Introduction

Nematodes are an important group of parasites in ruminants. Due to their high prevalence and pathogenicity, the Strongyloidea (which includes trichostrongylids and strongilids) is considered the most important parasite group. The life cycle of these nematodes consist of larval stages in the environment and adults in the definitive hosts, which are passively infected by pasture feeding (^{HONER; VIEIRA-BRESSAN, 1992}).

Clinical signs in animals with worm infections include hyporexia or anorexia and modification in the digestibility of food, due to damage caused to the digestive tract's lumen. These alterations are reflected by decreases in production and quality of milk and meat, in the host's work capacity and reproductive efficiency, and by the premature death of infected animals (^{HOLMES, 1985}).

The characteristics of tropical and sub-tropical climates can facilitate the development of parasitic agents (^{PERRY; RANDOLPH, 1999}). Lao PDR is located in a tropical area and has monsoon seasons; therefore, parasitic agents are considered to be a main cause of disease within production animals (thus reducing milk and meat production), as well as zoonosis in humans, as described previously (^{SATO et al., 2010}, ²⁰¹¹; ^{LATTES et al., 2011}).

Although intestinal parasite infections in production animals are a well-known problem, there are still very few studies on this matter in Thailand (^{RATANAPOB et al., 2012}) and in other countries in Southeast Asia, such as Lao PDR. The study area was located in Ban Lahanam, Savannakhet Province, a southern region of Lao PDR. Subsistence agriculture accounts for half of the country's GDP, and rice is the dominant agricultural product. Products from subsistence agriculture (such as goat and cattle) and also from forest exploitation (due to foreign demand for wild animals, and non-wood forest products for food and traditional medicines) are sold in city markets. This report describes the putative origin of human trichostrongylosis, a particularly important parasitic zoonosis in this region, and suggests haemonchosis and oesophagostomosis as possible zoonotic parasitic diseases in the studied area.

Materials and Methods

Study area and sampling

Faecal samples were collected from goats and cattle bred in the Ban Lahanam area (coordinates N16 16.120 E105 16.077), Savannakhet Province, Lao PDR, as detailed in the sampling map (Figure 1). Two field surveys were performed in August 2010, during the monsoon season. Samples were analysed at the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University (Thailand); and molecular studies were conducted at the School of Health Sciences, Faculty of Medicine, Niigata University (Japan) and at the Animal Laboratory for Medical Sciences at the School of Medicine, Universidade Federal do Tocantins (Brazil).

Figure 1. a) Map of Lao PDR and neighbouring countries. Star “???” corresponds to Vientiane (Capital of Lao PDR) and Hanoi (Capital of Vietnam). Savannakhet City (Lao PDR) and Mukdahan City (Thailand) are indicated by white circles“○”. b) Amplification of the hachured area of the “a” panel, with main routes (grey lines). Mekong River (thick black line) delineates Lao PDR/Thailand international border. Lahanam Village is indicated as a small white circle “○?”, near Banghiang River (blue line). c) Map of Lahanam Village showing study area. Black circles “•” indicate the 25 sampling spots, which correspond to all families owning goat/cattle in the study area. Streets are indicated by grey lines. Banghiang River is indicated by blue lines. 

The total number reported of goats and bovines in Ban Lahanam was 82 and 452, respectively (P. Tiengkham, personal communication, 2010). The sampling numbers of goats and bovines were estimated by the number of families surveyed in the area (32 households), that owned 23 goats and 29 bovines. They are raised extensively and share the same pasture area. Samples collected directly from the rectums, were taken randomly from the available animals (11 bovines and 14 goats) of 25 families that were breeding goats and/or cattle for meat production for their subsistence and selling in the local market, as shown in the sampling map (Figure 1c).

All study procedures were approved in advance by the Lao Medical Ethics Committee (172/NECHR).

Results and Discussion

The results showed that 93% of caprines and 36% of bovines were infected with trichostrongylids, with EPG averages of 1,728 and 86 for caprines and bovines, respectively. Trichuris spp. and Strongyloides spp. eggs were also observed in the faecal examinations.

