International Commission on Trichinellosis: Recommendations for genotyping Trichinella muscle stage larvae.

Being able to identify the species or genotype of Trichinella is of paramount importance not only for epidemiological studies but to better ascertain the source of outbreaks that still occur worldwide. This has become more critical in recent years given the increase in imported meat products and the relationship that wild animals play in the domestic and sylvatic transmission cycles. In contrast to a time when the genus Trichinella was considered monospecific, research in recent years has revealed that the genus consists of 9 species and at least 3 additional genotypes which have yet to be named. Except for a non-encapsulated clade consisting of Trichinella pseudospiralis, Trichinella zimbabwensis, and Trichinella papuae, all members of this genus are morphologically indistinguishable. Thus, identification has been relegated to using PCR and in special cases, DNA sequencing or restriction enzyme digestion. Rather than using a collection of PCR primers specific for each genotype, a single multiplex PCR previously developed for differentiating the major encapsulated and non-encapsulated genotypes has been adopted by the International Commission on Trichinellosis. Since the assay was first developed, other species have been named. Thus, DNA sequencing has been used to validate closely related genotypes. The ICT recommends genotyping be performed as described herein during all outbreaks and whenever Trichinella has been found in consumable foods.

Introduction

The purpose of these recommendations is to present methodology for identifying the species or genotype of Trichinella larvae by a multiplex polymerase chain reaction (PCR), or in rare cases, by other PCR-derived methods and DNA sequencing. These techniques can be applied to larvae collected from human biopsies or from muscle tissues of animal origin. In most cases, the multiplex PCR assay (Zarlenga et al., 1999, Zarlenga et al., 2001) can identify pure as well as mixed infections using pooled larvae. Based upon the multiplex PCR for pooled larvae, descriptions of assays using individual worms are also provided (Pozio and La Rosa, 2010) in the event that few worms are obtained. Further, analysis of individual worms may be necessary where mixed infections can cause incorrect diagnoses such as in the delineation of T. nativa and Trichinella-T6 which are endemic to North America above the −6 °C isotherm. However, the methods described below do not address sampling issues since these will be unique for each purpose and each application. It is advised that the users determine the intent of this methodology prior to sampling and PCR analysis to advance the desired outcome. Data interpretation can be assisted by being familiar with the biogeography and geographical distribution of these genotypes (Karadjian et al., 2017; Pozio and Zarlenga, 2013).

Trichinella morphology

The morphology of adult worms (male, total length 0.62 mm to 1.58 mm, width 25 μm to 33 μm; female, total length 0.952 mm to 3.35 mm, width 26 μm to 43 μm) and of new born larvae (average 110 μm in length, 7 μm in width) does not have any diagnostic importance, since the only stage which can be easily isolated and identified is the muscle larva. Muscle larvae are the infective stage of the parasite, known as first-stage larvae (L1), and subsequent developmental molts occur only after their penetration in the gut mucosa of a new host. The following morphological characters can sexually distinguish male and female muscle larvae. Male larva: total length 0.641 mm to 1.07 mm; width 26 μm to 38 μm; intestinal bulb typically close to the convex surface; in some larvae close to the concave surface; intestine crossing the gonad from the convex to the concave surface; in some larvae, crossing the gonad from the concave to the convex surface and then re-crossing to the concave surface; length of rectum approximately 40 μm to 50 μm. Female larva: total length 0.71 mm to 1.09 mm, width 25 μm to 40 μm; intestinal bulb generally close to the concave surface; intestine on the concave surface; in some larvae, intestine crossing the gonad from the concave to the convex surface and then re-crossing to the concave surface; length of rectum 17 μm to 35 μm; presence of a thickened subcuticular layer in the region of vulva primordium, i.e. on the convex surface at approximately 2/3 of the length along the stichosome. The recovery of nematodes belonging to genera other than Trichinella during routine meat inspection suggests that the persons performing the analyses need to be informed of the possibility of false positives and, consequently, the larva morphology should be kept in mind before proceeding with the molecular identification.

