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The contribution of red poultry mites (Dermanyssus

gallinae (Degeer 1778) (Acari: Dermanyssidae)) to the


cross contamination of poultry with Campylobacter spp.
and Salmonella spp.
and the acaricidal effect of carvacrol, thymol, bay oil and
neem oil on Dermanyssus gallinae

Research Project Veterinary Medicine University Utrecht


Astrid van Sauers

Supervisor: Dr. Sara A. Burt


Department of IRAS, Veterinary Public Health Division
February 2009.
Student: Astrid van Sauers
Student number: 0258571
Email address: A.R.vanSauers@students.uu.nl
Address: Ina Boudier Bakkerlaan 117-4, 3582XP, Utrecht
Phone: 0642135944

Supervisor: Dr. Sara A. Burt


Department: IRAS, Veterinary Public Health Division
Address: Yalelaan 2, 3584 CM Utrecht, Nieuw Gildestein
Phone: +31 30 2535350
Email: S.A.Burt@uu.nl

Keywords: Dermanyssus gallinae, campylobacter spp., Salmonella spp.; acaricidal effect;


thymol, carvacrol; bay oil; neem oil.

1
Contents
The contribution of red poultry mites (Dermanyssus gallinae (De Geer
1778) (Acari: Dermanyssidae)) to the cross contamination of poultry with
Campylobacter spp. and Salmonella spp.
Page

Summary 3
Introduction 4
Material and methods 9
Results 12
Discussion 13
Conclusion 15

The acaricidal effect of carvacrol, thymol, bay oil and neem oil on
Dermanyssus gallinae

Introduction 16
Material and methods 18
Results and discussion 20
Conclusion 30
Acknowledgments 31
References 32

2
Summary

Food borne infections are the cause of many illnesses and cost the
Netherlands tens of millions of euro’s every year. The most important food
borne pathogens are Campylobacter spp. and Salmonella spp. Poultry
products are important carriers of these pathogens. This is why it is important
to eradicate these pathogens from the poultry system. Poultry mites have
infested more than 80 % of the Dutch poultry and cost the poultry industry
millions every year according to the Animal Sciences Group. In this study
poultry mites were tested if they carried Campylobacter spp. and Salmonella
spp. and in this way contributed to the cross infection of poultry flocks. It was
concluded that the red poultry mite, Dermanyssus gallinae De Geer, does not
carry Campylobacter spp. and thus the infection of the mites can not be
correlated with the Campylobacter status of the farm. Correlation between the
Salmonella status of the flock and infection of the mites could not be tested in
this study for there were no flocks found positive for Salmonella.
The second aim of this study was to test the acaricidal effect of carvacrol,
thymol, bay oil and neem oil on Dermanyssus gallinae using direct contact
and vapour. For the contact test carvacrol and thymol had the highest mean
mortality percentage of a 100 % at 0.5 % suspension, followed by bay oil and
neem oil. Significant difference between acaricidal activity at different
temperatures (20°C and 30°C) were only found for bay oil at 0.5 % and neem
oil at 2 %. Carvacrol and thymol also had high mean mortality percentages
(approximately 85 %) in the vapour tests, but never 100 %; indicating that
contact with these substances is more effective than vapour. Looking at the
acaricidal effectiveness of carvacrol and thymol, they can be considered for
practical use in the future.

3
Introduction

Food borne infections are the cause for many illnesses and approximately 80
deaths in the Netherlands every year. They also cost the Dutch society tens of
millions of euros every year (1, 2, 3, and 4).
According to the RIVM and VWA, food-borne infections caused by
Campylobacter spp. and Salmonella spp. have dropped considerably the last
few years (1, 2, 3, and 4). But still eradication is not yet reached. It is
important for humane and economical reasons, but also for political (EU)
reasons to diminish these food-borne pathogens. Both Campylobacter spp.
and Salmonella spp. are the causative agents for half of the food borne
infections (5). According to the RIVM 40% of the reported food borne
diseases in 2007 had Campylobacter spp. as the causative agent, while
another 40% had Salmonella spp. as the causative agent. YOPI’s (the young,
old, pregnant and immunosuppressed) have a higher risk for getting a (more
severe) food borne disease.
Campylobacter spp. bacteria are gram negative, spiral, microaerophilic
bacteria and are motile with the use of flagella. Salmonella spp. bacteria are
rod-shaped, gram negative enterobacteria, also motile with the use of flagella.
In the Netherlands Campylobacter spp. is the most frequent agent for causing
diarrhea. Most illnesses are caused by C. jejuni spp. Symptoms include fever,
(bloody) diarrhea, vomiting, headache, stomach-ache and nausea. Mostly
people get infected by consuming infected food (especially meat products),
water or by direct fecal-oral contact with infected animals. Important
reservoirs include poultry, raw meat, unpasteurized milk, raw fish and
contaminated water. Dogs, cats and other animals can also be an important
reservoir (6). Campylobacter spp. are not carried over by means of vertical
transmission via eggs therefore infections in chickens usually occur by
horizontal transmission (7).
Salmonella spp. is divided in the typhus (S. enteridis subspecies
enterica serotypes Typhi and Paratyphi) and the non-typhus form. The most
common Salmonella involved in food-borne infections are the non-typhus
forms S. enteridis subspecies enterica serotypes Enteritidis and
Typhimurium. The non-typhus form causes diarrhea, vomiting, nausea, fever
and headache. Approximately 8% of the laying hens in the Netherlands are
infected with Salmonella spp. Eggs are the main cause for human
Salmonellose. Other important infection routes include contact with infected
animals (carriers). More than 80% of the Salmonella infections are caused by
eating infected raw food (8). Systemic infections are often caused by the
typhus form of Salmonella. Symptoms include high fever, anorexia, headache
and often diarrhea. This form of salmonellosis in humans often originates
from a source other than poultry.

