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Brief Communication |
Correspondence: 1 Corresponding Author: María Teresa Pérez-Gracia, Departamento de Atención Sanitaria, Salud Pública y Sanidad Animal, Facultad de Ciencias Experimentales y de la Salud, Universidad Cardenal Herrera–CEU, Moncada 46113, Valencia, Spain, e-mail: teresa{at}uch.ceu.es
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Abstract |
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 TOPSources and manufacturers Abstract References |
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Key Words: Feces • hepatitis E virus • pigs • production stages • swine • zoonoses
Hepatitis E virus (HEV) is the main causative agent of non-A non-B enterically transmitted hepatitis with an acute and self-limiting clinical presentation in humans.13,16 Mortality rates are low in the population, except for pregnant women, especially in their third trimester, where mortality rates increase dramatically and can reach 25%. HEV was provisionally classified as belonging to the Caliciviridae family from 1988 through 1998, but is now classified in the separate genus Hepevirus in a new family which has been named Hepeviridae. Although at least 4 major genotypes have been identified, only 1 serotype of HEV is recognized.18
Epidemic and sporadic epidemiological forms of hepatitis E have been reported. The former is related to drinking contaminated water in developing countries with poor sanitary conditions.20 The latter form has been reported between epidemics in these areas, in patients with a history of traveling to endemic areas, and in those in industrialized countries who have not traveled abroad (autochthonous hepatitis).14
Occurrence of autochthonous HEV infection in humans has led to the screening of domestic animals for the presence of the virus in developed countries, and a high seroprevalence has been found, especially in swine farms. In 1997 the first swine strain HEV was isolated10 and showed a high percentage of homology with an autochthonous human strain in USA (US-2). A year later, the experimental infection of macaques with a swine HEV strain demonstrated the ability of the virus to cross the species barrier, but only mild clinical signs and/or biochemical alterations were observed.11 Several swine HEV isolates were subsequently identified in pigs in Canada,22 Japan,12 Korea,4 New Zealand,5 the Netherlands,19 and the UK.2
The aim of this work was to study the prevalence of HEV in pig feces in Spain and determine the production stages in which HEV is shed by the highest number of pigs, in order to estimate the relative risk associated with each stage. To do this, 146 pigs were selected at random for the study from 21 farms. From January 2002 through August 2004 fecal samples were collected directly from the rectum and placed in sterile containers. Farms were of the following types: 5 farrow-to-weaning farms (farms that keep breeder sows, piglets, and weaners, with piglets moved to nurseries from weaning at 3 weeks up to 12 weeks), 11 grower-to-finish farms (in these farms, 12-week-old weaners are bought and fed until they reach 5 months), and finally 5 farrow-to-finish farms (pigs of all production stages are kept on the same farm). Pigs were divided into groups on the basis of their production stages: 18 suckling piglets (from birth to weaning); 24 weaners (pigs from weaning to 12 weeks old); 20, 20, and 28 pigs in the first, second, and third month of feeding respectively; 32 breeder sows (adult females nursing piglets); and 4 boars (adult males).
In addition, a sample from the raw manure ditch and a sample of drinking water (obtained directly from the trough in the pen) were taken from 16 farms. Samples from the manure ditch and samples of feces were diluted with phosphate-buffered saline (PBS) to 10% (w/v) suspensions. These were centrifuged at 2000 x g for 10 minutes at room temperature; the supernatant was either used immediately to extract RNA or kept frozen at –80°C until used.
RNA was extracted from 140 µl of each fecal, manure, or water sample using a commercial kita following the manufacturer's instructions. Two pairs of degenerated oligonucleotide primers were used to amplify a 348-base-pair (bp) fragment of open reading frame 2 (ORF-2) of HEV using an RT-nested PCR.7 These primers were based on 18 human HEV sequences and the swine HEV prototype strain from USA. A positive control from a naturally infected pig (GenBank accesion number AY323506) was included in each procedure. All assays were performed in different places to avoid the possibility of cross-contamination. The PCR products were separated by electrophoresis in 2% agarose gel and were detected by staining with ethidium bromide. Amplicons were purified and both strands sequenced using a sequencing reaction kit.b Sequences obtained in this study were deposited in the GenBank database under accession numbers DQ093564, DQ093566, DQ093567, DQ093568, DQ141118, DQ141119, DQ141120, DQ141121, and DQ141127. A binary logistic regression was performed to determine the effect of the production stage on the prevalence of HEV in pig feces and to calculate OR and their 95% CI. The fit of the final model was assessed by the Hosmer-Lemeshow goodness-of-fit test. Statistical analyses were performed using a statistical analysis software package.c The samples were considered positive to the presence of HEV when a band of 348 bp was seen in the agarose gel. HEV RNA was detected in fecal samples of 34 pigs (23.3%) although none of them exhibited clinical signs of infection.
