Familial Hepatitis E Outbreak Linked to Wild Boar Meat Consumption

An HIV‐infected patient was diagnosed with acute hepatitis E infection in our hospital. An epidemiological inquiry was performed to collect demographic, food and animal exposure variables in order to identify the potential route of transmission. The patient reported that his family traditionally hunted wild boar for food. All family members were analysed for hepatitis E virus infection. Additionally, route of transmission by wild boar meat consumption and prevalence of HEV infection among wild boar from the same hunting area were investigated. In all‐family members (n = 8), HEV‐RNA was amplified. Two wild boar meat slices consumed was analysed, showing the presence of HEV. The virus isolated was consistent with genotype 3, revealing 100% homology between family members and meat. Additionally, we tested nine wild boar hunted in the same hunting area. All of them were RNA‐HEV positive, isolating the same HEV genotype 3 viral strain. We demonstrated by phylogenetic analysis zoonotic transmission of HEV by wild boar meat consumption. The prevalence of HEV infection among wild boar found in our study suggests that this species is an important route of transmission to human.     

Detection of hepatitis E virus in archived German wild boar serum samples

Hepatitis E is a rare human disease in Central Europe commonly imported from endemic regions. For autochthonous infections a zoonotic transmission from pigs, deer and wild boar is assumed. Using three different RT-PCR protocols, hepatitis E virus (HEV) RNA was detected in 10 out of 189 (5.3%) serum samples collected in 1995/1996 from wild boars in Germany. Sequence analysis indicates a close relationship with genotype 3 isolates of pigs and humans from the Netherlands and Japan. The results indicate that HEV is present in Germany since more than 10 years and that wild boar may function as a reservoir for HEV.     

Localisation of swine hepatitis E virus in experimentally infected pigs

The distribution of intravenously inoculated swine hepatitis E virus (HEV) was assessed by in situ hybridisation for a period of 50 days. Evidence of apparent clinical disease was found in only one pig in the HEV infected group. The only gross lesion observed was mildly enlarged mesenteric lymph nodes at 50 days post infection (dpi). Histopathologically, mild lymphoplasmacytic infiltration and focal hepatocellular necrotic lesions were found in HEV-infected pigs. Swine HEV nucleic acids were detected by RT-PCR in the faeces at 3 dpi in 100% of the 18 pigs infected with the virus. Thereafter, the number of positives declined.The most consistent and intense signal was found in the liver of infected animals using in situ hybridisation. The positive cells were hepatocytes, Kupffer cells, bile epithelial cells and interstitial lymphocytes. Swine HEV RNA was localised in the cytoplasm of the hepatocytes, with a slightly granular pattern of staining, but hybridisation signals were not observed in degenerative or vacuolated hepatocytes. HEV was much less frequently detected in extrahepatic tissues such as lymph nodes, tonsil, spleen and small and large intestine. It was concluded that swine HEV had replicated primarily in the hepatocytes and infection resulted in subclinical infection with minimal histopathological changes in the liver.     

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.     

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.     

Retrospective Study Evaluating Seroprevalence of Hepatitis E Virus in Blood Donors and in Swine Veterinarians in Italy (2004)

Hepatitis E is an emerging viral disease in developed countries, with sporadic cases occasionally linked to the consumption of raw or undercooked pork, wild boar or deer meat. Cases due to transfusion or transplantation have also been reported. In developed countries, hepatitis E is considered a zoonosis and pig is the main reservoir. In the last few years, several studies conducted in Europe reported variable seroprevalence rates among the general population, ranging between 0.26% and 52.5%. A higher seroprevalence was described among workers who come in contact with pigs. The aim of this retrospective study was to evaluate the seroprevalence of anti‐HEV IgG and IgM antibodies in blood donors (170) and in pig veterinarians (83). Archival sera were collected in Italy in 2004. The observed seroprevalence was 9.64% and 8.82% in veterinarians and blood donors, respectively. Overall, only three sera from blood donors were positive for IgM, but no HEV‐RNA was detected.     