Principle of the methods

The PCR is a molecular biology technique that enables the amplification of specific nucleic acid fragments, of which the initial and terminal nucleotide sequences are known (oligonucleotide pair). If a species (or genotype) has its own characteristic DNA portion, due to its composition and/or dimension, it is possible to choose an oligonucleotide pair enabling its amplification. The PCR amplification is characterized by a high sensitivity and specificity.

A modification of the “conventional PCR” is the multiplex-PCR, where two or more oligonucleotide pairs are used. In this case, it is possible to amplify with a single PCR analysis more than one sequence at the same time.

Currently, 9 sibling species, namely T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae, T. zimbabwensis, T. patagoniensis, and 3 genotypes, Trichinella-T6, Trichinella-T8 and Trichinella-T9, have been identified in the genus Trichinella. The comparative analysis of three nucleotide sequences belonging to the internal transcribed spacers ITS1, ITS2 and expansion segment ESV of the rDNA repeat, allows the unequivocal identification of most epidemiologically relevant taxa: T. spiralis, T. nativa, T. patagoniensis, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae, T. zimbabwensis and Trichinella-T6. Since the size difference between T. nativa and T. patagoniensis amplicons is of only two base pairs, they can be distinguished by PCR amplification and sequencing of the ESV; T. britovi and Trichinella-T9 can be distinguished by PCR-RFLP of the CO I mitochondrial gene, and; T. britovi and Trichinella-T8 can be distinguished by ancillary PCR amplification of the ITS2.

The sizes of the ITS1, ITS2 and ESV fragments produced by the PCR amplification are shown in Table 1.

Table 1

Dimension of the expected amplification products (in base pairs) for each taxon.

Locus	T. spiralis	T. nativa	T. patagoniensis	T. britovi	Trichinella T8	Trichinella T9	T. pseudospiralis	T. murrelli	Trichinella T6	T. nelsoni	T. papuae	T. zimbabwensis
ESV	173	129	127	129	129	135	292–3601	129	129	155	240	264
ITS1				253	253	253			210
ITS2								316		404

A multiple band pattern (with 1, 2 or 3 bands) of variable size can be detected in this range.

Using the multiplex-PCR technique with 5 oligonucleotide pairs, it is possible to identify larvae with only one amplification assay to the species or genotype level. Specific instructions are provided in Appendix A.

Conflict of interest

The authors declare no conflict of interest.

Table A1

Set B PCR primer sequences, their codes and amplified target loci.

Primer sequences	Code	Amplified sequence
5′-GTT.CCA.TGT.GAA.CAG.CAG.T-3′5′-CGA.AAA.CAT.ACG.ACA.ACT.GC-3′	cp-I·Fcp-I.R	ESV
5′-GCT.ACA.TCC.TTT.TGA.TCT.GTT-3′5′-AGA.CAC.AAT.ATC.AAC.CAC.AGT.ACA-3′	cp-II·Fcp-II.R	ITS1
5′-GCG.GAA.GGA.TCA.TTA.TCG.TGT.A-3′5′-TGG.ATT.ACA.AAG.AAA.ACC.ATC.ACT-3′	cp-III·Fcp-III.R	ITS1
5′-GTG.AGC.GTA.ATA.AAG.GTG.CAG-3′5′-TTC.ATC.ACA.CAT.CTT.CCA.CTA-3′	cp-IV·Fcp-IV.R	ITS2
5′-CAA.TTG.AAA.ACC.GCT.TAG.CGT.GTT.T-3′5′-TGA.TCT.GAG.GTC.GAC.ATT.TCC-3′	cp-V·Fcp-V.R	ITS2

Table A2

Single sample Reaction mix: components and volumes.

2× PCR MasterMix	15 μL
Multi-Q-grade H2O	4 μL
Set B	1 μL
Total	20 μL

Table A3

PCR amplification profile.

Pre-denaturation	4 min/95 °C
Amplification	10 s/95 °C30 s/55 °C30 s/72 °C
Number of cycles	35
Final extension	3 min/72 °C

Table A4

Alignment of the ESV of T. nativa and T. patagoniensis. Primers are marked in grey.