In Thailand, a neighboring country with Lao PDR, paramphistomiasis can reach prevalences of 80%, caused mainly by P. cervi [2,11]. However, there are no reports of paramphistomes occurring in Lao PDR. Then, aiming to fill this lack of information, molecular identification of F. cobboldi and P. epiclitum from bovines was performed. This is the first description of the occurrence of these important parasites of livestock in this southeast Asian country.

Parasite materials were collected from bovines bred in Ban Lahanam, a previously described area [12-14] located in Savannakhet Province, Lao PDR at Lahanam public market. Twenty specimens were collected from 4 individual rumens; the parasites were stored in 70% ethanol for later study. Morphological analysis of the specimens collected in this study was carried out at Department of Helminthology, Faculty of Tropical Medicine, Mahidol University (Thailand), the molecular studies were done in the School of Health Sciences, Faculty of Medicine, Niigata University (Japan), Department of Tropical Medicine and Parasitology, Dokkyo Medical University (Japan) and Animal Laboratory, School of Medicine at Universidade Federal do Tocantins (Brazil).

Genomic DNA was extracted from single adult worms in a total of 11 specimens (P1 to P11) using a Wizard^® Genomic DNA Purification Kit (Promega, Madison, Wisconsin, USA) and used as templates. Identification of each species collected was carried out based on nucleotide sequence of cox1 amplified by PCR. A set of PCR primers were used for amplification with the following sequences; 5´-TGGTTTTTTGTGCATCCTGAGGTTTA-3´ (COI forward) and 5´-AGAAAGAACGTAATGAAAATGAGCAAC-3´ (COI reverse) [15]. PCR was performed with GoTaq^® Master Mix (Promega). The PCR products were sequenced by ABI3730XL sequencer (Applied Biosystems, Foster City, California, USA) at Macrogen Inc. (Seoul, Korea) and at Dokkyo Medical University (Japan). The obtained sequences were aligned and compared with the same gene sequences from GenBank database using BioEdit version 7.1.3 [16]. The phylogenetic tree was reconstructed by using the neighbor-joining method evaluated by a bootstrap test based on 1,000 resamplings using MEGA version 6.0 [17].

This is the first study reporting the occurrence of F. cobboldi and P. epiclitum in Laos, with molecular confirmation by sequencing of mitochondrial cox1 gene. Although paramphistomes mixed infections occur in endemic areas [18], the concomitant parasitism of 2 these species is newly reported in the region and may frequently be occurring.

Morphological analysis of the specimens collected in this study revealed that they are Paramphistomoidea by comparison with keys for trematoda [19], with the following characteristics: Two types were observed, one with the total length of 7 mm and the other 5 mm length, both presented a conical shape, thick, and muscular; oral sucker terminal, ventral sucker large, located at posterior end of the body (Fig. 1). Unfortunately, accurate measurements were not possible due to the collecting conditions and alcohol fixation, and the worms were difficult in morphological identification. On other hand, DNA was successfully extracted, and cox1 of 11 specimens were amplified producing amplicons of 446 base pairs, submitted for DNA sequencing (GenBank databases accession nos. from LC113915 to LC113925). Pairwise alignment of cox1 sequences were performed and elucidated the morphological types found (Fig. 2). With these morphological and genetic characteristics, this study confirmed the presence of 2 species, F. cobboldi and P. epiclitum in Lahanam, Lao PDR. These findings differ from other studies in Thailand where predominantly P. cervi was identified [2,11]. In our study, cox1 sequencing revealed higher identity with P. epiclitum and F. cobboldi, both newly identified in the region in this study. The analyses conducted using the maximum composite likelihood model [20] presented a homology of 87% between F. cobboldi and P. epiclitum found in this study. However, more molecular studies on the taxonomy of paramphistomes are necessary, once the small amount of information in the database jeopardizes the accuracy of the molecular identification. A review in the classification of paramphistomes using molecular data may help to reduce orders and regrouping taxa as considered by Sanabria and Romero [21].

There is lack of information on the pathogenesis of the 2 species found in this study and also no studies using experimental infections. However, in studies mainly with P. cervi infections, it is evident that there is importance of rumen flukes on livestock in endemic areas [22-25]. The migration of immature paramphistomes through the intestinal tract can cause acute parasitic gastroenteritis with high morbidity and mortality, particularly in young animals [1,6,22-25]. The damage in the intestinal tract caused by parasites reduce nutrient availability to the host through both reductions in voluntary food intake and/or reductions in the efficiency of absorbed nutrients [26] causing a direct impact on the production of meat and milk. In our study, according to the meat sellers at the local markets, these parasites are frequently seen in the meat sold in Savannakhet city, and it may be causing important of economic losses, as previously described with other paramphistome infections [6,25]. Another important point on paramphistomiasis is that the disease is often not diagnosed. Usually, the diagnosis of paramphistomosis is based on the history and clinical signs of the disease with confirmation done by detection of paramphistome eggs in fecal examination. However, this method often results in misdiagnosis [1,25,27,28]. Then, sequencing cox1 can be useful to make an accurate diagnosis once it can be done with different sources of parasite DNA, including environmental DNA as eggs in fecal samples [14,25, 29,30].

In this study, the occurrence F. cobboldi and P. epiclitum was determined for the first time in Lao PDR and its mitochondrial gene cox1 was successfully sequenced, adding basic information on the paramphistomes of southeast Asia. Though, more studies are needed to determine the rumen fluke species present in Savannakhet and its hosts to clear the natural history of these parasites of ruminants in the region and measure the impact of paramphistomosis in the life of the local people.