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- Material and methods
Please cite this paper as: Trevennec et al. (2012) Transmission of pandemic influenza H1N1 (2009) in Vietnamese swine in 2009–2010. Influenza and Other Respiratory Viruses 6(5), 348–357.
Background The pandemic of 2009 was caused by an H1N1 (H1N1pdm) virus of swine origin. This pandemic virus has repeatedly infected swine through reverse zoonosis, although the extent of such infection in swine remains unclear.
Objective This study targets small and commercial pig producers in North Vietnam, in order to estimate the extent of H1N1pdm infection in swine and to identify the risk factors of infection.
Methods Virologic and serologic surveillance of swine was carried out in 2009–2010 in pig farms (38 swabs and 1732 sera) and at a pig slaughterhouse (710 swabs and 459 sera) in North Vietnam. The sera were screened using a influenza type A-reactive ELISA assay, and positive sera were tested using hemagglutination inhibition tests for antibody to a panel of H1-subtype viruses representing pandemic (H1N1) 2009 (H1N1pdm), triple reassortant (TRIG), classical swine (CS), and Eurasian avian-like (EA) swine lineages. Farm-level risk factors were identified using a zero-inflated negative binomial model.
Results We found a maximal seroprevalence of H1N1pdm of 55·6% [95% CI: 38·1–72·1] in the slaughterhouse at the end of December 2009, 2 weeks after the peak of reported human fatalities with H1N1pdm. Farm-level seroprevalence was 29% [95% CI: 23·2–35·7]. In seropositive farms, within-herd seroprevalence ranged from 10 to 100%. We identified an increased risk of infection for farms that specialized in fattening and a decreased risk of infection in farms hiring external swine workers.
Conclusions Our findings suggest extensive reverse-zoonotic transmission from humans to pigs with subsequent onward transmission within pig herds.
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- Material and methods
H1N1pdm seroprevalence in swine at the Hanoi slaughterhouse rapidly increased during the winter of 2009, to peak at overall seroprevalence of 55·6% [95% CI: 38·1–72·1] of all animals tested by the end of December 2009 (Figure 3). As in many other parts of Asia, H1N1pdm infection was introduced to Vietnam in June–July 2009.22 The detailed epidemic curve of human H1N1pdm infections in North Vietnam is not yet available. However, the reported numbers of fatal H1N1pdm cases peaked in October–November 2009 (Figure 3) and thus appear to have preceded the peak of seroconversion in swine by 1–2 months. This may well reflect the delay between infection and seroconversion of pigs and also the time interval between their infection in the farms and their sale for slaughter. The farm-level seroprevalence for H1N1pdm was 29·5%, and within-herd seroprevalence in infected farms ranged from 10 to 100%. The data are therefore suggestive of extensive spill-over of H1N1pdm from humans to swine and efficient transmission of the virus within herds. The low seroprevalence of H1 viruses in swine prior to November 2009 would have facilitated explosive outbreaks of H1N1pdm infection in swine. However, the lack of geographic clustering of infected farms is more compatible with multiple discrete transmission events from humans to swine amplifying within each swineherd but not spreading extensively between swineherds. The geographic overlap in the occurrence of human fatal cases and seroprevalence of H1N1pdm in swine (Figure 1C) corroborates this assumption.
Seroprevalence in swine declined after the peak in December 2009–January 2010, suggesting that the H1N1pdm virus was not sustaining high-level virus transmission in swine. This may reflect the reduction of infection in the source (viz humans) but increasing herd immunity in swine may also contribute to this decline in virus activity in pigs. Pig production in Vietnam peaks prior to the Têt festival in February. The post-Têt decline in the susceptible pig population as well as commercial trade after Têt may have also contributed to the decline in seroprevalence. Seasonal factors may also play a role.
Our results are consistent with cases of human-to-swine H1N1pdm transmissions already observed in farms: Canada,23 Thailand,24 and Korea25 (three independent human-to-swine transmissions). Because we had only serological data, we could not determine whether these transmission events were single or several cross-species transmissions. While our data are suggestive of extensive transmission of H1N1pdm within swine herds, it is also suggesting that virus activity is not self-sustaining at high levels in pigs. Reassortants between H1N1pdm and swine viruses have already been isolated in Asia.4 If the spread of H1N1pdm in the Vietnamese swine population continues even at low frequency, this human virus may also reassort in Vietnam with swine viruses, as it has been recently observed for H3N2.26 Further investigation, including continuous monitoring, molecular epidemiology, and modeling, would be necessary to elucidate such questions.
