Somewhere on the moon battling worms:
can rotational grazing do the trick?
Naam:
Studentnummer:
Email:
Begeleider:
Email:
Datum:
Elise Johanna Wessels
3516679
[email protected]
Harm Ploeger
[email protected]
11 01 2013
Inhoudsopgave
0) Voorwoord
1) Abstract
2) Introduction
2.1 Hypothesis
2.2 Narrowing down the hypothesis: who are the players?
2.2.1 Strongylus lifecycle
2.2.2 Nematodirus battus lifecycle
2.3 Narrowing down the hypothesis; defining a low infection status
2.3.1 Effects of the weather and life cycle of nematodes on an EPG count
2.3.2 Age of the lambs
2.3.3 Expected EPG counts; a low infection status
2.4 Narrowing down the hypothesis; defining a rotational grazing plan
2.5 Narrowing down the hypothesis; the fully defined hypothesis
3) Materials and Methods
3.1 Survey
3.1.1 Development
3.1.2 Administering the survey
3.1.3 Respondent inclusion criteria
3.2 McMaster EPG
3.2.1 Collecting the samples
3.2.2 Performing the McMaster technique
4) Results
4.1 Results of the survey
4.1.1 Inclusion criteria
4.1.2 Clinical signs of infections with nematodes and anthelmintic treatments
4.2 Results of the McMaster
4.3 Comparing EPG counts of the lambs to the expected values
4.4 Finding a correlation between the number of days that the lambs spend on one
pasture and their EPG count
4.5 Finding a correlation between the length of time spent not using the pasture and
the mean EPG count of the lambs
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4.6 Finding a difference in EPG count for lambs that do, or do not go to a permanent
sheepfold or a pen during the night
5) Conclusion
6) Discussion
7) Literature
8)
Appendix I- The Survey, questions and results, PDF
Appendix II- Results EPG, PDF
3
0 Voorwoord
Vooraf zag ik deze onderzoeksperiode aan voor het lastigste onderdeel van de master
diergeneeskunde. Witte jassen, eindeloos labwerk, saaie onderzoeksvragen en lange dagen
zitten op stoelen om tegelijkertijd duimendraaiend te denkend over wetenschappelijke
vraagstukken.
Gelukkig bleek niets minder waar! Een enquête ontwikkelen vond ik een praktische en
uitdagende bezigheid. Binnen korte tijd in de wereld van schaapherders thuis te geraken
vergde veel van mijn sociale vaardigheden en wat is er leuker dan ‘s ochtends in de mist op de
prachtige heidevelden schapen te mogen bemonsteren? Daarnaast is het op een laboratorium
gewoon heel gezellig.
Na het oriënteren op een onderzoeksgebied en het zoeken naar een juiste begeleider volgt een
periode waarin een onderzoeksvraag gedefinieerd moet worden. Na inlezen, wikken en
wegen, strepen en schrijven is er hierna tijd voor het ontwikkelen van het onderzoeksplan. Als
dit uitgekristalliseerd is moet het uitgevoerd worden om vervolgens voor het documenteren
van de gevonden resultaten een zekere motivatie te ontwikkelen om opgedane kennis en
inzichten te delen met je beroepsgroep. Dus ook al is het praktische werk gedaan, daarop
volgt een lange laptoptijd, waarin je jezelf onderdompelt in cijfers, woorden, zinsconstructies
en conclusies.
Mijn dank voor de afronding van al deze perioden ben ik verschuldigd aan hen die mij hierin
zo fijn geholpen hebben. Voorop Harm Ploeger; worm- enthousiasteling, grote vriendelijke
reus, vakkundig docent. Verder Mario Wolff, Femke van Biezen, Jose Struik en Judith van
Andel. Jullie vriendschappelijke-, wetenschappelijke- en/ of liefdevolle hulp heeft mij
geholpen dit onderzoek te voltooien. En als laatste wil ik natuurlijk alle herders niet vergeten
die aan dit onderzoek hebben meegewerkt en mij hun schapen hebben laten bemonsteren.
Dank!
4
1 Abstract
It is not unlikely that in the future the use of anthelmintic treatment will not suffice for
treating nematode infections because of the resistance developed by these nematodes. Then,
other strategies will have to be followed to deal with nematode burdens. One method to do
this is to prevent sheep from getting infected by nematodes by using a rotational grazing
system in which sheep graze one area no longer than it takes the worms to develop from an
egg to an infectious L3- larvae, with as goal to interrupt the worm cycle.
In this research is tested whether following a rotational grazing programme brings the number
of EPG (Eggs Per Gram faeces) of lambs under 100 EPG for N. battus and under 500 EPG for
strongylus- type eggs. The rotational grazing plan that was followed included grazing of a
pasture no longer than 14 days and a waiting period before returning to a pasture of at least 2
weeks. The lambs, who were all under one year old, had been administered no anthelmintic
treatment shorter than 6 weeks prior to testing.
Concluding can be said that the hypothesis as stated in this research has been proved wrong
when it comes to strongylus- type eggs. Most lambs that followed a rotational grazing plan all
their life did not have an egg count lower than 500 EPG.
As to Nematodirus battus the hypothesis holds true, for only a small percentage of the lambs
sampled (6,7%) had counts higher than 100 EPG.
Unfortunately, when trying to explain why the hypothesis is not true for strongylus- type
worms, no correlations were found between the data from the questionnaire survey and the
results from the McMaster procedure.
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2 Introduction
On mainstream European sheep farms, the conventional method for combating worm
infestations with nematodes is the use of anthelmintic drugs. Research has shown that the use
of these drugs promotes insensitivity of the worms to specific anthelmintic drugs. In the
Netherlands, Borgsteede has shown that resistance within the nematode population already
exists for doramectin, albendazole and ivermectine (Borgsteede; 2007 & 2010). In the future,
this development will lead to difficulties in controlling nematode infestations. It is because of
this that a discussion has started in which there is room for the development of alternative and
durable methods for controlling worm infestations in sheep.