Results according to the production stage of pigs are shown in Table 1. Only 2 of the 3-week-old pigs out of 18 (11.1%) suckling piglets were shedding the virus. The prevalence increased in weaners with a result of 41.7%, reaching a maximum in the first month of feeding with a prevalence of 60%. The minimum rate was observed in the second month of feeding with 1 positive pig out of 20 (5%). Of the adult pigs, none of the 4 boars tested positive for HEV RNA, but there was a prevalence of 21% in the breeding sows. To determine the correlation between the presence of HEV in feces and production stages, odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated using binary logistic regression analysis. A P value of less than 0.05 was considered statistically significant. Table 1 shows the results of the binary logistic regression analysis. In this model, the first month of feeding (OR 19.5, 95% CI 3.59–106.08, P = 0.001) was recorded as the main significant risk stage for shedding HEV, followed by the weaners stage (OR 9.3, 95% CI 1.78–48.42, P = 0.008). The Hosmer-Lemeshow goodness-of-fit test showed a good fit for the final model (
2 = 2.87, P = 0.897).
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2 test, but no significant association was recorded. However, a large number of samples would be necessary to confirm these results. The percentage of infected pigs on farms found to be negative for HEV in manure was 21.4% (12 out of 56 pigs), whereas the percentage of farms found to be positive for HEV in manure was 6.2% (2 out of 32).
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It takes between 2 and 3 weeks from initial infection to the time when HEV is excreted in feces,11 and the viral shedding can last up to 7 weeks. Considering the fact that the maximum prevalence recorded in this work was found in the first month of feeding (around 13 to 16 weeks of age), it is reasonable to extrapolate that the majority of animals were infected at 3 to 6 weeks of age from contact with the sows. After an incubation period of 2 to 3 weeks, the virus would have spread during the weaner stage and reached maximum prevalence in the first month of feeding. The second and third months of feeding (just before slaughter) showed the lowest prevalence values (5% and 7.1% respectively). This could be due to the development of an effective immune response to HEV at this stage. An interesting point is the relatively high prevalence found in breeding sows (21.9%). It is known that during pregnancy sows may shed some infectious organisms, such as parasites and rotavirus, in higher titer.3,6,16 This may also be the case with HEV but, to the authors' knowledge, there is only 1 study of this stage: experimental infection of pregnant gilts8 found no clinical signs or documented transmission in utero to the offspring, even though HEV infection was successful and gilts shed HEV in feces. HEV antibodies were detected in 10- to 61-day-old pigs born to inoculated sows. As in other studies, no clinical signs were recorded in the pigs sampled, so it would seem that the infection is subclinical in pigs. Thus exposure in the form of personal contact with subclinically infected animals could result in HEV transmission from pigs to humans.
For practical purposes, the data on viral prevalence according to production stages obtained in this study could help to guide targeted sampling in the production stages most at risk, in order to minimize the cost of testing on a farm. In addition, it could also help prevent the involuntary transmission by infected animals to an HEV-free farm. There is no report of HEV RNA being detected in manure ditches and water troughs on swine farms in Europe. The values obtained in manure ditches were surprisingly high, with 8 out of 16 farms testing positive (50%) (Table 2). Only 1 water trough out of the 16 farms was found to be positive (6.2%). The virus could have reached the water trough though a pig's snout that had previously been in contact with a contaminated floor. No correlation has been recorded between the presence of HEV in pigs on a particular farm and the presence of HEV in the manure ditch of that farm. Furthermore, the percentage of infected pigs on farms found to be negative for HEV in manure was much higher (21.4%) than that on farms found to be positive for HEV in manure (6.2%). Thus, the analysis of only 1 type of sample would decrease the sensitivity of HEV detection on farms. The high presence of the virus in manure ditches raises concern about its use as soil fertilizer. A study of the viability of the virus15 reported detectable levels of HEV in sewage by RT-PCR over a 2-month period. Thus it would be desirable to undertake studies to determine the resistance of the virus in the environment in order to evaluate the potential risk to humans, especially when water is not tested for HEV. Correct treatment of sewage or its routine screening and treatment of waste materials may be needed to prevent HEV contamination of waterways or areas around farms.
Several studies performed in Japan,12 the UK,2 and the US10,11 support the contentions that zoonotic infection between pigs and humans may occur and that human and swine HEV may coexist in nonendemic areas. Recent studies suggest that autochthonous hepatitis E infection is underdiagnosed in developed countries. A study in which sera from 336 Spanish patients diagnosed for idiopathic acute hepatitis was reevaluated14 found seroprevalence of 8.9% using anti-HEV IgM. Other studies carried out in several European countries such as Austria,21 England,2 France,9 and Italy23 have found autochthonous cases with a high percentage of nucleotide similarity to porcine strains from the same geographic area. In summary, this work could serve to highlight the production stages in which pigs are in most danger of infecting other pigs. The presence of swine manure indicates the potential for spread to humans through contact with contaminated crops or in personnel that handle swine manure and spread this waste on agricultural fields.
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Acknowledgments |
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a. QIampViral RNA Kit, Qiagen, Valencia, CA.
b. ABIPRISM Dye Terminator Cycle Sequencing Ready Reaction kit, Perkin-Elmer, Applied Biosystems, Foster City, CA.
c. SPSS software package (version 12.0). SPSS Inc., Chicago, IL.
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