The differences between seroprevalences estimated in slaughterhouse and farms may be related to a number of possible biases including the clustering of animals at farm level, the age of animals, and geographic location. Pigs sent to the slaughterhouse are older than those collected in farms and have more opportunity to have been infected. The swine sampled in farms originate only from the Ha Noi province, while pigs sampled in the slaughterhouse come from a broader region of the Red River Delta.
There are a number of limitations in our study which is likely to underestimate the prevalence of H1N1pdm infection in swine. Serological testing of swine sera for H1N1pdm by HI tests was only carried out on sera that were positive in screening influenza type A ELISA assay. The sensitivity of such ELISA assays is likely to be less than ideal, and this would be lead to underestimation of the overall H1N1pdm seropositivity in swine.27 There is a proportion of sera (up to 22·5% in February 2010) that had evidence of influenza type A antibody detected in ELISA tests but were negative for the different antigenic variants of H1-subtype swine influenza viruses. This suggests that other subtypes of influenza may be circulating in swine in Vietnam. We included three H3N2 viruses in our panel of virus antigens, viz Eurasian avian-like H3N2; human-like H3N2 swine viruses isolated in Hong Kong in 1998 with A/Sydney/5/97-like hemagglutinin;11 and more recent human H3N2 viruses from 2008, with no evidence of virus activity which was surprising.11 H3-subtype viruses have been reported in swine in China9 and Thailand.24 More recently, H3N2 viruses (e.g., A/swine/Binh Duong/03_08/2010) have been isolated from swine in South Vietnam with H3 hemagglutinins that are closely related genetically and antigenically to human H3N2 viruses A/New York/365/2004 and A/Wyoming/3/200326 and to a recently isolated virus from Hong Kong A/swine/HK/2503/2011 (H3N2). Interestingly, in our study carried out in North Vietnam, none of the pigs have evidence of antibody to A/swine/HK/2503/2011 (H3N2).
Virus isolation attempts from 748 swabs collected during this study did not yield virus isolates. This may in part be related to freezing and thawing of these swabs and also poor-cold chain management as viral isolation could not be carried out at the local laboratory. On the other hand, another recent study of swine influenza in Vietnam found detectable virus only in two pooled swabs of 759 tested, both coming from the same farm.26 This and other studies suggest that virus isolation rates from swine are low and larger numbers of swabs need to be tested in order to be successful at isolating viruses. Availability of local virus isolates would have allowed us to use better matched strain for the HI serology testing, probably reducing the proportion of samples that were positive for influenza type A antibodies in the ELISA assay but negative in HI tests.
In farms, the risk of seropositive pigs was associated with the presence of external employees. This is in fact counterintuitive as one would expect that a more heterogeneous work-force will lead to increased risk of introduction of human H1N1pdm infection to swine. Unfortunately, our epidemiological survey data were not precise enough to propose a more detailed explanation for this observation; for example, are employed swine workers more respectful of biosecurity, do they use more self-protection, or are they less inclined to work when they are sick?
Between-farm transmission may occur either via humans (interspecies) or pigs (intraspecies). To our knowledge, no previous study has reported farm-level seroprevalence or risk factors of H1N1pdm in swine. A farm may be infected by infected humans, swine, or fomites. The relatively low proportion of seropositive farms, scattered locations (Figure 4), and the absence of spatial autocorrelation favor limited local diffusion from farm to farm. Thus, the observations are in favor of independent farm infections, possibly with infected humans being the major source of infection. However, the number of family members working on the farm, the employment of swine workers, the restriction of visitors, or the wearing of protective clothes/masks was not significantly associated with swine infection risk. The risk factor analyses highlighted an increased risk of farm infection for farms specialized in fattening. Such farms are characterized by the frequent purchase of growing pigs and larger numbers of finishing pigs. Regular introduction of new animals may contribute to the increased infection risk.