, we inoculated eight 4‐ to 6‐month‐old Holstein calves intravenously with lumpy skin disease virus (LSDV) and collected samples over a period of 42 days for analysis by virus isolation, real‐time PCR and light microscopy. Following inoculation, cattle developed fever and skin nodules, with the extent of infection varying between animals. Skin nodules remained visible until the end of the experiment on day post‐inoculation (DPI) 42. Viremia measured by real‐time PCR and virus isolation was not observed in all animals but was detectable between 6 and 15 DPI. Low levels of viral shedding were observed in oral and nasal secretions between 12 and 18 DPI. Several tissues were assessed for the presence of virus at DPI 3, 6, 9, 12, 15, 18 and 42 by virus isolation and real‐time PCR. Virus was consistently detected by real‐time PCR and virus isolation at high levels in skin nodules indicating LSDV has a tropism for skin. In contrast, relatively few lesions were observed systemically. Viral DNA was detected by real‐time PCR in skin lesions collected on DPI 42. Cattle developing anti‐capripoxvirus antibodies starting at DPI 21 was detected by serum neutralization. The disease in this study varied from mild with few secondary skin nodules to generalized infection of varying severity, and was characterized by morbidity with no mortality.     

Experimental Transmission of African Swine Fever (ASF) Low Virulent Isolate NH/P68 by Surviving Pigs

African swine fever (ASF) has persisted in Eastern Europe since 2007, and two endemic zones have been identified in the central and southern parts of the Russian Federation. Moderate‐ to low‐virulent ASF virus isolates are known to circulate in endemic ASF‐affected regions. To improve our knowledge of virus transmission in animals recovered from ASF virus infection, an experimental in vivo study was carried out. Four domestic pigs were inoculated with the NH/P68 ASF virus, previously characterized to develop a chronic form of ASF. Two additional in‐contact pigs were introduced at 72 days post‐inoculation (dpi) in the same box for virus exposure. The inoculated pigs developed a mild form of the disease, and the virus was isolated from tissues in the inoculated pigs up to 99 dpi (pigs were euthanized at 36, 65, 99 and 134 dpi). In‐contact pigs showed mild or no clinical signs, but did become seropositive, and a transient viraemia was detected at 28 days post‐exposure (dpe), thereby confirming late virus transmission from the inoculated pigs. Virus transmission to in‐contact pigs occurred at four weeks post‐exposure, over three months after the primary infection. These results highlight the potential role of survivor pigs in disease maintenance and dissemination in areas where moderate‐ to low‐virulent viruses may be circulating undetected. This study will help design better and more effective control programmes to fight against this disease.     

Risk Factors for Exposure to Influenza A Viruses,Including Subtype H5 Viruses,in Thai Free‐Grazing Ducks

Free‐grazing ducks (FGD) have been associated with highly pathogenic avian influenza (HPAI) H5N1 outbreaks and may be a viral reservoir. In July–August 2010, we assessed influenza exposure of Thai FGD and risk factors thereof. Serum from 6254 ducks was analysed with enzyme‐linked immunosorbent assay (ELISA) to detect antibodies to influenza A nucleoprotein (NP), and haemagglutinin H5 protein. Eighty‐five per cent (5305 ducks) were seropositive for influenza A. Of the NP‐seropositive sera tested with H5 assays (n = 1423), 553 (39%) were H5 ELISA positive and 57 (4%) suspect. Twelve per cent (74 of 610) of H5 ELISA‐positive/suspect ducks had H5 titres ≥ 1 : 20 by haemagglutination inhibition. Risk factors for influenza A seropositivity include older age, poultry contact, flock visitors and older purchase age. Study flocks had H5 virus exposure as recently as March 2010, but no HPAI H5N1 outbreaks have been identified in Thailand since 2008, highlighting a need for rigorous FGD surveillance.     

Seroprevalence and Risk Factors for Swine Influenza Zoonotic Transmission in Swine Workers from Northwestern Mexico

