| Trichinellosis is an important food-borne parasitosis and parasitic zoonosis all over the world. When raw or undercooked meat or its products which contain infective Trichinella larvae were ingested, trichinellosis should occur. In recent years, outbreaks of human trichinellosis have taken place in many countries, and trichinellosis was defined as a re-emerging disease for its epidemiological trends. From 2004 to 2009, at least 15 outbreaks of trichinellosis were reported in China. Pork was the predominant source of outbreaks of human trichinellosis in China,12 out of 15 outbreaks were caused by eating raw or undercooked pork. Trichinellosis can cause expensive economic loss, not only harms to public health, but also to farming, especially the industrialized hoggery.Eight species and 4 uncertain genotypes were recognised in the genus Trichinella till now. Because there were distinct differences among different species of Trichinella, for instance, morphology, geography distribution, the range of host, the resistance to freezing, the infecting ability to host and immunity response of host to different species, so the study on taxonomy of Trichinella is necessary. The aim of the study on Trichinella taxonomy is to offer valuable informations on the pathogen biology, the clinical treatment, the epidemiology, the prevention and control of trichinellosis in humans and animals. The range of Trichinella's hosts was very wide, and 8 species of Trichinella have been separated from wild animals. In China, only Trichinella spiralis (T1) and Trichinella nativa (T2) were reported till today, were there other species or genotypes of Trichinella exist in China still not sure.In order to find the new Trichinella isolates in China, epidemiological investigation was carried on to separate novel isolates from Qinghai and Henan province in this study. The male Kunming mice were inoculated with new Trichinella isolates for species keeping. Further more, these different isolates of Trichinella were identified by multiplex PCR method. To provide the evidence for the relationship among pathogenesis, distribution and hosts, the epidemiology and molecular biology research were carried into execution in this study.Materials and Methods1. Epidemiological investigation The prevalence rates of swine trichinellosis in Qinghai and Henan Province were observed by epidemiological investigation. The sampling points were slected by local Animal Disease Control Center (ADC) and Center for Disease Control and Prevention (CDC). Samples were taken from diaphragm muscle and 50g each.2. Samples inspection All the samples were numbered first, and quarantined by trichinelloscopy, then, all of them should be inspected again by artifical digestion method which recommended by International Commission on Trichinellosis (ICT).3. Artificial inoculation of experimental animals Two month-old clean Kunming mice (18g～20g) were obtained from the Experimental Animal Center of Medical College of Zhengzhou University. After a whole day fast, male Kunming mice were orally inoculated with new Trichinella isolate from the same sampling point for species keeping.4. Recovery of Trichinella muscle larvae The Kunming mice which infected with different isolates and international reference strains of Trichinella were killed 40 days later, and purified muscle larvae were collected by artifical digestion method. 5. Genomic DNA acquired and purity checked Genomic DNA was prepared according to phenyl-Chloroform method from muscle larvae. The purity of genomic DNA was checked by spectrophotometer (U-2001). OD value of A260 and A280 were detected by spectrophotometer first, and then the ratio of A260 to A280 was calculated for pure assessment. The purity of genomic DNA was fine when the ratio between 1.8-1.9.6. Identification by multiplex PCR The genomic DNA which acquired from new isolate and different Trichinella international reference strains were identified by the method multiplex PCR. When the gene was amplied,5 pairs of primers that designed by Zarlenga (1999) were used. The products of multiplex PCR were electrophoresed in 2% agarose gel and visualised under Gene Genius Imaging System.Results1. The investigation on swine trichinellosis in Qinghai province In this investigation,192 pieces of meat was collected from diaphragm muscle in Huzu and Delingha of Qinghai province. Samples were quarantined by trichinelloscopy and digestion method, and none trichinella was found. All the samples were negative, none of the Trichinella infective swine was found in parts of Qinghai province.2. The investigation on swine trichinellosis in Henan province In Henan province, sampling points were choosen from Anyang and Shangqiu city. Three groups of diaphragm muscle samples were collected from 278 swine. None Trichinella was found when quarantined by trichinelloscopy, but 18 positive samples were detected when the digestion method was used, the positive rate of all the samples was 6.47%(18/278). All the positive samples were collected from the swine that breeded in pig bed by local farmer, and the Trichinella infection rate of this kind of swine was 10.84%(18/166). The swine which from industrialized hoggery were free of Trichinella infection (0/112), and the differentiation was statistically significant (χ2=11.67, P<0.01) between local pig bed and industrialized hoggery of Trichinella infection rates. There was no difference of Trichinella infection rates in local pig bed between Anyang and Shangqiu (χ2=0.12,P>0.05).3. Infective intensity of Trichinella in swine When 18 positive samples were detected by artifical digestion method, about 51 larvae were found from 122.8g of pork, and the larvae per gram (lpg) is 0.42. For the 9 positive samples that from Anyang county, the lpg has a range from 0.11 to 1.41, and the average level of lpg is 0.34 (19/55.9). Among the 9 Trichinella infective samples which from Sui county, the minimum of lpg is 0.10, the maximum is 1.58, with 0.48 (32/66.9) as its average. Both maxima of lpg are higher than the lowest level, which threaten to the public health (1pg≥1). The difference of lpg between Anyang county and Sui county without statistically significant (Z=-0.05, P>0.05).4. Multiplex PCR Anyang isolate of Trichinella was identified by multiplex PCR method, and the result was observed by using electrophoresis. The Nanyang isolate and 5 Trichinella international reference strains were also analyzed as control at the same time. The special band (173bp) that belongs to Trichinella spiralis (T1, ISS3) was also showed by Nanyang and Anyang isolates, which was different from Trichinella nativa (T2,127bp), Trichinella britovi (T3,127bp and 253bp), Trichinella pesudospiralis (T4,310bp) and Trichinella nelsoni (T7,155bp and 404bp).Conclusions1. The swine infected with Trichinella are not found in part areas of Qinghai province. Though prevalence of swine trichinellosis is relatively high in rural part of Henan province, the intensity of infection is low.2. The Trichinella isolates recovered from pork in Anyang were identified as Trichinella spiralis (T1). |
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