Sonia Pina

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.

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus.

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.

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.

Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains

Haemophilus parasuis is the agent responsible for causing Glässers disease, but little is known about the pathogenic determinants of this major pig disease. Here we describe, for the pathogenic strain Nagasaki, the molecular characterization of 13 trimeric autotransporters as assessed by the presence of YadA C-terminal translocator domains which were classified into three groups. All passenger domains possess motifs and repeats characteristic of adhesins, hemagglutinins, and invasins with various centrally located copies of collagen-like repeats. This domain architecture is shared with two trimeric autotransporter proteins of H. somnus 129Pt. Genomic comparison by microarray hybridization demonstrated homologies among H. parasuis virulent strains and high divergence with respect to nonvirulent strains. Therefore, these genes were named vtaA (virulence-associated trimeric autotransporters). The sequencing of 17 homologous vtaA genes of different invasive strains highlighted an extensive mosaic structure. Based also on the presence of DNA uptake signal sequences within the vtaA genes, we propose a mechanism of evolution by which gene duplication and the accumulation of mutations and recombinations, plus the lateral gene transfer of the passenger domain, led to the diversity of this multigene family. This study provides insights to help understand the tissue colonization and invasiveness characteristic of H. parasuis pathogenic strains.

Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA)

Haemophilus parasuis is the etiological agent of Glässers disease in swine, characterized by fibrinous polyserositis, polyarthritis and meningitis. The lack of a vaccine against a broad spectrum of strains has limited the control of the disease. Recently, virulence associated trimeric autotransporters (VtaA) were described as antigenic proteins of H. parasuis. In this study 6 VtaA were produced as recombinant proteins and used to immunize snatch-farrowed, colostrum-deprived piglets. Immunized animals developed specific systemic and mucosal antibodies. The protective capacity of the anti-VtaA antibodies was evaluated by the inoculation of 3 × 10(8) or 6 × 10(6) colony forming units (CFU) of the highly virulent strain Nagasaki. Vaccinated animals had a delayed course of disease and 33 or 57%, respectively, of the animals survived the lethal challenge. The partial protection achieved with the recombinant VtaA supports their potential as candidates to be included in future vaccine formulations against H. parasuis.

Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.

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.

Identification of potentially virulent strains of Haemophilus parasuis using a multiplex PCR for virulence-associated autotransporters (vtaA)

Haemophilus parasuis is the aetiological agent of Glässers disease and is also a commensal of the upper respiratory tract of pigs. Trimeric autotransporter (vtaA) genes have been identified in H. parasuis and divided into three groups on the basis of the translocator domain sequence. In this study, group 3 vtaA genes were demonstrated by PCR in all 157 H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains; 52/54 (96%) group 1 vtaA negative field isolates were isolated from the nasal passages of healthy animals, whereas no group 1 vtaA negative field isolates were isolated from cases of Glässers disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate putative non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.