4
Infections in the poultry industry usually occur through horizontal
transmission between animals. Vertical transmissions (via eggs) are blocked
by eradicating infected breeder flocks (8, 9).
The approach of the Productschappen Vlees, Vee en Eieren (PVE) is striving
to complete eradication of Salmonella spp. in poultry products. It has recently
been established that all poultry meat should be Salmonella free by January
1st 2011, according to European guidelines (10). Therefore more severe
measures must be taken to eradicate Salmonella spp. in the poultry industry
(11, 12).

Poultry mites are considered a problem in the whole poultry system. So far
known eradication of the mites is still not achievable in the Netherlands or
anywhere else. They can become a serious pest severely affecting the health
of the flock. Mites can cause serious blood loss, high feed conversion,
weakness, weight loss, production losses, more susceptibility to diseases and
death in poultry. They can be a carrier of important poultry pathogens like E.
coli, Paramyxovirus 1(New Castle Disease), typhus, Salmonella spp. (13),
Borelia anserina, Pasteurella multocida (14), Mycobacterium spp. (15), and
Erysipelothrix rhusiopathiae (16). They are the cause of restlessness at night,
dermatitis, loss of feathers and feather picking/cannibalism. These mites can
also cause dermatitis, skin irritation and ear infections in humans (18). The
most common bird mite in the Netherlands is the red poultry mite,
Dermanyssus gallinae. This was the only mite found during sampling in this
study.
D. gallinae mites only spend their active feeding times on the bird and retreat
to nearby protected locations after feeding. A small portion of the poultry
mites will let themselves fall from the chicken after a meal. Poultry mites can
incidentally bite other animals than poultry, but will consume and produce
less. They can survive for months without feeding and complete its life cycle
in a week under favorable conditions (17). The mites are attracted to higher
carbon dioxide concentration (breath), movement and warmth. They always
try to stay very close to the chickens. The most optimal temperature and
humidity for development and reproduction for the mites are respectively
±25oC and ±76 % RH (17). Its lifecycle contains five stages respectively egg,
larva, protonymph, deutonymph, and the adult stage.
They will bite humans they encounter but cannot reproduce on humans (17).
Because laying hens have longer (longer than broiler flocks) lifecycles, mites
have a better opportunity to acquire a balance and growing into large
colonies. The mites need about five months to acquire a natural balance.

5
Fig 1.
Dermanyssus gallinae.

Laying hens in caging systems seem to have the highest rates of infection
according to research in 2005. This fact could have multiple reasons. For one,
caging systems have a (dry) manure belt which gives the mites an easier way
to migrate back to the chickens if they fall on the belt. The second reason is
that mites have more opportunities to hide from pesticides in caging systems.
Caging systems (87 %) were followed by free-range (83 %), birdhouse (82
%) and organic (78 %) systems (17).
Methods for control can be mechanical (i.e. vacuuming, burning), physical
(cryogenically, heating method), biological (silica, biodiesel, natural enemies,
mix of green soap, spiritus and chalk), physiological (garlic, vitamin B2) and
chemical. Methods with the best results and which are most used are the
biological (biodiesel, natural enemies and silica), some chemical substances
and by heating. The heating method takes 5 days, whereby the shed is heated
above 45°C. Above this temperature the eggs will also be destroyed, but it is
expensive, the shed inventory will be negatively influenced and the mites will
try to escape while the temperature increases. Chemical substances containing
cyfluthrin (Solvac), dichlorvos (Lurectron) or pyrethrin insecticides are also
used against mites. A negative effect of the long term and repetitive treatment of
the products is the risk of resistance against it, which has also been proven (17).
Pyrethrins are nerve poisons. They destroy the nerve conduction, which can
paralyze insects and mites. They will over stimulate the nervous system and
induce paralysis. Cyfluthrin is a synthetic pyrethroid, derived from pyrethrins.
Dichlorvos is a highly volatile organophosphate, which is also a nerve poison.
This substance gives paralysis by blocking acetylcholinesterase (19). Insect
Growth Regulators (IGR) also seem effective against mites, but these synthetic
products are not on the market currently.

6
Fig 2.
D.gallinae in the poultry shed.

There is use of illegal chemical products like nicotine, which seems to be


effective according to farmers using the substance. Nicotine is found not to be
significantly harmful for the health of the consumer or chickens, but is harmful
to the handler (20). Nicotine is a tobacco derived alkaloid, which has significant
contact toxicity to mammals. Nicotine and its derivates are used as pesticides.
Nicotine mimics the action of acetylcholine, which causes paralysis (21). A
biological method that is upcoming is the use of natural enemies like
Dutchy’s®. These predator mites eat the bird mites (22). Darkling beetles,
Alphitobius diaperinus, could also be used as effective predators of bird
mites, but they have too many harmful side effects. They can cause damage to
the inventory and be carriers for various pathogens like Salmonella and
Campylobacter (23). They lay their eggs in manure, so with the manure belt,
the manure is scraped away too fast and they can poorly survive. Chickens
are also an important predator of the beetles. These problems do not make the
darkling beetle ready for practical use (24).
Some of these methods can be quite effective, but eradication is still not
possible.
It is estimated that red poultry mites can cost a farm about 1.5 euros per
chicken (25). According to the Animal Sciences Group (ASG) bird mites
cause the sector tens of millions every year (26).

