Arch Virol DOI 10.1007/s00705-016-2778-y
BRIEF REPORT
Serological survey of hepatitis E virus infection in farmed and pet rabbits in Italy Ilaria Di Bartolo1 • L. De Sabato1 • A. Marata2 • N. Martinelli3 • C. F. Magistrali4 M. Monini1 • E. Ponterio1 • F. Ostanello5 • F. M. Ruggeri1
•
Received: 27 October 2015 / Accepted: 27 January 2016 Ó Springer-Verlag Wien 2016
Abstract The recent identification in rabbits of hepatitis E viruses (HEV) related to viruses infecting humans raises the question of the role of this species as possible HEV reservoir. A serological survey on rabbit HEV infection was conducted in Italy during 2013-2014, including both farmed and pet rabbits. We found an anti-HEV antibody seroprevalence of 3.40 % in 206 farmed rabbits (collected on 7 farms) and 6.56 % in 122 pets. RNA was extracted from IgG-positive sera and analyzed by HEV-specific realtime RT-PCR. None of the samples were positive, confirming that no viremia was present in the presence of IgG. Only one serum sample from a farmed rabbit was positive for IgM, but no HEV RNA was detected in it. Pet rabbit feces were also tested for HEV RNA, with negative results. This finding suggests that HEV is circulating in rabbits in Italy. Keywords IgM
Hepatitis E virus Zoonoses Rabbits IgG
& Ilaria Di Bartolo [email protected] 1
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
2
Macerata, Italy
3
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER), Brescia, Italy
4
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy
5
Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, BO, Italy
Hepatitis E virus (HEV) is a small RNA virus that causes hepatitis in humans. The disease varies from asymptomatic to acute hepatitis (mortality, 1 %) and may become chronic in immunosuppressed patients [1]. The HEV genome is approximately 7.5 kb in length and contains three open reading frames (ORFs). ORF1 encodes nonstructural proteins; ORF2, the viral capsid [2]; and ORF3, a small multifunctional protein [3]. The virus belongs to the family Hepeviridae, which was recently divided into the genera Piscihepevirus and Orthohepevirus [4]. The latter includes the species Orthohepevirus A, whose members infect several mammals, including humans, and are classified in four genotypes. HEV genotypes 1 and 2 are restricted to humans and cause waterborne outbreaks in low-income countries, whilst cases in developed countries are linked to travelling in endemic area. HEV genotypes 3 and 4 infect both humans and animals, causing sporadic human cases and small outbreaks in industrialized countries, and apparently asymptomatic infections in animals. Zoonotic transmission of genotype 3 and 4 HEV has been related to consumption of raw or undercooked meat and liver from pork, wild boar and deer [5]. Recently, HEV strains were found in both farmed and wild rabbits in several areas of China, the USA and France [6–9]. The circulation of HEV in rabbits has also been confirmed by the observed HEV seroprevalence, which ranges between 7 % and 57 % in different countries [10, 11]. The risk of zoonotic transmission of rabbit HEV to humans is supported by several lines of evidence, including the detection of an HEV strain closely related to rabbit HEV in a man [8]. Indeed, the rabbit HEV strains form a separate lineage of genotype 3 [10]. In Italy, genotype 3 HEV has been reported in pigs [12], wild boar [13], and deer [14] and in a pet rabbit [15], in addition to autochthonous sporadic cases in humans [16].