Prevention measures play an important role in this discussion. One of these measures is the
use of a rotational grazing system for sheep (Waller, P.J.; 1999: Eysker et al; 2005 ). In this
system, sheep graze one area no longer than it takes the worms to develop from an egg to an
infectious L3- larva. In this way, sheep do not come in contact with infectious L3- larvae, thus
the worm- cycle is interrupted and therefore the sheep do not get a worm infection, or at least
remain only lightly infected.
In the Netherlands this rotational grazing system is used by several shepherds. These
shepherds maintain golf courses, public parks and nature reserves with their sheep. They use
dogs to herd the flock, and they try to move every 14 days to a fresh grazing pasture.
2.1 Hypothesis
The fact that the flocks move to another grazing area almost every day creates the perfect
circumstances to test whether a rotational grazing plan is a sufficient measure to minimize the
level of infection of the sheep with nematodes. The goal of this research is therefore to answer
the following hypothesis;
Lambs up to one year old that have followed a rotational grazing programme since
birth show a low infection status with nematodes in November 2012.
Should this hypothesis be confirmed, economic benefit could be realised for sheep farmers,
because the cost of anthelmintic treatment could be diminished, as well as veterinary costs
and costs that accompany production losses. Also, a positive contribution will be made to the
research concerning resistance issues of the nematodes to anthelmintic treatment.
To properly answer this hypothesis, first it must be narrowed down to specifically defined
components.
2.2 Narrowing down the hypothesis: who are the players?
The specific kind of worm- eggs that this research focuses on are of Nematodirus battus and
of strongylus- type eggs. This is because these represent the most pathogenic worm species.
Strongylus-type eggs are produced by many gastrointestinal nematode species, including the
pathogenic species Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus
spp.
2.2.1 Strongylus lifecycle
Strongylus- type worms typically have a direct life cycle whereby eggs develop to an
infective L3 larva in approximately 5 days for Haemonchus contortus to 10 days for
trichostrongylus- species in optimal circumstances. After ingestion, larvae penetrate the
mucosa of the abomasum or small intestine. Here they moult two times to become L5. The
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prepatent period is 2- 3 weeks (Taylor, M.A., et al.; 2007; pp 158-164). Clinical problems can
be seen mostly in spring and summer as a result of the increase of larval numbers on pasture
in these seasons. The larvae remain infectious on pasture for a range of several weeks to
months, sometimes even surviving the winter in temperate regions. Immunity is acquired
slowly (Taylor, M.A., et al.; 2007; p165).
2.2.2 Nematodirus battus lifecycle
Development to L3 takes place within the eggshell, hatching requires a temperature of 10 °C
or more, but only after a prolonged period of chill. This normally results in a massive
hatching at the end of spring. Sometimes some N. battus eggs deposited in spring are capable
of hatching in the autumn of that same year resulting in infectious L3 larvae in autumn.
After ingestion, larvae penetrate the mucosa of the ileum and moult into L4 directly after. L5
inhabits the lumen of the intestines. The prepatent period is 14- 16 days.
Clinical signs such as diarrhoea, followed by dehydration occur as a result of the disruption of
the intestinal mucosa by the larvae. The signs already occur during the prepatent period,
therefore for an early diagnosis an egg count is useless.
Eggs of N. battus can survive up to 2 years on pasture. (Taylor, M.A., et al.; 2007; pp 167168). This could explain an infection with N. battus in traditionally farmed lambs when new
lambs are grazed on a specific pasture every year. In herded sheep an infection with N. battus
might be harder to link to specific causes such as grazing mis- management, because the
sheep tend to walk around freely.
2.3 Narrowing down the hypothesis: defining a low infection status
As there is no literature on what EPG count is likely to be expected in November, an
estimation has been made based on the time of year combined with the weather, the age of the
lambs and the life cycle of the Nematodirus battus and the strongylus- type worms. This
estimation is then combined with the EPG count that, based on literature, is the limit value
where clinical signs and production losses indicate that anthelmintic treatment should be used.
This leads to a range of EPG counts that indicate several stages of worm infections sheep can
be subjected to.
2.3.1 Effects of the weather and life cycle of nematodes on an EPG count
September, October and November, the autumn of 2012, experienced a mean temperature of
10.5 °C, this is similar to the long- term average in the Netherlands. The long- term average is
based on the period 1981- 2010. September 2012 was colder than usual. The mean
temperature of October was similar to the long- term average but in the second half of
October an unusual period of warmth occurred. There were seven days in the fall the
temperature fell below 0°C, namely on September 23rd, October 28th and November 5th, 15th,
18th, 19th and 20th, these seven days are also similar to the long- term average in the
Netherlands. A prolonged period of chill occurred from the 5th to the 12th of October with
temperatures ranging from 0°C to 4°C. As to the rain, 215 mm was measured throughout the
entire fall, this is less than average, which is 243 mm. September and November were relative
dry months, on the contrary to October which experienced more rain than the long- term
average. The sun also shone more than the long- term mean. Registered were 348 hours of
sunshine, which is more than the average of 320 hours. Especially September was
exceptionally sunny (http://www.knmi.nl, 10 Dec 2012).
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Conclusively, the fall of 2012 in the Netherlands was relatively sunny and dry with a
temperature comparable to the long- term mean.
A possible effect of the weather of autumn 2012 on the development of the Nematodirus
battus larvae is a rise in EPG, for these larvae need a stretch of chill followed by a rise in
temperature for the infectious L3 to hatch from the egg and this is exactly what happened in
the Netherlands on the 5th to the 12th of October 2012 (Taylor, M.A., et al.; 2007; pp 167168).
A likely effect of this weather on the population of strongylus- type larvae on pastures in the
Netherlands is hard to predict. While larvae of Haemonchus contortus do not develop and
hatch well in cold temperatures, larvae of Teladorsagia and Trichostrongylus appear to
tolerate relatively low temperatures while hatching. Especially exposure to temperatures
below 0°C decrease chances of survival for H. contortus and as there were seven days with
temperatures below 0°C in the autumn of 2012, this could result in a decrease in EPG counts
for H. contortus and thus for the general strongylus- type EPG counts. For migrating from the
faeces to herbage that the sheep will eat, strongylus-type larvae need a film of moisture.
Because the fall of 2012 was relatively dry, this could prevent larvae from migrating, which
would result in a decrease in EPG count. However, as no prolonged periods of drought were
registered, the effect of the relative dryness of autumn can be expected to be minimal on the
possibilities for migrating.
2.3.2 Age of the lambs
Based on the age of the lambs, EPG counts are expected to be low for both N. battus and
strongylus- type eggs. This because these lambs were born late 2011 or in the beginning of
2012 and therefore in November 2012 had reached an age of approximately one year old. At
this age, the lambs will have reached an advanced stage of natural resistance to the
nematodes. Only a small portion of the herd, namely fifteen percent, will discard
approximately 50 percent of the total egg count (Kloosterman, A., et al.; 1992)
2.3.3 Expected EPG counts: a low infection status
To not cause clinical signs and/ or production losses, in traditionally farmed sheep, the egg
count of N. battus should be lower than 100 EPG (De Wormenwijzer, WUR). As this is a
typical spring parasite, it should not be expected to get close to this count in November.
However, related to the weather in the fall of 2012, with a cold stretch (5th -12th of October)
followed by an unusual warm week (17th – 24th) in October, the count might be expected to be
100 EPG or even higher.
For strongylus- type nematodes to not cause disease and/ or production losses, the egg count
should be less than 500 EPG (De Wormenwijzer, WUR). Based on the weather a more precise
prediction could not be made, the mean temperature was only slightly above the 10°C
required for development of the larvae while on the other hand the cool and dry climate of the
fall could result in a longer survival- period of already existing larvae on pasture.
Based on the age of the lambs EPG counts of both strongylus- type nematodes and N. battus
should be low because lambs should have built up natural resistance after one year. If
occasional high measurements occur, these could originate from the 15 percent of lambs that
still may discard high levels of nematode eggs.
Concluding: ‘De wormenwijzer’ of the University of Wageningen states that sheep with
nematodirosis may start showing clinical signs and/ or production losses from a count of 100
8
EPG if lambs remain untreated or are not moved to another pasture. Only several lambs that
were sampled showed severe diarrhoea and some of them were thin, but most of them showed
no sign of disease or loss of production. This combined with the estimation made based on the
age of the lambs, the weather of the autumn of 2012 and the life cycle of the nematodes leads
to the estimation that EPG ranges of N. battus were expected to vary from 0 to the cut-off
value of 100 EPG.
‘De wormenwijzer’ of the University of Wageningen states that sheep infected with
strongylus- type worms may start showing clinical signs and/ or production losses when they
have reached a count of 500 EPG. But as there were few lambs showing clinical signs, and
the weather and the age of the lambs is taken into account, in this research EPG counts were
expected to be considerably lower that this cut- off value of 500 EPG.
2.4 Narrowing down the hypothesis: defining a rotational grazing plan
With a rotational grazing plan is meant that shepherds move their herds from pasture to
pasture, where the sheep graze one area no longer than it takes the worms to develop from an
egg to an infectious L3- larva. In this way, sheep do not come in contact with infectious L3larvae, thus the worm- cycle is interrupted and therefore the sheep do not get a worm
infection. In order to keep the EPG count low, Taylor says: “[With these methods] sheep only
graze a paddock for 3.5 to 4 days and are then moved to the next paddock. (…) Return to the
original paddock must not occur at an interval of less than five weeks” (Taylor, M.A., et al.;
2007; pp 157- 158).
In this research herds are moved to a fresh area at least every 14 days, and grazed pastures are
not revisited by sheep for at least 14 days. These values were partly chosen for practical
reasons, because none of the respondents fitted the criteria as stated above. On the other hand,
it was argued that the shortest development time between egg and infective larva on the grass
was 14 days under Dutch summer conditions. (De Wormenwijzer, WUR, Eysker, M., et al.;
2005)
2.5 Narrowing down the hypothesis: the fully defined hypothesis
Resulting from the last paragraphs the fully defined hypothesis reads:
Lambs up to one year old that have followed a rotational grazing programme since
birth, in which is included that the lambs are moved to a fresh pasture at least every
14 days and that the pasture is not used for grazing sheep for at least two weeks
before returning the lambs, show an egg count lower than 500 EPG and 100 EPG of
respectively strongylus- type worms and N. battus in November 2012.
9
3 Materials and Methods:
In this research a questionnaire was sent out to 51 shepherds in the Netherlands. After they
had completed and returned it, some of these shepherds were visited to collect faecal samples
from 20 of their lambs. These samples were then subjected to a McMaster procedure to count
and categorize worm eggs and Eimeria oocysts.
3.1 Survey
3.1.1 Development
When designing a questionnaire, distinct questions should be kept in mind. What would you
like to know? From which group of people would you like to know this? What is the purpose
of your quest? To make these intentions clear, the research design of the questionnaire should
have a logic behind it that makes it possible to draw valid and sound conclusions and the
research technique of the questionnaire, the method for collecting data, should deal with the
measurement and quantification of the data available.
According to Oppenheim (1992) there are several steps to designing and administering a
questionnaire. Firstly, consider the aim of the study. Secondly, variables concerning this aim
must be distinguished. For these, a set of questions, scales and indicators will have to be
formulated. Then check whether the design of the questionnaire answers to the hypothesis that
is the basis of this study. If so, decide on the research instruments or research technique.
Following this, one must do a pilot to rule out any ambiguities or flaws. Now design the
samples: think about how to deal with non-responders, whether a sample has to be
representative and if you need a control group. Finally, do the fieldwork. Afterwards, process
the data and perform statistical analysis to test the hypothesis. Now conclude with writing the
research report.
Following the steps that Oppenheim suggested, the aim of this study is clearly defined in the
hypothesis: Lambs up to one year old that have followed a rotational grazing programme
since birth show a low infection status with nematodes in November.
In this questionnaire, a set of variables was designed considering the descriptive side of the
hypothesis. Variables that were distinguished in this part of the questionnaire included:
- the overall details of the company that owns the sheep
- the herds rotational grazing system
- the process of combining the lambs with the sheep
- issues concerning nematode- infections within the herd
- the anthelmintic treatment in the past year of the lambs that are included in this research
Then a set of questions was designed that linked these variables, in order to make a cross
sectional design. This combination of questions will lead to a mix of a descriptive kind of
questionnaire, which is filled with questions considering numbers and proportions and an
analytic kind of questionnaire, that deals with ‘why’ or ‘how’ questions. The choice for the
distribution of open versus closed questions was based upon this quote: ‘All closed questions
should start their careers as open ones […]’, by A.N. Oppenheim (Oppenheim, A. N.; 1966;
p46). The open question should be closed afterwards in the following questions. This
approach will limit the loss of information to a minimum for it will minimize the bias that
occurs with prefab closed questions, while it still gives a measurable answer.
10
For all questions a scale or indicator was designed, to make interpretation measurable. In this
questionnaire mostly selected response- questions were designed because this type of question
forces the respondent to make a choice. However, this type of question only works well if the
most probable answers are well piloted beforehand (Gillham, B.; 2000; p30).
After this, boundaries of each question were marked. What would be the influence of nonresponders? What would be a representative sample of answers per question? Is a control
group necessary for this question to be representative?
Non- responders will be noted in the results as missing values, they will not reflect on the
research results. This also goes for respondents that have only partly finished the survey.
3.1.2 Administering the survey
Administering the entire questionnaire was done after a pilot was performed. This pilot had as
goal to eliminate any confounded variables or biases, such as hidden additional causes and
error (Oppenheim, A. N.; 1992; pp 21-22).
The questionnaire was sent to the participating shepherds through the online questionnaire
design software Survey Monkey. This was considered the best option to administer the
questionnaire because prior to this survey contact with the shepherd already had been
established by telephone. In this prior conversation the goal of this research had been made
clear and participation was pledged. Also, in this way shepherds were enabled to fill in the
questionnaire well before the herds were visited by the researcher, so there was a certainty
that visited herds were already documented in the survey and that the herd fitted the inclusion
criteria.
There are known disadvantages to administering a questionnaire by (e)mail instead of faceto face. The greatest disadvantage known is the low responder- rate (Oppenheim, A. N.; pp
34). In this case, out of 51 sent surveys, 26 were returned fully completed, 8 were returned
only partly completed. Another big disadvantage, according to A. N. Oppenheim (1966) is
that respondents can’t add additional explanation to a question. However, in this survey this
problem is mostly tackled by a box that the shepherd can tick that says: ‘otherwise, namely’.
The questionnaire that was eventually sent to the shepherds can be seen in Appendix I.
3.1.3 Respondent inclusion criteria
Not all respondents fitted the inclusion criteria. Filters were used on several questions.
Eventually 18 respondents out of 26 were selected. They all had at least 25 lambs under the
age of one year old, male or female. None of the selected respondents had used anthelmintic
treatment shorter than 8 weeks prior to our visit not including incidental treatments. All of the
respondents did not graze their sheep on one area longer than 14 days successively. Most of
them waited at least 8 weeks before returning to a previously grazed area. Six out of 15
shepherds waited less than 8 weeks before returning to a pasture to graze. One of them
returned to the same pasture only as soon as two weeks later. The research was originally
designed with shepherds returning to a pasture no sooner than eight weeks, however in reality
this criterium resulted in very few respondents that could be included. Therefore the
hypothesis states that a minimum of a two- week return period is used as criterium.
3.2 McMaster EPG
3.2.1 Collecting the samples
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Rubber gloves were used for collecting the faeces from the rectum of the sheep. The samples
were stored in plastic bags that were closed and then inversed to prevent air to get in contact
with the faeces. These bags then were stored in a cooling box during transport to the
laboratory to keep the samples cool so that the eggs inside the faeces could not hatch.
3.2.2 Performing the McMaster technique
Three grams of faeces were weighed in a 50 ml falcon- tube. This was then topped with 42 ml
saturated saline solution. This suspension was grinded in a mortar until the faeces had
dissolved. The suspension was then strained and put back in a falcon tube. This tube was
swerved 10 times and a sample was taken from the middle of the tube with a pipette. This
sample was then pipetted in one McMaster frame of 0.15 ml. Then swerving of the falcon
tube was repeated to fill the second McMaster frame of 0.15 ml. The frame was then left to
set for 10 minutes to allow the eggs to surface on the saline solution.
With a 10x 10 microscope, eggs were counted per species, except for strongylus- type eggs,
which were all counted together. Oocysts of Eimeria were also counted. Per faeces sample
two McMaster frames were counted individually. The volume of the two frames together is
0.3 ml. This is 1/150 of approximately 45 ml. (42 ml saline solution plus 3 gram faeces) Since
three grams of faeces was used, the amount of eggs per gram was calculated by multiplying
the eggs counted in the two frames together by 50.
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4 Results
4.1 Results of the survey
Results of the survey are numerous. The full report can be found in Appendix I. From these
results only those relevant to answer the hypothesis are discussed here. All respondents have
been given a number to ensure their anonymity.
Out of 51 sent surveys, 26 were returned fully completed and 8 were returned only partially
completed. Out of these 26 respondents 18 were selected to participate in this research based
on their answers in the survey. Out of the 18 selected respondents, 4 respondents were ruled
out for different reasons after contacting them. One because he had applied massive
anthelmintic treatment the day after he filled in the questionnaire. The second because she had
just combined all her flocks and put them on a winter pasture, thereby abandoning her
rotational grazing schedule. The third wished to not further participate in this research. The
fourth was too far away to fit into any routing.
Eventually 14 respondents were visited for collecting faecal samples. On one of the farms
two different flocks were available. These are processed in the results as two different flocks
with a similar management, thus bringing the eventual number of herds tested to 15.
4.1.1 Inclusion criteria
Inclusion criteria were based on several things. The number of available lambs under the age
of one year old, male or female, exceeded 20. All participants did not graze their sheep on one
area longer than 14 days successively. Most of them waited at least 8 weeks before returning
to a previously grazed area. Also, none of the selected respondents had used anthelmintic
treatment shorter than 8 weeks prior to our visit not including incidental treatments. The
precise numbers are shown in table 1.