7
Mite colonies remain in the sheds throughout rearing cycles. As a
consequence of this persistence, the possibility for horizontal transmission of
pathogens like Campylobacter spp. and Salmonella spp. and therefore
infection of sequential cycles of poultry is possible. Because of the high
priority to diminish Campylobacter spp. and Salmonella spp. from the poultry
industry, eradicating possibly infected mites can be another way to reach that
goal. It is the aim of this study to find out if poultry mites collected from
poultry farms carry Campylobacter spp. or Salmonella spp. and if this can be
connected to the Campylobacter and Salmonella status of the flock.

8
Material and Methods

Our goal was to see if bird mites collected from commercial poultry farms
carry Campylobacter spp. and Salmonella spp. Ten poultry sheds were
sampled (coded I to X). Different samples were taken from each visited farm,
each labelled according to the shed number.
Mite samples were composed of mites collected from 3 locations in the shed
by scooping mites into sterile plastic tubes. The tubes were labelled according
to shed number.
The poultry flock status was also checked by taking faecal samples.
Faecal samples were collected by using sterile overshoes (2 pairs per shed).
Each one was well smeared with faecal excreta. In the case of caging systems
the overshoes were wiped over the manure-belt in every row by hand. The
pairs of overshoes were divided over two sterile stomacher bags in the end
and labeled. One pair of overshoes was used for the determination of the
Salmonella status of the flock. The other pair was used for determination of
the Campylobacter status of the flock. The Campylobacter status was also
determined by using caecal excreta (according to PVE procedures). These
were collected in sterile plastic tubes using sterile spatula or spoons. The
sensitivity of two methods for determining the Campylobacter status was
compared.
Samples were not cooled during transfer to the laboratory and were used for
tests the same day of sampling. Cooling was not necessary because
Campylobacter spp. is very sensitive to cooling and enrichment was desired.
In the lab the mite samples were transferred into two sterile plastic bags and
crushed lightly by rolling a glass bottle over the bag. Mite and overshoes
samples were tested for Campylobacter spp. and Salmonella spp. Caecal
samples were tested for Campylobacter spp. only.

For the detection of Campylobacter spp. the following protocol was used.
1. Preston Broth (PB) without horse blood (Nutrient broth base nr 2 Oxoid
CM067 with Preston Campylobacter supplement SR0117) was used as
selective enrichment medium. Under microaerophilic conditions,
incubation was done for 48 hours at 41.5oC with shaking.
Microaerophilic conditions were accomplished by using a gas generator
kit (Oxoid CampyGen CN0025) in an anaerobic jar (Oxoid AG0025).
The sachet contains ascorbic acid, which produces carbon dioxide out
of the absorbed oxygen.
 To the sterile stomacher bag containing the overshoes, 250 ml PB
was added and massaged for 90 seconds in a stomacher machine. It
was also massaged by hand to ensure that the exterior of the
overshoes was moistened.
 Mites were put into a bag were 10 ml PB was added.

9
 The caecal samples were inoculated onto Campylobacter blood-free
agar (CCDA) (Oxoid CM0739 with SR0155) plates using an
inoculation loop. These were incubated under microaerophilic
conditions for 48 h at 41. 5oC. After that proceeded to the
morphology and motility check.
2. From PB, 10 µl was inoculated onto Campylobacter blood-free agar
(CCDA) (Oxoid CM0739 with SR0155) using an inoculation loop.
These were incubated under microaerophilic conditions for 48 h at
41.5oC. Typical Campylobacter colonies produced on this medium are
flat, glossy and effuse. Well-spaced colonies resemble droplets of fluid.
3. Typical colonies were chosen for morphology and motility check using
hanging drop under light microscope. Campylobacter species are
highly motile slender rods with curved or spiral morphology. Motility
is characterized by darting or corkscrew like movements.
4. Filter strips impregnated with 0.1% N, N, N', N'-tetramethyl-1,4-
phenylenediammonium-dichloride (Merck-Schuchardt, S04089 752)
were used for oxidase testing. Campylobacter species are oxidase
positive.
5. If all of the above showed reactions typical for Campylobacter,
serological agglutination was carried out (using M46 Microgen®
Campylobacter).
6. Preservation of the isolates for possible future study was done at -80oC
using Microbank™ vials (Pro-Lab Diagnostics®).

For the detection of Salmonella spp. the following protocol was used.
1. Buffered Peptone water (BPW) (Oxoid CM0509) was used as non-
selective enrichment medium. Incubation was done for 24 hours at
37oC with shaking.
 To the sterile stomacher bag containing the overshoes, 250 ml
BPW was added and massaged for 90 seconds in a stomacher
machine. It was also massaged by hand to ensure exterior of
overshoes was moistened.
 The mites were put into a bag were 10 ml BPW was added.
2. Three drops of suspension from BPW was transferred (after careful
mixing/shaking) onto modified semi-solid Rappaport Vassiliadis agar
MSRV (Oxoid CM0910 with selective supplement SR0161). These
were incubated for 24 h at 41.5oC. Motile microorganisms (like
Salmonella) show a halo of growth originating from the inoculation
spot.
3. Bacteria taken from the edge of typical motile growth were transferred
to Xylose-Lysine-Desoxycholate agar (XLD, Oxoid CM0469) and
incubated for 24 h at 37oC. Salmonella colonies may have large, glossy
black centres or may appear as almost completely black colonies.

10
4. Typical colonies on XLD were continued for the following biochemical
tests: Triple sugar iron agar (TSI), Lysine decarboxylase (LDC) and
Ureum agar (U). These were incubated for 24 h at 37oC. These typical
colonies were also cultured on Tryptone soy agar (TSA) for 24 h at
37oC for possible agglutination testing. Typical for Salmonella in TSI
is an alkaline (red) slant and acid (yellow) butt, with or without H2S
production (blackened agar). The LDC test will be positive changing to
an alkaline condition, indicated by a purple colour. And the U tests will
be negative (yellow, no colour change) for Salmonella.
5. If all of the above biochemical reactions were typical for Salmonella,
colonies on TSA were used for serological agglutination (using
Enteroclon anti Salmonella Gr. D TR1203).
6. Preservation of the isolates for possible future study was done at -80oC
using Microbank™ vials (Pro-Lab Diagnostics®).

Positive control tests were run simultaneously. Campylobacter jejuni ATCC®


33291 (Oxoid Culti-loop C1400L) and Salmonella enteritidis ATCC® 13076
(Oxoid Culti-loop C8200L) were used for the positive control tests.

11
Results

Table 1
Results of Campylobacter spp. and Salmonella spp. detection in the samples taken from the
poultry farms
I II III IV V VI VII VIII IX X
Poultry Farms

Species Samples
Mites - (a) - - - - - - - - -

Campylobacter Overshoes + (b) + + + - - + - - +


spp.
Caecal + + + + + + + - + +
excreta

Mites - - - - - - - - - -
Salmonella
spp. Overshoes - - - - - - - - - -
(a)
- represents sample which was found negative.
(b)
+ represents sample which was found positive.

Campylobacter spp. was detected on every farm, except on farm VIII. Looking
at the growth on the CCDA plates, there was significant difference between
caecal and overshoes samples. Campylobacter spp. were not detected in
overshoes samples in 3 of 9 cases, although it was detected in the caecal faeces.
Campylobacter spp. detection in caecal faeces is more sensitive, than the
detection in overshoes samples. Detection of Campylobacter spp. in caecal
faeces is 30% more sensitive than detection in overshoes samples. Salmonella
spp. were not detected on any of the farms.
Extra mite samples were received from an unknown farm. These mites were
known to have fed on possible Salmonella-positive chickens. These mites were
found to be Salmonella spp. negative. There were no overshoe samples taken
from this farm. So the Salmonella status of the flock was not checked.
Darkling beetle (Alphitobius diaperinus) samples were also taken from
Campylobacter positive farm № IV. These were Campylobacter spp. and
Salmonella spp. negative.

12
Discussion

Campylobacter spp. were detected on almost every farm. The farms sampled
were all egg-laying farms. As for the fact that Campylobacter spp. are not
transmitted vertically, there is no direct motive to eradicate Campylobacter spp.
from these farms.
Salmonella spp. was not detected on any of the ten sampled farms. This seems
reasonable because only eight percent of the Dutch laying hens are Salmonella
spp. positive.
The mites are attracted to higher CO2 concentration (breath), movement and
warmth. They always stay close to the chickens. The most optimal temperature
and humidity for development and reproduction for the mites are respectively
±25oC and ±76 % relative humidity. The mites mostly live in dark crevices near
the chickens perches or nest (18).
The lifestyle of the mites might have influence on them being able to become
infected with these pathogens. They do not live on the ground were faeces are
dropped and so normally do not come into contact with fresh faeces. One of the
ways for poultry mites to get contaminated with Campylobacter or Salmonella is
trough a blood meal. To make this happen the chickens would have to be
systemically infected with these gastrointestinal organisms. Another way is to
get infected is through direct contact with fresh contaminated faeces. A small
portion of mites will fall after a blood meal or while migrating and can then
come into contact with fresh faeces. Because Campylobacter spp. and
Salmonella spp. are transmitted through faeces, there is little chance of mites
becoming infected with these pathogens. And it is unknown if and for how long
Campylobacter could survive in or on the mites. It is also unknown if
Campylobacter would still be detectable in the mites. It has been experimentally
demonstrated that mites can be a vector of Salmonella by acquiring bacteria
through the blood meal or cuticular contact. Up to 14 days after experimental
infection Salmonella survived and multiplied inside the mite. Mites could also
contaminate the host (chicken) blood with Salmonella after a blood meal (13).
Studies also confirmed that chickens could get infected by eating infected blood
mites (27).
Experimentally infected darkling beetles (Alphitobius diaperinus) can be a
potential vector for Salmonella and Campylobacter (28). In this study however
one sample of darkling beetles from a Campylobacter positive farm were found
to be negative for Campylobacter. A possible explanation is that the
Campylobacter numbers were too low to be detected. It is described in another
study (29, 30) that some insects have antimicrobial effects. This could also be
the case for Campylobacter in darkling beetles.
There were mites sampled from dry faeces in this study. It is expected that these
faeces were too dry and cold for Campylobacter spp. to survive in.
Campylobacter spp. are thought to be environmentally fragile and easily killed

13
by freezing, heating and oxygen. A higher Campylobacter incidence was found
at higher relative humidity (RH) and temperatures in sheds (31, 32). The
negative result of Campylobacter in the darkling beetles sample in this study
could also be explained by the critical temperature or humidity levels for
Campylobacter in the beetles.
If ventilation and heating systems are not properly managed this could lead to
problems with the RH. Especially in winter some farmers complained of having
problems with their ventilation. They also stated that mite infestation was higher
at places in the shed where it was warmer and less ventilated. Optimal
ventilation would lead to temperatures too low, but low ventilation could lead to
a high RH. High RH will lead to a higher contamination of Campylobacter (31,
32) and Salmonella (33) but also higher mite prevalence.
By changing the climate in the shed, it could be possible to create an unpleasant
environment for the mites and other pathogens. This could be done by lowering
the relative humidity and temperature, respectively by drying the air and cooling
the shed. This would result in less lifecycle-development and less food intake
for the mites.

14
Conclusion
The collected mites were found to be negative for Campylobacter and
Salmonella, although most farms were found to be infected with Campylobacter
spp. So mites were found not to be carriers of Campylobacter spp. Also, no
correlation was found between the status of the flock and the infection of mites.
There was marked difference between the sensitivity of determining the
Campylobacter status of the flocks by means of examining. Campylobacter spp.
were not detected in overshoes samples in 3 of 9 cases, although it was detected
in the caecal faeces. Therefore caecal samples can be stated 30 % more sensitive
than overshoes samples in detecting Campylobacter spp.
As for Salmonella spp. the mites were not infected, but the flocks were also to
be found negative on their Salmonella status. Therefore there can be no
conclusion stated about the correlation between the status of the flock and
infection of the mites for Salmonella. Since 2000 the PVE has been trying to
reduce the level of Salmonella infection in the poultry system. The PVE’s plan
of approach has been used especially in the broiler flocks. The fact that in this
study all ten arms were found to be negative for Salmonella shows that the
Dutch poultry is well on track with its Salmonella status.

15
The acaricidal effects of carvacrol, thymol, bay oil and neem oil
on Dermanyssus gallinae.
Introduction

In the Netherlands there are but a few legal acaricidal means to kill bird mites in
the commercial poultry system and even fewer are effective. So there is a high
demand for other ways to kill bird mites.
A major issue is the residue problem. Studies (34, 35) have shown that hens
treated with N-methyl carbamate pesticides (e.g. Carbaryl and Propoxur) will
produce eggs with measurable pesticide residue for weeks. Carbaryl was
frequently used for treating mites in the Netherlands, until it was banned in 2001
by the Board for the Authorisation of Plant Protection Products and Biocides
(CTB). Carbamate pesticides are nerve poisons, which can also be toxic to
humans. These insecticides can cause cholinesterase inhibition poisoning by
reversibly inactivating the enzyme acetylcholinesterase. For the chemical
control against mites, cyfluthrin (Solvac) and dichlorvos (Lurectron) are
approved in the Netherlands. Other chemicals like pyrethrin insecticides are also
used, but are not registered for use against mites.
Other problems are resistance (36, 37 and 38) against repetitively used
chemicals, environmental concerns, risks to human health (handler), undesirable
effects on nontarget organisms and economic reasons. Resistance of poultry
mites to pyrethroids have been found in France (36). In that study the
concentration of permethrin required to kill 50 percent of the mites on five
tested farms, who had experienced problems with the control of mite
populations, was between eight and 40 times the concentration required on the
control farm. The control farm did not experience problems with control of the
mite populations.
Safe products will be a welcome acaricidal alternative.
Research shows acaricidal effects of the essential oils and neem oil (39, 40, 41,
42, 43 and 44). The acaricidal effect of bay oil was also tested on Dermanyssus
gallinae (44).
West Indian Bay essential oil is extracted from the Pimenta racemosa tree, a
plant originating from the Caribbean. It is traditionally used in local folk
medicine. Bay oil is also known to have antifungal (45) activity and antibacterial
(46, 47) activity. Antibacterial activity was tested on Escherichia coli,
Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis and
Staphylococcus aureus.
Neem oil is a vegetable oil pressed from the fruits and seeds of Neem
(Azadirachta indica), an evergreen tree which is endemic to the Indian
subcontinent. Like bay oil, neem is traditionally used in local folk medicine and
cosmetics. Neem oil consists of many components, of which azadirachtin
especially is known to have insecticidal properties (48). Azadirachtin derivatives

16
belong to the group of triterpenoids and are found in greatest concentrations in
the seed core. Besides its effect on insects, neem can also be used to control
nematodes, phytopathogenic fungi and snails (48, 49, 50, and 51).
Neem is not toxic to warm-blooded organisms and is harmless to the majority of
beneficial organisms. Neem has a low risk of pest resistance developing,
because many of the active extracts differ in their mode of action (48).
The essential oils carvacrol and thymol are known to have antimicrobial (52,
53), acaricidal (39, 43, 40 and 42) and insecticidal (54) activity. Thymol and
carvacrol are two major components of certain herbs such as oregano and
thyme.
In this study the acaricidal activities of carvacrol, thymol, bay oil and neem oil
are tested against Dermanyssus gallinae. These substances are tested for their
acaricidal effect via the route of contact and vapour (thymol and carvacrol only).

17
Material and Methods

Mites, in this case D. gallinae, were sampled from different poultry farms. The
mites were separated from the dust by using a pin. In this way about ten or
twenty mites (nymphs and adults) were separated for each sample. These were
kept at 5°C until they were needed for this test.
Different agents were used to dissolve the substances. Alcohol (99.8%), Tego
(90g/l) and DMSO (dimethyl sulfoxide (DMSO), 78.13 g/mol) in different
concentrations were tried. But the solvent results varied for the different agents.
Finally, in order to compare the substances with each other, acetone was used as
a common solvent. Each test was repeated three times at two different
temperatures, namely 20 and 30 °C.
Mortality was determined 24 h. post-treatment using a pin. The mites were
considered dead if they did not move after poking.

