Identification of Three Toxocara species, T. canis in Dogs, and T. cati and T. malaysiensis in cats, in Vietnam by PCR-RFLP Analysis and DNA Sequencing

Toxocara canis and T. cati are common roundworms parasitizing dogs and cats, respectively. However, a recent study detected T. malaysiensis , but not T. cati , in cats from some parts of Ha Noi and Nam Dinh Provinces raising the question of whether T. cati is present in cats in Vietnam. This study was conducted to determine the composition of Toxocara species in dogs and cats in Vietnam. One hundred and twenty-seven Toxocara adult worms were collected from dogs and cats and were analyzed by PCR-RFLP assay and DNA sequencing of a partial section of the cox1 gene. As a result, all samples amplified by PCR showed bands about 430 bp in length. PCR products digested by MseI from isolates from cats and dogs showed four and two restriction patterns, respectively. The six different patterns were chosen to sequence. Based on the restriction patterns and sequencing results, two Toxocara species were identified in the cats, namely T. cati (110/111 isolates) and T. malaysiensis (1/111 isolates) collected from Ha Noi and Hai Duong, respectively; and T. canis was identified in the dogs (16/16 isolates). In addition, the present study indicated intra-specific sequence variations of Toxocara spp. in dogs and cats. In conclusion, the study confirmed the presence of both T. cati and T. malaysiensis in cats, and T. canis in dogs in Vietnam, and suggests further large-scale investigations to fully understand the distribution and genetic variations of Toxocara spp. in cats and dogs in Vietnam.


Introduction
Toxocara canis and Toxocara cati are common roundworms parasitizing canids and felids, respectively.Both of them are also etiological agents causing human toxocariasis, a zoonotic parasitic disease reported worldwide (Magnaval et al., 2001).In 2001, a new Toxocara species named T. malaysiensis was described in cats from Malaysia, and since then, it has been detected in China and Vietnam (Gibbons et al., 2001, Li et al., 2006;Le et al., 2016).While T. canis is the unique Toxocara species found only in dogs (Nguyen Thi Quyen et al, 2016;Le et al., 2016), Toxocara species composition in cats has been questioned.In Vietnam, Toxocara spp. is one of the most common roundworms in dogs and cats (Nguyen Thi Hoang Yen et al., 2020;Nguyen Thi Hoang Yen et al., 2022).The prevalence of Toxocara spp. in dogs (34.6-47.8%)has generally been found to be greater than in cats (21.0-37.7%)(Anh et al., 2016;Nguyen et al., 2020;Nguyen et al., 2022).T. cati has been considered the predominant species in cats in Vietnam (Le et al., 2016).However, a recent study that detected T. malaysiensis, but not T. cati, in domestic cats from Ha Noi and Nam Dinh Provinces (Le et al., 2016) raised the question of whether T. cati is present in cats from Vietnam.
Traditionally, Toxocara species are identified mainly based on their morphological characteristics and predilection sites in a particular host species (Kim et al., 2020).However, misidentification may occur because of morphological similarities between species, such as the shape of the esophagus with a ventriculus, dentigerous ridges on the lips, a Vshaped cervical alae, spicules, and position of the vulva opening (Gibbons et al., 2001); meanwhile, molecular techniques using ribosomal and mitochondrial DNA markers have helped to obtain more accurate identifications (Jacobs et al., 1997;Zhu et al., 2001;Gasser et al., 2006;Pawar et al., 2012;Mikaeili et al., 2017;Wang et al., 2018;Fava et al., 2020).PCRlinked restriction fragment length polymorphism (PCR-RFLP) developed by Fava is a time and cost-saving method for distinguishing the three Toxocara species, namely T. canis, T. cati and T. malaysiensis, because it only requires a single restriction enzyme (MseI) (Fava et al., 2020).Thus, in this study, PCR-RFLP and DNA sequencing were performed to differentiate and confirm Toxocara species collected from dogs and cats in Northern Vietnam.

Collection of roundworms and sample processing
One hundred and twenty-seven adult worms of Toxocara spp.were collected from naturally infected dogs and cats (111 from 36 cats and 16 from 6 dogs) at veterinary clinics by anthelminthic expulsion or local abattoirs by necropsy around Ha Noi (Nam Tu Liem and Gia Lam districts), and one Toxocara adult worm was collected from a domestic cat in Tien Dong commune, Tu Ky district, Hai Duong province.They were preliminarily identified based on macro-morphology, as Toxocara spp.have a ventriculus intercalated between the esophagus and the intestine, and males have a finger-like tail, which is distinguished by tapering to a point in Toxascaris leonina males (Bowman, 2014).The roundworms were then thoroughly washed several times in saline solution and kept in 70% ethanol for molecular analyses.

