Seroprevalence of Hepatitis E virus in slaughter pigs and practices of abattoir workers in Kampala, Uganda

Seroprevalence of Hepatitis E virus in slaughter pigs and practices of abattoir workers in Kampala, Uganda

Moses Katagwa¹, Marvin Apollo Ssemadaali^1,2, Gabriel Tumwine¹, Samuel Majalija¹, Lawrence Mugisha^1,2,3,&

¹College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P. O Box 7062, Kampala, Uganda, ²Department of Microbiological Sciences, North Dakota State University, Fargo, North Dakota, USA, ³Conservation and Ecosystem Health Alliance (CEHA), P.O. Box, 34153, Kampala, Uganda

^&Corresponding author
Lawrence Mugisha, Makerere University, Department of Wildlife and Animal Resources Management (WARM), Conservation & Ecosystem Health Alliance (CEHA) P.O. Box 34153, Kampala, Uganda

Introduction    

Hepatitis E Virus (HEV) is a major public health concern in developing countries with sub-standard sanitation conditions [1, 2]. HEV has also been reported as an endemic in many industrialized and developed countries [3]. In humans, HEV is the primary cause of acute hepatitis [4] with mortality rates up to 4% in immune-competent individuals. Higher mortality rate of up to 20% have been reported among pregnant women [5], while immune-compromised individuals develop chronic HEV infection [1, 6]. HEV is classified into five genotypes [7] and of the 5 genotypes, HEV genotypes 3 and 4 are zoonotic and are particularly harboured by the domestic pigs, wild boars and other animal species [8-11], while genotype 5 is exclusively known to infect chickens [12]. The risk for human infection with HEV genotypes 3 and 4 is associated with exposure to infectious body fluids of infected animals and consumption of contaminated undercooked pork, and other associated infected animal products [13]. In developed countries, HEV genotypes 1 and 2 infections have been diagnosed in travellers returning from endemic countries[14]. However, sporadic autochthonous cases of genotypes 3 and 4 have also been reported in individuals without any travel history to endemic regions [15-17]. Besides humans, infections of pig herds with HEVgenotype 3 and 4 have been reported in developed countries [18, 19]. In developing countries, several HEV outbreaks have been reported [20-22], and in 2007, outbreaks of HEV in humans in Uganda caused 160 deaths and over 10,196 illnesses [23]. The human population at risk of acquiring the zoonotic HEV that has been reported include; pig farmers, pork consumers, veterinarians and workers at pig abattoirs [24-26]. Uganda has the largest pig population in Eastern Africa, approximately 3.3million pigs (UBOS, 2008); and pig farming has greatly improved food security and livelihoods. Consequently, the ready pork market in Kampala attracts pig traders from different districts of Uganda. Hence, the objectives of this study were to determine the seroprevalence of HEV in slaughtered pigs; and to assess abattoir workers´ knowledge and practices toward HEV in Wambizzi pig abattoir -Kampala, Uganda.

Methods     

The cross-sectional study was carried out at Wambizzi pig abattoir in Nalukolongo in Kampala, Uganda. The study population consisted of Wambizzi abattoir workers, as well as pigs brought for slaughter. Blood samples were collected from 182 randomly slaughtered pigs between June and December, 2015. Samples were transported in cool boxes to Mulago Hospital Immunology Laboratory and serum was extracted, stored at -80°C until it was analyzed. Eighty (80) abattoir workers in Wambizzi present and consented at the time of study were interviewed using a structured questionnaire to assess their level of knowledge and occupational practices that might expose them to zoonotic HEV. Written consent was obtained from individual participants prior to being interviewed.

ELISA Assays for detection of anti-HEV antibodies: Pig collected from the blood samples, were screened for the presence of HEV-IgG antibodies using a commercially available ID Screen® Hepatitis E Indirect ELISA kit (IDVet Innovative Diagnostics, France) following manufacturer´s instructions. Briefly, 190μl of dilution buffer was added to each micro well; then 10μl of the negative and positive controls were added to wells A1, A2, B1, B2 and C1, C2, D1, D2 respectively. For the remaining wells, 10μl of each respective pig serum was added, and incubated at 21°C for 45minutes. The microplate was washed three times with 300μl of a wash solution. Then, 100μl of the diluted conjugate was added to each well, and incubated at 21°C for 30 min. The microplate was then washed three times with 300μl of the wash solution. Then, 100μl of the substrate solution was added to each well, and incubated in the dark for 15minutes at 21°C. The colour-developing reaction was stopped by adding 100μl of the stop solution to each well. Reading of plates was done at 450nm with a microplate ELISA reader (Results were interpreted

Data analysis: data was entered into Microsoft Excel 2010 and analysed using SPSS (Statistical software version 20) computer programs. Chi-square test was performed to establish the relationship between different variables. 95% CI were computed, and test results were considered significant for p< 0.05.

Ethical consideration: this study was under a large study titled Capacity building for Integrated Management of Transboundary Animal Diseases and Zoonosis (CIMTRADZ) project at COVAB that was peer reviewed and approved by the College of Health Sciences -Institutional Review Board (CHS-IRB), College of Veterinary Medicine, Animal resources and Biosecurity Institutional Review Board (COVAB-IRB), and Uganda National Council of Science and Technology.

Results     

Seroprevalence of anti-HEVantibodies among slaughter pigs by Indirect ELISA: one hundred and eighty two (182) serum samples from slaughtered pigs at Wambizzi abattoir were analysed for anti-HEV antibodies. Of the total number of pigs sampled, 23.6% or 43/182 (95% CI 18.1-30.2) were seropositive for anti-HEV. The analysis revealed higher seroprevalence rates of 14/38 (36.8%), 6/19(31.6%) and 5/14 (35.7%) of HEV antibodies in slaughtered pigs from Kampala, Masaka, and Wakiso districts respectively (Figure 1). In addition, the seroprevalence of HEV antibodies was higher in slaughter pigs (27.2%) older than 6 months.

Socio-demographic characteristics of study respondents: a total of 80 human abattoir workers were interviewed during the study period and was composed of 50% males and 50% females, of which only 5% had attained tertiary education as summarised in Table 1.

Knowledge about zoonotic HEV among Wambizzi abattoir attendants: most of the demographic social factors in the study population were not associated with the level of awareness/knowledge on the zoonotic HEV except for the education that was significantly associated (p < 0.027). The majority of the respondents 76 (95%) were not aware of HEV as a zoonotic disease, pigs as reservoir for the virus and risk factors for disease transmission (Table 2).

Practices that could predispose abattoir workers to HEV: fifty eight percent (58%) of the respondents in the study reported to have eaten under cooked pork, (80%-90%) as well as not wearing personal protective gear and most of the times in direct contact with blood, saliva and/or faeces from slaughtered pigs.

Discussion     

In the present study, the overall seroprevalence of Hepatitis E virus (HEV) antibodies among slaughtered pigs was 23.6% (95% CI 18.1-30.2). This is the first known study to document anti-HEV antibodies in pigs from Uganda. This provides evidence that pigs are exposed to HEV and could be a potential reservoir for disease transmission to humans. HEV in slaughter pigs has been previously reported elsewhere including the United Kingdom [27]. Our report of HEV antibodies in slaughtered pigs at slaughter houses raises potential public health concerns for zoonotic transmission of HEVgenotypes 3 and 4 strains from pigs to humans. However, this study did not characterise the circulating genotypes in pigs. The seroprevalence of HEV antibodies in pigs observed in this study is comparable to 24.4% reported in Zaria state in Nigeria [28]. Higher HEV seroprevalence (55.6% and 97.0%) in pigs have been previously reported in Nigeria [29, 30]. The difference in the seroprevalence could be attributed to various factors, including; farming systems, geographical location as well as methodological differences [31, 32]. Findings from this study also demonstrate a variation in the seroprevalence of HEV antibodies in pigs from different districts with the highest being documented in urban districts. Similarly, a study done in Canada showed significant variations of HEV seroprevalence between the different geographical regions [33]. In this study, the observed differences could be due to the dynamics of transmission of HEV in pigs and husbandry practices in different farming systems from which pigs are selected and brought for slaughter at Wambizzi in Kampala. The high anti-HEV antibodies from urban districts could probably be due to the fact that pigs reared within these areas are kept in confinement buildings/ pens which hastens the spread of the virus amongst pigs within the same pen environment. Pigs housed in the same pen are routinely exposed to saliva, nasal secretions and urine of multiple sty-mates daily [34], and this results in rapid spread of the virus in a very short time. Additionally, besides greater contact frequency, pigs in the same enclosure experience more exposure to pig manure which increases the transmission rate [31]. Within-pen environmental transmission of HEV occurs between pigs in the same enclosure [35]. On the other hand, pigs reared in rural districts like Kamuli and Mubende are left to freely move and not always kept under confinement thus the low rate of spread of the virus due to less contact frequency between pigs, minimal exposure to infectious wastes, faeces, and urine and nasal secretions [31]. It should be noted, however, that the free ranging pigs could be exposed to infectious wastes of infected wild animals, rodents and other primate [25, 36, 37] which can be encountered in rural districts. This study reports a higher seroprevalence of HEV in pigs of six months and above than in pigs below 6 months as previously reported in other studies [19, 38, 39], and this could be due to long-term exposure of pigs to the virus [40].

Strikingly, the study revealed that the majority of Wambizzi abattoir workers (95%) did not know the existence of the zoonotic HEV, the possible source of the virus and how the virus is transmitted. They did not even know that they are occupationally exposed to the risk of acquiring the zoonotic HEV. This raises a serious concern on the health and safety of the pig abattoir workers and undocumented exposure to the public of this zoonotic HEV through contact with pigs by products and wastes. The high risk exposure is further potentiated by documented behavioural practices in this study whereby 58% of the respondents were reported to have eaten under cooked pork. In addition, 80-90% of the respondents were not using personal protective gear regularly hence directly exposed to blood, saliva, and/or faeces from slaughtered pigs. Not surprisingly therefore, Uganda has recently reported several HEV outbreaks in several districts in Northern Uganda. The most recent report of HEV outbreak in Karamonja region in 2014 recorded 1,390 confirmed cases of HEV; whereby 15 individuals died, of which 50% were pregnant mothers [41]. Previously high seroprevalence of HEV (25.5%) and evidence of recent infection (37.3%) was documented in children aged 0-15 years during 2007 HEV outbreak in Uganda [41]. Between 2007 and 2009, Uganda had the largest HEV outbreak in the world, an epidemic that caused more than 10,196 cases and 196 deaths [23]. This provides clear evidence that Uganda is a hotspot for HEV epidemics and concerted efforts are required to identify the source/ reservoirs for infection to prevent the disease in human population. Previous studies have shown that pig handlers and abattoir attendants and, farmers are at great risk of infection with HEV [42-44] due to exposure with HEV containing infectious material and eating undercooked pork [45-48]. This study provides an insight on the serological existence of anti-HEV antibodies in slaughtered pigs, practices that predispose abattoir workers to HEV and potential risks of HEV transmission to public.

Conclusion     

Findings from this study show that there is a considerable proportion of HEV seropositivity in slaughtered pigs at Wambizzi abattoir that originated from different parts of Uganda. This demonstrates that pigs are a potential reservoir for HEV transmission and more studies are needed to understand the epidemiology of the virus in pigs and characterise genotypes circulating in pigs. Other potential reservoir sources of the HEV should be explored and documented to facilitate interventions for control measures.

Competing interests     

The authors declare no competing interest.

Authors´ contributions     

Katagwa Moses, Mugisha Lawrence, Samuel Majalija and Marvin Ssemadaali designed the study. Katagwa Moses conducted the field and laboratory work. Mugisha Lawrence, Samuel Majalija supervised the data collection and analysis. Katagwa Moses, Samuel Majalija and Gabriel Tumwine did the data analysis. Katagwa Moses, Lawrence Mugisha wrote the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgments     

This research was supported by Krista deBoer .We also acknowledge the support from Marshall, Barnes and the HED-USAID through Capacity building in Integrated Management of Transboundary Animal Diseases and Zoonoses (CIMTRADZ) project at Makerere University.

Tables and figure     

Table 1: socio-demographic characteristics of respondents

Table 2: knowledge of abattoir workers toward Zoonotic HEV, Wambizzi slaughter house

Figure 1: seroprevalence of Hepatitis E virus per district of origin

References     

1. Purcell, Emerson. Hepatitis E: an emerging awareness of an old disease. Journal of hepatology. 2008; 48(3): 494-503. PubMed | Google Scholar


2. Arankalle. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 199 J Infect Dis. 1995; 171(2): 447-50. PubMed | Google Scholar


3. Aggarwal R, Krawczynski K. Hepatitis E: an overview and recent advances in clinical and laboratory research. Journal of gastroenterology and hepatology. 2000; 15(1): 9-20. PubMed | Google Scholar


4. Gerolami R. Treatment of severe acute hepatitis E by ribavirin. Journal of Clinical Virology. 2011; 52(1): 60-62. PubMed | Google Scholar


5. Boccia D. High mortality associated with an outbreak of hepatitis E among displaced persons in Darfur, Sudan. Clinical Infectious Diseases. 2006; 42(12): 1679-1684. PubMed | Google Scholar


6. Péron. Acute autochthonous hepatitis E in western patients with underlying chronic liver disease: a role for ribavirin? Journal of hepatology. 2011; 54(6): 1323-1324. PubMed | Google Scholar


7. Pischke S, Wedemeyer H. Chronic hepatitis E in liver transplant recipients: a significant clinical problem? Minerva Gastroenterol Dietol. 2010 Jun; 56(2): 121-8. PubMed | Google Scholar


8. Xia. Swine and rabbits are the main reservoirs of hepatitis E virus in China: detection of HEV RNA in feces of farmed and wild animals. Arch Virol. 2015; 160(11): 2791-8. PubMed | Google Scholar


9. Pavio N, Meng XJ, Renou C. Zoonotic hepatitis E: animal reservoirs and emerging risks. Veterinary research. 2010; 41(6): 46. PubMed | Google Scholar


10. Teo CG. The two clinico-epidemiological forms of hepatitis E. J Viral Hepat. 2007; 14(5): 295-7. PubMed | Google Scholar


11. Park WJ. Hepatitis E virus as an emerging zoonotic pathogen. Journal of veterinary science. 2016; 17(1): 1-11. PubMed | Google Scholar


12. Huang F. Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. Journal of General Virology. 2004; 85(6): 1609-1618. PubMed | Google Scholar


13. Yugo DM, Meng XJ. Hepatitis E virus: foodborne, waterborne and zoonotic transmission. International journal of environmental research and public health. 2013; 10(10): 4507-4533. PubMed | Google Scholar


14. Miyamura T. Hepatitis E virus infection in developed countries. Virus Res. 2011; 161(1): 40-6. PubMed | Google Scholar


15. Dalton HR. Hepatitis E Virus: Time to Change the Textbooks. Dig Dis. 2016; 34(4): 308-16. PubMed | Google Scholar


16. Kuniholm MH. Epidemiology of hepatitis E virus in the United States: results from the Third National Health and Nutrition Examination Survey, 1988-1994. J Infect Dis. 2009; 200(1): 48-56. PubMed | Google Scholar


17. Mizuo H. Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. J Clin Microbiol. 2002; 40(9): 3209-18. PubMed | Google Scholar


18. Colson. Hepatitis E Virus of Subtype 3a in a Pig Farm, South-Eastern France. Zoonoses Public Health. 2015; 62(8): 593-8. PubMed | Google Scholar


19. de Oya NJ. Widespread distribution of hepatitis E virus in Spanish pig herds. BMC research notes. 2011; 4(1): 412. PubMed | Google Scholar


20. Kmush B. Epidemiology of hepatitis E in low-and middle-income countries of Asia and Africa. in Seminars in liver disease. Theme Medical Publishers. 2013. Google Scholar


21. Guthmann JP. A large outbreak of hepatitis E among a displaced population in Darfur, Sudan, 2004: the role of water treatment methods. Clinical Infectious Diseases. 2006; 42(12): 1685-1691. PubMed | Google Scholar


22. Isaäcson M. An outbreak of hepatitis E in Northern Namibia, 1983. The American journal of tropical medicine and hygiene. 2000; 62(5): 619-625. PubMed | Google Scholar


23. Teshale EH. Evidence of person-to-person transmission of hepatitis E virus during a large outbreak in Northern Uganda. Clinical Infectious Diseases. 2010; 50(7): 1006-1010. PubMed | Google Scholar


24. Adjei AA. Unexpected elevated alanine aminotransferase, aspartate aminotransferase levels and hepatitis E virus infection among persons who work with pigs in accra, Ghana. Virol J. 2010; 7: 336. PubMed | Google Scholar


25. Meng. Hepatitis E virus: animal reservoirs and zoonotic risk. Vet Microbiol. 2010; 140(3-4): 256-65. PubMed | Google Scholar


26. Meng. Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of clinical microbiology. 2002; 40(1): 117-122. PubMed | Google Scholar