High HEV presence in four different wild boar populations in East and West Germany

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.     

Seroprevalence of hepatitis E virus in dogs in Switzerland

Hepatitis E virus (HEV) is the causative agent of an acute and in most cases self‐limiting hepatitis. Of the four major HEV genotypes that infect humans, genotype 3 and 4 are zoonotic and have been identified in humans but predominantly in pigs and wild boar, which are considered the main reservoirs. However, the known host range of zoonotic HEV may be increasing to comprise additional species, including companion animals. Several studies have identified contact with dogs as a risk factor for HEV infection in humans, yet information on the occurrence of HEV in Swiss dogs is lacking. To examine a possible risk of exposure, this study was designed to assess the seroprevalence of HEV in 84 Swiss dogs. Serum and plasma samples collected from four veterinary clinics were screened for HEV‐specific antibodies by HEV‐antibody ELISA test kit. In addition, information of 22 dogs regarding the country of origin, the type of dog feed and any history of hunting was recorded. Samples from seropositive animals were also screened for the presence of HEV RNA by quantitative real‐time RT‐PCR (qRT‐PCR). Overall, 38% (32 of 84) of the dogs tested seropositive for anti‐HEV, indicating exposure to HEV. Among the 22 dogs for which information was available, HEV‐specific antibodies were detected in three of five dogs that were born abroad, in one of two dogs that were fed a raw meat‐based diet, and in one hunting dog. No viral RNA could be detected in any of the serum and plasma samples; thus, the genotype of the strains remained undetermined. This study provides further evidence for canine exposure and susceptibility to HEV and highlights the need to further assess the risks of HEV transmission to humans with contact to dogs.     

The high prevalence of hepatitis E virus infection in wild boars in Ibaraki Prefecture,Japan

Hepatitis E virus (HEV) is known as a causative agent of zoonosis and food poisoning. Pigs and some species of wild animals, including wild boar, are known to be a reservoir of HEV. In this study, we investigated the situation regarding HEV infection in wild boars in Ibaraki Prefecture, Japan. Serum, liver and feces samples from 68 animals were collected, and the presence or absence of HEV genomic RNA and HEV antibodies were analyzed. The viral genome was detected in samples from 7 (10.3%) animals, with all HEVs classified as genotype 3, subtype 3b. HEV antibodies were detected in samples from 28 (41%) animals. This report demonstrates for the first time the high prevalence of HEV infection in wild boars in Ibaraki Prefecture.     

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.     

Hepatitis E virus and related viruses in wild,domestic and zoo animals: A review

Hepatitis E is a human disease mainly characterized by acute liver illness, which is caused by infection with the hepatitis E virus (HEV). Large hepatitis E outbreaks have been described in developing countries; however, the disease is also increasingly recognized in industrialized countries. Mortality rates up to 25% have been described for pregnant women during outbreaks in developing countries. In addition, chronic disease courses could be observed in immunocompromised transplant patients. Whereas the HEV genotypes 1 and 2 are mainly confined to humans, genotypes 3 and 4 are also found in animals and can be zoonotically transmitted to humans. Domestic pig and wild boar represent the most important reservoirs for these genotypes. A distinct subtype of genotype 3 has been repeatedly detected in rabbits and a few human patients. Recently, HEV genotype 7 has been identified in dromedary camels and in an immunocompromised transplant patient. The reservoir animals get infected with HEV without showing any clinical symptoms. Besides these well‐known animal reservoirs, HEV‐specific antibodies and/or the genome of HEV or HEV‐related viruses have also been detected in many other animal species, including primates, other mammals and birds. In particular, genotypes 3 and 4 infections are documented in many domestic, wildlife and zoo animal species. In most cases, the presence of HEV in these animals can be explained by spillover infections, but a risk of virus transmission through contact with humans cannot be excluded. This review gives a general overview on the transmission pathways of HEV to humans. It particularly focuses on reported serological and molecular evidence of infections in wild, domestic and zoo animals with HEV or HEV‐related viruses. The role of these animals for transmission of HEV to humans and other animals is discussed.     