DNA extraction and polymerase chain reaction (PCR)
Total genomic DNA was extracted from adult worms of Toxocara spp.by the alkaline lysis method with a few slight changes.Firstly, a piece (about 50mg) of the worm was cut and transferred into a 1.5-mL Eppendorf tube, followed by the addition of 1,800µL of 50mM NaOH.The tubes were incubated at 95°C overnight on a block heater.After that, 200 µl of 1 M Tris-HCl (pH 8.0) was added into each tube.Next, the mixture was vortexed thoroughly and centrifuged at 14,000 x g for 10 minutes.Finally, the supernatant was transferred into a new tube and then stored at -20°C until analysis (Nguyen et al., 2016).
All PCR reactions were carried out in a total volume of 50μL using 2μL of template DNA, 25 μl Mastermix 2X_100_tracking dye (Phusa Biochem LTD.Company, Can Tho, Vietnam), 2 μl of each primer (10 pmol), and 19 μl distilled water.The amplification conditions followed those described in a previous study (Fava et al., 2020).PCR products were electrophoresed on a 1.0% agarose gel in TAE at 100V for 30 minutes.Gels were stained with GelRed® (Biotium, Fremont, CA) and the bands were visualized under UV light.

DNA sequencing analysis
PCR amplicons were purified and six isolates were chosen for sequencing in both the forward and reverse strands using the same primers employed in the PCR by the Suran Medical and Scientific Solutions Join Stock Company (Hanoi, Vietnam).DNA sequences were aligned using the Geneious Prime Biomatters Company and compared with sequences from the GenBank database via the BLAST search tool.

Results
A total of 127 samples were analyzed by PCR assay, and all of them showed a band of around 430bp (Figure 1 (2020), the former pattern was expected to be T. canis based on the specific band of 210bp and the canid host; and the latter pattern seemed to be T. malaysiensis (Fava et al., 2020).
Analysis of the DNA sequences revealed some variations in the sequences of the restriction sites for MseI in both the cat and dog isolates (Figure 4).MseI recognizes sites with the sequence 5'-T * TAA-3'.The sequence of the cox1 gene of T. malaysiensis contained three cutting sites for MseI (black rectangles, Figure 4), thus four fragments of 44, 51, 121, and 210bp were collected after digestion.For T. cati, the isolate named Tspcat22 contained four cutting sites, and produced five fragments of 22, 44, 63, 109, and 188bp.Two isolates, Tspcat52 and Tspcat56, had three cutting sites but at different positions, giving two different restriction patterns of four fragments 22, 44, 172, and 188bp; and 22, 95, 121, and 188bp, respectively (green rectangles, Figure 4).The fragments of 172 and 188bp were very close because they only  differed in size by 16bp, while the 22-bp fragment was not clearly observed on the gel electrophoresis.
In the case of T. canis, the partial cox1 gene of the sequence of Tspdog1 contained two cutting sites for MseI, producing three fragments, 95, 121, and 210bp after digestion.The isolate named Tspdog4 had three cutting sites, producing four fragments of 44, 51, 121, and 210bp (red rectangles, Figure 4).Of note, we recognized that the restriction fragment pattern of Tspcat1 (T.malaysiensis) was similar to that of Tspdogs4 (T.canis).Unfortunately, when comparing the PCR-RFLP results and sequencing, we recognized that the 22-bp band was unclearly observed in the electrophoresis results.Positions of the restriction enzyme sites in the amplified cox1 region for the different species of Toxocara and the sizes of the fragments in each isolate are represented in Table 1.In comparison to sequences deposited in GenBank, only one isolate from cats was identified as T. malaysiensis with 99.53% identity to the reference sequence (GenBank