123
I. Bartolo et al.
This study investigated HEV circulation among farmed and pet rabbits in Italy. For this purpose, a serological survey was conducted on 328 sera to detect both IgG and IgM anti-HEV antibodies. HEV RNA was investigated in 122 pet rabbit fecal samples. During 2013-2014, 206 sera were collected from asymptomatic rabbits reared on seven farms (one in southern Italy and six in northern Italy, designated A-H). Animals were caged in small groups (3-5 animals), except for adult males, which were caged individually. In 2013, 122 paired sera and feces were collected from pet rabbits aged between 6 months and 10 years, which presented to veterinary clinics located in central Italy as healthy (no. 66) or affected by non-hepatic diseases (i.e., stypsis, Encephalitozoon cuniculi infection, and tumors). For farmed rabbits, blood sampling was part of a routine monitoring program, aimed to detect antibodies against myxomatosis and rabbit hemorrhagic disease. For pet rabbits, blood was collected for control purposes when animals were brought to the veterinary clinics for routine check up or because they were affected by non-hepatic diseases. HEV antibodies were detected by western blotting (WB), ELISA and immunoperoxidase (IPA) assays based on a recombinant swine genotype 3 HEV capsid protein [17]. This antigen, lacking the first 111 amino acids, was expressed using the baculovirus BacD111ORF2HEV in Sf9 insect cells [17]. WB and ELISA tests were performed as described previously [17], using anti-rabbit IgG (1:3000, Pierce) or anti-rabbit IgM (1:500) [18] (kindly provided by IZSLER) secondary antibodies. Rabbit sera were diluted 1:100 and tested in triplicate. To improve the specificity of the assays, the serum samples were pre-incubated with uninfected Sf9 cell crude protein extract and then incubated with the partially purified HEV capsid protein. The same test was conducted against a heterologous baculovirus-expressed antigen (bovine norovirus VLPs, data not shown) [19] that was used in a negative control test. The cutoff value (COV) was defined as the optical density (OD) mean value ? 2 standard deviations (SD). For IPA assays, conducted on a group of sera positive in ELISA and/or in WB, Sf9 cell monolayers in a 96-wells plate infected with BacD111ORF2HEV or mock-infected were fixed and incubated with rabbit sera (diluted 1:50 up to 1:500). Replication foci were visualized by reaction with amino ethylcarbazole. Total RNA was extracted from 10 % fecal suspension (w/v) or from sera using an RNeasy Mini Kit (QIAGEN). HEV RNA was detected by HEV real-time RT-PCR as described previously [20]. Detection of anti-HEV antibodies was conducted by WB and ELISA, and positive sera were tested by IPA assay. Based on the results obtained, considering sera positive in at least one test (ELISA and/or WB), anti-HEV antibodies
123
were detected in 7 of 206 (3.40 %) sera from farmed rabbits and in eight of 122 (6.56 %) sera from pets (Table 1). The tests showed some lack of concordance (Table 1). Therefore, the seroprevalence decreased to some extent, i.e., to 1.94 % (4/206) for farmed animals and to 3.28 % (4/ 122) for pets, if considering samples positive only in WB (Table 1). Differences between ELISA and WB results for HEV antibody detection have already been described [21], and these are possibly linked to a lower specificity of the ELISA. Positive sera were further tested by IPA assays; however, none of the seven sera that were positive only by ELISA and only four of the eight WB-positive sera were positive by IPA. These data suggest that this newly developed IPA assay could be a more sensitive test for antibody detection, but further studies are needed to validate the test. Overall, the observed seroprevalence suggests that rabbit HEV is circulating in Italy, but with a low prevalence. Considering the farmed rabbits, among the seven farms sampled, only farms A and C housed anti-HEV positive animals, with a herd prevalence of 4.82 % (4/83) and 7.69 % (3/39), respectively. The seroprevalence ranged from 0 to 7.69 % among farms (Table 2). No differences other than their geographical location were observed among the farms. Farm A was located in southern Italy, while the others were from two distinct areas in northern Italy, and only breeding animals were housed on farm A, where 45 does were tested. Two of the IgG-positive rabbits from farm A were does, while the other five IgG-positive animals from farms A and C were bucks (2 years old) and fattening, respectively. Interesting, samples from farm A were collected in two different months in 2014, March and December. Both groups were positive, with no difference (p [ 0.05) in prevalence, suggesting that spreading of the virus did not Table 1 Detection of HEV antibodies in sera from farm and pet rabbits by WB, ELISA and IPA tests WB Farm animals
ELISA
IPA
?
?
?
1/206 (0.49)
?
-
?
1/206 (0.49)
?
-
-
2/206 (0.97)
-
?
-
3/206 (1.46)
-
-
-
199/206 (96.60)
? ?
? -
?
1/122 (0.82) 2/122 (1.64)
?
-
-
1/122 (0.82)
Total positive Pets
Total positive
No. of samples/total (%)
7/206 (3.40)
-
?
-
4/122 (3.28)
-
-
-
114/122 (93.44) 8/122 (6.56)
Hepatitis E virus infection in rabbits in Italy Table 2 Detection of HEV antibodies in sera, grouped by farms and categories Farm
No. of anti-HEV positive sera/total Does
Bucks
Fattening
nd
Prevalence (%)
A
2/45
2/38
-
-
4/83 (4.82)
B
-
-
-
0/9
0/9
C D
-
0/4 0/21
3/35 -
-
3/39 (7.69) 0/21
E
-
-
0/13
-
0/13
F
-
0/10
0/11
-
0/21
G
-
-
0/20
-
0/20
Total tested
45
73
79
9
206
nd, unknown; 2-4 months old
increase appreciably during the nine months of the study. Since HEV transmission by the fecal-oral route on farms is sustained by HEV-shedding animals, the maintenance of a low seroprevalence on the HEV-positive farm might be due to good hygienic conditions and housing of rabbits in feces-free cages, limiting virus spread. RNA was extracted from IgG-positive sera and analyzed by HEV-specific real-time RT-PCR. None of them were positive, confirming that no viremia was present in presence of serum IgG. One of the does was positive for the IgM, but its serum was negative for HEV viral RNA by HEV-specific real-time RT-PCR. The absence of HEV RNA might be due to long storage of the sera, but the detection of IgM in this sample confirms a recent infection. Eight of 122 (6.56 %) sera from pet rabbits also tested positive in at least one test for HEV IgG antibodies. None of them contained specific IgM. The seropositive animals were aged from 2 to 10 years. Two of them were infected with Encephalitozoon cuniculi, one animal was affected by tumors and five were asymptomatic. One animal died a few months after the study. Apparently, none of the positive animals showed hepatic symptoms. No HEV RNA was detected from the 122 fecal samples; this could be explained by the absence of a recent infection, as revealed by the lack of IgM-positive animals. Overall, this study indicates that HEV is present in both farmed and pet rabbits in Italy. The detection rate of antiHEV IgG antibodies confirmed that this virus circulates in rabbits with a low seroprevalence, in agreement with the lack of HEV RNA detection in the pet rabbit fecal samples investigated. Several studies have reported high seroprevalence rates, ranging from 23.2 % in China [22] to 57.3 % in Mongolia [13] or HEV-RNA detection in both wild and farm rabbits in France, with a prevalence of 7 % and 23 %, respectively [8]. Our results differ, and this is somewhat unexpected because the Italian rabbit meat
production and farming sector is larger than in other EU countries. The world production of rabbit meat is close to 1 million tons, 54 % of which is produced in the EU, where Italy (44 %), France (25 %) and Spain (17 %) are the main producers [23]. Factors other than farm size, e.g., housing and husbandry systems or stocking density, might have influenced the low HEV seroprevalence observed in Italy compared to other European countries. Further studies should evaluate the correlations between HEV prevalence in rabbits and farming risk factors such as management, dimension, and hygiene procedures. Moreover, since rabbit HEV strains belong to the zoonotic genotype 3, the possible occupational risk of infection in farmers, veterinarians and slaughterhouse workers should be also considered. Recently, HEV was reported in a 7-year-old pet rabbit in Italy, which died for Pasteurella spp. infection. The HEV strain detected belonged to genotype 3 and was closely related to human strains circulating in Europe [15]. In Italy, rabbits represent 2.1 % of pet animals [24], which have increased in the last few years. If zoonotic transmission is proven, the close contact between pets and humans might represent an important risk factor for HEV transmission that has not yet been evaluated. Acknowledgments The research leading to these results has received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreement no. 278433 PREDEMICS. Compliance with ethical standards Conflict of interest None of the authors have a conflict of interest that could influence the results of the paper. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
References 1. Arends JE, Ghisetti V, Irving W, Dalton HR, Izopet J, Hoepelman AI, Salmon D (2014) Hepatitis E: an emerging infection in high income countries. J Clin Virol 59:81–88 2. Panda SK, Thakral D, Rehman S (2007) Hepatitis E virus. Rev Med Virol 17:151–180 3. Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H (2009) ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 90:1880–1891 4. Smith DB, Simmonds P, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA (2014) Consensus proposals for classification of the family Hepeviridae. J Gen Virol 95:2223–2232 5. Pavio N, Meng XJ, Doceul V (2015) Zoonotic origin of hepatitis E. Curr Opin Virol 10:34–41 6. Geng Y, Zhao C, Song A, Wang J, Zhang X, Harrison TJ, Zhou Y, Wang W, Wang Y (2011) The serological prevalence and genetic diversity of hepatitis E virus in farmed rabbits in China. Infect Genet Evol 11:476–482
123
I. Bartolo et al. 7. Cossaboom CM, Cordoba L, Sanford BJ, Pineyro P, Kenney SP, Dryman BA, Wang Y, Meng XJ (2012) Cross-species infection of pigs with a novel rabbit, but not rat, strain of hepatitis E virus isolated in the United States. J Gen Virol 93:1687–1695 8. Izopet J, Dubois M, Bertagnoli S, Lhomme S, Marchandeau S, Boucher S, Kamar N, Abravanel F, Guerin JL (2012) Hepatitis E virus strains in rabbits and evidence of a closely related strain in humans, France. Emerg Infect Dis 18:1274–1281 9. Geng Y, Zhang H, Li J, Huang W, Harrison TJ, Zhao C, Zhou Y, Lian H, Wang Y (2013) Comparison of hepatitis E virus genotypes from rabbits and pigs in the same geographic area: no evidence of natural cross-species transmission between the two animals. Infect Genet Evol 13:304–309 10. Zhao C, Ma Z, Harrison TJ, Feng R, Zhang C, Qiao Z, Fan J, Ma H, Li M, Song A, Wang Y (2009) A novel genotype of hepatitis E virus prevalent among farmed rabbits in China. J Med Virol 81:1371–1379 11. Lhomme S, Dubois M, Abravanel F, Top S, Bertagnoli S, Guerin JL, Izopet J (2013) Risk of zoonotic transmission of HEV from rabbits. J Clin Virol 58:357–362 12. Di Bartolo I, Ponterio E, Castellini L, Ostanello F, Ruggeri FM (2011) Viral and antibody HEV prevalence in swine at slaughterhouse in Italy. Vet Microbiol 149:330–338 13. Caruso C, Modesto P, Bertolini S, Peletto S, Acutis PL, Dondo A, Robetto S, Mignone W, Orusa R, Ru G, Masoero L (2015) Serological and virological survey of hepatitis E virus in wild boar populations in northwestern Italy: detection of HEV subtypes 3e and 3f. Arch Virol 160:153–160 14. Di Bartolo I, Ponterio E, Angeloni G, Morandi F, Ostanello F, Nicoloso S, Ruggeri FM (2015) Presence of hepatitis E virus in a RED Deer (Cervus elaphus) population in central Italy. Transbound Emerg Dis. doi:10.1111/tbed.12353 15. Caruso C, Modesto P, Prato R, Scaglione FE, De Marco L, Bollo E, Acutis PL, Masoero L, Peletto S (2015) Hepatitis e virus: first description in a pet house rabbit. A new transmission route for human? Transbound Emerg Dis 62:229–232
123
16. La Rosa G, Muscillo M, Vennarucci VS, Garbuglia AR, La Scala P, Capobianchi MR (2011) Hepatitis E virus in Italy: molecular analysis of travel-related and autochthonous cases. J Gen Virol 92:1617–1626 17. Ponterio E, Di Bartolo I, Orru G, Liciardi M, Ostanello F, Ruggeri FM (2014) Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein. BMC Vet Res 10:133 18. Cooke BD, Robinson AJ, Merchant JC, Nardin A, Capucci L (2000) Use of ELISAs in field studies of rabbit haemorrhagic disease (RHD) in Australia. Epidemiol Infect 124:563–576 19. Zakhour M, Maalouf H, Di Bartolo I, Haugarreau L, Le Guyader FS, Ruvoen-Clouet N, Le Saux JC, Ruggeri FM, Pommepuy M, Le Pendu J (2010) Bovine norovirus: carbohydrate ligand, environmental contamination, and potential cross-species transmission via oysters. Appl Environ Microbiol 76:6404–6411 20. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR (2006) A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 131:65–71 21. Thiry D, Mauroy A, Saegerman C, Thomas I, Wautier M, Miry C, Czaplicki G, Berkvens D, Praet N, van der Poel W, Cariolet R, Brochier B, Thiry E (2014) Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium. Vet Microbiol 172:407–414 22. Liu P, Bu QN, Wang L, Han J, Du RJ, Lei YX, Ouyang YQ, Li J, Zhu YH, Lu FM, Zhuang H (2013) Transmission of hepatitis E virus from rabbits to cynomolgus macaques. Emerg Infect Dis 19:559–565 23. Unaitalia (2012) La filiera cunicola. http://www.unaitalia.com/ Filieracunicola.aspx. Last accessed Dec 2014 24. EURISPES (2011) Rapporto Italia 2011. http://eurispes.eu/con tent/rapporto-italia-2011
BRIEF REPORT
Serological survey of hepatitis E virus infection in farmed and pet rabbits in Italy Ilaria Di Bartolo1 • L. De Sabato1 • A. Marata2 • N. Martinelli3 • C. F. Magistrali4 M. Monini1 • E. Ponterio1 • F. Ostanello5 • F. M. Ruggeri1
•
Received: 27 October 2015 / Accepted: 27 January 2016 Ó Springer-Verlag Wien 2016
Abstract The recent identification in rabbits of hepatitis E viruses (HEV) related to viruses infecting humans raises the question of the role of this species as possible HEV reservoir. A serological survey on rabbit HEV infection was conducted in Italy during 2013-2014, including both farmed and pet rabbits. We found an anti-HEV antibody seroprevalence of 3.40 % in 206 farmed rabbits (collected on 7 farms) and 6.56 % in 122 pets. RNA was extracted from IgG-positive sera and analyzed by HEV-specific realtime RT-PCR. None of the samples were positive, confirming that no viremia was present in the presence of IgG. Only one serum sample from a farmed rabbit was positive for IgM, but no HEV RNA was detected in it. Pet rabbit feces were also tested for HEV RNA, with negative results. This finding suggests that HEV is circulating in rabbits in Italy. Keywords IgM
Hepatitis E virus Zoonoses Rabbits IgG
& Ilaria Di Bartolo [email protected] 1
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy
2
Macerata, Italy
3
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER), Brescia, Italy
4
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy
5
Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell’Emilia, BO, Italy
Hepatitis E virus (HEV) is a small RNA virus that causes hepatitis in humans. The disease varies from asymptomatic to acute hepatitis (mortality, 1 %) and may become chronic in immunosuppressed patients [1]. The HEV genome is approximately 7.5 kb in length and contains three open reading frames (ORFs). ORF1 encodes nonstructural proteins; ORF2, the viral capsid [2]; and ORF3, a small multifunctional protein [3]. The virus belongs to the family Hepeviridae, which was recently divided into the genera Piscihepevirus and Orthohepevirus [4]. The latter includes the species Orthohepevirus A, whose members infect several mammals, including humans, and are classified in four genotypes. HEV genotypes 1 and 2 are restricted to humans and cause waterborne outbreaks in low-income countries, whilst cases in developed countries are linked to travelling in endemic area. HEV genotypes 3 and 4 infect both humans and animals, causing sporadic human cases and small outbreaks in industrialized countries, and apparently asymptomatic infections in animals. Zoonotic transmission of genotype 3 and 4 HEV has been related to consumption of raw or undercooked meat and liver from pork, wild boar and deer [5]. Recently, HEV strains were found in both farmed and wild rabbits in several areas of China, the USA and France [6–9]. The circulation of HEV in rabbits has also been confirmed by the observed HEV seroprevalence, which ranges between 7 % and 57 % in different countries [10, 11]. The risk of zoonotic transmission of rabbit HEV to humans is supported by several lines of evidence, including the detection of an HEV strain closely related to rabbit HEV in a man [8]. Indeed, the rabbit HEV strains form a separate lineage of genotype 3 [10]. In Italy, genotype 3 HEV has been reported in pigs [12], wild boar [13], and deer [14] and in a pet rabbit [15], in addition to autochthonous sporadic cases in humans [16].
