Bachelor of Veterinary Medicine

Bachelor of Veterinary Medicine Research Project 2 Corprological prevalence and intensity of gastrointestinal parasitism in the working Title ponies of the Kampong Chan and Kandal provinces, Cambodia.

3865 Word count

Abstract

Working ponies of Cambodia provide the backbone of transportation for both people and goods, as well as having a key role in the agriculture and tourism industries. The aim of this study was to determine prevalence of and risk factors for gastrointestinal parasitism in the working ponies of Cambodia. Information on age, sex, use, body condition score, husbandry and anthelmintic practices were recorded to assess if there was a significant association with parasite burden. Level of parasitism was measured using the MacMaster faecal egg counting protocol via analysis of fresh faeces. Prevalence of Strongyle infections was 77.6%, 13.45% of these were categorised as ‘high’ burden (>1000epg). Parascaris equorum was present in 6.7% of the study population, Oxyuris equi and Anoplocephala perfoliata were both identified in 1.8% of ponies. No associations between husbandry factors and parasite burden were identified. Geldings showed a lower Strongyle burden than mares or stallions (p<0.001) and stallions showed a lower Strongyle burden than mares (p=0.002); no other animal demographic factors were statistically significant. Timing of previous anthelmintic treatment was significantly associated with parasite burden, as was type of treatment used. Ponies treated in the previous month had significantly lower FEC than those treated 1 month to 1 year previously (p<0.001) and those never wormed (p<0.001). Ponies treated with natural products had a lower burden than those treated with ivermectin (p<0.001) or nothing at all (p=0.004). Further research into seasonal fluctuations of parasite burden and efficacy of regularly used anthlemintics including natural anthelmintic techniques, which many owners reported using in Cambodia, is crucial. The results from this study can help with the implementation of a more targeted and sustainable approach to gastrointestinal parasite control for the working equids in this region.

Acknowledgements

The author is extremely grateful to World Horse Welfare for funding this research, many thanks go to Vicki Campling and Alana Chapman at World Horse Welfare for putting my mind at ease and ensuring the project ran as smoothly as possible. The author would like to extend gratitude to Clare Muir for her help with the project proposal and to Kristien Verheyen for her constant advice and guidance throughout the course of the study. A special thank you must go to everybody at the CPWO for their untiring help with sample collection, processing, translation and making this research feasible. Finally, the author would like to extend gratitude to the pony owners of Cambodia for their participation in this study. Abbreviations BCS Body condition score CI Confidence interval CWPO Cambodia Pony Welfare Organisation epg Eggs per gram FEC Faecal egg count FECRT Faecal Egg Count Reduction Test FAO Food and Agriculture Organisation of the United Nations GI Gastrointestinal RVC The Royal Veterinary College SD Standard deviation 1. Introduction The Cambodia Pony Welfare Organisation have been operating out of the capital city, Phnom Penh, since 2007 providing routine veterinary care to many communities in the surrounding area on a rotational basis. CPWO have a ‘prevention is better than cure’ mantra, working alongside community ambassadors expanding knowledge and skills within the local communities by training farriers and saddlers, and running workshops on issues such as colic, parasitism, lameness and tack fitting 1 . Working equids worldwide are pivotal to the livelihoods of millions of people, particularly those in developing and lower income countries 2, 3..They are extremely versatile, filling a wide range of roles including transportation of people, water, goods, and construction materials as well as use in tourism and agricultural industries 4. Research has shown that such equids facilitate opportunities for vulnerable groups including women and landless communities5, contributing an estimated 80% of power input on farms in developing countries 6. Regardless of their crucial role, working equids are often overlooked by government agricultural policies, disease surveillance and preventive medicine programs. This situation exposes working equids to higher risk of disease, poor welfare and misguided husbandry practices 7. Harmful or ineffective therapeutic techniques are often employed by owners due to the lack of education, income and limited availability of veterinary assistance. Coupled with harsh climatic conditions, this compromises the health and welfare of working equids worldwide 8. Subsequently, these animals are more likely to suffer from infectious diseases, parasitism, wounds and dental disease than their counterparts in developed countries8,9,10 . Many studies across the developing world have identified the high prevalence of gastrointestinal parasites and polyparasite infestations in working equids11,12, 13 . It is also widely accepted that high worm burden predisposes to colic, anaemia, diaorrhea, reduced productivity and weight loss. Consequently, the importance of controlling gastrointestinal parasitism in such communities is evident, yet there is little structure, organisation or implementation of parasite control schemes in developing countries12.

Cambodia is a rapidly developing country with population of 15.33 million and with a Gross National Income (GNI) of $1,020 per capita per annum, with 17.7% of the population living in relative poverty14. It is estimated that there are 10,000 ponies in Cambodia 15 which are arguably indispensable to the local community. This is especially true as pony owners also supplement income by providing delivery services within their communities for those without their own means of transportation.