Thymol contact test with DMSO as solvent


To evaluate the effect of thymol on D. gallinae, pure samples of thymol were
tested at three concentrations and two temperatures (20 and 30°C). Thymol (5-
methyl-2-isopropylphenol, EEC 201 944 8, Sigma®) was used in three
concentrations namely 1, 0.5 and 0.25 %. Thymol is not easily soluble in water,
which is why it was emulsified in aqueous dimethyl sulfoxide (DMSO, 78.13
g/mol) 1% at 60°C. The negative and positive controls were, respectively,
DMSO at 1% and Bogena Birdspray (Permethrin 5 g/L, Beaphar®, spray 6
times for enough absorption on the filter paper).
Filter paper (qualitative circles 90 mm Ø, 4, Whatman®, which can absorb
about 1 ml fluid) was rolled up and placed in a screw-topped sterile 15 ml
plastic tube (Greiner Bio one®).
1 ml of fluid was added to the filter paper in the flacon. Then ten mites (D.
gallinae) were added to the filter paper. The tubes were laid on their side at
specific temperature for 24 hours.

Contact comparison test with acetone as solvent


Bay oil (C. Melchers Essential Oils®) and neem oil (Neem Nimba Margosa
Indian-Lilac Nim Mambo®) were also used for the toxicity test. Acetone was
used as solvent for all substances. All the substances were easily soluble in
acetone. After dissolving, the 1 ml of the solution was pipetted on a filter paper
(qualitative circles 90 mm Ø, 4, Whatman®, which can absorb about 1 ml fluid).
The acetone was allowed time to evaporate I a fume cupboard for 1 minute.
Thereafter the substance remained on the filter paper and this was rolled up in a
tube (sterile PP-tube, 15 ml, Greiner bio one®). To compare all the substances,
they were uniformly solved in acetone. The 20 mites were placed in the tube for
24 hours at two temperatures (20 °C and 30 °C). For a negative control only
acetone was placed on a filter paper and given time to evaporate. The positive

18
control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®, for enough
absorption spray 6 times on the filter paper). For this test four concentrations
were used namely 2.0, 1.0, 0.5 and 0.25 %. Carvacrol and thymol were tested in
concentrations of 1, 0.5 and 0.25 %. Neem was tested in concentrations, starting
with the 1 % concentration. When this concentration seemed inadequate the 2 %
concentration was used. For bay oil concentrations of 1.0 and 0.5 % were used.

Vapour test
In this test 0.5 % w/v thymol was used for the vapour test. Filter paper
(qualitative circles 10 mm Ø, Whatman®, which can absorb about 0.125 ml)
was put on the bottom of a tube (sterile PP-tube, 50 ml, Greiner bio one®). Then
the substance was added. Carvacrol (98 %) was tested on mites. For the damp
test 0.1 ml was poured on a filter paper (qualitative circles 10 mm Ø,
Whatman®, which can absorb about 0.125 ml) on the bottom of the tube (sterile
PP-tube, 15 ml, Greiner bio one®). After that 20 mites were added on then side
of the tube, so that they would not fall to the filter paper. After 24 hours at 20 °C
and 30 °C the mortality rate was checked.

Statistical analysis
SPSS version 16.0 software was used to compare results by analysis of variance
(ANOVA) between the tested oils, different concentrations, temperatures and
controls. The Tukey honestly significant differences Post Hoc test was
performed on the data to determine significant differences among the various
concentrations of the tested oils.

19
Results and discussion

Table 2.
Mortality percent and mean percent mortality with standard deviation (SD) of the toxicity
tests of different concentrations of thymol (w/v 1% DMSO) against Dermanyssus gallinae at
20 and 30 °C after 24 hours of exposure.

Mean Mean
mortality mortality
N (%) and (%) and p
Substance Mortality (%) at 20 °C SD Mortality (%) at 30 ° C SD
Negative control* 5 10 5 6.6±2.8 5 0 5 3.3±2.8 0.230
Positive control** A 100 100 100 100 100 100 100 100
Thymol (%) 1.00 A 100 100 100 100 100 100 100 100
0.50 A 100 100 100 100 100 100 100 100
0.25 A 100 100 90 98±5.8 100 100 100 100 0.374

*the negative control was DMSO at 1%.


**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).
N- Indicates no significant difference between mortality means of pairs.
p Indicates the significant difference between the two temperatures’ mortality rates.

100
90
80
70
Mean mortality %

60
20°C
50
30°C
40
30
20
10
0
Neg* Pos** 0.25 0.50 1.00
Thymol %

Fig 1. Mean mortality percentage with SD of the thymol contact test with DMSO as
solvent. Thymol was dissolved in 1 % DMSO. The mortality of Dermanyssus gallinae
was tested at 20 and 30°C for 24 hours.
*the negative control was DMSO at 1%.
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

20
Table 3.
Mortality percent and mean percent mortality with SD for the toxicity tests of carvacrol, bay
oil, thymol and neem oil on Dermanyssus gallinae at 20 and 30 °C after 24 hours of exposure.
Acetone was used as solvent.

