Funding: Singapore Ministry of Education Academic Research Fund (AcRF) Tier 1 grant
Diarrhoea is a common symptom of gastrointestinal infection which has caused approximately 1.5 million child-deaths annually, especially in developing nations. It can be caused by a wide range of pathogens including viruses, bacteria, protozoa and helminths. Of these, Cryptosporidium and Giardia are two of the most dominant and dangerous pathogen which infect not only humans, but also domestic animals and wildlife globally. While there may be several modes of transmission leading to cryptosporidiosis and giardiasis, infection typically occurs due to the ingestion of water contaminated with faeces from Cryptosporidium and Giardia-infected hosts.
A study by UNICEF in 2012 revealed that, in Thailand, at least one of two children below the age of five was suffering from diarrhoea in the two weeks preceding the survey. Researches have revealed high prevalence of Cryptosporidium and Giardia infection especially amongst Thai children as well as the immuno-compromised. However, little is known about the prevalence of these parasites in the drinking water resources in Thailand. To date, only 5 of such studies are available in Thailand. Of these, 3 were based in Bangkok and its vicinity in Central Thailand where the surface waters are highly polluted and are generally unsuitable for treatment for drinking purposes. The other 2 were conducted in Southern Thailand – of which, one of the study was to assess the levels of Cryptosporidium and Giardia contamination following the destruction of drinking water infrastructures due a tsunami event while the other investigated Cryptosporidium and Giardia contamination in random water resources with no consideration for any spatial or temporal factors to understand the source and distribution of these pathogens.
The aim of this project was to investigate the prevalence of Cryptosporidium and Giardia in an area of Thailand where the water resources represent not only crucial drinking water sources locally to the region but also to Bangkok and many parts of Central Thailand. In addition, the seasonal effects (dry vs wet season) on the level of Cryptosporidium and Giardia contamination in these water resources were also investigated. Additionally, we also studied the prevalence of Cryptosporidium and Giardia in cattle, a potential and important host to the zoonotic variety of both protozoa.
Unlike previous studies, we have considered both the spatial and temporal aspects when designing the sampling plan. A catchment-scale study (a pre-requisite to water quality management in the Clean Water Act of the United States and the Water Framework Directive of the European Union) was conducted within the Kuang River basin. The Kuang River is one of the main tributaries to the Ping River which, in turn, is one of the most significant tributaries to the Chao Phraya, the most important river in Thailand. Samples were collected systematically along the main reach of the Kuang River and its tributaries. With the catchment-scale approach, both point- and non-point sources of these microbial contaminants can be identified. Similarly, specific hazardous or at-risk areas within a drainage basin can also be flagged such that necessary precautions can be taken to remedy the situation and protect the water supply.
The hydroclimatic factors play vital roles in determining the levels of contamination in water bodies. Rain events may mobilise and transport contaminants from sources in the terrestrial environment to surface waters, thus increasing the contamination levels. Conversely, the increase of volume in surface water bodies during wet spells may also cause dilution to the contaminants and thereby decrease the concentration. Northern Thailand has very distinct dry-wet periods and through our survey, we were able to observe the seasonal trends of the prevalence and intensity of Cryptosporidium and Giardia contamination at the study site. This information is especially vital to water resource managers, water treatment engineers, policy makers etc. such that necessary plans and actions can be made to ensure safe drinking water can be provided to the local consumers.
Cattle are known to be important hosts to the zoonotic species of both protozoa, namely Cryptosporidium parvum as well as Giardia duodenalis (syn. lamblia, intestinalis). With considerable cattle farming activities (meat and dairy production) in the region, it is also important to know the prevalence of both protozoa in the meat and dairy cattle found within the study area such that a link between the hosts and water bodies can be made.
Water samples. The detection and enumeration of Cryptosporidium and Giardia in water samples are processed according Method 1632.1 by the United States Environmental Protection Agency. Briefly, the steps included – (i) filtration; (ii) elution (wash); (iii) concentration; (iv) purification (immunomagnetic separation); (v) staining and (vi) immunofluorescence assay microscopy – in this particular order. Steps (i) to (vi) were carried out JC under the supervision Professor Yvonne Lim (YL) from the Department of Parasitology, University of Malaya (UM).
