Poster presented in Med-Vet-Net Association 5th International Scientific Conference (OneHealth: Zoonoses - Emerging Threats)

June 27^th, 2017

Garcia N., Rodriguez-Lazaro D., Hernandez M., Navarro A. and Goyache J.

Hepatitis E virus (HEV) is the main causative agent of acute hepatitis in humans. Nowadays hepatitis E is considered an important emerging zoonosis in developed countries where human cases are mainly related to animals, primarily pigs (1). The origin of this infection in humans seems to be the contact with infected animals and the consumption of raw or undercooked meat (1) (Figure 1). HEV has been circulating in Spain since many years ago (2-4), but little relevant data is available about the presence of the HEV in the whole food chain.

OBJECTIVE
Assess the presence of the HEV in the Spanish pork food chain, from the origin to the point of sale of the product in order to determine the possible transmission routes and therefore to propose prevention and control strategies.

METODOLOGY
SEROLOGY: Hepatitis E multi species indirect ELISA kit validated for swine based in recombinant genotype 3 capsid antigens (ID.vet, Montepellier, France).
MOLECULAR DETECTION: RNA extraction from faeces and livers was performed using QIAamp® viral RNA mini kit and RNeasy Midi kit (QIAGEN) and analysed by Real time RT-PCR aimed to the ORF3 region (Jothikumar et al., 2006).
STATISTICS: The proportion of positive samples to HEV (and Wilson’s 95% confidence interval) were calculated by the geographical origin of the pigs, and the possible association between both was assessed using chi-square and Fisher exact tests. Calculations were carried out using the SPSS software V.20 (IBM Inc., Chicago, Il, USA) and WINPEPI (PEPI-for-Windows) V.11.35 software.

ANALYZED SAMPLES
FARMS: Serum samples from 895 sows taken in 29 pig farms from four different provinces of Spain (Toledo, Huesca, Zaragoza and Teruel) were analysed for the presence of anti-HEV antibodies. Samples were taken between 2013 and 2015.
SLAUGHTERHOUSES: A cross sectional study on pigs being slaughtered in 2015 was undertaken through a sampling strategy with a national coverage. 20 Spanish slaughterhouses were selected according to their slaughter capacity, which represents the 50% of national pig production, and were located in different regions within the country.
A total of 241 serum, 245 faecal and 246 liver samples from 241 healthy pigs from 29 different provinces were collected.

RESULTS AND DISCUSION
FARMS: Positive animals were detected in all the provinces sampled, being statistically significant higher in Toledo than in Huesca (Chi2 p=0,0007) and Zaragoza (Chi2 p<0,001). Overall, 653 out of 895 animals were positive to HEV, which represent and individual seroprevalence of 72,9% (95% CI=69,9-75,8); the herd prevalence was 68,9% (CI=50,7-82,7). This results confirmed previous studies performed in other regions of Spain and underlined the widespread of HEV in swine farms (2-4).
SLAUGHTERHOUSES: From a total of 726 samples analysed, 21,9% (95% IC=16,9-27,7), 19,6% (95% IC= 16,9-27,7) and 71,3% (95% IC=65,2-76,9), resulted positive in liver, faeces and sera samples, respectively (Figure 2). Results indicate that the virus is circulating among the majority of the Spanish herds and that near 20% of the animals were infected, as the virus RNA was present in liver as well as in faeces.
Although the elevated presence of HEV have been demonstrated in the Spanish swine population since the 80’s, only few studies have been performed and they were geographically restricted (2-4). This study comprised animals from the whole pig production chain, and confirm that HEV is endemic in the Spanish pig industry, as is the case in other European countries (2-4).

CONCLUSIONS
In conclusion, the present work shows the active HEV circulation in the Spanish pig production chain from the origin (farms) to the slaughterhouses, which could represent a threat to Public Health.
The next step of the work will be to determine the presence of HEV in pigs products in order to establish the risk of pork consumption, especially in raw products as “morcilla”, “chorizo”, and ham.

ACKNOWLEDGEMENTS
This work is funded by the INIA project entitled “Integrated monitoring and control of Toxoplasma gondii and enteric viruses in food supply chains” RTA 2014-00024-C04-04.
TAVS-CM R&D Grant Programme of Regional Interest of the Madrid Regional Government 2013. S2013/ABI-2747. 2014-2018.

REFERENCES
Meng (2011). Virus Res., 161 (1): 23-30
Clemente-Casares et al. (2003). Emerg. Infect. Dis., 9 (4): 448-54
Berto et al. (2012). BMC Res. Notes, 5: 190
Casas et al. (2009). Vet. Microbiol., 135 (3-4): 248-252
Jothikumar et al., 2006. J. Virol. Methods., 131(1): 65-71

	Servicio de Zoonosis Emergentes, de Baja Prevalencia y Agresivos Biológicos (NED). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).
	Unidad de Microbiología. Departamento de Biotecnología y Ciencia de los Alimentos. Universidad de Burgos (UBU).
	Laboratorio de Biología Molecular y Microbiología. Instituto Tecnológico Agrario de Castilla y León (ITACyL).