Discussions
As mentioned in the introduction, there has not been enough molecular evidence for the presence of T. cati in cats in Vietnam.T. malaysiensis was discovered in Malaysia (Gibbons et al., 2001) and China (Li et al., 2006), and more recently in Vietnam (Le et al., 2016).In a previous study, T. malaysiensis was identified as a unique species in the absence of T. cati in Vietnamese domestic cats (Le et al., 2016).However, the research scale in the previous study was limited to two communes (Thanh Oai and Thuong Tin) in Hanoi and a village (Nghia Hung) in Nam Dinh Province.Similarly, samples used in this study were also collected on a small scale, mainly around Ha Noi.In contrast to a previous report (Le et al., 2016), T. cati was found to be the predominant species of the Toxocara population in cats (110/111 isolates); and the sample collected in Hai Duong Province was the only T. malaysiensis isolate.This difference in Toxocara spp. in cats may be due to the small sample size from limited areas or may depend on the geographical locations.For the Toxocara population in dogs, there was only one species identified in Vietnam as published previously (Le et al., 2016;Nguyen Thi Quyen et al., 2016).
In addition, we recognized that there were intra-specific sequence variations in the cox1 region among the isolates (in both cats and dogs).This was the reason why the Tspdog4 sample was initially recognized to be T. malaysiensis based on the PCR results.We first expected that there may be cross-infection, hybridization, or back-crossing between Toxocara species as the result of the detection of two T. canis worms in cats (Fava et al., 2020).However, the sequencing results demonstrated there was no crossinfection, and since we only included the cox1 gene, a mitochondrial marker, we were not able to identify hybrids.Moreover, there was no unification between PCR-RFLP in this study when compared with the research of Fava et al. (2020), due to the replacement of nucleotides in restriction sites for MseI (Figure 4), resulting in different restriction patterns.Intra-specific sequence variations were reported in T. cati around 0-3.6% for cox1 (He et al., 2017), not exceeding 2% and 4% for the ITS gene in T. cati and T. canis, respectively (Fogt-Wyrwas et al., 2013).This also explains why there were three and two different restriction patterns in T. cati and T. canis in this study, respectively.Hence, the results in this study show that it is necessary to use both the PCR-RFLP assay and DNA sequencing techniques to differentiate Toxocara species in dogs and cats, as PCR-RFLP helps recognize the similar restriction patterns, which can minimize the number of samples for sequencing.

Conclusions
This study confirmed the presence of two Toxocara species (T.cati and T. malaysiensis) in cats, and only one Toxocara species (T.canis) in dogs in Hanoi and Hai Duong provinces, Vietnam.It is necessary to extend the research scale in order to fully evaluate the distribution as well as sequence variations of the Toxocara spp.population in cats in this country.
).After digestion by the MseI enzyme, six restriction fragment patterns were observed from the PCR products, four patterns in the Toxocara worms from cats, and two other patterns in the Toxocara worms from dogs.The first pattern had three clear bands about 210, 120, and 40bp (Figure 2A, Tspcat1); the second pattern had three clear bands with two close bands about 190bp and one band about 44 bp (Figure 2A, Tspcat2-Tspcat6, Tspcat13; Figure 2B, Tspcat52); the third one had four clear bands about 190, 110, 60, and 40 bp (Figure 2B, Tspcat22, Tspcat47); and the last one had three clear bands about 190, 120, and 100bp (Figure 2B, Tspcat56).According to the restriction patterns reported by Fava et al. (2020), the first pattern in this study was expected to be T. malaysiensis based on the specific band of 210 bp and the felid host; the following three patterns were expected to be T. cati characterized by 190 bp bands.In the case of the dog Toxocara worms, two restriction patterns of fragments were observed, one had three bands of 210, 120, and 100bp (Figure 3, Tspdog1, Tspdog3); while the other consisted of three bands of about 210, 120, and 50bp (Figure 3, Tspdog4).In comparison to the restriction patterns reported by Fava et al.

Figure 3 .
Figure 3. PCR-RFLP band patterns of the cox1 region using the endonuclease MseI.Lane M: Marker; Lanes Tspdog1, 4 and 3 show the digested products of Toxocara worms from dogs.Black arrow shows the 500bp position.

Figure 4 .
Figure 4. Alignment of the cox1 sequences (426 bp) representing the distinct genetic variants among the Toxocara isolates from cats and dogs.Rectangles are the locations of the MseI restriction sites: black represents T. malaysiensis, green represents T. cati, and red represents T. canis.

Table 1 .
Positions of the restriction enzyme sites in amplified in the cox1 region for different species of Toxocara and the sizes of fragments in each isolate