Bachelor of Veterinary Medicine

Research Project 2

Title

Corprological prevalence and intensity of

gastrointestinal parasitism in the working

ponies of the Kampong Chan and Kandal

provinces, Cambodia.

Word count3865

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.

1 3 5 7 9 11 13 15 17 19 21 23 250

5

10

15

20

25

30

35

40

45

50

Histogram

AGE (years)

Freq

uenc

y

Figure 1: Frequency and distribution of age in the working ponies of the Kampong Chan

and Kandal provinces, Cambodia (n=223).

1 1.5 2 2.5 3 3.5 4 More0

102030405060708090

100

BCS

Freq

uenc

y

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 200O. 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 treatment Frequency

Prevalence (%)

Median FEC (epg)

FEC Range (epg)

Interquartile range (epg)

< 1 month 45 20 0 0-850 0-1001 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., HÖGLUND, J., LJUNGSTRÖM, 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., Hernández-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

outEvery day          Every 2 days               Once a week              Less than once a

week

Companions  Alone                      As a group

Use of animal Transport Ridden                     Breeding                 Agriculture

Are you aware of

anthelmintics? Aware                                           Not aware

How important is

anthelmintic

treatment? Clare muirVery important- crucial      Quite important but not crucial          Not 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.