Detection of hepatitis E virus RNA in rats caught in pig farms from Northern Italy

Hepatitis E virus (HEV) strains belonging to the Orthohepevirus genus are divided into four species (A–D). HEV strains included in the Orthohepevirus A species infect humans and several other mammals. Among them, the HEV‐3 and HEV‐4 genotypes are zoonotic and infect both humans and animals, of which, pigs and wild boar are the main reservoirs. Viruses belonging to the Orthohepevirus C species (HEV‐C) have been considered to infect rats of different species and carnivores. Recently, two studies reported the detection of HEV‐C1 (rat HEV) RNA in immunocompromised and immunocompetent patients, suggesting a possible transmission of rat HEV to humans. The role of rats and mice as reservoir of HEV and the potential zoonotic transmission is still poorly known and deserves further investigation. To this purpose, in this study, the presence of HEV RNA was investigated in the intestinal contents and liver samples from 47 Black rats (Rattus rattus) and 21 House mice (Mus musculus) captured in four pig farms in Northern Italy. The presence of both Orthohepevirus A and C was investigated by the real‐rime RT‐PCR specific for HEV‐1 to HEV‐4 genotypes of Orthohepevirus A species and by a broad spectrum hemi‐nested RT‐PCR capable of detecting different HEV species including rat HEV. The intestinal content from two Black rats resulted positive for HEV‐C1 RNA and for HEV‐3 RNA, respectively. None of the House mice was HEV RNA positive. Sequence analyses confirmed the detection of HEV‐C1, genotype G1 and HEV‐3 subtype e. The viral strain HEV‐3e detected in the rat was identical to swine HEV strains detected in the same farm. Liver samples were negative for the detection of either rat HEV or HEV‐3.     

Virus isolated and immunofluorescence in different organs of pigs infected with hemagglutinating encephalomyelitis virus

Hemagglutinating encephalomyelitis virus (HEV; also designated vomiting and wasting disease virus) was inoculated oronasally in 26 colostrum-deprived pigs. Anorexia and vomition were seen after an incubation period of 4 to 6 days. In pigs killed during the incubation period or within 2 days after the onset of the clinical signs, HEV could be isolated regularly from the tonsils and the respiratory tract, irregularly from the digestive tract, rarely from the blood, and never from lymph nodes and spleen. The brainstem almost always contained virus after clinical signs appeared, but was only one positive during the incubation period. Olfactory bulb, cerebrum, cerebellum, and vagal nerve were also frequently virus positive in pigs which were ill when killed. The results of the examination by immunofluorescent antibody technique indicated that HEV multiplies in the epithelium lining the respiratory tract and the tonsillar crypts, in neuroepithelium of the nasal mucosa, and in neurons of the digestive tract. The neuronotropism of HEV was also shown by the presence of fluorescence in the perikaryon of neurons in the brainstem and in the trigeminal ganglion without the involvement of other cell types. The presence of viral antigens in the perikaryon of trigeminal sensory ganglion cells in pigs killed during the incubation period was considered as positive evidence for viral spread via nerves.     

Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus.

Cesarean-derived, colostrum-deprived pigs (n = 23) were inoculated intranasally and subcutaneously with a low cell culture passage of type 2 porcine circovirus. In 11 pigs, a persistent fever that lasted 7-17 days began 12-15 days after inoculation with virus. Additional signs of disease in those 11 pigs included depression (11 of 11 pigs), palpable enlargement of inguinal, prefemoral, and popliteal lymph nodes (11 of 11), icterus (6 of 11), and hyperpnea (2 of 11). The remaining 12 pigs had fever that occurred intermittently for 2-4 days between days 12 and 20 postinoculation. Overt signs of disease in those pigs were limited to palpable enlargement of inguinal and popliteal lymph nodes (9 of 12 pigs). When compared with control pigs of similar age, the average daily rate of weight gain for all pigs inoculated with virus was less over a 2-week period that began 2 weeks post inoculation. At postmortem examination, lymph node enlargement was seen in 14 of 14 pigs euthanized between days 20 and 28 postinoculation. Lymph node enlargement was especially prominent in pigs that developed a persistent fever. Microscopic lesions noted in pigs that developed a persistent fever included cellular depletion in lymphoid tissues; hepatic cell necrosis; and lymphogranulomatous inflammation of lymph nodes, Peyer's patches of the intestine, liver, kidney, and heart. Virus was isolated with varying frequency from nasal, rectal, or tonsil swab specimens, buffy coat, serum, urine, and lung lavage fluid obtained antemortem or postmortem. Virus was isolated from or viral DNA was detected in a variety of tissues obtained postmortem up to 125 days postinoculation. Antibody against type 2 porcine circovirus usually was detected in serum between 15 and 20 days postinoculation; however, antibody against virus was not detected in serum from 4 pigs euthanized 20-24 days postinoculation. Direct contact with pigs inoculated with virus 42 days previously resulted in transmission of virus to 3 of 3 control pigs.     