The majority of ponies in Cambodia are of the traditional Khmer stock and are extremely versatile, having been used for riding and the transportation of goods for generations. Such ponies are usually kept tethered or loose boxed underneath the family home alongside other livestock. Currently, it is unknown which gastrointestinal parasites colonise the working equids of Cambodia and the extent to which this burden may impact on health and welfare. However, owners perceive anthelmintic use to be of great benefit to their animals, often presenting their equids to the CPWO for the sole purpose of requesting anthelmintic treatment. Anthelmintic protocols in the country are arbitrary and poorly regulated; treatment is given to all ponies presented to the CPWO on their routine visits to communities with ivermectin being the only anthelmintic available in Cambodia. Worming is not based on faecal egg counts and there are no records kept of worming history, which is complicated further by the high rate of pony trading between owners. The objectives of this study were to ascertain the prevalence of gastrointestinal helminthiasis in the working ponies of Cambodia, and to assess risk factors for gastrointestinal parasite burden by considering a range of husbandry, demographic and owner awareness factors. It aimed to consider the efficacy of anthelmintic administration on parasite burden and effect of natural anthelmintics, which have been employed by the people of Cambodia for centuries, and may be a crucial and much overlooked means to gastrointestinal parasite control in rural areas where other medication is unattainable.

2. Hypotheses Hypotheses were generated after reference to similar studies and acknowledgement of accepted protocols employed in developed countries for the reduction of equid gastrointestinal parasitism 16, 17. The following hypotheses were tested: 1. Statistically significant association exists between main work and gastrointestinal parasitism. 2. Statistically significant association exists between gastrointestinal parasitism and husbandry; a) Tethered/Stabled, b) mucking out, c) kept with other equids or alone, d) diet. 3. Statistically significant association exists between gastrointestinal parasitism and a) age, b) sex c) BCS. 4. Statistically significant association exists between owner awareness of gastrointestinal parasitism, and level of worm burden. 5. There is a statistically significant association between frequency of anthelmintic treatment and gastrointestinal parasitism. 6. There is a statistically significant association between type of anthelmintic used (ivermectin, natural or never treated) and gastrointestinal parasitism. 3. Materials and methods

3.1 Study area and period Data collection took place in the area surrounding Phnom Penh; communities in the Kampong Cham and Kandal provinces and one equitation centre in Phnom Penh were accessed. Ponies brought to the CPWO on community visits and those at riding centres visited were included in the sample population. The study period commenced on the 22nd June 2015 and was completed on the 11th July 2015. Sampling consisted of completion of a questionnaire and collection of a faecal sample.

3.2 Animals and sample collection With verbal consent from owners, animals encountered on routine visits were deemed eligible for inclusion into this study. Aging by dentition was carried out following a standardised technique 18, 19 and Body Condition Score (BCS) was determined using a 1-5 scale 20. Faecal samples were collected rectally, however if animals were deemed too dangerous to handle samples were obtained from the ground if possible. Samples of at least 3 faecal balls were obtained to overcome uneven egg distribution 21 and samples were collected in labelled zip-lock bags which were then stored in a cool box containing ice packs to ensure the contents remained below 4oC and were analysed within 24 hours, 17, 22, 23 .

3.3 Questionnaire Owners were asked a series of questions, using a standardised questoinnaire (see appendix) by a member of the CPWO, with verbal consent obtained in advance. Questions considered the main use of the pony, husbandry factors, signalment, owner awareness of parasitism, along with frequency and type of anthelmintic treatment used.

3.4 Sample analysis The McMaster FEC technique was used to analyse all samples collected 24. The faecal sample was first mixed to provide representative sample, 4g of faeces were weighed out and a saturated salt solution was added so total the volume was equal to 60mls. The mixture was shaken for 5 minutes to homogenise faecal material and ensure suspension of parasite eggs, the solution was then strained to remove organic matter. A fraction of this filtrate was then pipetted into a McMasters counting chamber slide, before leaving to stand for another 5 minutes enabling parasite eggs to rise in the saturated solution. Samples were viewed on the 10x objective magnification. All eggs observed in both of the chambers were counted and identified with the aid of reference material 25, 26, 27. Total FEC was calculated with each egg visualised representing 50epg. Differentiation between Cyathostomins and Strongyloids was not undertaken as this could not be reliably verified.

3.5 Statistical analysis Data were entered into Microsoft Excel before being transferred to GraphPad Prism 6 for statistical analysis. Statistical significance was set at p<0.05 for all analyses carried out. Prevalence of species identified (Strongyle, Parascaris equorum, Oxyuris equi, and Anoplocephala perfoliata) was calculated, with strongyle burden then being categorised as absent (0epg), low (<500epg), medium (501-1000epg) or high (>1000epg) 29. All continuous data sets were non-normally distributed, which was established using statistical (D’Agostino) and graphical tests for normality, hence analysis was carried out with non- parametric tests. The Mann-Whitney U test was used to assess significant relationship between two category variables (gender and housing) and parasite burden. The Kruskal Wallis test was applied to assess relationship between variables with more than two categories; frequency of mucking out, diet, anthelmintic frequency and type of anthelmintic used. Association between continuous parameters (BCS and age) and FEC were analysed using Spearman’s test for correlation. Chi squared test was used to compare strongyle infection intensity categories and the following categorical variables; gender, housing, frequency of mucking out, anthelmintic frequency, anthelmintic type and diet. Spearman’s test for correlation was used to compare strongyle infection intensity to continuous variables of age and BCS.

4. Ethical approval The Royal Veterinary College’s Ethic’s and Welfare committee granted ethical approval for this study, (approval number 2015/T77).

5. Results

5.1 Descriptive results Samples were collected from 223 ponies, 45.3% of which were mares, 46.2% stallions and 8.5% geldings. Age was not normally distributed with a negative skew (Figure 1), (range 6 months- 25 years, median age 4.5, interquartile range 7). BCS was not normally distributed with a positive skew (Figure 2), (range 1.5 to 4, median 2, interquartile range 0.5). Most ponies were used for transportation of goods (74%), 11.2% were ridden, 10.8% were being broken in, and one stallion was used purely for breeding purposes.

Figure 1: Frequency and distribution of age in the working ponies of the Kampong Chan and Kandal provinces, Cambodia (n=223).

Figure 2: Frequency and distribution of BCS in the working ponies of the Kampong Chan and Kandal provinces, Cambodia (n=223).

The majority of ponies were stabled (96%), and only 4% tethered. All owners reported that their ponies’ ate grass, 88% of ponies were fed bran, 17.5% banana tree, 6.7% porridge and 19.7% were fed rice as part of their diet.

Most owners reported they clear their ponies’ living areas of faeces every day (80.2%), 3.6% mucked out on a weekly basis and 17% of owners not at all.

All owners were reportedly aware of gastrointestinal parasites, all deeming them to be ‘very important’ to their ponies’ health and welfare. Most ponies had been wormed previously (66.8%): 11.2% in the previous month, 54.7% in the previous year, none had been wormed in the week preceding this study. To the best of the current owners’ knowledge, 34.1% of ponies had never received anthelmintic treatment. Ivermectin had been administered previously to 55.2% of the study and 15.7% had been treated with natural wormers prepared by the owner. Parasite species identified in this study were; strongyle, Parascaris equorum, Oxyuris equi and Anoplocephala perfoliata.

Table 1: Frequency and prevalence of gastrointestinal parasite species in ponies of the Kampong Chan and Kandal provinces, Cambodia (n=223).

Species Frequency Prevalence (%) Median epg Strongyles 173 77.6 200 O. equi 4 1.8 0

P. equorum 15 6.7 0 A. perfoliata 4 1.8 0

Strongyle infection was present in 77.6% of ponies (95% CI 72.1-83.1%). Strongyle epg was non-normally distributed with a positive skew, (median 200epg, interquartile range 50-600epg). Table 2 shows infection intensity categories for strongyle burden. Table 2: Frequency and prevalence of Strongyle infection intensity groups in the working ponies of the Kapong Chan and Kandal provinces, Cambodia (n=223). Infection Intensity Frequency Prevalence (%) 95% Confidence Interval Zero 50 3.65 1.2-6.1 Low (<500) 109 48.9 42.55.5 Medium (501-1000) 34 15.2 10.5-19.9 High (>1000) 30 13.45 9-17.9

Prevalence of O.equi was 1.8% (95% CI 0.06-3.54%), all four ponies affected had a burden of 100epg. Prevalence of P.equorum was 6.7% (95%, CI 3.42-9.98%), (range 50-750epg, mean of 100epg.) Prevalence of A. perfoliata was 1.8% (95% CI 0.06-3.54%), However further interpretations of this result were not made due to the low sensitivity of the McMaster FEC method for detecting this species.

5.2 Associations between animal demographics, BCS and parasite infection. The majority of stallions (78.4%) had a positive FEC (range 0-2200epg, median 150epg, interquartile range 50-550epg). Gastrointestinal parasites affected 84.7% of mares, (range 0- 2200epg, median 400epg, interquartile range 100-850epg). All geldings had a FEC of 0. Strongyle infestation was significantly lower in geldings when compared to stallions (p<0.001) and mares (p<0.001). Significantly higher strongyle burden was seen in mares compared to stallions (p=0.002). P. equorum infestation did not vary significantly between genders (p=0.98).

No statistically significant correlations between BCS and FEC was seen for strongyles or P. equorum (p=0.18 and p=0.04, respectively). No statistically significant association between age and strongyle or P. equorum burden was seen (p=0.57 and p=0.07, respectively).

5.3 Associations between variables: General husbandry factors No significant difference between diet and strongyle (p=0.26), P.equorum (p=0.20) or O.equi (0.18) was found. No significant difference between housing type (stabled or tethered) and FEC was found (p values; strongyles=0.50, P.equorum=0.25, O.equi=0.02). Frequency of mucking out did not significantly affect FEC (p values; strongyles=0.16, P.equorum=0.27, O.equi=0.17). No statistically significant difference between FEC and a ponies’ main use was found (p values; strongyles=0.15, P.equorum=0.29, O.equi=0.17).

