Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.     

Pandemic A/H1N1/2009 influenza virus in swine, Cameroon, 2010

Although swine origin A/H1N1/2009 influenza virus (hereafter "pH1N1″) has been detected in swine in 20 countries, there has been no published surveillance of the virus in African livestock. The objective of this study was to assess the circulation of influenza A viruses, including pH1N1 in swine in Cameroon, Central Africa. We collected 108 nasal swabs and 98 sera samples from domestic pigs randomly sampled at 11 herds in villages and farms in Cameroon. pH1N1 was isolated from two swine sampled in northern Cameroon in January 2010. Sera from 28% of these herds were positive for influenza A by competitive ELISA and 92.6% of these swine showed cross reactivity with pandemic A/H1N1/2009 influenza virus isolated from humans. These results provide the first evidence of this virus in the animal population in Africa. In light of the significant role of swine in the ecology of influenza viruses, our results call for greater monitoring and study in Central Africa.     

Seroprevalence and risk factors for influenza A viruses in pigs in Peninsular Malaysia

Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.     

1株猪源H9N2亚型流感病毒的分子特征

旨在进一步了解山东省猪流感的流行情况及其病原特征,笔者于2019年春季,在山东省泰安某屠宰场采集130份猪鼻拭子,进行病毒分离鉴定;并对分离病毒进行全基因组测序和分子特征分析;用禽H9N2亚型标准抗原联合血凝抑制方法检测2018—2019年从山东省8个地区猪场采集的1 527份猪血清样品中的猪流感病毒抗体。结果显示：分离到1株H9N2亚型流感病毒,命名为A/swine/Shandong/TA009/2019（H9N2）。分离病毒与A/environment-air/Kunshan/NIOSH-BL20/2018（H9N2）和A/environment-air/Kunshan/NIOSH-BL25/2018（H9N2）遗传关系最近,其基因片段的核苷酸相似性均在99.5%以上。分离病毒的HA和NA基因属于Y280-like分支,PB2和M基因属于G1-like分支,PB1、PA、NP和NS基因属于SH/F98-like分支。分离病毒HA蛋白裂解位点处的氨基酸序列为“PSRSSR/GL”,符合低致病性禽流感病毒的分子生物学特性。HA蛋白的216位为L,具有结合人源唾液酸α 2,6-Gal的能力。血清学分析结果显示,9份血清中H9N2抗体呈阳性,其总阳性率为0.59%。综上：本研究分离到1株猪源H9N2亚型流感病毒,并在猪血清中检测到H9N2抗体,提示应加强对猪流感的流行情况及其病原特征的持续监测。     

Molecular Characterization of a H9N2 Subtype Swine Influenza Virus

The objective of this study was to explore the epidemic situation and pathogenic characteristics of swine influenza virus (SIV) in Shandong Province. In the spring of 2019, 130 swine nasal swab samples were collected from a slaughterhouse in Tai'an city, Shandong Province for virus isolation and identification. The whole genome of isolated virus was sequenced and analyzed. Meanwhile, 1 527 swine serum samples were collected from swine farms in 8 regions of Shandong province and their anti-SIV antibody were detected by HI assay using standard avian H9N2 antigen. The results showed that a H9N2 subtype influenza virus strain was isolated and named as A/swine/Shandong/TA009/2019(H9N2). The homology analysis showed that the isolated virus had close genetic relationship with A/environment-air/Kunshan/NIOSH-BL20/2018(H9N2) and A/environment-air/Kunshan/NIOSH-BL25/2018(H9N2), and the nucleotide homology of the gene fragments were above 99.5%. Phylogenetic analysis results demonstrated that HA and NA genes of the isolated virus belong to the Y280-like lineage, PB2 and M genes belong to the G1-like lineage, and PB1, PA, NP and NS genes belong to the SH/F98-like lineage. The cleavage site in HA protein is “PSRSSR/GL”, which was in accordance with the molecular biological characteristics of low pathogenic avian influenza virus.The position 216 of HA protein is L, and it has the ability to bind human-derived sialic acid α 2,6-Gal. The results of HI showed that 9 among 1 527 serum samples were positive with a positive rate of 0.59%. The isolated virus was swine-derived H9N2 virus, and serological investigations revealed that H9N2 subtype virus infection was present in swine herds in Shandong Province. The results of this study suggest that continuous surveillance of the SIV epidemiological situation and its pathogenic characteristics should be strengthened.     