Case report: Porcine circovirus type 2 infection in an European wild boar (Sus scrofa) in the state of Brandenburg, Germany

This case represents the first case of Porcine Circovirus Type 2 (PCV-2)--infection in a free living European wild boar associated with morphological lesions, which are regarded as characteristic for Postweaning Multisystemic Wasting Syndrome (PMWS) in domestic pigs. The animal, an approximately 10 month old male, was found dead in a rural area within the state of Brandenburg, Germany. The closest commercial pig farm is located in 3 km distance from the spot where the carcass was found. At necropsy, the animal was found to be in a runted condition. Morphological investigation revealed two lesion complexes. Firstly, lymphatic depletion was present in different organs. Mainly the white pulp of the spleen was affected, where lymph follicles and periarteriolar lymphatic sheaths were nearly completely depleted of lymphoid cells. The former lymphatic areas could only be identified by the presence of histiocytic cells. Secondly, there were widely distributed lesions indicative of a bacterial septicemia i.e. purulent-necrotizing lymphadenitis, pulpous hyperplasia of the spleen, miliary lytic liver necroses and foci of fibrinous pneumonia. Within the lesions, bacterial colonies were found (short Gram-negative rods). Bacteriology revealed a septicemic Salmonella choleraesuis var. Kunzendorf--infection. Virologically, the animal was tested with negative results for Classical Swine Fever Virus and PRRSV. The unusual depletion of the lymphatic tissue mainly in the spleen led to the suspicion of a PCV-2 infection. Typical circoviral particles were found by negative-contrast electron microscopy in samples from spleen and lymph nodes. Using a commercial antiserum against Porcine Circovirus, positive staining was found by fluorescence microscopy in tonsils, spleen and lymph nodes. Finally, the virus was identified to be PCV-2 by species-specific PCR. The presented case rises the questions if PCV-2 is endemic in the European wild boar population at least in certain areas, if it is of pathogenetic importance for wild boars and if the virus present in wild boars is identical to that present in domestic pigs with PMWS.     

Experimental infection of pregnant gilts with swine hepatitis E virus

To determine the effect of swine hepatitis E virus (HEV) infection on pregnant gilts, their fetuses, and offspring, 12 gilts were intravenously inoculated with swine HEV. Six gilts, who were not inoculated, served as controls. All inoculated gilts became actively infected and shed HEV in feces, but vertical transmission was not detected in the fetuses. There was no evidence of clinical disease in the gilts or their offspring. Mild multifocal lymphohistiocytic hepatitis was observed in 4 of 12 inoculated gilts. There was no significant effect of swine HEV on fetal size, fetal viability, or offspring birth weight or weight gain. The offspring acquired anti-HEV colostral antibodies but remained seronegative after the antibodies waned by 71 days of age. Swine HEV infection induced subclinical hepatitis in pregnant gilts, but had no effect on the gilts' reproductive performance, or the fetuses or offspring. Fulminant hepatitis associated with HEV infection was not reproduced in gilts.     

Epidemiology of Mycobacterium bovis infections of pigs and wild boars using a molecular approach

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis.