Bibiana Peralta

Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm

The objective of the present study was to determine the pattern of Hepatitis E virus (HEV) infection in a naturally infected, farrow-to-finish herd. For that purpose, a prospective study was conducted in randomly selected 19 sows and 45 piglets. Blood samples were collected from sows at 1 week post-farrowing and from piglets at 1, 3, 6, 9, 12, 15, 18 and 22 weeks of age. Furthermore 3 or 5 animals were necropsied at each bleeding day (but at 1 week of age), and serum, bile, liver, mesenteric lymph nodes and faeces taken. HEV IgG, IgM and IgA antibodies were determined in serum and viral RNA was analysed in all collected samples by semi-nested RT-PCR. Histopathological examination of mesenteric lymph nodes and liver was also conducted. From 13 analysed sows, 10 (76.9%) were positive to IgG, one to IgA (7.7%) and two to IgM (15.4%) antibodies specific to HEV. In piglets, IgG and IgA maternal antibodies lasted until 9 and 3 weeks of age, respectively. IgG seroconversion occurred by 15 weeks of age while IgM and IgA at 12. On individual basis, IgG was detectable until the end of the study while IgM and IgA antibody duration was of 4-7 weeks. HEV RNA was detected in serum at all analysed ages with the highest prevalence at 15 weeks of age. HEV was detected in faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15 weeks of age. This peak coincided with the occurrence of hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes and faeces, and also with highest IgG and IgM OD values at 15 weeks. Finally, different HEV sequences from this farm were obtained, which they clustered within 3 different groups, together with other Spanish sequences, all of them of genotype 3. Moreover, the present study also indicates that the same pig can be infected with at least two different strains of HEV during its productive life. This is the first study characterizing HEV infection in naturally infected pigs with chronological virus detection and its relationship with tissue lesions throughout the productive life of the animals.

Effect of Acidified Feed on the Prevalence of Salmonella in Market-age Pigs

Two trials were carried out to determine the effect of feed acidification upon Salmonella carriage in market‐age pigs. In the first trial, the administration for the last 14 weeks of the fattening period of a commercial pelleted feed added with 0.6% lactic acid plus 0.6% formic acid (Lac‐Formic‐1.2) was compared to an unacidified standard diet (STD). A second experiment was carried out in two herds of growing pigs (Herd I, 3000 pigs; Herd II, 900 pigs) in which three different diets were assayed during the last 8–9 weeks of the fattening period: a diet containing 0.8% formic acid (Formic‐0.8), a diet containing 0.4% lactic acid plus 0.4% formic acid (Lac‐Formic‐0.8) and a STD. In the first experiment, serological evolution of the infection was examined by ELISA and microbiological cultures (rectal samples and mesenteric lymph nodes) were also done. Feed intake by pen and the individual weight of the animals were also measured. In the second trial, blood, rectal samples and mesenteric lymph nodes were collected at slaughter in both herds (30 pigs per experimental group). In the first experiment, the acidified diet (Lac‐Formic‐1.2) reduced Salmonella carriers in mesenteric lymph nodes (Fisher’s exact P < 0.01). In the second trial, Lac‐Formic‐0.8 diet significantly reduced Salmonella seroprevalence compared to the STD (P = 0.001) in both herds. Also Lac‐Formic‐0.8 and Formic‐0.8 diets in Herd II showed a lower faecal excretion and Salmonella carriage in mesenteric lymph nodes than the STD (P < 0.05). Our results suggest that the administration of a combination of lactic and formic acids at the levels used in this study could be used to reduce Salmonella prevalence in finishing pigs.

Longitudinal study of hepatitis E virus infection in Spanish farrow-to-finish swine herds

Hepatitis E is a zoonotic disease and is highly prevalent in European swine livestock. There is a need to compare the infection dynamics of hepatitis E virus (HEV) between herds with the same production system and determine the percentage of animals that could arrive infected at slaughter age. Therefore, a longitudinal study was performed in six Spanish farrow-to-finish affected farms. Twenty piglets per farm were monitored from nursery to slaughter. RT-PCR and serology techniques were applied to analyze longitudinally collected sera and/or faecal samples. Liver and bile samples were also taken at the abattoir. Anti-HEV IgM were firstly detected at 7 weeks of age in 5 farms whereas at 13 weeks of age in 1 farm (farm 2). At slaughter age 50-100% of pigs had seroconverted to anti-HEV IgG in the former 5 farms whereas in the other herd only 5% of pigs were IgG seropositive (farm 2). Six out of 96 livers and 5 out of 80 biles analyzed were HEV positive at the abattoir (total percentage of infected animals: 11.5%). All these positive animals had already seroconverted except 2 pigs of farm 2. Hence, pigs can be seronegative at slaughter age being infected during the latest fattening period. Manipulation of HEV-infected livers or other organs from pigs could be considered a possible route of transmission in Spanish abattoirs. This study represents the first longitudinal survey on swine HEV infection dynamics conducted in different herds.

Evidence of widespread infection of avian hepatitis E virus (avian HEV) in chickens from Spain

In the present work, 262 serum samples and 29 faeces pools from chickens coming from 29 healthy flocks were analysed by RT-PCR for detection of avian HEV and by ELISA using an aHEV derived antigen for detection of anti-HEV IgG. Additionally, other 300 randomly selected serum samples were also analysed by RT-PCR. Seven serum samples were positive to RNA detection. Sequence analysis of both the helicase and the capsid genes revealed that the Spanish isolates were clustered together and close related to those strains from the United States isolated from farms with HSS. On the serology study, 26/29 flocks had at least one positive animal (89.7%) and chickens older than 40 weeks were found to have higher seropositivities compared to the rest of age groups. Within positive farms, the proportion of positive animals ranged from 20% to 80%. This is the first report of aHEV sequences in chickens from Europe. Further studies are needed to elucidate the clinical significance of avian HEV infections in Europe.

