Parasitol Res (2016) 115:4707–4709 DOI 10.1007/s00436-016-5240-x

SHORT COMMUNICATION

Survey of Trichinella infection from domestic pigs in the historical endemic areas of province, central

Peng Jiang1 & Xi Zhang1 & Li Ang Wang1 & Lu Hong Han1 & Mei Yang1 & Jiang Yang Duan1 & Ge Ge Sun1 & Xin Qi1 & Ruo Dan Liu1 & Zhong Quan Wang1 & Jing Cui1

Received: 16 July 2016 /Accepted: 30 August 2016 /Published online: 7 September 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract The aim of this work was to investigate the current of the nematode genus Trichinella (Murrell and Pozio 2011). situation of Trichinella infection from domestic pigs in the Human trichinellosis has been documented in 55 countries of historical endemic areas of Henan province, central China. A the world and is considered as an emerging/re-emerging dis- total of 823 diaphragm samples from the indoor-raised pigs ease, especially in developing countries (Bruschi 2012;Cui were collected in five cities of Henan during 2014–2015 and et al. 2006a). In China, Trichinella infection in animals and examined by artificial digestion method. The overall preva- humans has been documented in 33 out of 34 provinces/ lence of Trichinella infection in pigs was 0.61 % (5/823). autonomous regions/municipals (Liu and Boireau 2002; Trichinella larvae were detected in 0.91 % (5/550) of pigs Wang et al. 2007; Cui et al. 2011). Henan province was located from Nanyang city of Henan. The larval burden in infected in central China and considered as a historical endemic area for animals was 0.03 larvae per gram (lpg) of muscles with a range trichinellosis; 16 outbreaks consisting of 521 cases were re- from 0.02 to 0.05 lpg. The larvae were identified as Trichinella ported during 1984–2003 in this province. Pork is the predom- spiralis by multiple PCR. Our study confirms the existence of inant source of outbreaks of human trichinellosis in Henan; swine trichinellosis in Henan, but the infection level was under 75 % (12/16) of outbreaks were caused by eating raw or the minimum level for defining infectious sources for humans. undercooked pork (Wang and Cui. 2001;Wangetal.2006). However, the prevalence of swine Trichinella infection in All 16 outbreaks of trichinellosis occurred in southwestern, Henan need to be further evaluated with a large scale of pork western, and central parts of Henan. The etiological agent of samples for ensuring meat food safety. Trichinella infection in swine from Henan was identified as Trichinella spiralis (Fu et al. 2009;Wangetal.2012). Keywords Trichinella spiralis . Domestic pigs . Artificial However, no outbreaks of human trichinellosis were re- digestion . Multiplex PCR . China cordedinHenanfrom2004to2015(Wangetal.2014). Currently, it is unknown whether this is due to the absence of Trichinella infection in pigs in Henan. The aim of this work Introduction was to investigate the present situation of Trichinella infection in indoor-raised pigs from the historical endemic areas of Trichinellosis is a serious parasitic zoonosis acquired by eating Henan province. Trichinella spp. larvae collected from dia- raw or undercooked meat contaminated with infective larvae phragm muscles of infected swine were identified at the spe- cies level by a multiplex-PCR analysis.