Summary A cross‐sectional study was conducted to evaluate the transmission of swine influenza through occupational exposure and to assess some risk factors for zoonotic transmission in workers from commercial farms in Mexico. Seroprevalence to swine influenza subtypes was determined by hemagglutinin inhibition assay and was higher in exposed (E), in comparison with unexposed (UE) participants (P < 0.05). Percentages of seropositivity between UE and E were 28.57% and 19.35% to A/NewCaledonia/20/99 (H1N1), 68.25% and 33.87% to A/Panama/2001/99‐like (H3N2), 1.58% and 12.9% to A/Sw/England/163266/87 (H3N2), respectively. No antibodies were detected against A/Sw/Wisconsin/238/97 (H1N1) in the UE subjects, and only 3.22% were positive in the E group (P < 0.05). A significant association between elevated antibody titres to swine influenza virus (SIV) H3N2 and the exposition to swine [OR 3.05, 95% (CI) 1.65–5.64] and to geographic location [OR 8.15, 95% (CI) 1.41–47.05] was found. Vaccination appeared as a protective factor [OR 0.05, 95% (CI) 0.01–0.52]. Farms with high number of breeding herd were associated with increased anti‐SIV antibodies in the E group [OR 3.98, 95% (CI) 1.00–15.86]. These findings are relevant and support the evidence of zoonoses in swine farms and point out the need to implement preventive measures to diminish the occurrence of the disease and the potential emergence of pathogenic reassortant strains.     

Early Detection of Foot‐And‐Mouth Disease Virus from Infected Cattle Using A Dry Filter Air Sampling System

Foot‐and‐mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to the aerogenous nature of the virus. In the current study, air from rooms housing individual (n = 17) or two groups (n = 4) of cattle experimentally infected with FDMV A24 Cruzeiro of different virulence levels was sampled to assess the feasibility of applying air sampling as a non‐invasive, screening tool to identify sources of FMDV infection. Detection of FMDV RNA in air was compared with first detection of clinical signs and FMDV RNA levels in serum and oral fluid. FMDV RNA was detected in room air samples 1–3 days prior (seven animals) or on the same day (four animals) as the appearance of clinical signs in 11 of 12 individually housed cattle. Only in one case clinical signs preceded detection in air samples by one day. Overall, viral RNA in oral fluid or serum preceded detection in air samples by 1–2 days. Six individually housed animals inoculated with attenuated strains did not show clinical signs, but virus was detected in air in one of these cases 3 days prior to first detection in oral fluid. In groups of four cattle housed together, air detection always preceded appearance of clinical signs by 1–2 days and coincided more often with viral shedding in oral fluid than virus in blood. These data confirm that air sampling is an effective non‐invasive screening method for detecting FMDV infection in confined to enclosed spaces (e.g. auction barns, milking parlours). This technology could be a useful tool as part of a surveillance strategy during FMD prevention, control or eradication efforts.     

A Refined Guinea Pig Model of Foot‐and‐Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds

An antiviral containment strategy for foot‐and‐mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre‐emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV‐infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T‐1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T‐1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID₅₀ of the GP‐adapted FMDV strain O₁ Manisa 1 h after the first administration. The efficacy of T‐1105 was compared with that of prophylactic vaccination with a highly potent double‐oil emulsion‐inactivated O₁ Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T‐1105‐treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T‐1105‐treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T‐1105‐treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T‐1105‐treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T‐1105 conferred a substantial clinical and virological protection against infection with O₁ Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.     

Complete genome sequence of a novel influenza A H1N2 virus circulating in swine from Central Bajio region,Mexico

The aim of this study was to perform the complete genome sequence of a swine influenza A H1N2 virus strain isolated from a pig in Guanajuato, México (A/swine/Mexico/GtoDMZC01/2014) and to report its seroprevalence in 86 counties at the Central Bajio zone. To understand the evolutionary dynamics of the isolate, we undertook a phylogenetic analysis of the eight gene segments. These data revealed that the isolated virus is a reassortant H1N2 subtype, as its genes are derived from human (HA, NP, PA) and swine (M, NA, PB1, PB2 and NS) influenza viruses. Pig serum samples were analysed by the hemagglutination inhibition test, using wild H1N2 and H3N2 strains (A/swine/México/Mex51/2010 [H3N2]) as antigen sources. Positive samples to the H1N2 subtype were processed using the field‐isolated H1N1 subtype (A/swine/México/Ver37/2010 [H1N1]). Seroprevalence to the H1N2 subtype was 26.74% in the sampled counties, being Jalisco the state with highest seroprevalence to this subtype (35.30%). The results herein reported demonstrate that this new, previously unregistered influenza virus subtype in México that shows internal genes from other swine viral subtypes isolated in the past 5 years, along with human virus‐originated genes, is widely distributed in this area of the country.     