Mean Mean
mortality mortality
N (%) and (%) and p
Substance %ⁿ Mortality (%) at 20 °C SD Mortality (%) at 30 °C SD
Negative
control* B 0 0 5 5 5 3±2.8 10 5 5 10 5 8±2.8 0.101
Positive
control** A 100 100 100 100 100 100 100 100
Carvacrol 1.00 A 95 100 70 88±16 100 100 100 100 0.277
0.50 A 100 80 95 92±10.4 100 100 100 100 0.238
0.25 AC 90 90 80 87±5.8 95 100 90 95±5 0.132
Thymol 1.00 A 100 100 100 100 100 100 100 100
0.50 A 100 95 95 97±3 100 100 100 100 0.116
0.25 AC 90 85 80 85±5 100 95 90 95±5 0.070
Bay oil 1.00 C 60 60 100 73±23 70 70 85 75±8.7 0.912
0.50 E 10 10 30 17±11.5 50 90 70 70±20 0.016ª
Neem oil 1.00 BD 20 5 10 12±7.6 10 40 10 20±17 0.488
2.00 ED 5 10 20 12±7.6 40 50 40 43±5.7 0.005ª
ⁿ Percentage of used substance.
N- Indicates the mortality means between pairs that are not significantly different (p ≥ 0.05).
ª Indicates a significant difference between the two temperatures’ mortality rates (p ≤ 0.05).
*the negative control was only acetone, placed on a filter paper and given time to evaporate
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

21
100
90
80
Mean mortality %

70
60 Carvacrol
Bay oil
50
Thymol
40 Neem oil
30
20 b
b
10
0
Neg* Pos** 0.25 0.50ª 1.00
Concentration %

Fig 2. Contact test with acetone as solvent at 20°C. Mean mortality of the substances
dissolved in acetone in three concentrations (0.25, 0.5 and 1%) at 20°C for 24 hours. In
this figure the mean percent mortality of the four substances at different concentrations
can be compared.
ªDue to lack of effectiveness of neem oil, the concentration for this substance was doubled to 2 %.
b Indicates a significant difference between the two temperatures’ mortality rates (p ≤ 0.05).
*the negative control was only acetone, placed on a filter paper and given time to evaporate
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

22
100
90
80
b
Mean mortality %

70
60 Carvacrol
Bay oil
50
b Thymol
40 Neem oil
30
20
10
0
Neg* Pos** 0.25 0.50ª 1.00
Concentration %

Fig 3. Contact test with acetone as solvent at 30°C. Mean mortality of the substances
dissolved in acetone in three concentrations (0.25, 0.5 and 1%) at 30°C for 24 hours. In
this figure the mean percent mortality of the four substances at different concentrations
can be compared.
ªDue to lack of effectiveness of neem oil, the concentration for this substance was doubled to 2 %.
b Indicates a significant difference between the two temperatures’ mortality rates (p ≤ 0.05).
*the negative control was only acetone, placed on a filter paper and given time to evaporate
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

23
100
90
80
Mean mortality %

70
60 20°C
30°C
50
40
30
20
10
0
Neg* Pos** 0.25 0.50 1.00
Carvacrol %

Fig 4. Carvacrol was dissolved in acetone in three concentrations (0.25, 0.5 and 1%) at 20
and 30°C. In this figure the mean percent mortality for the two temperatures can be
compared.
*the negative control was only acetone, placed on a filter paper and given time to evaporate
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

24
100
90
80
Mean mortality %

70
60
20°C
50
30°C
40
30
20
10
0
Neg* Pos** 0.25 0.50 1.00
Thymol %

Fig 5. Thymol was dissolved in acetone in three concentrations (0.25, 0.5 and 1%) at 20
and 30°C. In this figure the mean percent mortality for the two temperatures can be
compared.
*the negative control was only acetone, placed on a filter paper and given time to evaporate
**the positive control was Bogena Birdspray (Permethrin 5 g/L, Beaphar®).

Table 4.
Shown here is the mean percent mortality and SD at 20 and 30 °C after 24 hours of exposure
with different quantities of thymol (0.5%).

Mean Mean
Mortality (%) at mortality Mortality(%)at mortality
p
Substance N 20 °C (%) and SD 30 °C (%) and SD

Negative control* 5 10 0 5±5 0 10 5 5±5 1.000

Thymol ª(ml) 1.00 A 90 95 95 93±3 90 70 70 77±11.5 0.072

0.50 A 90 100 90 93±5.7 90 50 40 60±26.5 0.100

0.25 A 50 100 90 80±26.5 60 40 50 50±10 0.140


ª Thymol 0.5%, 1% DMSO was used.
*the negative control was DMSO at 1%.
N- Indicates the mortality means between pairs that are not significantly different (p ≥ 0.05).
p Indicates the significant difference between the two temperatures’ mortality rates.

25
100

90

80

70
Mean mortality %

60

20°C
50
30°C
40

30

20

10

0
Neg* 0.25ml 0.50ml 1.00ml
Thymol (0.5 %, 1% DMSO)

Fig 6. In this figure the mean percent mortality of different quantities of thymol (dissolved in
1% DMSO) at different temperatures is compared.
*the negative control was DMSO at 1%.

Table 5.
Shown here is the mean percent mortality and SD at 20 and 30 °C after 24 hours of exposure
with 0.1ml of carvacrol (98%).
Mean mortality Mean mortality
Substance Mortality (%) at 20 °C (%) and SD Mortality (%) at 30 °C (%) and SD p
Negative control* 0 5 10 5±5 5 0 10 5±5 1.000
Carvacrol ª 0.1ml 80 60 95 78±17.5 90 85 90 88±3 0.386
ª in this test 0.1ml of carvacrol (98%) was used.
*the negative control was DMSO at 1%.
p Indicates the significant difference between the two temperatures’ mortality rates.

