PhD Thesis defense by Alejandro Navarro Gómez at the VISAVET Centre of the Complutense University of Madrid

July 22^nd, 2022

Hepatitis E (HE) is one of the main acute viral hepatitis worldwide, and can represent an important problem in Public Health. The virus responsible for this disease is the hepatitis E virus (HEV), belonging to the Hepeviridae family. To date, 8 different genotypes of HEV are known. The genotypes that affect humans are genotypes 1-4, although the possible zoonotic potential of genotypes 5, 7 and 8 has been described. In developed countries, the causes of HE in humans are genotype 3 and 4, generally associated with the consumption of contaminated and under cooked food. The main animal reservoir of HEV, apart from wild boar and deer, is the pig. In our country there is no surveillance plan against HEV in pigs, nor official and harmonized diagnostic techniques for its detection. The infection in pigs is asymptomatic and there are few studies carried out in Spain, which means that it is underdiagnosed. Therefore, the main objective of this thesis is to know the situation of HEV in the production chain of the pig sector. To achieve this objective, six studies divided into three chapters have been carried out.

Chapter I is made up of three studies that aim to clarify some aspects of the current situation of HEV in our country throughout the entire pig production chain. The studies were carried out in both white and Iberian pigs. The first study evaluated the seroprevalence and prevalence of HEV in serum samples from white and Iberian pig farms. The sera were analyzed by ELISA and RT-qPCR techniques. An overall seroprevalence of 80.3% (95% CI 77.7-82.6%) and a prevalence of 2.69% (95% CI 1.31-5.45%) were obtained.

In the second study, the seroprevalence and the prevalence of the virus were evaluated in slaughterhouses distributed throughout the country. The results obtained in the sera analyzed by the ELISA technique also showed a high mean seroprevalence in Spanish slaughterhouses (73.8%, 95% CI 71.5-76.0%). Samples analyzed by RT-qPCR technique (serum, liver, cecal content), showed a high presence of the virus reaching prevalences of up to 24.1% (95% CI 21.5-26.8%).

In the third study, the presence of the genetic material of the virus in products made from raw materials from pigs was investigated. The products (chorizo, black pudding, compango and morcón), which were purchased in different establishments in the Community of Madrid and analyzed by RT-qPCR, revealed a high number of positive products (33.9%, 95% CI 29.3 - 38%).

Chapter II comprises the fourth study carried out in this doctoral thesis. This study assessed the presence of HEV and its organic distribution in apparently healthy pigs. Ten different samples (blood, cecal content, heart, kidney, liver, ribs, bacon, diaphragm, lean meat and loin head) belonging to 45 animals were analyzed. A total of 73.3% (95% CI 58.9 – 84.0%) of the sera from the animals tested were positive for the antibody detection by ELISA. Viral RNA was detected in 6 of the 10 sample types analyzed by RT-qPCR.

Chapter III includes the last two studies carried out in this doctoral thesis. In the fifth study, liver transudate was evaluated as an alternative sample to serum, since this sample is sometimes difficult to obtain, for the detection of antibodies against HEV using the ELISA technique. For this purpose, serum and liver transudate samples from 44 white pigs, 35 wild boars and 46 Iberian pigs were compared. The transudates were analyzed undiluted and at different dilutions (1:2, 1:10 and 1:20). A higher degree of concordance was obtained, both in qualitative and quantitative results, compared to serum samples (gold standard), with liver transudate at a 1:10 dilution.

In the sixth study, detection of HEV RNA by RT-qPCR in serum was compared with undiluted liver transudate. For this purpose, sera and liver transudates from 35 white pigs, 5 Iberian pigs and 18 wild boars were analyzed. The results obtained rule out liver transudate as an alternative to serum for detecting HEV by RT-qPCR.

The data obtained in this doctoral thesis reveal the high exposure of both white and Iberian pigs to HEV throughout their productive life, as well as the presence of the virus in the different matrices studied (organs and finished products), suggesting the need to implement surveillance and control measures against HEV and to intensify the existing hygienic-sanitary measures in all phases of the production system. The results also reveal that tissues of muscular origin are not risk products in relation to HEV. Finally, liver transudate at a 1:10 dilution can be considered a good alternative to the use of serum for the detection of antibodies against HEV by ELISA.

- Piden implantar medidas de vigilancia frente a la hepatitis E en el porcino de España - animalshealth.es