5.4 Associations between variables: owner awareness of anthelmintics. All owners claimed to be aware of anthelmintic treatment and all deemed it to be ‘very important, crucial’ to their ponies health and welfare, therefore no further investigations were made into effect of this variable on parasite burden in the study population.

5.5 Associations between variables: Timing of previous anthelmintic treatment and type of treatment used. Ponies treated less than 1 month before sample collection had statistically significantly lower parasite burden than those treated 1 month - 1 year before sample collection (p<0.001) and those never treated (p<0.001). Worm burden did not significantly differ between ponies receiving anthelmintic treatment 1 month- 1 year ago, compared to those never treated (p=0.12) (Table 3).

Table 3: Frequency, prevalence and FECs of ponies treated at different times prior to sample collection, Kapong Chan and Kandal provinces, Cambodia (n=223). Previous Prevalence Median FEC FEC Range Interquartile treatment Frequency (%) (epg) (epg) range (epg)

< 1 month 45 20 0 0-850 0-100 1 month- 1 year 122 54.7 225 0-2200 0-650

Never 56 25 300 0-2200 50-750

Ponies receiving natural anthelmintics showed a significantly reduced FEC than those treated with ivermectin (p<0.001) and those never treated before (p=0.004). Ponies treated with ivermectin had significantly reduced FEC compared to those that had never received anthelmintic treatment (p=0.002). Table 4. Frequency of ponies treated with ivermectin or with natural preparations at different times prior to sample collection.

Treatment <1 month 1 month- 1 year Total Ivermectin 25 97 122 Natural 20 25 45 Total 45 122 167

Of all ponies treated with natural preparations, 44.5% received treatment in the month prior to sampling, whereas only 20% of ponies treated with ivermectin received it in the month prior to sampling (Table 4).

6. Discussion of results The results of this study have shown a high parasite burden in the working ponies in the Kampong Chan and Kandal provinces, Cambodia. Due to the significant pathogenicity of species present 25, it follows that improvement of gastrointestinal parasite control would benefit the working ponies of Cambodia. The strongyle prevalence in this study (77.6%) was lower compared to prevalence estimates from similar studies; strongyle prevalence has been calculated at 99% and 100% in Ethiopia 28, 30, 81% in the Kashmir Valley 31, 88% in Lesotho 13 and 93% in Vietnam 32, but similar to that seen in Southern Ethiopia (73.4%) by Seyou et al33 (73.4%) and in Sweden by Lind et al (78%) 34. Prevalence of ponies with a ‘high’ strongyle burden in this study (13.45%) is lower than similar studies by Getachew et al and Upjohn et al, in who found ‘high’ burdens in 55% and 49% of working equids, respectively13, 35. Strongyle speciation involving larval culture was beyond the scope of this study, however studies have shown that ‘typical strongyle’ eggs usually represent both small and large strongyles35. In recent studies employing larval culture 35, 36 and post mortem collection of worms34, 37 species such as S. edentatus, S. equinus, S. vulgaris, T. tenuicolis (as well as other less pathogenic species) and various cyathostomins were isolated from ponies with Strongylid eggs present in FEC. Large strongyle species may cause significant morbidity including colic, reduced performance, anaemia, ischaemic infarction, ileus, gut perforation and death25, 39. Cyathostomins (small strongyles), which usually represent 75-100% of total strongyle count 39 , are less pathogenic than large strongyles but are none the less considered to be of consequence due to high prevalence and propensity to develop anthelmintic resistance 40, 41 substantial infestations may lead to protein losing enteropathies, diarrhea, colic, weight loss and mortality 41. This study revealed no demographic or husbandry risk factors associated with Strongylosis, which mirrors results by Sheferaw et al in Ethiopia, 201330. We would expect increased frequency of mucking out, and reduced access to grazing to reduce parasite burden42, however, this was not seen in the study population, possibly due to uniform husbandry techniques across all ponies with the majority being kept stabled (96%) and mucked out daily (80.2%).

Prevalence of P.equorum was seen at 6.7% in the sample population, lower than previous studies; a prevalence of 21.4% was seen in Ethiopia33, 22% in Lesotho 13 and 29% in Saudi Arabia 44 but similar to that seen in Kashmir Valley, 9% 31 .Wide variation in P. equorum infestation is likely due to disparities in climate, age, susceptibility, and workload which is harder to recognise in the wide range of species counted as ‘typical’ strongyle eggs. High infestation with P. equorum may lead to enteritis and diarrheoa 38 and reduction in working capacity. Typically, ponies under the age of 6 months are predisposed to P. equorum 45, 46, 47, however this was not seen in this study, or in those by Upjohn et al 13 or Getachew et al35, this may be attributable to immunosuppression in overworked adults, or failure to develop adequate immunity when young. O.equi was found at a prevalence of 1.8%, similar studies reported prevalence of 2.1% in Ethiopia 30, 6.2% in Lesotho 13, 9.4% in Kashmir 31 and 8.8% in Saudi Arabia 44. O. equi eggs are deposited in the perineal area of infected animals, therefore faecal egg counts can be a lot lower than actual infection intensity. Apart from causing perineal itching and hair loss, O.equi is of little clinical significance and control is not a priority. No association was seen between O.equi burden and animal demographic or husbandry factors, contrary prior studies in Lesotho which found a decrease in O.equi prevalence with age13. The reasons for finding no significant husbandry or demographic risk factors associated with O. equi or P. equorum burden are unclear, however low number of infected animals (n=4, n=15, respectively), may have led to a lack of power to identify significant risk factors.

The lower prevalence of gastrointestinal parasites in this study compared to others may be attributable to climatic and geographical disparity in distribution of parasite species, and more consistent anthelmintic use in this sample population frequently visited by the CPWO. Sampling occured at the end of the dry season (which extends from October to May) when parasite levels are usually at their lowest28 and may not be representative of burdens throughout the year. Wetter weather has shown to cause higher parasite burdens and hence higher FECs hence information gained on the impact of seasonality would be beneficial in devising a more targeted anthelmintic program. Previous studies have suggested that anthelmintic treatment should be reserved for those ponies showing signs of parasitism (lower BCS, diarrhea, recurrent colic) 16, 36. However, this study showed no association between BCS and FEC, mirroring results by Valdez-Cruz et al37in Mexico, but contrary to findings by Seyoum et al in Southern Ethiopia33, suggesting that ponies were not being adversely affected despite high parasite burden. Hence, reserving anthelmintic treatment for those ponies showing outward clinical signs of parasitism (in the absence of regular FECs to target higher shedders) may be a prudent means to decreasing anthelmintic use.

Parasite burden was reduced in geldings when compared to mares and stallions. The majority of working ponies in Cambodia are entire and geldings were only present in riding schools, therefore this result may be due to other management differences involved with keeping ponies in riding schools, which were not evaluated in this study. All owners reported to be aware of gastrointestinal parasitism as a health risk for their ponies and also deemed anthelmintic therapy to be ‘crucial’ when asked. Previous study groups run by the CPWO focusing on parasitism and questionnaire bias may be attributable to this unanimous opinion. Ponies wormed within a month of sampling showed significantly lower burden than those wormed over a month ago, inferring that integrity of treatment is still intact, but level of efficacy is unclear as faecal egg count reduction test (FECRT) was beyond the scope of this study. Interval treatment programs yield reductions in large strongyle burden, but exacerbate anthelmintic resistance, especially in cyathostomins 40, 41 therefore, more emphasis should be put on altering management practices12, 46 to reduce burden and slow development of resistance. Treatment with ivermectin did not reduce Strongyle burden significantly when compared to natural preparations. Owners using natural preparations appear to be treating more regularly (table 4), and hence more recently, in turn giving rise to lower burdens than seen in ponies receiving less frequent ivermectin from the CPWO. Further investigation into frequency of treatment with natural preparations is necessary to comment on this result further. Ivermectin has been the only widely available anthelmintic to the CPWO due to cost and supply issues in this area of the world. Results from this study imply that anthelmintic resistance to ivermectin may be occurring, highlighting the necessity for research into altered management practices and natural preparations (the efficacy of which has been demonstrated in other countries49, 50, 51). Reduced efficacy of ivermectin may be due to using out of date drugs, ivermectin not licensed for ponies, incorrect storage or underdosing. Ivermectin used by the CPWO was licensed for equids and used at the correct dose rate based on weigh tape measurements, however under dosing is a significant risk when using weigh-tapes 52. Investigation into storage and use by dates of ivermectin used in the study population was not undertaken. Owner over-reporting of treatment due to social desirability bias 53 may have been present, causing FECs for untreated ponies to be included in the treated category, hence reducing the accuracy to assess treatement with ivermectin as a risk factor for parasite burden. The above factors need to be clarified in order to comment further on the efficacy of ivermectin from these results.

7. Limitations Sample storage may have intermittently exceeded the recommended 4oC; egg hatching time is reduced below 6oC but may occur within 24hours over 20o C54, 22, 23. Samples were stored in airtight bags and cool boxes, but adequate cooling became a difficult objective with daily temperature exceeding 30oC and the necessity to open the cool box regularly during sample collection. This has the potential to lead to egg hatching before samples were analysed, hence artificially lowering FEC. Mixing samples when collecting faeces from the ground was always a risk and may have decreased ability to identify risk factors, although it never knowingly occurred. Many owners who presented their ponies for sampling regularly attend CPWO meetings and therefore may not have been representative of the total population and are inherently more likely to be aware of their ponies needs. However some owners who had never attended a meeting before did present their ponies for this study. Owners may have over reported anthelmintic treatment frequency, mucking out frequency, or altered their opinion on the importance of anthelmintic use when asked, in an attempt to please CPWO staff. Such questionnaire bias may have decreased this study’s ability to identify risk factors. Sending out anonymous questionnaires or non-CPWO team members asking questions may reduce such bias 53,56. The Mac Master technique has low sensitivity, with other methods giving more reliable results (FECPAK and FLOTAC methods) 55. Lowered sensitivity may have caused underestimation of parasite burden, yet this technique was deemed most suitable given the field conditions, user experience and equipment available.

7. Recommendations Due to the blanket nature of current anthelmintic practices, recommendation of a more structured approach is paramount. Egg shedding is overdispersed; 20% of horses shed 80% of eggs present 57, 58, emphasising the need for FEC to target highest shedders, which is logistically difficult given the already stretched time and resources of the CPWO. Investigation into seasonal variation of gastrointestinal prevalence is advised to aid strategic timing of treatment, based on previous studies, a rise in FEC in the rainy season is expected28. Faecal Worm Egg Count Reduction Test (FECRT) would also be beneficial to ascertain the level of resistance present to ivermectin and drive changes in current anthelmintic regimes. Locally sourced natural anthelmintic preparations are used worldwide and it is common for owners in developing countries to treat their animals in this way. Efficacy of such treatments in Ethiopia has been demonstrated 50, 51, hence preparations used in Cambodia should be investigated as potentially invaluable techniques in reducing parasite burden.

8. Conclusions

Working ponies of the Kampong Chan and Kandal provinces of Cambodia demonstrated a high level of gastrointestinal parasitism. Current anthelmintic regimes in this region vary greatly, and there is scope for a more organised and integrated approach to anthelmintic use to curb development of anthelmintic resistance. Further studies into seasonal fluctuation of parasite burden in Cambodia is recommended, and investigation into efficacy and safety of natural anthelmintics is advised.

References

1) Cambodiaponywelfare.org, (2016). About Us. [online] Available at: http://www.cambodiaponywelfare.org/about-us [Accessed 10 Sep. 2015].

2) Perry, B., Randolph, T. and McDermott, J. (2002). Investing in animal research to alleviate poverty. International Livestock Research Institute.

3) Thornton, P. (2002). Mapping poverty and livestock in developing countires. International Livestock Research Institute (ILRI), Nairobi (Kenya). 4) Pritchard, J. (2010). Animal traction and transport in the 21st century: Getting the priorities right. The Veterinary Journal, 186(3), pp.271-274.

5) Garuma, S., Lemecha, F. and Sisay, A. (2007). Study on gender distribution of ownership of animal-drawn carts and it's effect on women's life in Adami Tulu and Dugda Bora districts. Draft Animal News, Centre for Tropical Veterinary Medicine (CTVM), Edinburgh.

6) Pearson, R. (2005). Contributions to Society: Draught and Transport. Encyclopedia of Animal Science.

7) Stringer, A. (2014). Infectious Diseases of Working Equids. Veterinary Clinics of North America: Equine Practice, 30(3), pp.695-718.

8) Pritchard, J., Lindberg, A., Main, D. and Whay, H. (2005). Assessment of the welfare of working horses, mules and donkeys, using health and behaviour parameters. Preventive Veterinary Medicine, 69(3-4), pp.265-283.

9) de Aluja, A. (1998). The welfare of working equids in Mexico. Applied Animal Behaviour Science, 59(1-3), pp.19-29.

10) du Toit, N., Burden, F. and Dixon, P. (2008). Clinical dental findings in 203 working donkeys in Mexico. The Veterinary Journal, 178(3), pp.380-386.

11) Burden, F., Du Toit, N., Hernandez-Gil, M., Prado-Ortiz, O. and Trawford, A. (2009). Selected health and management issues facing working donkeys presented for veterinary treatment in rural Mexico: some possible risk factors and potential intervention strategies. Trop Anim Health Prod, 42(4), pp.597-605.

12) Crane, M., Khallaayoune, K., Scantlebury, C. and Christley, R. (2011). A randomized triple blind trial to assess the effect of an anthelmintic programme for working equids in Morocco. BMC Vet Res, 7(1), p.1.

13) Upjohn, M., Shipton, K., Lerotholi, T., Attwood, G. and Verheyen, K. (2010). Coprological prevalence and intensity of helminth infection in working horses in Lesotho. Trop Anim Health Prod, 42(8), pp.1655-1661.

14) Data.worldbank.org, (2016). Cambodia | Data. [online] Available at: http://data.worldbank.org/country/cambodia [Accessed 26 Jan. 2016].

15) Change For Animals Foundation CFAF, (2016). Change For Animals Foundation CFAF. [online] Available at: http://www.changeforanimals.org/#!pony-welfare-in- cambodia/c4pz [Accessed 22 Jan. 2016].

16) Nielsen, M., Vidyashankar, A., Andersen, U., DeLisi, K., Pilegaard, K. and Kaplan, R. (2010). Providing an accurate faecal egg count in horses: analysis of faecal sample storage. Veterinary Record, 166(2), pp.54-54.

17) Nielsen, M. (2012). Sustainable equine parasite control: Perspectives and research needs. Veterinary Parasitology, 185(1), pp.32-44. 18) Evans, P. and Jack, N. (2007). Ageing Horses by their teeth. 1st ed. [ebook] Available at: http://extension.usu.edu/files/publications/publication/AG_Equine_2007-06pr.pdf [Accessed 12 Mar. 2015].

19) Richardson, J. (1997). Ageing horses - an illustrated guide. In Practice, 19(9), pp.486- 489.

20) CARROLL, C. and HUNTINGTON, P. (1988). Body condition scoring and weight estimation of horses. Equine Veterinary Journal, 20(1), pp.41-45.

21) Lester, H., Bartley, D., Morgan, E., Hodgkinson, J. and Matthews, J. (2012). The spatial distribution of strongyle eggs in horse faeces. Journal of Equine Veterinary Science, 32(10), pp.S33-S34.

22) Ogbourne, C. (1972). Observations on the free-living stages of strongylid nematodes of the horse. Parasitology, 64(03), p.461.

23) Mfitilodze, M. and Hutchinson, G. (1987). Development and survival of free-living stages of equine strongyles under laboratory conditions. Veterinary Parasitology, 23(1- 2), pp.121-133.

24) Rvc.ac.uk, (2016). McMaster egg counting technique: Principle. [online] Available at: http://www.rvc.ac.uk/Review/Parasitology/EggCount/Principle.htm [Accessed 26 Jan. 2016].

25) Bowman, D. and Georgi, J. (2009). Georgis' parasitology for veterinarians. St. Louis, Mo.: Saunders/Elsevier.

26) The working equid veterinary handbook. (2013). Whittet books.

27) Thiepont D, Rochette F, Vanparijs OFJ: Diagnosing helminthiasis by corprological examination. Beerse, Belgium, Janseen, Research Foundatio, 1979.

28) Getachew, M., Feseha, G., Trawford, A. and Reid, S. (2008). A survey of seasonal patterns in strongyle faecal worm egg counts of working equids of the central midlands and lowlands, Ethiopia. Trop Anim Health Prod, 40(8), pp.637-642.

29) Soulsby, E. and Monnig, H. (1968). Helminths, arthropods, & protozoa of domesticated animals (6th edition of Monnig's Veterinary helminthology & entomology). London: Bailliere, Tindall & Cassell.

30) Sheferaw, D. and Alemu, M. (2013). Epidemiological study of gastrointestinal helminths of equines in Damot-Gale district, Wolaita zone, Ethiopia. J Parasit Dis, 39(2), pp.315- 320.

31) Pandit, B., Shahardar, R. and Jeyabal, L. (2008). Prevalence of gastrointestinal parasitic infestation in equines of Kashmir Valley.

32) , W., Geurden, T. and Do, P. (2001). Strongyle infection in horses from North Vietnam. Revue d Elevage et de Medecine Veterinaire des Pays Tropicaux, 54, pp.29-31. 33) Seyoum, Z., Tesfaye, M. and Derso, S. (2015). Prevalence, intensity and risk factors of infestation with major gastrointestinal nematodes in equines in and around Shashemane, Southern Ethiopia. Trop Anim Health Prod, 47(8), pp.1515-1521.

34) LIND, E., HÃ–GLUND, J., LJUNGSTRÃ–M, B., NILSSON, O. and UGGLA, A. (1999). A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts. Equine Veterinary Journal, 31(1), pp.68-72.

35) Getachew, M., Trawford, A., Feseha, G. and Reid, S. (2009). Gastrointestinal parasites of working donkeys of Ethiopia. Trop Anim Health Prod, 42(1), pp.27-33.

36) Kyvsgaard, N., Lindbom, J., Andreasen, L., Luna-Olivares, L., Nielsen, M. and Monrad, J. (2011). Prevalence of strongyles and efficacy of fenbendazole and ivermectin in working horses in El Sauce, Nicaragua. Veterinary Parasitology, 181(2-4), pp.248-254.

37) Valdez-Cruz, M., HernÃ¡ndez-Gil, M., Galindo-RodrÃguez, L. and Alonso-DÃaz, M. (2012). Gastrointestinal nematode burden in working equids from humid tropical areas of central Veracruz, Mexico, and its relationship with body condition and haematological values. Trop Anim Health Prod, 45(2), pp.603-607.

38) Pereira, J. and Vianna, S. (2006). Gastrointestinal parasitic worms in equines in the Paraba Valley, State of Sao Paulo, Brazil. Veterinary Parasitology, 140(3-4), pp.289-295.

39) Ballweber, L. (2001). Veterinary parasitology. Boston: Butterworth-Heinemann.

40) Lester, H., Spanton, J., Stratford, C., Bartley, D., Morgan, E., Hodgkinson, J., Coumbe, K., Mair, T., Swan, B., Lemon, G., Cookson, R. and Matthews, J. (2013). Anthelmintic efficacy against cyathostomins in horses in Southern England. Veterinary Parasitology, 197(1-2), pp.189-196.