Use of an enzyme-linked immunosorbent assay for detection of infection with pseudorabies virus on a herd basis.

The use of an ELISA that can differentiate between swine infected with pseudorabies virus (PRV) and swine vaccinated with a specific PRV vaccine was evaluated on an individual and herd basis, and a system for interpreting ELISA results on a herd basis was developed. In 17 herds, recently introduced replacement gilts, seronegative for PRV, were vaccinated with a thymidine kinase- and glycoprotein X (gpX)-deleted vaccine. After vaccination, blood samples were collected from these gilts approximately every 1 to 2 months for up to 19 months. Serum samples were analyzed for antibodies to gpX antigen, using a commercially available ELISA kit according to the manufacturer's protocol. Herd status was determined as positive, suspect, or negative, according to the serum sample:negative control (S:N) values of the samples collected from the herd. From the 17 herds, 130 evaluations were performed. On 49 (38%) of the 130 herd evaluations, 1 or more gilts had suspect test results. Additional testing was required in 19 (39%) of these 49 herd evaluations to determine the PRV infection status of the herd. Status of herds having gilts with suspect results and no positive results was usually negative after retesting. Herds having gilts with positive results were unlikely to have negative status after retesting.     

Influenza A viruses: epidemiologic study in fatteners in Spain (1987-89).

2,979 sera were collected from slaughtered swine in two geographic areas of Spain from 1987 to 1989. They were tested for antibodies against an H1N1- and H3N2-influenza virus by haemagglutination-inhibition tests (HI). The percentage of positive sera was higher in area I (78%-69.2%) than in area II (63.1%-60.4%) for both viruses respectively. The coexistence of high titres to both H1N1- and H3N2-influenza virus became apparent in cold months simultaneously in each area, although influenza viruses circulated in the Spanish swine population for two years. Also this study suggests the possible circulation of A/Texas/1/77-like strains in Spain, results which have not been reported before.     

Seroprevalence of porcine respiratory coronavirus in selected Korean pigs

A total of 446 serum samples from 88 herds in Korea were examined for antibody to porcine respiratory coronavirus (PRCV) using blocking enzyme-linked immunosorbent assay (ELISA). All serum samples were collected from 24- to 26-week-old finishing pigs between December 1998 and June 1999. By ELISA, 237 out of 446 sera tested (53.1%) and 54 out of 88 sampled herds (61.3%) were positive against PRCV. Of 446 sera from 88 herd tested, 185 (41.5%) serum samples from 22 (25%) herds were seronegative against PRCV and transmissible gastroenteritis virus infection. Our data suggested that seropositive herds for PRCV are distributed diffusely throughout South Korea.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.     

Serological Surveillance of Influenza A Virus Infection in Swine Populations in Fujian Province,China: No Evidence of Naturally Occurring H5N1 Infection in Pigs

Several highly pathogenic H5N1 avian influenza viruses were isolated from swine populations in Fujian Province, China, since 2001. Because it is thought that H5N1 infection in pigs might result in virus adaptation to humans, we surveyed swine populations in Fujian Province in 2004 and 2007 for serological evidence of the infection. Twenty‐five pig farms covering all nine administrative districts of Fujian Province were sampled and a total of 1407 serum specimens were collected. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection and only a few cases of H9 infection. The negative results for H5 infection were further verified by micro‐neutralization tests. By contrast, H1 influenza virus infections were prevalent in swine in both surveys according to the results of enzyme‐linked immunosorbent assay (ELISA). The H3 infection rate was reduced dramatically in 2007 compared with 2004, when examined by HI and ELISA. In summary, the results imply that the swine populations in Fujian Province had not been affected greatly by the H5N1 avian influenza virus, given that there is no serological evidence that H5N1 influenza virus has infected the pig populations. The reported isolates represent only sporadic cases.     