123
I. Bartolo et al.
This study investigated HEV circulation among farmed and pet rabbits in Italy. For this purpose, a serological survey was conducted on 328 sera to detect both IgG and IgM anti-HEV antibodies. HEV RNA was investigated in 122 pet rabbit fecal samples. During 2013-2014, 206 sera were collected from asymptomatic rabbits reared on seven farms (one in southern Italy and six in northern Italy, designated A-H). Animals were caged in small groups (3-5 animals), except for adult males, which were caged individually. In 2013, 122 paired sera and feces were collected from pet rabbits aged between 6 months and 10 years, which presented to veterinary clinics located in central Italy as healthy (no. 66) or affected by non-hepatic diseases (i.e., stypsis, Encephalitozoon cuniculi infection, and tumors). For farmed rabbits, blood sampling was part of a routine monitoring program, aimed to detect antibodies against myxomatosis and rabbit hemorrhagic disease. For pet rabbits, blood was collected for control purposes when animals were brought to the veterinary clinics for routine check up or because they were affected by non-hepatic diseases. HEV antibodies were detected by western blotting (WB), ELISA and immunoperoxidase (IPA) assays based on a recombinant swine genotype 3 HEV capsid protein [17]. This antigen, lacking the first 111 amino acids, was expressed using the baculovirus BacD111ORF2HEV in Sf9 insect cells [17]. WB and ELISA tests were performed as described previously [17], using anti-rabbit IgG (1:3000, Pierce) or anti-rabbit IgM (1:500) [18] (kindly provided by IZSLER) secondary antibodies. Rabbit sera were diluted 1:100 and tested in triplicate. To improve the specificity of the assays, the serum samples were pre-incubated with uninfected Sf9 cell crude protein extract and then incubated with the partially purified HEV capsid protein. The same test was conducted against a heterologous baculovirus-expressed antigen (bovine norovirus VLPs, data not shown) [19] that was used in a negative control test. The cutoff value (COV) was defined as the optical density (OD) mean value ? 2 standard deviations (SD). For IPA assays, conducted on a group of sera positive in ELISA and/or in WB, Sf9 cell monolayers in a 96-wells plate infected with BacD111ORF2HEV or mock-infected were fixed and incubated with rabbit sera (diluted 1:50 up to 1:500). Replication foci were visualized by reaction with amino ethylcarbazole. Total RNA was extracted from 10 % fecal suspension (w/v) or from sera using an RNeasy Mini Kit (QIAGEN). HEV RNA was detected by HEV real-time RT-PCR as described previously [20]. Detection of anti-HEV antibodies was conducted by WB and ELISA, and positive sera were tested by IPA assay. Based on the results obtained, considering sera positive in at least one test (ELISA and/or WB), anti-HEV antibodies
123
were detected in 7 of 206 (3.40 %) sera from farmed rabbits and in eight of 122 (6.56 %) sera from pets (Table 1). The tests showed some lack of concordance (Table 1). Therefore, the seroprevalence decreased to some extent, i.e., to 1.94 % (4/206) for farmed animals and to 3.28 % (4/ 122) for pets, if considering samples positive only in WB (Table 1). Differences between ELISA and WB results for HEV antibody detection have already been described [21], and these are possibly linked to a lower specificity of the ELISA. Positive sera were further tested by IPA assays; however, none of the seven sera that were positive only by ELISA and only four of the eight WB-positive sera were positive by IPA. These data suggest that this newly developed IPA assay could be a more sensitive test for antibody detection, but further studies are needed to validate the test. Overall, the observed seroprevalence suggests that rabbit HEV is circulating in Italy, but with a low prevalence. Considering the farmed rabbits, among the seven farms sampled, only farms A and C housed anti-HEV positive animals, with a herd prevalence of 4.82 % (4/83) and 7.69 % (3/39), respectively. The seroprevalence ranged from 0 to 7.69 % among farms (Table 2). No differences other than their geographical location were observed among the farms. Farm A was located in southern Italy, while the others were from two distinct areas in northern Italy, and only breeding animals were housed on farm A, where 45 does were tested. Two of the IgG-positive rabbits from farm A were does, while the other five IgG-positive animals from farms A and C were bucks (2 years old) and fattening, respectively. Interesting, samples from farm A were collected in two different months in 2014, March and December. Both groups were positive, with no difference (p [ 0.05) in prevalence, suggesting that spreading of the virus did not Table 1 Detection of HEV antibodies in sera from farm and pet rabbits by WB, ELISA and IPA tests WB Farm animals
ELISA
IPA
?
?
?
1/206 (0.49)
?
-
?
1/206 (0.49)
?
-
-
2/206 (0.97)
-
?
-
3/206 (1.46)
-
-
-
199/206 (96.60)
? ?
? -
?
1/122 (0.82) 2/122 (1.64)
?
-
-
1/122 (0.82)
Total positive Pets
Total positive
No. of samples/total (%)
7/206 (3.40)
-
?
