Comparative genetic characterization of Porcine Circovirus type 2 samples from German wild boar populations

PCV-2 is involved in "postweaning multisystemic wasting syndrome" (PMWS), "porcine dermatitis and nephropathy syndrome" (PDNS), respiratory and reproductive disorders, and thereby plays a crucial role in today's swine production world-wide. The virus is apparently ubiquitous in domestic pigs and has also been demonstrated in wild pigs. Up to now, a characterization of PCV-2 samples from wild pigs, which might help to estimate the possible role of wild pigs as sources of domestic pig infection, has not been carried out. Spleen samples from 16 PCV-2-positive wild pigs from hunting grounds of four regions in Germany were used for the analysis of the viral genome. In one sample, the complete sequence of the genome was determined. In the other, a 742 nucleotide fragment from the highly variable capsid sequence of the ORF2 was sequenced. Analysis of the sequences led to the identification of three PCV-2 strains. One strain, representing 14 of the 16 samples, was closely related with Chinese, but not with German strains. The genome of this strain was shortened by one nucleotide by a deletion close to the end of ORF2. The deletion led to a shift of the stop-codon and to the insertion of a further codon. Two further strains differed in up to 4.7% of nucleotides and up to 10.5% of amino acids (aa). These strains were aligned with clusters of PCV-2 samples from mainly French and German domestic origin.     

Natural and experimental hepatitis E virus genotype 3 - infection in European wild boar is transmissible to domestic pigs

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0121-8) contains supplementary material, which is available to authorized users.     

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar.     

种公猪精液伪狂犬病野毒的筛查

本文基于1 100头基础母猪的规模化场进行公猪精液伪狂犬病病毒筛查,农业部规定伪狂犬病病原学检测方法是通过ELSIA方法检测猪伪狂犬病gE野毒抗体。由于抗体检测具有滞后性,导致正在发生感染的猪群g E抗体检测往往呈阴性,但此时通过病原学检测精液已经正在排毒。本文互补了ELSIA抗体检测方法与荧光定性PCR检测病原的优劣,成功实现种公猪精液伪狂犬病病毒的筛查。     

Aujeszky's disease virus infection patterns in European wild boar

Evidence of exposure (i.e. seroprevalence) to Aujeszky's disease virus (ADV) is high among wild boars from south-central Spain. This research aims to determine the presence of ADV by molecular detection, and to describe the patterns of ADV infection in wild boars. Tonsils (TN) and trigeminal ganglia (TG) for ADV molecular detection, and sera were collected from wild boars (n = 192) in 39 hunting estates from south-central Spain (2004/2005). A nested polymerase chain reaction (PCR) for a fragment of the ADV surface glycoprotein B was performed on collected tissues. Individual status of presence of viral DNA was tested against explanatory variables by means of a Generalized Linear Mixed Model (GLIMMIX) analysis. Viral detection prevalence was 30.6 ± 6.7%. Although there was an increasing pattern with age and females presented higher prevalences, no statistically significant influence of sex and age was found for viral presence. Molecular testing in TN and TG allowed classifying infection status into (i) ADV negative (in both TN and TG), (ii) only positive in TN, (iii) only positive in TG and (iv) positive in both TN and TG. ADV DNA was statistically more frequently evidenced in TN in females than in males. With the exception of one individual, all wild boars with presence of ADV DNA in TN and TG or only in TG reacted positive in the ELISA. In contrast, animals with only ADV DNA in TN serorreacted positively and negatively. Interestingly, 45% of the PCR positive wild boars (n = 59) were seronegative in the serological test, all of them with viral DNA only in TN. Our results provide evidence for latency of ADV in wild boars and stress the fact that antibody detection based tests may fail to detect a proportion of recently infected animals. This is of great concern since current management schemes in our study promote animal translocation for hunting purposes, with the associated risk of under-detecting ADV infected individuals when using serology to screen for ADV infection.     