A cross-sectional study of swine influenza in intensive and extensive farms in the northeastern region of the state of São Paulo,Brazil

Swine influenza (SI) is a seasonal infectious disease highly important to the world pig industry. Loss of daily weight gain, increased costs for the prevention and treatment of secondary infections are the main economic losses associated with the presence of this disease. However, some epidemiological features of SI remain quite unclear. This study focused on assessing the prevalence of swine influenza virus (SIV) infection in intensive and extensive pig herds and associating risk factors. A set of 601 blood samples of five intensive farrow-to-finish farms and 361 blood samples from 56 extensive farms were analyzed using an indirect ELISA kit CIVTEST SUIS INFLUENZA®, Hipra (Amer, Spain), in order to detect anti-SIV antibodies. In total, 24.13 % of samples from intensive herds were positive, while no positive samples were detected in extensive rearing herds. Sow and weaning piglets had the highest prevalence values. In the intensive rearing system, occurrence of reproductive disorders and exposure to recently introduced animals were positively associated with the disease occurrence in swine herds. The findings highlight the importance of sows in the epidemiology of the disease and bring information about risk factors involved in the occurrence of swine influenza in intensive herds.     

The first detection of influenza in the Finnish pig population: a retrospective study

Background

Swine influenza is an infectious acute respiratory disease of pigs caused by influenza A virus. We investigated the time of entry of swine influenza into the Finnish pig population. We also describe the molecular detection of two types of influenza A (H1N1) viruses in porcine samples submitted in 2009 and 2010.This retrospective study was based on three categories of samples: blood samples collected for disease monitoring from pigs at major slaughterhouses from 2007 to 2009; blood samples from pigs in farms with a special health status taken in 2008 and 2009; and diagnostic blood samples from pigs in farms with clinical signs of respiratory disease in 2008 and 2009. The blood samples were tested for influenza A antibodies with an antibody ELISA. Positive samples were further analyzed for H1N1, H3N2, and H1N2 antibodies with a hemagglutination inhibition test. Diagnostic samples for virus detection were subjected to influenza A M-gene-specific real-time RT-PCR and to pandemic influenza A H1N1-specific real-time RT-PCR. Positive samples were further analyzed with RT-PCRs designed for this purpose, and the PCR products were sequenced and sequences analyzed phylogenetically.

Results

In the blood samples from pigs in special health class farms producing replacement animals and in diagnostic blood samples, the first serologically positive samples originated from the period July–August 2008. In samples collected for disease monitoring, < 0.1%, 0% and 16% were positive for antibodies against influenza A H1N1 in the HI test in 2007, 2008, and 2009, respectively. Swine influenza A virus of avian-like H1N1 was first detected in diagnostic samples in February 2009. In 2009 and 2010, the avian-like H1N1 virus was detected on 12 and two farms, respectively. The pandemic H1N1 virus (A(H1N1)pdm09) was detected on one pig farm in 2009 and on two farms in 2010.

Conclusions

Based on our study, swine influenza of avian-like H1N1 virus was introduced into the Finnish pig population in 2008 and A(H1N1)pdm09 virus in 2009. The source of avian-like H1N1 infection could not be determined. Cases of pandemic H1N1 in pigs coincided with the period when the A(H1N1)pdm09 virus was spread in humans in Finland.     