41) Lester, H. and Matthews, J. (2013). Faecal worm egg count analysis for targeting anthelmintic treatment in horses: Points to consider. Equine Vet J, 46(2), pp.139-145.

42) Evans, S. (2001). Field management for horses and ponies. London: J.A. Allen.

43) Taylor, M., Coop, R. and Wall, R. (2007). Veterinary parasitology. Oxford: Blackwell Pub.

44) AL Anazi, A. and Alyousif, M. (2011). Prevalence of non-strongyle gastrointestinal parasites of horses in Riyadh region of Saudi Arabia. Saudi Journal of Biological Sciences, 18(3), pp.299-303.

45) Reinemeyer, C. (2009). Diagnosis and control of anthelmintic-resistant Parascaris equorum. Parasites Vectors, 2(Suppl 2), p.S8.

46) Duncan, J. (1985). Internal parasites of the horse and their control. Equine Veterinary Journal, 17(2), pp.79-82.

47) Urquhart, G. (1987). Veterinary parasitology. Essex, England: Longman Scientific & Technical 48) Fao.org, (2016). 7. Treatment and control strategies. [online] Available at: http://www.fao.org/wairdocs/ilri/x5492e/x5492e09.htm#7.1.6 control of gastro intestinal nematodes [Accessed 22 Jan. 2016].

49) Payne, S., Kotze, A., Durmic, Z. and Vercoe, P. (2013). Australian plants show anthelmintic activity toward equine cyathostomins in vitro. Veterinary Parasitology, 196(1-2), pp.153-160

50) Peachey, L., Pinchbeck, G., Matthews, J., Burden, F., Mulugeta, G., Scantlebury, C. and Hodgkinson, J. (2015). An evidence-based approach to the evaluation of ethnoveterinary medicines against strongyle nematodes of equids. Veterinary Parasitology, 210(1-2), pp.40-52

51) Scantlebury, C., Peachey, L., Hodgkinson, J., Matthews, J., Trawford, A., Mulugeta, G., Tefera, G. and Pinchbeck, G. (2013). Participatory study of medicinal plants used in the control of gastrointestinal parasites in donkeys in Eastern Shewa and Arsi zones of Oromia region, Ethiopia. BMC Vet Res, 9(1), p.179.

52) Ellis, J. and Hollands, T. (1998). Accuracy of different methods of estimating the weight of horses. Veterinary Record, 143(12), pp.335-336.

53) Fisher, R. (1993). Social Desirability Bias and the Validity of Indirect Questioning. J CONSUM RES, 20(2), p.303.

54) Nielsen, M., Vidyashankar, A., Andersen, U., DeLisi, K., Pilegaard, K. and Kaplan, R. (2010). Effects of fecal collection and storage factors on strongylid egg counts in horses. Veterinary Parasitology, 167(1), pp.55-61.

55) Levecke, B., Rinaldi, L., Charlier, J., Maurelli, M., Bosco, A., Vercruysse, J. and Cringoli, G. (2012). The bias, accuracy and precision of faecal egg count reduction test results in cattle using McMaster, Cornell-Wisconsin and FLOTAC egg counting methods. Veterinary Parasitology, 188(1-2), pp.194-199.

56) Baker, T. and de Vaus, D. (1986). Surveys in Social Research. Contemporary Sociology, 15(5), p.760.

57) WOOD, E., MATTHEWS, J., STEPHENSON, S., SLOTE, M. and NUSSEY, D. (2012). Variation in fecal egg counts in horses managed for conservation purposes: individual egg shedding consistency, age effects and seasonal variation. Parasitology, 140(01), pp.115-128.

58) Kaplan, R. and Nielsen, M. (2010). An evidence-based approach to equine parasite control: It ain't the 60s anymore. Equine Veterinary Education, 22(6), pp.306-316. Appendix 1) Questionnaire for owners Sample Number:...... Owner’s Name:...... Animal’s Name Gender Mare Stallion Gelding Length of ownership Main food source Grass Hay Concentrate

Housing Stabled Tethered Loose

Frequency of mucking Every day Every 2 days Once a week Less than once a out week Companions Alone As a group

Use of animal Transport Ridden Breeding Agriculture

Are you aware of Aware Not aware anthelmintics? How important is anthelmintic Very important- crucial Quite important but not crucial Not treatment? Clare muir important Previous anthelmintic treatment <1 week ago <1month ago <1 year ago >1 year ago Never What product was used? Thank you for your participation. This is a research project carried out by a final year vet student (Charis Holmes) from The Royal Veterinary College (RVC), London, in partnership with World Horse Welfare and The Cambodia Pony Welfare Organisation (CPWO). Data collected in this study will be used to assess the level of gastrointestinal parasite burden in the ponies of Cambodia, and what the factors affecting this are. No personal data will be shared to any other parties and owner identity will remain anonymous. Verbal consent is required for participation of the animal into this study.