-
4/122 (3.28)
-
-
-
114/122 (93.44) 8/122 (6.56)
Hepatitis E virus infection in rabbits in Italy Table 2 Detection of HEV antibodies in sera, grouped by farms and categories Farm
No. of anti-HEV positive sera/total Does
Bucks
Fattening
nd
Prevalence (%)
A
2/45
2/38
-
-
4/83 (4.82)
B
-
-
-
0/9
0/9
C D
-
0/4 0/21
3/35 -
-
3/39 (7.69) 0/21
E
-
-
0/13
-
0/13
F
-
0/10
0/11
-
0/21
G
-
-
0/20
-
0/20
Total tested
45
73
79
9
206
nd, unknown; 2-4 months old
increase appreciably during the nine months of the study. Since HEV transmission by the fecal-oral route on farms is sustained by HEV-shedding animals, the maintenance of a low seroprevalence on the HEV-positive farm might be due to good hygienic conditions and housing of rabbits in feces-free cages, limiting virus spread. RNA was extracted from IgG-positive sera and analyzed by HEV-specific real-time RT-PCR. None of them were positive, confirming that no viremia was present in presence of serum IgG. One of the does was positive for the IgM, but its serum was negative for HEV viral RNA by HEV-specific real-time RT-PCR. The absence of HEV RNA might be due to long storage of the sera, but the detection of IgM in this sample confirms a recent infection. Eight of 122 (6.56 %) sera from pet rabbits also tested positive in at least one test for HEV IgG antibodies. None of them contained specific IgM. The seropositive animals were aged from 2 to 10 years. Two of them were infected with Encephalitozoon cuniculi, one animal was affected by tumors and five were asymptomatic. One animal died a few months after the study. Apparently, none of the positive animals showed hepatic symptoms. No HEV RNA was detected from the 122 fecal samples; this could be explained by the absence of a recent infection, as revealed by the lack of IgM-positive animals. Overall, this study indicates that HEV is present in both farmed and pet rabbits in Italy. The detection rate of antiHEV IgG antibodies confirmed that this virus circulates in rabbits with a low seroprevalence, in agreement with the lack of HEV RNA detection in the pet rabbit fecal samples investigated. Several studies have reported high seroprevalence rates, ranging from 23.2 % in China [22] to 57.3 % in Mongolia [13] or HEV-RNA detection in both wild and farm rabbits in France, with a prevalence of 7 % and 23 %, respectively [8]. Our results differ, and this is somewhat unexpected because the Italian rabbit meat
production and farming sector is larger than in other EU countries. The world production of rabbit meat is close to 1 million tons, 54 % of which is produced in the EU, where Italy (44 %), France (25 %) and Spain (17 %) are the main producers [23]. Factors other than farm size, e.g., housing and husbandry systems or stocking density, might have influenced the low HEV seroprevalence observed in Italy compared to other European countries. Further studies should evaluate the correlations between HEV prevalence in rabbits and farming risk factors such as management, dimension, and hygiene procedures. Moreover, since rabbit HEV strains belong to the zoonotic genotype 3, the possible occupational risk of infection in farmers, veterinarians and slaughterhouse workers should be also considered. Recently, HEV was reported in a 7-year-old pet rabbit in Italy, which died for Pasteurella spp. infection. The HEV strain detected belonged to genotype 3 and was closely related to human strains circulating in Europe [15]. In Italy, rabbits represent 2.1 % of pet animals [24], which have increased in the last few years. If zoonotic transmission is proven, the close contact between pets and humans might represent an important risk factor for HEV transmission that has not yet been evaluated. Acknowledgments The research leading to these results has received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreement no. 278433 PREDEMICS. Compliance with ethical standards Conflict of interest None of the authors have a conflict of interest that could influence the results of the paper. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