Inactivation of Avian Influenza Viruses on Porous and Non‐porous Surfaces is Enhanced by Elevating Absolute Humidity

This study was to evaluate the effect of absolute humidity (AH), a combined factor of temperature and relative humidity (RH), on inactivation of avian influenza viruses (AIVs) on surfaces. Suspensions of the H9N2 or H6N2 AIV were deposited onto carrier surfaces that were either porous (pine wood) or non‐porous (stainless steel, synthetic rubber and glass). The inoculated carriers were incubated at 23, 35 or 45°C with 25% or 55% RH for up to 28 days. After incubation, virus was recovered and quantified by chicken embryo assays. The time required to obtain a log₁₀ reduction in virus infectivity (D‐value) was estimated using a linear regression model. At AH of 5.2 g/m³ (23°C & 25% RH), both viruses survived up to 14 days on the porous surface and for at least 28 days on the non‐porous surfaces. The corresponding D‐values for H9N2 and H6N2 were 1.49 and 6.90 days on the porous surface and 7.81 and 12.5 days on the non‐porous surfaces, respectively. In comparison, at AH of 9.9 g/m³ (35°C & 25% RH) or 11.3 g/m³ (23°C & 55% RH), the D‐values for H9N2 and H6N2 dropped to ≤0.76 day on the porous surface and to ≤1.81 days on the non‐porous surfaces. As the AH continued to rise from 11.3 to 36.0 g/m³, the D‐value for both viruses decreased further. The relationship between D‐value and AH followed a form of y = ax^−b for both viruses. The D‐values for H9N2 virus were significantly lower (P < 0.05) than those for H6N2 virus. Exposure to ammonia gas at concentrations of 86 and 173 ppm did not significantly alter test results. The findings give evidence that increasing the AH in poultry buildings following an outbreak of disease could greatly reduce the length of time required for their decontamination.     

Frequent infection of wild boar with atypical porcine pestivirus (APPV)

The recently identified atypical porcine pestivirus (APPV ) was demonstrated to be the causative agent of the neurological disorder “congenital tremor” in newborn piglets. Despite its relevance and wide distribution in domestic pigs, so far nothing is known about the situation in wild boar, representing an important wild animal reservoir for the related classical swine fever virus. In this study, 456 wild boar serum samples obtained from northern Germany were investigated for the presence of APPV genomes and virus‐specific antibodies. Results of real‐time RT ‐PCR analyses revealed a genome detection rate of 19%. Subsequent genetic characterization of APPV (n  = 12) from different hunting areas demonstrated close genetic relationship and, with exception of APPV from one location, displayed less than 3.3% differences in the analysed partial NS 3 encoding region. Furthermore, indirect E^rns ELISA revealed an antibody detection rate of approx. 52%, being in line with the high number of viremic wild boar. Analysis of fifteen wild boar samples from the Republic of Serbia by E^rns antibody ELISA provided evidence that APPV is also abundant in wild boar populations outside Germany. High number of genome and seropositive animals suggest that wild boar may serve as an important virus reservoir for APPV .     

Pathology of Trichuris suis Infection in Pigs Fed an Inulin‐ and a Non‐inulin‐containing Diet

Pathology of the colon in pigs infected with Trichuris suis and fed an inulin and a non‐inulin containing diet was studied to investigate the inflammatory response induced and the influence of inulin on this. Twenty‐nine pigs were allocated into four groups (N‐7, I‐7, N‐9 and N/I‐9). Groups N‐7, N‐9 and N/I‐9 were given a diet with non‐fermentable carbohydrates, and group I‐7 was fed a diet including fermentable carbohydrates (inulin). After 2 weeks, all pigs were inoculated with 2000 T. suis eggs. Seven weeks post‐infection (p.i.), groups N‐7 and I‐7 were killed and group N/I‐9 changed to inulin diet to study the influence of inulin on already established T. suis. Nine weeks p.i., groups N‐9 and N/I‐9 were killed. Trichuris suis were collected from the colon for enumeration. Tissues from the colon of each pig were taken for histological and immunohistochemical quantitative and semi‐quantitative evaluations of heterophils, eosinophils, mast cells, IgA⁺, IgG⁺, IgM⁺ and CD3⁺ cells. The findings were compared with tissues from seven uninfected pigs of the same age. Pigs fed inulin had significantly fewer worms compared with pigs on the non‐fermentable carbohydrate diet. The number of inflammatory cells in the colonic mucosa was increased in all T. suis‐infected pigs compared with uninfected controls. The numbers of eosinophils, mast cells, IgA⁺, IgG⁺ and CD3⁺ cells were significantly lower in pigs fed inulin than pigs fed non‐fermentable carbohydrate. Whether this is a direct effect of inulin or a secondary effect due to the lower parasitic burden in inulin fed pigs is not known.