* Zhong Quan Wang [email protected] Materials and methods * Jing Cui [email protected] Study area and animals 1 Department of Parasitology, Medical College, University, 40 Daxue Road, Zhengzhou 450052, People’sRepublic The survey of Trichinella infection from domestic pigs was of China conducted in five cities of the historical trichinellosis endemic 4708 Parasitol Res (2016) 115:4707–4709 areas in southwestern, western, and central parts of Henan: from Nanyang city, and the prevalence of Trichinella infection Nanyang, , , , and . in swine from Nanyang was 0.91 % (5/550). The lpg of in- The diaphragm samples were collected from domestic pigs fected pork samples was 0.02, 0.04, 0.05, 0.02, and 0.02, slaughtered at home for home consumption only. All pigs respectively, and the average of larval burden in infected pork were raised in small pig farms in suburb or backyard farms was 0.03 lpg. of rural and mountainous areas in above five cities of Henan. Six larvae were isolated from five pork samples. The amplicon of the expected size (173 bp) for T. spiralis was Pork samples revealed in multiplex-PCR assays containing larval DNA from Trichinella larvae in pork samples (Fig. 1). Our results A total of 823 diaphragm samples were collected from the showed that the Trichinella isolates from naturally infected indoor-raised pigs in five cities of Henan province. Diaphragm pig in Henan were T. spiralis. samples (50 g ∼ 100 g of each animal) were collected in winter during 2014–2015. Discussion Parasitological test In Henan province, Trichinella infection in domestic pigs was Diaphragm samples were trimmed free from fat and fascia, first recorded in 1957. Since 1980s, the prevalence of and 5 g of muscle tissues were removed from each pig dia- Trichinella infection in swine slaughtered at abattoirs of phragm and combined to make a pool of 50 g of muscles. The Henan gradually increased, and the prevalence was 13.85 % pooled samples were digested by the magnetic stirring method (1 825/13 177) in 1983, 34.24 % (1 243/3 630) in 1986, and as previously described (Gamble et al. 2000;Lietal.2010). 1.38 % (2 379/172 516) in 1991, respectively (Wang et al. When a Trichinella positive sample was detected, an addition- 2014). The highest prevalence of Trichinella infection in do- al of about 40 g of muscles from each diaphragm comprising mestic pigs was 50.4 % (126/250) in Xinye county of the pooled positive sample was individually tested by the ar- Nanyang city, southwestern Henan (Chang et al. 1991). The tificial digestion to identify the pigs harboring Trichinella lar- first outbreak of human trichinellosis with 34 cases occurred vae. In brief, the muscle samples were minced, mixed with in Nanyang in 1984. Since then, the endemic areas of artificial digestion fluid [0.33 % pepsin (1:31,000, Sigma, trichinellosis extended towards to northern Henan and other USA)-1 % HCl-0.9 % NaCl] at 43 °C for 4 h. A 1:30 ratio 15 outbreaks of human trichinellosis occurred in western and of meat to digest solution (e.g., 1 g of muscle in 30 ml of digestion fluid) was used to facilitate rapid and complete di- gestion. The digests were allowed to settle, and larvae were suitably diluted and placed in gridded Petri dish. Larvae were then counted under an Olympus light microscope (Tokyo, Japan) with ×40 magnification, and results were expressed as muscle larvae per gram (lpg) of tissue.

Molecular identification of collected larvae

All the larvae collected after digestion were identified at the species level by multiplex PCR (Zarlenga et al. 1999). The reference Trichinella isolates used in this study were T. nativa (ISS10), T. britovi (ISS100), T. pseudospiralis (ISS13), and T. nelson (ISS29), which were obtained from the Trichinella Reference Center (IRC; Rome, Italy).