27. Grierson S. Prevalence of hepatitis E virus infection in pigs at the time of slaughter, United Kingdom, 2013. Emerging infectious diseases. 2015; 21(8): 1396. PubMed | Google Scholar


28. Alkali B. Serological evidence and public health implication of hepatitis E virus infection in pigs found in Zaria, Kaduna State. African Journal of Microbiology Research. 2015; 9(46): 2270-2274. PubMed | Google Scholar


29. Clement M. Serological Evidence and Risk Factors Associated With Hepatitis E Virus Infection in Pigs and Human at an Intensive Piggery Complex, Lagos Nigeria. Journal of Healthcare Communications. 2016. Google Scholar


30. Owolodun OA. First report of hepatitis E virus circulation in domestic pigs in Nigeria. Am J Trop Med Hyg. 2014; 91(4): 699-704. PubMed | Google Scholar


31. Rutjes S. Seroprevalence of hepatitis E virus in pigs from different farming systems in The Netherlands. Journal of food protection. 2014; 77(4): 640-642. PubMed | Google Scholar


32. Martinelli N. Prevalence of hepatitis E virus antibodies in pigs in Northern Italy. Infect Ecol Epidemiol. 2011; 1: 10. PubMed | Google Scholar


33. Yoo D. Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus. Clinical and diagnostic laboratory immunology. 2001; 8(6): 1213-1219. PubMed | Google Scholar


34. Kasorndorkbua C. Routes of transmission of swine hepatitis E virus in pigs. Journal of clinical microbiology. 2004; 42(11): 5047-5052. PubMed | Google Scholar


35. Andraud M. Direct contact and environmental contaminations are responsible for HEV transmission in pigs. Veterinary research. 2013; 44(1): 1. PubMed | Google Scholar


36. Meng X, Lindsay D, Sriranganathan N. Wild boars as sources for infectious diseases in livestock and humans. Philosophical Transactions of the Royal Society of London B: Biological Sciences. 2009; 364(1530): 2697-2707. PubMed | Google Scholar


37. Vitral CL. Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil. Memórias do Instituto Oswaldo Cruz. 2005; 100(2): 117-122. PubMed | Google Scholar


38. Liu X. Seroprevalence and molecular characteristics of hepatitis E virus in household-raised pig population in the Philippines. BMC veterinary research. 2015; 11(1): 1. PubMed | Google Scholar


39. Krumbholz A. Age-related and regional differences in the prevalence of hepatitis E virus-specific antibodies in pigs in Germany. Veterinary microbiology. 2013; 167(3): 394-402. PubMed | Google Scholar


40. Liang H. The prevalence of hepatitis E virus infections among swine, swine farmers and the general population in Guangdong Province, China. PLoS One. 2014; 9(2): e88106. PubMed | Google Scholar


41. Patel RC, Kamili S, Teshale E. Hepatitis E virus infections in children age 0-15 Uganda outbreak, 2007. Journal of Clinical Virology. 2015; 73: 112-114. PubMed | Google Scholar


42. Junaid SA, Agina SE, Abubakar KA. Epidemiology and associated risk factors of hepatitis e virus infection in plateau state, Nigeria. Virology (Auckl). 2014 May 27; 5: 15-26. PubMed | Google Scholar


43. Krumbholz A. Prevalence of hepatitis E virus-specific antibodies in humans with occupational exposure to pigs. Medical microbiology and immunology. 2012; 201(2): 239-244. PubMed | Google Scholar


44. Galiana C. Occupational exposure to hepatitis E virus (HEV) in swine workers. The American journal of tropical medicine and hygiene. 2008; 78(6): 1012-1015. PubMed | Google Scholar


45. Yapa CM. First reported outbreak of locally acquired hepatitis E virus infection in Australia. Med J Aust. 2016; 204(7): 274. PubMed | Google Scholar


46. Colson P. Pig liver sausage as a source of hepatitis E virus transmission to humans. Journal of Infectious Diseases. 2010; 202(6): 825-834. PubMed | Google Scholar


47. Purcell, Emerson S. Hidden danger: the raw facts about hepatitis E virus. Journal of Infectious Diseases. 2010; 202(6): 819-821. PubMed | Google Scholar


48. Li T. Hepatitis E virus transmission from wild boar meat. Emerging infectious diseases. 2005; 11(12): 1958. PubMed | Google Scholar