Prevalence of and risk factors for influenza in southern Ontario swine herds in 2001 and 2003

This research included 2 prevalence studies and a risk-factor investigation conducted in 2001 at 93 sites with sows only, finishers only, or both. In 2001, 1300 serum samples from sows in 65 herds and 720 serum samples from finisher pigs in 72 herds were tested for antibodies to swine influenzavirus (SIV) of H1N1 subtype with an enzyme-linked immunosorbent assay (ELISA). In 2003, 1140 serum samples from sows in 76 herds were tested for antibodies to SIV of H3N2 subtype with a hemagglutination-inhibition assay based on A/Swine/Colorado/1/77 and A/Swine/Texas/4199-2/98 isolates. The apparent pig-level H1N1 seroprevalence in 2001 was 61.1% and 24.3% in sows and finishers, respectively. The apparent pig-level seroprevalence in 2003 for H3N2 A/Sw/CO/1/77 and A/Sw/TX/4199-2/98 in sows was 0.6% and 0.7%, respectively. The factors associated with sow-herd H1N1 positivity included pig or farm density at different geographic levels, an external source of breeding pigs, number of animals on site, and decreasing proximity to other barns. Higher-parity sows had higher odds of seropositivity, but there was significant random variability in this association among herds. The odds of finisher-herd SIV positivity were higher with large herd size, high pig farm density, and farrow-to-finish type of farm. Finisher herds were SIV-positive only if source sow herds were positive. Simultaneously, 45% of finisher herds were SIV-negative although sow source herds were positive.     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.     

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.     

长沙市对伪狂犬病野毒感染的调查

为了解长沙市规模猪场伪狂犬病流行情况,对市内47个规模猪场送检的1534猪血清样品和22个已免疫伪狂犬基因缺失苗规模场送检的1300份血清样品,用ELISA方法对猪伪狂犬病野毒感染抗体（gpI抗体,下同）和免疫抗体（gB抗体,下同）进行检测。结果表明,长沙市规模猪场猪伪狂犬病gpI抗体阳性率为20.9%,场阳性率为44.7%;22个免疫猪伪狂犬基因缺失苗规模猪场猪伪狂犬病gpI抗体阳性率下降1.8%,场阳性数下降4.6%。结果表明,通过利用猪伪狂犬病基因缺失疫苗科学免疫,同时配合伪狂犬野毒抗体检测技术可以控制和净化规模猪场猪伪狂犬病流行。     

Characterization of influenza a outbreaks in Minnesota swine herds and measures taken to reduce the risk of zoonotic transmission

Influenza A virus infections commonly cause respiratory disease in swine and can be transmitted between people and pigs, with potentially novel strains introduced into herds and spilling back into the human population. The goals of this study were to characterize influenza infections in Minnesota pigs and assess biosecurity measures used by swine workers. Veterinarians submitting influenza-positive swine samples to the University of Minnesota Veterinary Diagnostic Laboratory between October 2007 and April 2009 were surveyed regarding disease-related information and biosecurity procedures at each farm. Influenza-positive samples were submitted year-round, peaking in spring and fall. H1N1 was the most commonly detected subtype (56%), followed by H3N2 (14%) and H1N2 (12%). Most positive submissions were associated with illness in growing pigs (median age 8.8 weeks, IQR 5-15). Median morbidity and mortality were 25% (IQR 10-48) and 2% (IQR 0.5-3.5), respectively. Vaccination of sows and growing pigs was conducted at 71% and 7.9% of the swine farms, respectively. Specialized footwear was reported as the most common form of protective equipment used by workers. Employee vaccination for seasonal influenza was 19%. The sow vaccination rate in this study is consistent with national data, although growing pig vaccination is lower than the national average. Seasonal and age trends identified here may provide diagnostic guidance when growing pigs experience respiratory disease. Inconsistent use of protective equipment and employee vaccination at swine farms indicates the need for further discussion and research of approaches to minimize interspecies influenza transmission on swine farms.     

Comparison of a commercial enzyme-linked immunosorbent assay with hemagglutination inhibition assay for serodiagnosis of swine influenza virus (H1N1) infection.

A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.     

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago,West Indies

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.