26
100
90
80
Mean mortality %

70
60
20°C
50
30°C
40
30
20
10
0
Neg* 0.1ml
Carvacrol 98%

Fig 7. In this figure the mean mortality percentage and SD is given for carvacrol (98%) 0.1 ml
at different temperatures.
*the negative control was DMSO at 1%.

The result of the tested oils after 24 h showed that thymol and carvacrol were
the most effective oils, followed by bay oil and neem oil. The results of the
acaricidal effects of thymol and carvacrol in this study corroborate with the
results of cited articles (39, 40, 42 and 43). The results also indicate that contact
with the substance is more effective than the vapour of the tested substance, in
the case of thymol and carvacrol. There was significant difference in mortality
rates between the thymol vapour test (1 ml thymol 0.5 % in 1 % DMSO) and 1
ml thymol solution (thymol 0.5 % in 1% DMSO). There is also significant
difference in mortality rates between the thymol vapour test (1 ml thymol 0.5 %
in 1 % DMSO) and the thymol solution (0.5 % thymol) in test were acetone was
used as solvent.
The difference between the contact and the vapour test with carvacrol was not
significant. The vapour test had lower mean mortality rates, although higher
amounts of carvacrol were used (approximately 0.098ml) in comparison to the
contact test (were the highest amount of carvacrol (1 %) used was 0.0098ml).
The difference in results may lie in the fact that in the contact test the substance
is spread evenly and in the vapour test there may not be an amount of vapour
high enough to provide adequate results. Using a solvent to increase volume
may also increase vaporization. In the case of carvacrol, a higher temperature
was more effective in all the tests. In the tests with thymol this was also the case,
except for the vapour test. In the vapour test the higher temperature gave a lower

27
mortality, when you would expect more evaporation and thus higher mortality at
higher temperatures. This was the case in a study about the inhibition of
Salmonella enterica by carvacrol vapour at different temperatures (52). Perhaps
the effective substances in thymol are less active at higher temperatures. In the
contact test where acetone was used as solvent, there was no significant
difference in acaricidal activity between the positive control, the different
concentrations of carvacrol and the different concentrations of thymol results.
This indicates high acaricidal effectiveness for carvacrol and thymol.
There was a significant difference between the negative control results and the
different concentrations of carvacrol and thymol.
For the practical use of these oils against bird mites, further research is
necessary on safety issues of these oils on human health, non target organisms
and formulations for improving the acraicidal potency and stability.
The acaricidal effect of bay oil was tested on Dermanyssus gallinae in a study.
Higher concentrations per cm² were tested. A 100 % mean mortality was
reached using a concentration of 0.07 mg/ cm² (44). In this study the lowest
concentration per cm² was 1.57*105, which resulted in a mean mortality of 70
%. There was a significant difference in acaricidal activity of bay oil and the
negative and positive controls. There was no significant difference between
carvacrol 0.25 %, thymol 0.25 % and bay oil 1.00 % in the test were acetone
was used as solvent. This indicates that the used bay oil concentrations were
probably too low and thus not as effective as thymol and carvacrol in the same
concentrations. Further research should be performed to assess potential safety
issues of this compound related to human health, the acaricidal mode of action
and formulations to improve the acaricidal potency and stability for practical use
against bird mites. For the control of bird mites, research for reducing the cost of
these essential oils is necessary.
The effectiveness of neem oil was less than expected. In this study the highest
mean mortality rate was 43 % for 2 % (w/v neem) at 30°C after 24 h. In a study
about the acaricidal effects of active extracts of neem oil, the extracts gave
mortality rates up to a 100 %. One of the extracts had an LT90 (time needed for
90 % mortality) after 20 h. at a concentration of 3 % (41). In this study crude
neem oil was tested at lower concentrations. There was no significant difference
in acaricaidal activity of neem oil and the negative control.
Neem is safe for non target organisms, the environment and has low resistance
development in pests, but it also has characteristics which may hinder its use as
a pesticide. The harvest is labour intensive, processing and use often have
technical problems and the knowledge is not extensive. Neem products are
harder to obtain, use and are often more expensive (48). In tropical conditions
the period for storage of raw materials is limited. There are but a few countries
that produce them on a large scale. Neem could be introduced to countries with
high usage potential and ability to produce and process neem themselves.

28
During the contact test it cannot be excluded that vapour of the substance also
effects mortality rate in closed tubes. These oils are tested only in closed tubes;
their effect should still be tested in the open for practical usage. In a study about
acaricidal activity of plant essential oils on Dermanyssus gallinae (44) a
significant difference in acaricidal activity between exposure in closed
containers and exposure in open containers was found. This indicates that the
vapour of the substances may be the most important factor for its acaricidal
activity.
These results suggest a significant acaricidal effect of essential oils on
Dermanyssus galinae. However there is no knowledge about the effects of these
oils on non target organisms and the environment.

29
Conclusion
These results show the acaricidal effectiveness of the essential oils and neem oil,
especially for carvacrol and thymol on Dermanyssus gallinae. These also show
the opportunity for usage of these natural products against D. gallinae. Further
research is needed to assess the safety of these natural oils on non target
organisms like chickens and humans (handler).

30
Acknowledgements
The author would like to thank Dr. Francisca Velkers, Dr. Paul Overgaauw and
Drs. Sible Westendorp for their assistance. The author would also like to thank
Drs. Herman Cremers for his help in determination of the bird mites.

31
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