我国鸡群禽戊型肝炎病毒RT-PCR检测及部分ORF2基因序列分析

为从核酸水平证实我国鸡群中禽戊型肝炎病毒(HEV)的存在,本实验从山东省某鸡场患有肝脾肿大综合征的病鸡中采集10份粪便和8份胆汁样品,利用RT-PCR方法检测其中禽HEV ORF2基因片段,并将阳性PCR产物克隆测序。结果显示:18份粪便和胆汁样品中,13份为禽HEV RNA阳性;其序列间的同源性为97%～99%,与GenBank中登录的参考序列同源性为76.6%～98.1%;进化树显示与欧洲地区的禽HEV在同一分支,属于禽HEV基因3型。禽HEV ORF2基因的检出为进一步了解禽HEV对我国家禽养殖业的危害以及在我国的流行情况奠定了基础。     

屠宰猪肝和血清中乙型肝炎病毒及戊型肝炎病毒的检测

应用1对乙型肝炎病毒（HBV）S基因保守区的引物，采用PCR方法从屠宰猪肝、血清中检测到了HBV，序列分析表明，扩增片段与已发表的HBVS基因的同源性高达98％～100％。电镜负染色样品观察结果表明，在HBV表面抗原ELISA检测强阳性反应的血清样品中存在有形态、大小与人HBV Dane颗粒和小球状颗粒相似的病毒粒子。针对戊型肝炎病毒（HEV）（）RF2／ORF3重叠区设计了简并引物，采用巢式RT-PCR对屠宰猪肝和血清样品进行了检测。结果表明，部分屠宰猪肝中存在HEV。     

Serosurvey of Aujeszky's disease virus infection in European wild boar in Spain

Serum samples from 693 hunted wild boar (Sus scrofa) were analysed by means of a blocking ELISA technique, and the mean (se) prevalence of antibodies to Aujeszky's disease virus was 44 (4) per cent. All the seropositive wild boar were from south central Spain, except for one from central Spain, close to the main positive area. In this area, where large game species are increasingly managed for hunting, the seroprevalence was affected by the type of management. More intensively managed populations had a higher prevalence than wild boar living in natural situations, and the seroprevalence increased with the age of the animals; the seroprevalence was higher in females in all age groups. The seroprevalence in males more than one year old peaked after the breeding season, whereas females of the same age had a higher and constant seroprevalence throughout the year.     

Detection of infection with classical swine fever virus in wild boar: a comparison of different laboratory diagnostic methods

The aim of this study was to evaluate two commercially available ELISAs for routine diagnosis of classical swine fever virus (CSFV) in wild boar. For this, 222 tissue samples from wild boar were tested in the ELISAs and the results were compared to those obtained using standard methods. First, frozen spleen sections were examined by direct immunofluorescence, and organ suspensions were prepared and tested for CSFV antigen samples were simultaneously examined with the Chekit-ELISA (Dr. Bommeli AG) and the Herd-Chek-ELISA (IDEXX). From the 222 organ suspensions examined in cell culture 102 were positive for CSFV, while no virus could be isolated from the remaining 120 samples. Taking virus isolation as a standard, the Chekit-ELISAs showed a sensitivity of 97%, and the Herd-Chek-ELISA of 72.5%. Both ELISAs revealed high specificities ranging between 99 and 100%. No correlation was found between false negative results obtained in one or in both of the ELISAs with the positive findings in the immunofluorescence test and in the PLA, nor with the clinical reports. Due to the fact that a big number of samples can be processed in a short time with accurate results, the Chekit-ELISA may be considered useful for routine testing of wild boar samples for CSFV.     

RT-nPCR检测屠宰场胆汁中猪戊型肝炎病毒

戊型肝炎病毒(hepatitis E virus, HEV)基因组为单股正链RNA,约 7.5 Kb,具有3个互相重叠的开放读码框架(open reading frame,ORF).自5′末端到 3′末端依次排列着 5′UTR、ORF1、ORF3、ORF2、3′UTR以及 150～200 或更多的PolyA尾,其中ORF3与ORF1和 ORF2 部分重叠, 或完全包含在ORF2中[1].     