Table 1: Inclusion criteria for research, derived from answers to the questionnaire.
Herd
Number of
number lambs available
(ewe lambs)
Maximum number Minimum number
of days spent on of weeks waited
one pasture
before returning to
pasture
Number of weeks
between last
anthelmintic
treatment and
sampling
1
2
3
4
5
6
7
8
9
10
11
12
13
14
14
7
1
14
14
2
4
4
6
10
3
3
3
3
8
19
18
8
8
20
18
10
6
20
21
4
No treatment done
7
130
60
25
400
400
70
40
120 (ram lambs)
250
30
184
102
50
52
8
8
3
6
6
9
8
6
8
12
8
10
7
2
13
15
31
1
8
No treatment done
4.1.2 Clinical signs of infections with nematodes and anthelmintic treatments
According to the questionnaire, five shepherds out of fifteen claimed they did not have
problems with infections with nematodes. Nonetheless, only two of these five shepherds
(herds 13 and 15) used no anthelmintic treatment at all. Surprisingly, herd number eight
indicated that even though he had been confronted with signs of nematodirosis, he had not
used any anthelmintic treatment so far. The remaining three shepherds who, despite having
seen no visible signs of illness, used anthelmintic treatment are herds number one, three and
14. According to the answers from the questionnaire, herds 3 and 14 made use of standard
anthelmintic group treatments that they both administered in August 2012. This could explain
why they did not observe clinical signs of illness in their lambs. The reason herd number one
used anthelmintic treatment remains unknown.
Ten shepherds indicated that they used anthelmintic treatment in 2012. In the questionnaire,
they were asked what they did when they observed one or more of the following clinical
signs; diarrhoea, extreme weight loss, anaemia and/ or loss of condition of the fleece.
On seeing diarrhoea, four shepherds collected faeces samples for a McMaster egg count and
four shepherds administered anthelmintics without doing an egg count first. Two shepherds
indicated that they never saw diarrhoea as a clinical sign. On seeing anaemia, three shepherds
administered anthelmintics immediately and five performed McMaster egg counts. Two
shepherds indicated that they had not seen anaemia in their lambs. On seeing severe weight
loss, two shepherds administered a vitamin boost, it is unknown which one. Four shepherds
performed McMaster egg counts and one shepherd administered an anthelmintic treatment.
Three shepherds answered that they had not observed severe weight loss in their sheep. When
seeing the condition of the fleece getting worse, one shepherd did nothing, while four
shepherds performed McMaster egg counts. Five shepherds indicated they had not seen this
problem in their sheep.
When on location for sampling the sheep, shepherds were asked a few questions to doublecheck their answers in the questionnaire. Surprisingly, in contrast with what shepherds
answered in the questionnaire, when treating their sheep against nematodirosis all shepherds
mentioned they used cydectin.
4.2 Results of the McMaster
Only the results of the Strongylus- type eggs and the Nematodirus battus eggs are used in this
display of results. The other measured variables, Eimeria oocyst, Strongyloides, Nematodirus
spathiger, Nematodirus filicollis and the Monieza expansa are not further discussed in this
research because according to Anne M. Zajac (2006), these are species that are not considered
important pathogens. Nonetheless, these data are left included in Appendix II. An overview of
the results of strongylus- type eggs and Nematodirus battus is shown in table 2. The complete
results of the McMaster EPG count can be found in Appendix II.
Table 2: Result EPG counts and standard deviations of the mean of strongylus- type
eggs and N. battus.
Herd
numbe
r
strongylus- type Standard deviation of
eggs mean EPG the mean for
count
strongylus- type eggs
Nematodirus
battus mean EPG
count
Standard deviation
of the mean for
Nematodirus battus
14
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1645
1637,5
380
487,5
2,5
605
185
3555
575
1227,5
600
0
4687,5
45
1357,5
1471
1635,8
373,6
418,3
10,9
350
115,2
1925,1
522,4
835,7
392,7
0
2026,4
49,8
1174,0
12,5
83
25
60
0
0
80
35
2,5
0
20
0
42,5
2,5
7,5
26,8
117,9
46,1
94,3
0
0
103
42,1
10,9
0
29,2
0
89,8
10,9
32,7
Shown in table 2 is the mean EPG count per herd and the standard deviation of the mean.
Number two is grey, because even though this shepherd had enough lambs available for
sampling, only 12 out of 60 could be isolated from the herd. However, for this is 20 percent of
the entire population of lambs in this herd, this EPG count can still be expected to represent
the worm status of all lambs in this herd. Number 12 is grey because from this herd only four
sheep were tested because the shepherd had just subjected his sheep to anthelmintic treatment
only four weeks earlier. When in these four sheep not one worm- egg was found, the
McMaster procedure was discontinued.
Also shown in table 2 is that the mean is not a correct factor to use for potential conclusions
in a research with so few participants because the standard deviation of most herds is very
large, possibly biasing the results. This large deviation of the mean is caused by individual
sheep within one herd that have widely separated EPG values. This could be explained by the
fact that fifteen percent of the herd is responsible for approximately 50 percent of the total egg
count (Kloosterman, A., et al.; 1992). When a few of the lambs that belong to the 15 percent
category are sampled, they will enlarge the standard deviation.
4.3 Comparing EPG counts of the lambs to the expected values
Here the EPG counts of the lambs are compared to the expected values of 500 EPG and 100
EPG for respectively strongylus- type worms and N. battus.
Shown in table 3 are the total number of sheep sampled and the total number of sheep with an
egg count higher than the expected value. Also shown are the percentages of sheep per herd
that have egg- counts higher than 500 and 100 EPG for respectively strongylus- type eggs and
N. battus.
Table 3:
Herd
Total
number lambs
sampled
Number of sheep
with egg count
>500 EPG for
strongylus- type
eggs.
Sheep with
egg counts
>500 EPG for
strongylustype eggs (%)
Number of sheep
with egg count
>100 EPG for N.
battus
Sheep with
egg counts
>500 EPG
for N.
battus (%)
15
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
20
12
20
20
20
20
20
20
20
20
20
4
20
20
20
15
10
4
8
0
12
0
19
10
14
10
0
20
0
15
75
83
20
40
0
60
0
95
50
70
50
0
100
0
75
0
4
1
2
0
0
6
1
0
0
0
0
3
0
1
0
33
5
10
0
0
30
5
0
0
0
0
15
0
5
Herd number two, who had only 12 lambs tested, can be considered as the other flocks of
sheep, for it concerns percentages. Herd number 12 has zero percentage of the sheep above
the cut-off value because anthelmintic treatment with cydectin was administered to these
lambs four weeks prior to our visit. Numbers five and 14 simply have zero percentage of
lambs that exceeded the expected EPG count values.
As shown in the results, on herd level nine out of 15 herds had mean values that exceeded the
expected value of 500 EPG for strongylus- type worms. From the standard deviation of the
mean and the calculated percentage of sheep with egg counts higher than the expected values
can be concluded that in some herds there are many lambs with egg counts that are way too
high. On individual sheep level in nine out of 15 herds 50 percent or more of the sheep had
worm egg levels counting higher than 500 EPG. In two out of 15 herds less than 50% of
sheep had worm egg counts exceeding 500 EPG. In four herds no sheep had values exceeding
the cut- off value, however herd number 12 should be excluded from this numeration because
these sheep had been treated with anthelmintics shortly before sampling.
Conclusively, in 11 of 14 herds sheep were found with EPG counts higher than the expected
value. In total 51% of lambs were found to have egg- counts over 500 EPG. Thus it is not
true that the EPG counts of the lambs for strongylus- type eggs are within the range that is to
be expected according to the initial hypothesis.
For N. battus the results are different. 100 Percent of herds showed a mean that remained
under the expected count of 100 EPG. This would indicate that there was no problem
concerning Nematodirus battus infections in Dutch herded sheep in November 2012. Still, in
seven out of 15 herds sheep were found with EPG counts higher than 100 EPG, but in most of
these herds only a small percentage of the sheep exceeded this value. Herds two and seven
had the highest percentage of sheep exceeding the cut-off value. Nearly 30 percent of their
sheep had egg counts higher than 100 EPG and the range of found EPG within these sheep
goes up to 350 EPG. This result indicates clinical problems with N. battus for only part of the
herd.
Conclusively, it is true that the mean EPG counts of the herds for Nematodirus battus are
within the range of 0-100 EPG. 18 Individual sheep had counts exceeding 100 EPG, these 18
16
sheep represent 6,7 % of 267 sheep sampled. This being a low percentage, it can be concluded
that the EPG counts found in this research are indeed lower than the expected 100 EPG.
This raises the question why the number of strongylus- type eggs found is this high this late in
the season in sheep that theoretically should have low worm burdens. To answer this, data
from the survey is compared to data found in the EPG counts.
4.4 Finding a correlation between the number of days that the lambs spend on
one pasture and their EPG count
The answer to this could point out whether there is a maximum number of days that the lambs
can spend on a pasture before their EPG exceeds the directive EPG count. When several
herds have exceeded this period, this could explain why the EPG counts found are so much
higher than expected according to the hypothesis. An expiration date for pasture- use is not
expected to be found, because the periods the herds spend on the pasture varies from two days
to 14 days, which is very short for the worm to complete the transformation from egg to L3
and for the sheep to start shedding eggs. When an expiration date for pasture- use is found,
this can be employed for future advising of sheep farmers and/ or shepherds. However, as
shown in the graph 1, where the maximum days spent on the same pasture is set out against
the EPG count, such a relation does not exist.
The highest number, with an EPG mean of 4687,5 is herd number 13, who only spends a
maximum of three days on one pasture. This is in sharp contrast to herds number nine and 11,
who spend as much as 14 days on one pasture before moving to a fresh pasture. However,
both their EPG counts remain under 500 EPG.
Strongylus gemiddeld EPG
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
Strongylus gemiddeld EPG
0
5
10
dagen op zelfde graasgrond
15
Graph 1: Maximum days spent on the same pasture versus EPG count
Concluding from graph 1 no significant correlation exist between the number of days that the
lambs spend on a pasture and their EPG count.
4.5 Finding a correlation between the length of time spent not using the pasture
and the mean EPG count of the lambs
The answer to this could point out if this factor in the rotational grazing scheme is crucial to
keeping the EPG low as well as give an indication for a minimal number of weeks that the
17
lambs should not graze on a pasture to get it relatively cleaned of worms. When the herds that
spent the shortest time returning to pasture are found to have the highest worm burdens, this
could explain why the hypothesis is not confirmed. Also, when a correlation is found, this
information can in the future be used to design a rotational grazing advice for shepherds and
sheep farmers in temperate regions. Because in ideal circumstances strongylus- type larvae
can survive on pasture for weeks to months and are sometimes even able to survive the
winter, ideally herds have to wait until the winter has passed to return to a pasture. This is
obviously not the case in this research. However, as seasonal circumstances were not ideal for
survival of the larvae on pasture, a correlation between the length of time spent not using the
pasture and the mean EPG count of the lambs is expected to be found, certainly because the
variation in time spent not using the pasture within the herds ranges from two weeks to 12
weeks.
In graph 2 can be seen that herd eight and 13 are the high rankers, but herds four, five and
seven have spent a similar time between grazing one pasture and they have mean EPG counts
that are below 500 EPG.
Herd three and 14 have minimal amount of time between visiting one pasture. This does not
affect their EPG count as much as one might expect as Taylor, M. A. states that returning
sheep to the original pasture must not occur at an interval of less than five weeks in order to
effectively interrupt the worm cycle (Taylor, M.A., et al.; 2007; pp 157- 158). These herds
mean EPG count unexpectedly remains well under 500. Notable is that both these herds have
a similarity. They each have a sheepfold to come home to every night. Only one other herd
had a permanent shelter to come home to, except this is not a sheepfold but a paddock. This
herd is number six, who has a mean EPG count of 605. However the shepherd of this flock
noted in the survey that he waits at least nine weeks before returning to the same pasture,
which is a lot more than the two and three weeks of herds number three and 14.
Strongylus gemiddeld EPG
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
Strongylus gemiddeld EPG
0
2
4
6
8
10
tussengraastijd in weken
12
14
Graph 2: Correlation between the length of time spent not using the pasture and the
mean EPG count of the lambs
In relation to the chart shown above in answer to the question 5.1.3 can be said that in spite of
what was expected, no correlation exists between the time spent not using the pasture and the
mean EPG count of the lambs.
18
4.6 Finding a difference in EPG count for lambs that do, or do not go to a
permanent sheepfold or a pen during the night
Finding this difference could explain whether the high EPG values found are biased by the
fact that three herds were used that come home to a permanent shelter every night. Coming
home to a permanent sheepfold could be attributing to experiencing a high worm burden
because these sheep tend to walk the same route every morning and night, whereby inevitably
faeces is dropped and some grass and shrubbery is nibbled at every day. This would in
practice mean that the sheep return to the same pasture every day and therefore the worm
cycle in these sheep is never interrupted.
5000
4500
4000
3500
3000
2500
schapen met stal/kooi
2000
schapen zonder stal/kooi
1500
1000
500
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Graph 3: Sheep that do, or do not go to a permanent sheepfold during the night versus
their mean EPG count
As can be seen in the graph 3, the three herds that have permanent shelters for the night show
relatively low EPG counts.
Thus the answer to the question is negative, there is no systematic difference found in EPG
counts for lambs that do, or do not go to a permanent sheepfold or a pen during the night.
19
5 Conclusion
Concluding, the hypothesis as stated in this research has been proved wrong when it comes to
strongylus- type eggs. Most lambs (51%) that have followed a rotational grazing plan all their
life do not have an egg count lower than 500 EPG.
As to Nematodirus battus the hypothesis holds true, for only 6,7% of all sampled sheep was
found to have higher counts than the cut-off value of 100 EPG.
Unfortunately, when trying to explain why the hypothesis is not true for Strongylus- type
worms, no correlations were found between the data from the survey and the results from the
McMaster procedure. Possible explanations for this fact can be found in the number and
timing of the anthelmintic treatments that have been administered to the herds. Also the low
total number of participants in this research plays an important role in drawing the final
conclusions. A larger number of participants could provide clearer conclusions.
20
6 Discussion
There is always room for improvement or a different perspective, so as to help understand
results better. The following paragraphs point out where in this research these improvements
or different point of views are needed.
Narrowing down the hypothesis: Nothing as unpredictable as the weather
The fall of 2012 in the Netherlands was relatively sunny and dry with a temperature
comparable to the long- term mean. However, while Holland may be a small country with a
moderate sea- climate, suggesting a stable climate without great fluctuations, relatively big
climatic differences do occur within the country. The herds that were visited were located in
all corners of the country, from the dikes in Zeeland to the hills in Limburg and from the
heather in Drenthe to the sand dunes of Den Haag. As the climate between these areas differ,
this brings a bias to the climate factor.
Questionnaire: piloting is essential
Nothing as difficult as speaking a language everybody understands. Piloting the questions of
the survey helped to evaluate what most shepherds found a logical and understandable use of
words. However, it proved impossible to overcome all difficulties in understanding the
questionnaire. This is reflected in the multiple use of the box ‘other, namely’. And even
though this box was put there for that reason, it is a difficult box to process in the results.
Most answers given in the ‘other, namely’ box were written answers. After consulting the
shepherds concerned, these written answers were then also processed as ticked boxes, to be
able to use them in the statistical analysis. A few written answers could not be processed into
a tick box, these answers were left blank in the final analysis.
Results: small number of flocks (N)
The abomination of data between expected and found values could possibly be explained by
the small N that is used in this research. This was caused by the sheer absence of more
shepherds with sheep that fitted the inclusion criteria. Therefore, a suggestion for further
research is to expand the test group to other Northern European countries. Other suggestions
for expanding this research is to relax some of the inclusion criteria, or include all shepherds
in the research and then make groups based on their answers. However, with this last
suggestion, one could end up with more groups instead of bigger groups.
Results: explaining unexpected high EPG counts
There could be several possible reasons for the high EPG counts in spite of following a
rotational grazing plan. Taylor, et al. (2007, p167) states that calves can also be hosts of N.
battus. Thus, high EPG counts of N. battus in herds could be explained by switching ruminant
grazing on the pastures. This has as a result that the pasture is not laid fallow and therefore
eggs will continue to be deposited on the pasture and then hatching to infective L3 larvae
resulting in an instant infection of the sheep when they return to graze.
Taylor, et al. (2007, p167) also says that deer and horses can be host to strongylus- type
worms. As herded sheep tend to graze on nature reserves and other remote areas, infection
through larvae from deer or passing horses is an option. The chance that there are many
infective larvae from deer and/ or horses in nature reserves however is small, as these animals
are small in number and diffused over a large area. And because the infection with strongylus-
21
type worms in most sheep is quite above the estimate, it is not probable that this comes from
ingesting an infective larvae from deer here and there.
An explanation for finding high levels of Nematodirus battus in November is mentioned in
paragraph 2.2.3. Sometimes eggs of Nematodirus battus that are deposited in spring are
capable of hatching in autumn of that same year resulting in infectious L3 larvae in autumn.
And because the prepatent period is 14- 16 days, this could result in high faecal egg counts in
November. However, when this would be true, the sheep were expected to show signs of
disease at the time of sample taking, because clinical signs already may occur during the
prepatent period.
A last explanation has a different perspective. An assumption made in this research is that
herded sheep are extensively held, grazing large areas with relatively few sheep. When
infected with worms, this does not pose a great threat because of their widely scattering of the
eggs with the faeces. In reality was observed, while collecting the samples, that the sheep
were packed together tight in flexinets to graze a pasture as efficient as possible. This practice
results in high grazing pressure. When sheep get infected with worms somewhere, only one
flaw in management will suffice, they deposit their acquired eggs not widely but on a small
area. This results in a high worm pressure in this area. On returning the sheep, and again
packing them tight in flexinets, the remaining infective larva will immediately be ingested by
the sheep, thus maintaining the worm cycle.