Results

A total of 823 diaphragm samples from the indoor-raised pigs Fig. 1 Agarose gel separation of multiplex-PCR products using DNA of in five historical endemic areas of Henan province were col- Trichinella isolate from naturally infected pig in Henan, central China. Lane M: molecular weight markers; Lane 1: Henan Trichinella isolate lected and examined by artificial digestion method. The over- Lane 2 T. nativa Trichinella from a naturally infected pig; : isolate (ISS10) control; all detection rate of larvae in meat samples was Lane 3: T. britovi (ISS100) control; Lane 4: T. pseudospiralis (ISS13); 0.61 % (5/823). All the five positive samples were collected Lane 5: T. nelsoni isolate (ISS29) control Parasitol Res (2016) 115:4707–4709 4709 central parts of Henan during 1984–2003 (Wang et al. 2006). Acknowledgments This work was supported by the National The largest one outbreak of trichinellosis, with >600 infec- Natural Science Foundation of China (No. 81572024) and the National Undergraduate Innovation Training Programs of China tions and >300 clinical cases, occurred in Henan between (No. 201610459075). December 1995 and February 1996 (Cui et al. 1997). The prevalence of Trichinella infection in pork sold at the market was 4.27 % in Henan in 1997 (Wang et al. 2014). The geo- References graphic distribution of human trichinellosis was correlated well with the high prevalence of swine trichinellosis (Wang Bruschi F (2012) Trichinellosis in developing countries: is it neglected? J et al. 1998). The abattoir surveys of swine trichinellosis Infect Dev Ctries 6:216–222 were performed by direct microscopic examination, and Chang J, Zhao QF, Li Y, Li GL, Chen JS, Liu D (1991) The survey of the results showed that the prevalence of Trichinella infection human trichinellosis in Nanyang city of Henan Province. Chin J of Parasitol and Parasit Dis S1:142–143 in swine slaughtered at abattoirs began to decrease since Cui J, Wang ZQ (2011) An epidemiological overview of swine 2000s, with 1.52 % (7 451/491 401) in 2002, 0.014 % (137/ trichinellosis in China. Vet J 190:323–328 977 500) in 2004, and 0.25 % (754/304 621) in 2005, respec- Cui J, Wang ZQ, Hu DS (2006b) The epidemiology of swine – tively (Wang et al. 2014). trichinellosis in China during 1999-2004. Helminthologia 43:21 26 Cui J, Wang ZQ, Kennedy MW (2006a) The re-emergence of The results of the present study showed that the prevalence trichinellosis in China? Trends Parasitol 22:54–55 of Trichinella infection in indoor-raised pigs in Henan was Cui J, Wang ZQ, Wu F, Jin XX (1997) Epidemiological and clinical 0.61 % (5/823), and all the five positive samples were collect- studies on an outbreak of trichinosis in central China. Ann Trop – ed from Nanyang, indicating that porcine trichinellosis still Med Parasitol 91:481 488 Cui J, Wang ZQ, Xu BL (2011) The epidemiology of human trichinellosis existed in one historical endemic area of Henan. However, in China during 2004-2009. Acta Trop 118:1–5 the level of swine T. spiralis infection in Henan was low, Fu BQ, Liu MY, Yao CY, Li WH, Li YG, Wang YH, Wu XP, Zhang DL, and all the five positive samples had larval loads <1 lpg, which Cai XP, Blaga R, Boireau P (2009) Species identification of Trichinella – was under the minimum level for defining infectious sources isolates from China. Vet Parasitol 159:214 217 Gamble HR, Bessonov AS, Cuperlovic K, Gajadhar AA, van Knapen F, for humans (Gamble et al. 2000). So, there are no outbreaks of Noeckler K, Schenone H, Zhu X (2000) International Commission human outbreaks in this province since 2003. on Trichinellosis: recommendations on methods for the control of Swine trichinellosis in Henan is transmitted mostly through Trichinella in domestic and wild animals intended for human con- – garbage. Pigs infected with Trichinella are predominately sumption. Vet Parasitol 93:393 408 Li F, Cui J, Wang ZQ, Jiang P (2010) Sensitivity and optimization of from small backyard farms where animals are raised under artificial digestion in the inspection of meat for Trichinella spiralis. poor hygienic conditions and from rural and mountainous Foodborne Pathog Dis 7:879–885 areas where they range freely at pasture (Cui et al. 2006b). Liu M, Boireau P (2002) Trichinellosis in China: epidemiology and con- – Recently, to interrupt the transmission of trichinellosis, control trol. Trends Parasitol 18:553 556 Murrell KD, Pozio E (2011) Worldwide occurrence and impact of human measures, including improvement of pig feeding and eradica- trichinellosis, 1986-2009. Emerg Infect Dis 17:2194–2202 tion of rats, were applied in Henan. The majority of pigs were Wang ZQ, Cui J (2001) Epidemiology of human trichinellosis in China raised in industrialized pig farms under good hygienic and during 1964-1999. Parasite 8:S63–S66 sanitary conditions. Even in rural and mountainous areas of Wang ZQ, Cui J, Shen LJ (2007) The epidemiology of animal trichinellosis in China. Vet J 173:391–398 Henan, the domestic pigs were raised in piggery facilities (Cui Wang ZQ, Cui J, Xu BL (2006) The epidemiology of human and Wang. 2011). The overall low level of T. spiralis infec- trichinellosis in China during 2000-2003. Acta Trop 97:247–251 tions in pigs in Henan is likely due to improvements in pig Wang ZQ, Li LZ, Jiang P, Liu LN, Cui J (2012) Molecular identification Trichinella production practices such as shifting production to indoor fa- and phylogenetic analysis of isolates from different prov- inces in mainland China. Parasitol Res 110:753–757 cilities, the adoption of commercial grain forages as food Wang ZQ, Xu BL, Cui J, Zhang HW, Jiang P (2014) Trichinellosis. In: sources, the shortening of swine feeding times, and the man- Ma YX, Shang LY,Xu BL (eds) Analysis and prevention of parasitic datory inspection of pork. However, in order to ensure pork diseases in Henan Province (1949-2014). Henan people’s publishing – food safety, the prevalence of Trichinella infection in swine in house, Zhengzhou, pp 221 239 Zarlenga DS, Chute MB, Martin A, Kapel CM (1999) A multiplex PCR Henan need to be further evaluated with a large scale of pork for unequivocal differentiation of all encapsulated and non- samples. encapsulated genotypes of Trichinella. Int J Parasitol 29:1859–1867