Anti-HEV antibodies in domestic animal species and rodents from Spain using a genotype 3-based ELISA.

A truncated ORF2 capsid HEV antigen derived from a genotype 3 strain was developed in insect cells and insect larvae, and compared with the Sar55 antigen and a commercial ELISA. The antigen expressed in insect cells showed a better correlation with Sar55 (kappa value (k)=0.84) than the insect larvae antigen (k=0.69), and a better reproducibility as indicated by the intra and interplate variation coefficients. Commercial ELISA designed for human diagnosis but adapted to animal use using specific secondary antibodies demonstrated to have a very low sensitivity. The insect cell expressed antigen was used to develop an ELISA to detect anti-HEV-IgG in serum samples of different domestic animal and rodents. Seropositivity in the studied animal populations was 71.4% for pigs, 0.60% for goats, 1.92% for sheep, and 11.11% for cats. None of the 1170 cattle samples or 166 rodent samples analyzed was positive.

Retrospective serological study on hepatitis E infection in pigs from 1985 to 1997 in Spain.

The objective of the present work was to ascertain the date in which hepatitis E virus (HEV) was introduced in the Spanish pig population. For this, a serological retrospective study was carried out using archived sera. A total of 2871 serum samples gathered between 1985 and 1997 and collected in 208 farms of Spain were tested for anti-HEV IgG by an in-house ELISA. Of the 2871 sera analyzed by ELISA, 1390 were positive for anti-HEV antibodies (48.4%, 95% CI: 46.9-49.9%) and that corresponded to 204/208 farms (98%, 95% CI: 96.1-99.9%) having at least one positive pig. Our results show that HEV was present and widespread in Spanish swine farms at least since 1985. Any significant changes in prevalence were detected from 1 year to another and therefore, HEV infection in swine should be considered endemic in Spain.

Genetic characterization of the complete coding regions of genotype 3 hepatitis E virus isolated from Spanish swine herds.

The complete coding regions of five hepatitis E virus isolates of swine origin from two different pig farms and the complete genome sequence of two of these strains were obtained and compared to other full length or partial HEV sequences. Based on the nucleotide sequence, the examined Spanish isolates were 87.1-99.7% similar among them being the closest known strain a Mongolian porcine strain (swMN06-C1056) which shares 84.5-86.1% of the nucleotide sequence, and are also close to other HEV porcine strains from Japan. Two isolates from the same farm presented an 87 nucleotide insertion in the poly-proline hinge unique among all HEV isolates known so far. Comparison with partial HEV sequenced strains indicates that the isolates described in this study form a cluster containing human and porcine HEV strains from Europe, being the only representatives of the subtype 3f that were completely sequenced. Evolutive pressure analysis indicates that microevolution of HEV seems to be driven by negative selection. Further studies should be carried out in order to clarify the HEV origin and evolution.

Pigs orally inoculated with swine hepatitis E virus are able to infect contact sentinels.

The purpose of the present study was to explore the most likely natural route of infection of swine hepatitis E virus (HEV) by oral inoculation of pigs and to investigate the potential infection by direct contact exposure. A preliminary experiment was performed to assess the infectiousness of the bile used as source of virus. Once confirmed, 16 pigs were inoculated via oral drop with an HEV positive bile suspension containing 2x10(5) genome equivalents per pig. Nine animals were kept as contact sentinels and 12 more pigs were used as negative controls. A number of pigs from the three groups were euthanized at 16, 32 and 64 days post-inoculation. From the HEV inoculated group, three pigs shed virus in faeces, two had virus RNA in bile at necropsy and two seroconverted. In the contact group, two animals showed presence of HEV RNA in bile. This study demonstrates that pigs orally inoculated with a single HEV dose got infection, although few animals had evidence of infection. Moreover, the virus was successfully transmitted to direct contact exposed pigs.

Different feed withdrawal times before slaughter influence caecal fermentation and faecal Salmonella shedding in pigs.

The effects of different pre-slaughter feed withdrawal times (FWT) on the gastrointestinal tract (GIT) weight and the gut environment of pigs and Salmonella shedding were investigated. Trial 1 evaluated the effects under experimental conditions (feed withdrawal for 18, 30 and 36 h) and trial 2 under commercial conditions (15 and 30 h). In trial 1, the GIT weight tended to decrease (P=0.07), the caecal pH increased (P<0.0001), short-chain fatty acids (SCFA) decreased (P<0.001) and percentage of branched-chain fatty acids (BCFA) increased as FWT increased. Similar results were observed in trial 2, but Enterobacteriaceae numbers and Salmonella positive pigs tended to increase whereas lactobacilli decreased (P<0.0005) as FWT increased. The increase in FWT involved changes in the gut microbial ecosystem that could be associated with the trend of increased caecal Enterobacteriaceae and Salmonella in faeces, and may represent a higher risk of carcass contamination in cases of laceration of viscera.

Applying phylogenetic analysis to viral livestock diseases: moving beyond molecular typing.

Changes in livestock production systems in recent years have altered the presentation of many diseases resulting in the need for more sophisticated control measures. At the same time, new molecular assays have been developed to support the diagnosis of animal viral disease. Nucleotide sequences generated by these diagnostic techniques can be used in phylogenetic analysis to infer phenotypes by sequence homology and to perform molecular epidemiology studies. In this review, some key elements of phylogenetic analysis are highlighted, such as the selection of the appropriate neutral phylogenetic marker, the proper phylogenetic method and different techniques to test the reliability of the resulting tree. Examples are given of current and future applications of phylogenetic reconstructions in viral livestock diseases.