Detection of hepatitis E virus RNA in saliva for diagnosis of acute infection

Diagnosis of acute hepatitis E virus (HEV) infection is established by detection of anti‐HEV IgM antibodies by ELISA or by amplification of serum viral RNA. Here, we evaluate the diagnostic value of testing HEV RNA in saliva to identify patients with acute HEV infection. Prospective proof‐of‐concept study including patients with acute hepatitis. Whole blood and neat saliva samples were obtained from all patients. Saliva samples were processed and analysed for HEV RNA by RT‐PCR within 2 hr after collection. A total of 34 patients with acute hepatitis and 12 healthy donors were included in the study. HEV RNA in serum was confirmed by RT‐PCR in eight of these patients (23.5%; 95% CI: 12.2%–40.2%). HEV was isolated in the saliva of eight of 34 patients (23.5%; 95% CI: 12.2%–40.2%). All patients with HEV RNA amplified in saliva had detectable HEV RNA in serum. HEV was isolated neither in the saliva of any of the 26 patients without detectable HEV RNA in serum nor in healthy donors. Our study suggests that acute HEV infection could be diagnosed by assessing viral load in saliva.     

Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany

The aim of this study was to evaluate the epidemiological situation of swine influenza virus (SIV) infections in different wild boar populations in Germany based on a serological surveillance in some Bundeslaender (federal states) in connection with virological investigations in wild boar shot in Northern Germany (Mecklenburg-Western Pomerania, district of Nordvorpommern). Altogether, 1245 sera from wild boar were tested using the hemagglutination inhibition test. The established seroprevalence rate was low (on average 5.2%). Antibodies were only detected against the subtypes H1N1 and H3N2 showing differences between wild boar populations and age classes. The virological investigation of samples derived from lungs of wild boar shot in Mecklenburg-Western Pomerania, district of Nordvorpommern (n=242), revealed that the virus prevalence (two virologically positive animals, 0.8%) was very low. Based on serological typing, the isolated SIV was identified as subtype H3N2. Molecular biological investigations of the hemagglutinin (HA) and neuraminidase (NA) genes confirmed this result. This study suggests that SIV infections in wild boar seem to be no serious threat for domestic pigs.     

东北边境地区野猪及放养杂交野猪戊型肝炎流行病学调查

为了解东北边境地区野猪及放养杂交野猪群体猪戊型肝炎病毒(HEV)感染情况,于2015—2018年在吉林省、黑龙江省的中朝、中俄边境和内蒙古自治区境内加格达奇周边地区采集6月龄以上杂交野猪血清、粪便或肛拭子样品共520份,采集野猪血清和粪便样品共248份。ELISA检测、RT-nPCR检测、全基因组测序、同源性及进化分析结果显示,杂交野猪和野猪感染HEV的血清抗体总阳性率为34.1%(136/399);核酸总阳性率为1.56%(12/771),12份核酸阳性样品均来自杂交野猪,病毒基因组ORF2部分核苷酸序列同源性为85.4%～100.0%,属于基因4型,4a、4b亚型。对4a亚型的1份阳性样品(LJG-18)进行病毒全基因组扩增测序,其核苷酸序列与日本的人源毒株JKO-ChiSai98C同源性最高,为94.9%,与吉林省猪源毒株Ch-S-1同源性为90.2%。结果表明:东北边境地区放养杂交野猪群具有较高的HEV血清抗体阳性率,HEV流行毒株以4a亚型为主。本试验针对我国野猪及放养杂交野猪群体开展猪戊型肝炎流行病学调查,为该病的流行情况提供了新的科学数据,对我国养猪业健康发展和公共卫生安全具有重要意义。