Molecular identification of Trichinella species by multiplex PCR: new insight for Trichinella murrelli

In order to identify Trichinella at the species level, the commonly used test is a multiplex PCR, allowing the discrimination of nine out of the twelve taxa described so far. This test is based on five primer pairs amplifying fragments of the large subunit rDNA. Each taxon produces one or two bands of different sizes, resulting in a specific band pattern. By multiplex PCR, Trichinella murrelli shows two bands of 127 bp and 316 bp. However, a third band of 256 bp can occur. This band can lead to misidentification, since it is similar to the 253 bp band displayed by Trichinella britovi. BLAST analysis confirmed that the 256 bp band is from T. murrelli. The aim of this short note is to inform analysts that T. murrelli larvae may display either two- or three-band patterns.


Introduction
Trichinella spp. are the causative agents of trichinellosis, a foodborne zoonotic disease acquired through the consumption of raw or undercooked meat infected by larvae in the muscle cells. The main sources of human infection are domestic pigs and wild boars [6,15]. Meat inspection of susceptible livestock (backyard and freeranging pigs, horses) at slaughterhouses and game at game handling establishments is an important measure for preventing human infection [8]. On a routine basis, this inspection is internationally regulated with direct detec-tion of larvae achieved through artificial digestion of infected muscle samples [2,3,11,16,23]. The isolation of Trichinella larvae from muscles of infected animals allows the removal of infected carcasses from the food chain and enables the identification of larvae at species or genotype level in order to acquire valuable epidemiological information to control these zoonotic pathogens [8,21]. To date, nine species and three genotypes have been recognized within the Trichinella genus [12]. Eight of these taxa have been proven to be infectious to humans, while the remaining four are considered as potentially infective to humans [21]. Species/genotypes within these taxa are morphologically indistinguishable (sibling species), and their identification relies on the use of biochemical or molecular assays [14,20,24].
The North American species Trichinella murrelli [17] is known to circulate freely among wild carnivore mammals in the United States [9,18,22] and Canada [7], however this zoonotic pathogen has also been documented in domestic dogs and horses [4,10,19,21]. Although T. murrelli has not been recorded in European wildlife, this pathogen was the causative agent of a severe human outbreak, which occurred through the consumption of raw horse-meat imported from Connecticut (USA) to France in 1985 [1,5].
The most common molecular test for Trichinella taxon identification is a multiplex PCR analysis, which allows unequivocal identification of nine ofthe 12 recognized taxa on the basis of the generation of one-or two-band patterns [20]. This test is based on the use offive primer pairs amplifying the internal transcribed spacers ITS1 and ITS2 and the expansion segment V region (ESV) of the large subunit ribosomal DNA [24]. According to this method, T. murrelli shows a double-band pattern of 127 bp and 316 bp.
In 2016, as part of proficiency testing to identify the species/genotype of Trichinella larvae, the National Reference Laboratories (NRLs) for Parasites in European Union member states reported a three-band pattern for T. murrelli larvae instead of the expected two-band pattern [20,24]. The aim of this work was to investigate whether the extra band belongs to T. murrelli or is a faint band caused by slightly modified protocols (Table 1).

DNA isolation
The DNA was extracted using the DNA IQ System Kit (PROMEGA, DC6701) and the Tissue and Hair Extraction Kit (PROMEGA, DC6740) with a few modifications. Briefly, 20 mL of incubation buffer with DTT and proteinase K were added to larvae and incubated at 55°C for 30 min shaking at 1,400 vibrations per min. Then, 40 mL of lysis buffer with DTT and 4 mL of paramagnetic resin were added. The entire solution was incubated at 25°C for 5 min in a thermoblock without vibration, with a single vortexing step performed at mid time. Tubes were then placed in a magnetic separation stand for 1 min. The liquid phase was discarded. Then 100 mL of lysis buffer were added and resin particles were re-suspended before tubes were replaced on a paramagnetic stand and the liquid phase removed. The samples were washed four times using 100 mL washing buffer. The particles were then air-dried for 15 min and samples were eluted using 50°mL of elution buffer for 5 min at 65°C shaking at 1,400 vibrations per min.

Results and Discussion
Following multiplex PCR amplification, T. murrelli larvae displayed two-or three-band patterns independently of the isolate and the laboratory where the test was performed. A three-band pattern of 127 bp, 256 bp and 316 bp was observed by the French NRL (Figure 1), whereas a two-band pattern (127 bp and 316 bp) or a three-band pattern (127 bp, 256 bp and 316 bp) were found by the European Union Reference Laboratory for Parasites (EURLP) in Rome. Using the same multiplex PCR analysis protocol, T. britovi larvae displayed the expected band pattern of 127 bp and 253 bp (Figure 1).
Since the 256 bp band produced by T. murrelli was unexpected, a uniplex PCR was performed to identify which couple of primers allowed the amplification of the extra band. The 256 bp band amplified with primer pair II for ITS1 (Figure 2) was sequenced and identified by BLAST. The result revealed 99.6% identity with T. murrelli (GenBank accession number KC006421). Only one base was different and corresponded to the last base of the forward primer-annealing region (Figure 3). It follows that the complementarity of the forward primer is not 100% and this may explain the intermittent amplification of the 256 bp product. Slightly different PCR conditions may affect annealing, resulting in two-or three-band patterns.
The appearance of a third unexpected band using DNA of T. murrelli larvae by the multiplex PCR analysis described by Zarlenga et al., (1999) [24] may be the cause of misinterpretation, leading the analyst to suppose a T. murrelli/T. britovi hybrid or cross DNA contamination of the purified DNA sample under analysis. Incorrect identification of T. murrelli larvae occurred in 2016 during the proficiency testing organized by the EURLP for the NRLs to identify Trichinella larvae at the species level. Seven (33%) of the 21 participating laboratories failed to identify T. murrelli by multiplex PCR due to the appearance of the unexpected band of 256 bp (Final Report PT-Tm 1/2016; www.iss.it/dinary/crlp/cont/ Final_report_PT_Tm_2016.pdf). The appearance of the extra band of 256 bp in T. murrelli was previously documented [18], but since this band was generated intermittently, it was not considered diagnostic of T. murrelli and was consequently ignored.