Thoughts on standardized EPG counts
Although not mentioned in the sector ‘results’, when visiting the herds visual observations
were made considering the health of the flock. This is not mentioned in the result because no
hard evidence has been gathered. For this study 267 sheep have been sampled. Most of them
looked perfectly healthy. No weight loss, no signs of extreme thirst, no trouble with the
fleece. Only a small number of them, approximately 5 percent, showed signs of acute
diarrhoea, and approximately 10 percent showed signs of a diarrhoeic disease that had already
passed. This could be seen as dirty faeces- packed wool under the tail of the sheep. These
percentages are estimated by the researchers. Considering these observations and the result of
the McMaster EPG counts, perhaps the cut- off values described by ‘De Wormenwijzer’ are
too sharply defined, possibly indicating disease sooner than that it actually becomes a
problem. A possible explanation for this difference in finding high EPG counts and not
seeing physical signs of illness is that the definition of the cut- off value is meant for a
different target group, meaning sheep that are traditionally farmed. The farmers of these sheep
have as goal to raise as many heavy lambs as possible, for every kilo earns the farmer money.
This fact makes it understandable to treat lambs with anthelmintics before clinical signs
appear and thus before production losses occur, therefore the cut- off value of the McMaster
test should be sharply defined, preferably before clinical signs appear.
Another possible explanation between finding high EPG counts and sampling seemingly
healthy sheep is that not all eggs counted cause pathologic signs. An example of this is
Chabertia ovina, a worm that produces eggs that affect the total EPG count of strongylustype worms, while the clinical effects of infection with this nematode are minimal. Eggs of
this nematode are slightly larger than eggs of other strongylus- type worms like Haemonchus
contortus.
Further research
22
Expanding this research to a larger N could be helpful in showing the relationship between
EPG counts and rotational grazing schemes. When this research is expanded, inclusion
criteria should be stricter. Based on what Taylor says, pastures should be grazed only 3.5 to
four days instead of sometimes even 14 days. However, this is for warmer regions of the
world than the Netherlands and also, according to the lifecycle of nematodes, this period is
very short. Eysker, M., et al. (2005) has done research on development of nematodes
specifically in the Netherland, and he states that it takes two weeks before dangerously high
pasture infectivity levels are reached in summer and three weeks in May, which has a lower
temperature. Based on this information, pastures can be perfectly well grazed up to 14 days.
However, the return time should be expanded to a longer period than a minimum of two
weeks, for in his research Eysker says that it taker at least three months before pasture
infectivity had decreased to low levels again
Even though the hypothesis as stated in this research is not confirmed, still a combination of
rotational grazing schemes and anthelmintic treatment could be tested in order to find out if
that is a good way to minimize use of anthelmintic drugs in the future. Then research could
also be done on how much costs are saved and what kind of investment has to be done to
realise the implementation of a rotational grazing plan in the traditional sheep farming
industry. This is important for the realization and implementation of such a new management
plan.
Another adjustment that should be done when this research is expanded, is having a further
determination of the eggs performed to see which nematodes actually have infected the sheep
and whether these can be expected that time of the year and whether they can cause clinical
signs. That research has been done parallel to this research by Femke van Biezen who
identified the eggs found in this research by developing them into larvae. For further
information read her full report.
A last change that I would advise for adjusting this research is taking the anthelmintic
treatments that the lambs have received into account. These treatments have possibly been
great confounders in this research because all of them have different kinetics and dynamics
and different influences on the sheep and sometimes even the pasture they graze on.
23
7) Literature
Borgsteede, F.H.M., Dercksen, D.D., Huijbers, R.; Doramectin and albendazole resistance in
sheep in The Netherlands; (2007); Veterinary Parasitology, 144 (1-2), pp 180-183.
Borgsteede, F., Verkalk, J., Moll, L., Dercksen, D., Vellema, P., Bavinck, G.; How
widespread is resistance to invermectin among gastrointestinal nematodes in sheep in the
Netherlands?; (2010); Tijdschrift voor Diergeneeskunde, 135 (21), pp 782-785.
Eysker, M., et al.; Consequences of the unusually warm and dry summer of 2003 in The
Netherlands: Poor development of free living stages, normal survival of infective larvae and
long survival of adult gastrointestinal nematodes of sheep; Veterinary Parasitology 133, Issue
4; (2005); pp 313-321
Eysker, M., N. Bakker, F.N.J. Kooyman, H.W. Ploeger; The possibilities and limitations of
evasive grazing as a control measure for parasitic gastroenteritis in small ruminants in
temperate climates; Veterinary Parasitology 129; (2005); pp 155-164
Gillham, B.; Developing a questionnaire; Continuum International Publishing Group; (2000);
p 30
KNMI: http://www.knmi.nl/klimatologie/maand_en_seizoensoverzichten/seizoen/her12.html;
Klimaatdata- en advies, De Bilt, consulted on 10- 12- 2012
Kloosterman, A., H.K. Parmentier, H.W. Ploeger; Breeding cattle and sheep for resistance to
gastrointestinal nematodes; (1992); Parasitology Today, vol. 8, no. 10; pp 333-334
Oppenheim, A. N.; Questionnaire Design, Interviewing and Attitude Measurement; Basic
Book Inc.; (1992); pp 8- 9 & 21-22
Oppenheim, A. N.; Questionnaire Design and Attitude Measurement; Heinemann Educational
Books Ltd.; (1966); pp 46 & 34
Taylor, MA., RL Cool, RL Wall; Veterinary Parasitology; Blackwell Publishing; (2007);
ISBN: 978- 4051- 1964- 1; pp 157-168
Waller, P.J.; International approaches to the concept of integrated control of nematode
parasites of livestock; International Journal for Parasitology 29; (1999), p155-164
Wormenwijzer, WUR: http://www.wageningenur.nl/nl/show/Wormenwijzer-1.htm, consulted
on 15-12- 2012
Zajac, A. M.; Gastrointestinal Nematodes of small Ruminants: life Cycle, Anthelmintics and
Diagnosis; Vet Clin Food Anim 22; (2006); pp 529- 535
24