Evaluation of two PCR-based techniques for molecular epidemiology in Finland, a high-endemic area with four sympatricTrichinellaspecies

Abstract

Summary:Trichinella larvae collected from wildlife, domestic and synanthropic animals in Finland were identified to species by two molecular techniques: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and the recently described multiplex PCR. The RAPD-PCR was very sensitive to the sub-optimal preservation muscle larvae and resulting in weak and smeared bands on the gels for such material. However, the same samples yielded easily recognizable bands in the multiplex PCR; this latter technique is then recommended for epidemiological studies, especially when the preservation of the samples is sub-optimal. For larvae in good condition the unequivocal bands obtained by multiplex was the easiest identifiable. this is the first record of a mixed infection between T. spiralis and T. nativa in a naturally infected host. Raccoon dogs were the only host species from which all of the four Trichinella species were detected. Trichinella spiralis was found in both domestic animals and wildlife, but none of the sylvatic Trichinella species were detected in domestic pig. Because of the exceptionally high regional prevalence in wildlife (Oivanen & Oksanen, 1994;Oksanen et al, 1998) and recurrent infections in domestic swine (Oivanen & Oksanen, 1994), southern Finland presents an ideal opportunity for studies on the molecular epidemiology of Trichinella.The aims of the present study were to evaluate two techniques for molecular epidemiology in an area with sympatric species of Trichinella. It is the first time this multiplex PCR technique is used for an epidemiological study. MATERIALS AND METHODSM uscle samples were collected in Southern Finland (1993Finland ( -1997 from nine host species (bears, wolves, foxes, lynx, raccoon dogs, wild boars, domestic pigs, rats and cats). After initial examination, 87 muscle samples positive for Trichinella were stored at -20° C. However, all samples from rats were used for other purposes prior to the present study and were therefore thawed twice.Muscle larvae were released by HC1 pepsin digestion and recovered larvae were stored in 70 % ethyl-alcohol and stored at -20° C. Prior to molecular analysis, larvae were rehydrated in a series of alcohol and single larvae were finally placed in 5 µl sterile water.The condition of the larvae, was determined from the appearance of the larvae. Coiled and partly uncoiled larvae were described as in moderate condition and

Use of mitochondrial RNA genes for the differentiation of fourTrichinellaspecies by multiplex PCR amplification

“…Previous PCR-based methods using mitochondrial genes are either more laborious, like PCR-RFLP (Nagano et al , 1999), or can only differentiate T. spiralis and T. britovi (Borsuk et al , 2003). Nevertheless, mixed species infection has been observed in wild boars infected by T. spiralis and T. britovi (Pozio et al , 1997) and T. spiralis and T. pseudospiralis (Nockler et al , 2006), and in foxes, cats and racoon dogs infected by T. spiralis and T. nativa , T. spiralis and T. britovi , or T. britovi and T. nativa (Kapel et al , 2001). When mitochondrial multiplex PCR was applied to samples containing a mixture of genomic DNA of T. spiralis , T. nativa , T. britovi or T. pseudospiralis species, the specific amplified profile could lead to their precise identification (fig.…”

Comparative analysis of Trichinella spiralis and Trichinella nativa proteins by two-dimensional gel electrophoresis

“…There are 11 genotypes of Trichinella , four of which are confirmed to exist in Finland (Kapel et al 2001;Oivanen et al 2002). While some of these species have distinct morphological or biological characteristics (e.g., capsule formation and resistance to freezing), differential diagnostics of these under 1-mm-long intramuscular worms is today based on molecular biological methodsspecies cannot be differentiated only by appearance.…”