References 1. Arends JE, Ghisetti V, Irving W, Dalton HR, Izopet J, Hoepelman AI, Salmon D (2014) Hepatitis E: an emerging infection in high income countries. J Clin Virol 59:81–88 2. Panda SK, Thakral D, Rehman S (2007) Hepatitis E virus. Rev Med Virol 17:151–180 3. Yamada K, Takahashi M, Hoshino Y, Takahashi H, Ichiyama K, Nagashima S, Tanaka T, Okamoto H (2009) ORF3 protein of hepatitis E virus is essential for virion release from infected cells. J Gen Virol 90:1880–1891 4. Smith DB, Simmonds P, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA (2014) Consensus proposals for classification of the family Hepeviridae. J Gen Virol 95:2223–2232 5. Pavio N, Meng XJ, Doceul V (2015) Zoonotic origin of hepatitis E. Curr Opin Virol 10:34–41 6. Geng Y, Zhao C, Song A, Wang J, Zhang X, Harrison TJ, Zhou Y, Wang W, Wang Y (2011) The serological prevalence and genetic diversity of hepatitis E virus in farmed rabbits in China. Infect Genet Evol 11:476–482
123
I. Bartolo et al. 7. Cossaboom CM, Cordoba L, Sanford BJ, Pineyro P, Kenney SP, Dryman BA, Wang Y, Meng XJ (2012) Cross-species infection of pigs with a novel rabbit, but not rat, strain of hepatitis E virus isolated in the United States. J Gen Virol 93:1687–1695 8. Izopet J, Dubois M, Bertagnoli S, Lhomme S, Marchandeau S, Boucher S, Kamar N, Abravanel F, Guerin JL (2012) Hepatitis E virus strains in rabbits and evidence of a closely related strain in humans, France. Emerg Infect Dis 18:1274–1281 9. Geng Y, Zhang H, Li J, Huang W, Harrison TJ, Zhao C, Zhou Y, Lian H, Wang Y (2013) Comparison of hepatitis E virus genotypes from rabbits and pigs in the same geographic area: no evidence of natural cross-species transmission between the two animals. Infect Genet Evol 13:304–309 10. Zhao C, Ma Z, Harrison TJ, Feng R, Zhang C, Qiao Z, Fan J, Ma H, Li M, Song A, Wang Y (2009) A novel genotype of hepatitis E virus prevalent among farmed rabbits in China. J Med Virol 81:1371–1379 11. Lhomme S, Dubois M, Abravanel F, Top S, Bertagnoli S, Guerin JL, Izopet J (2013) Risk of zoonotic transmission of HEV from rabbits. J Clin Virol 58:357–362 12. Di Bartolo I, Ponterio E, Castellini L, Ostanello F, Ruggeri FM (2011) Viral and antibody HEV prevalence in swine at slaughterhouse in Italy. Vet Microbiol 149:330–338 13. Caruso C, Modesto P, Bertolini S, Peletto S, Acutis PL, Dondo A, Robetto S, Mignone W, Orusa R, Ru G, Masoero L (2015) Serological and virological survey of hepatitis E virus in wild boar populations in northwestern Italy: detection of HEV subtypes 3e and 3f. Arch Virol 160:153–160 14. Di Bartolo I, Ponterio E, Angeloni G, Morandi F, Ostanello F, Nicoloso S, Ruggeri FM (2015) Presence of hepatitis E virus in a RED Deer (Cervus elaphus) population in central Italy. Transbound Emerg Dis. doi:10.1111/tbed.12353 15. Caruso C, Modesto P, Prato R, Scaglione FE, De Marco L, Bollo E, Acutis PL, Masoero L, Peletto S (2015) Hepatitis e virus: first description in a pet house rabbit. A new transmission route for human? Transbound Emerg Dis 62:229–232
123
16. La Rosa G, Muscillo M, Vennarucci VS, Garbuglia AR, La Scala P, Capobianchi MR (2011) Hepatitis E virus in Italy: molecular analysis of travel-related and autochthonous cases. J Gen Virol 92:1617–1626 17. Ponterio E, Di Bartolo I, Orru G, Liciardi M, Ostanello F, Ruggeri FM (2014) Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein. BMC Vet Res 10:133 18. Cooke BD, Robinson AJ, Merchant JC, Nardin A, Capucci L (2000) Use of ELISAs in field studies of rabbit haemorrhagic disease (RHD) in Australia. Epidemiol Infect 124:563–576 19. Zakhour M, Maalouf H, Di Bartolo I, Haugarreau L, Le Guyader FS, Ruvoen-Clouet N, Le Saux JC, Ruggeri FM, Pommepuy M, Le Pendu J (2010) Bovine norovirus: carbohydrate ligand, environmental contamination, and potential cross-species transmission via oysters. Appl Environ Microbiol 76:6404–6411 20. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR (2006) A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J Virol Methods 131:65–71 21. Thiry D, Mauroy A, Saegerman C, Thomas I, Wautier M, Miry C, Czaplicki G, Berkvens D, Praet N, van der Poel W, Cariolet R, Brochier B, Thiry E (2014) Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium. Vet Microbiol 172:407–414 22. Liu P, Bu QN, Wang L, Han J, Du RJ, Lei YX, Ouyang YQ, Li J, Zhu YH, Lu FM, Zhuang H (2013) Transmission of hepatitis E virus from rabbits to cynomolgus macaques. Emerg Infect Dis 19:559–565 23. Unaitalia (2012) La filiera cunicola. http://www.unaitalia.com/ Filieracunicola.aspx. Last accessed Dec 2014 24. EURISPES (2011) Rapporto Italia 2011. http://eurispes.eu/con tent/rapporto-italia-2011