Faecal samples. The survey of research sites and the design of sampling plans were carried out by AZ, JC and Honours year undergraduate student, Nabila Mukhaidin (NM), also from the Department of Geography, National University of Singapore. The collection of faecal samples from cattle was carried out AZ, JC and NM.
Microscopy work was carried out by NM under the supervision of YL. Gene sequence-based work including DNA extraction, PCR, gel electrophoresis etc. were carried out by NM under the supervision of YL as well as Associate Professor Gavin Smith (GS) and Dr. Ian Mendenhall (IM), both from the Duke-NUS Graduate Medical School, Singapore.
Spatial prevalence. Samples were collected from 52 sampling sites on Kuang River and its tributaries, namely, Lai River, Pong River and San River. Additionally, samples were also collected from 8 sampling sites from the Mae Taeng-San Sai canal. The research site spans across the districts of San Sai, San Kamphaeng, Mae On and Doi Saket of the Chiang Mai province as well as the districts of Ban Thi, Pa Sang and the capital district (mueang amphoe) of the Lamphun province.
All 4 rivers at the study area were found to be contaminated with varying levels of Cryptosporidium and/orGiardia. More than half (52%; n = 27/52) of the river sampling sites were tested positive for Cryptosporidium or Giardia. Half of these samples (n = 26/52) contained Giardia while Cryptosporidium were detected in 25% (n = 13/52) of the river water samples (Figure 1a). Cryptosporidium-Giardia co-contamination occurred in nearly one quarter (23%; n = 12/52) of the monitored river sampling sites.
Neither Cryptosporidium nor Giardia was detected in the water samples collected from any of the sampling sites of the Mae Taeng-San Sai canal for either the dry or wet season.
Seasonal variation. During the dry season, Cryptosporidium or Giardia were detected in 21% (n = 11/52) of the river/stream sampling sites while the samples containing either protozoa nearly doubled (40%; n = 21/52) during the wet season. Giardia cysts were detected more frequently compared to Cryptosporidium oocysts for both dry and wet seasons. For the dry season samples, 14% contained Cryptosporidium (0.25 – 0.65 oocysts/10 L) while Giardia were detected in 18% of the samples (0.25 – 2.94 cysts/10 L). Meanwhile, for the wet season samples, 15% were tested positive for Cryptosporidium contamination (0.37 – 4.00 oocysts/10 L) and at least 38% contained Giardia (0.28 – 13.89 cysts/10 L). Figure 1b shows the summary of the season variation of Cryptosporidium and Giardia at the study site.
A summary of the concentration of (oo)cysts in all the sampling sites for both the dry and wet weather are presented in Table 1 while Figure 2 shows the locations of the sampling sites and the availability of Cryptosporidium and Giardia in the research area.
A total of 126 faecal samples were collected from Brahman beef cattle (n = 64) and Holstein-Friesian dairy cattle (n = 62). All samples were collected from the province of Chiang Mai. Dairy
cattle samples were collected directly from 4 farms in the San Sai district while samples from beef cattle were collected from free-ranging herds in the grazing fields along the Mae Taeng-San Sai
Dairy cattle from the study area had a marginally higher overall parasitic infection with 97% faecal samples tested positive for at least one parasite compared to the 94% detected in the faecal
samples from the beef cattle. Entamoeba (53%) was the most prevalent of the gastrointestinal parasites detected in beef cattle followed by the Eimeria (42%), Paramphistomum
(33%), strongyle (25%), Buxtonella sulcata (23%), Giardia (13%), Fasciola (8%), Cryptosporidium (3%) and Dicrocoelium (2%). Some of these
gastrointestinal parasites found in beef cattle were also detected in dairy cattle; of which, the most common was, again, Entamoeba (98%), followed by Eimeria (18%),
Paramphistomum (13%), Buxtonella sulcata (5%) and strongyle (3%). Dicrocoelium, Fasciola, Giardia and Cryptosporidium were not detected in any dairy
cattle samples. Co-infections were observed in 56% (n = 36) of the meat cattle samples and 38% (n = 24) in dairy cattle. Beef cattle showed noticeably higher infection for all parasites with the
exception of Entamoeba where infection in dairy cattle were almost double that in beef cattle. The results of the prevalence of these parasites are presented in Figure 3.