When comparing the EPG data of cattle and goats (Figure 2), the overall distribution showed differences of infection intensity between the two species, with a median of 0 and 1,425 and a maximum value of 350 and 6,300 for cattle and goats, respectively. The data showed less variability for cattle (150) than goats (875). Even with different breeding (Caninde, Bhuj and Anglo-Nubian) values can be higher for goats, but not for cattle, as shown in other studies: ^{Costa et al. (2000)} with a mean EPG varying from 302 to 397 (lower than our average of 1,728 EPG), and other studies in goats conducted in Thailand and Bangladesh, with averages of 1,176 and 263 EPG, respectively (^{RATANAPOB et al., 2012}; ^{HASSAN et al., 2011}). Our results were higher than other studies with periparturient animals, such as that of ^{Sweeny et al. (2011)}, where a high worm egg count in Dorper lambs was associated with a lower body condition score. However, the animals did not present any clinical signs, perhaps due to the resilience to nematode infection, as reviewed by ^{Chiejina and Behnke (2011)} in Nigerian West African dwarf goats.

Figure 2. Boxplot comparing EPG data of cattle (bov) and goats (capr). The distribution of data shows the difference of infection intensity between the two species, with a median of 0 and 1,425 and a maximum value of 350 and 6,300 for cattle and goats, respectively. Data for cattle showed less variability (150) than goats (875). 

Coproculture results showed infection with Haemonchus spp. (69%), Trichostrongylus spp. (16%) and Oesophagostomum spp. (15%) in caprines, and Haemonchus spp. (37%), Cooperia spp. (8%), Trichostrongylus spp. (48%) and Oesophagostomum spp. (6%) in bovines. These results are similar to other studies, but prevalence of Oesophagostomum spp. (15%) in goats was much higher than that observed by ^{Ahid et al. (2008)} in Brazil at 7.3%. It was also higher than that found in Norway, where L3 of Oesophagostomum/Chabertia was only encountered at a very low frequency, with values < 1% (^{DOMKE et al., 2012}).

Infection intensity is greater in caprines, with a much higher EPG count average than bovines. These results strongly suggest that larvae from goat faeces contaminate vegetables and fomites, and act as a main route of infection due to the interaction and close proximity of animals living near human domiciles.

After performing necropsy on one goat, adult worms were recovered and identified under a light microscope, using lactophenol diaphanisation. DNA was then extracted and ITS2 was successfully amplified and sequenced (DDBJ/EMBL/GenBank accession nos. AB908958-AB908966). The sequencing confirmed a morphological diagnosis of Trichostrongylus, Oesophagostomum and Haemonchus (Figure 3). Previous analysis of ITS sequences comparing human cases of trichostrongylosis with worms collected from goats showed 100% similarity (Figure 3), indicating humans were infected with L3 of Trichostrongylus(^{SATO et al., 2011}). Since it was not possible to obtain worm DNA from cattle, we could not determine if cattle play a role in Trichostrongylus transmission to humans; however, due to the low level of nematode infection in bovines, as observed by EPG count (Figure 2), we may consider that low levels of environmental contamination by L3 result in a lower risk of human infection.

Figure 3. Phylogenetic tree using sequenced ITS2 of the recovered and morphologically diagnosed worms. The phylogenetic tree was reconstructed using the neighbour-joining method, and worms were photographed after phenol diaphanisation. The sequencing data (left side) confirmed the morphological diagnosis of Trichostrongylus, Oesophagostomum, and Haemonchus (right side). 

While Trichostrongylus infection in humans has already been reported in this area (^{SATO et al., 2010}, ²⁰¹¹), there are no reports of oesophagostomosis and haemonchosis among humans in Laos. There is a high possibility of infection in Lahanam Village, since the way of infection could be similar for these species, and there are studies showing Haemonchus (^{GHADIRIAN; ARFAA, 1973}) and Oesophagostomum (^{BLOTKAMP et al., 1993}) infection in humans. More studies are required involving DNA confirmation of Strongyloidea eggs found in human faeces, because the severity of the pathogenicity of Haemonchus and Oesophagostomum can be more important than that caused by Trichostrongylus.

The infection rate of trichostrongylids in caprines is high, and infective larvae may spread to all areas in close proximity to houses, and also through contact with fomites, or nearby where vegetables are cultivated and eaten raw. There is a high potential for active transmission not only of Trichostrongylus spp., as described previously, but also of other animal nematodes in humans, such as Haemonchus and Oesophagostomum, as previously described (^{GHADIRIAN; ARFAA, 1973}; ^{BLOTKAMP et al., 1993}). We recommend that these infections be considered an important issue for public health concern in Laos and other Southeast Asian countries.