Research Internship Veterinary Medicine

Name Cilia de Roest Student number 3383075 Curriculum Master

Department Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious diseases and Immunology Faculty of Veterinary Medicine, Utrecht University

Supervisors Prof. Dr. F. Jongejan (Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, and, Department of Veterinary Tropical Diseases, University of Pretoria, South Africa)

Dr. Hein Stoltsz (Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Title Prevalence of ectoparasites on chickens (Gallus gallus domesticus) in the Mnisi Area of Mpumalanga Province, South Africa.

Abstract

Chickens from 13 villages in the Mnisi area are thoroughly examined over a period of four weeks to detect the presence of ectoparasites. Ectoparasite collection indicated that chickens in 92% of the households are infected with ectoparasites. One species of the order Siphonaptera (), six species of the order Phtiraptera (lice) and three species of the order Acarina (ticks and mites) are recorded. Among all ectoparasites found, fleas were the most prevalent. Fleas isolated in 100% of the villages were only sticktight fleas ( gallinae). Lice were isolated in 85% of the villages investigated. Menopon gallinae is found in 77% of all investigated villages and thereby most abundant. Followed by Gallacanthus cornutus (38%), Goniocotes gallinae (31%), Lipeurus caponis (23%) and finally Stenocrotaphus gigas and Eomenacanthus stramineus, both occurring in 15% of the villages investigated. From the order Acarina, two tick and one mite species are found. The tick species, found in 85% of the villages, are Amblyomma hebraeum and Haemaphysalis elliptica (from both tick species only immature stadia are recorded). The mite species found in 6% of all investigated household is Ornithonyssus bursa. The purpose of this study is to examine and identify the currently occuring ectoparasites on chickens in rural villages in the Mnisi area.

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1. Introduction (occasionally) defaecating. This may lead

to serious diseases in various , Poultry plays an important role in including humans.(4,5) improving the nutritional status and income of many small farmers and those The Mnisi area in the Mpumalanga with small land holdings. Besides the province of South Africa has many villages nutritional and economic roles, poultry where Shangaan people still live traditional also plays a role in traditional religious lifestyles. There are several domestic customs.(1) animals such as cattle, donkeys, goats, dogs and chickens. The chickens (Gallus By living on the host, ectoparasites gallus domesticus) are mostly free-ranging can cause (especially when in large and sometimes provided with night sheds numbers) several disorders. In poultry, made from local materials such as wire ectoparasites are considered as a common mesh and old corrugated iron. In some cause of growth retardation, lowered cases housing is not provided and the birds vitality and poor body conditions.(2) sleep on trees or roof tops. There is no Weight loss at the rate of 711 gram per documented information on prevalence of chicken and a reduced egg productivity at ectoparasites in poultry in the Mnisi area. the rate of approximately 66 eggs per In South Africa little has been done on chicken per year is observed, caused by ectoparasites affecting free-range chickens, ectoparasites.(3) The ectoparasites can hence it is imperative to determine the instantly influence health and welfare by prevalence of several ectoparasites, that causing irritation, discomfort, tissue possible also are dangerous to man. damage, blood loss, toxicosis, allergic reactions and dermatitis. These The aim of this study is therefore to consequences can all lead to reduced examine and identify the currently quality and quantity of meat and egg occuring ectoparasites on chickens in rural production.(2) villages in the Mnisi area. A questionnaire survey is conducted with the primary Aside from these consequences, purpose of determining the prevalence and many ectoparasites are known to be control of ectoparasites. vectors of several pathogenic agents, which are typically transmitted by the parasites to the hosts while feeding or

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2. Materials and methods parasites with nocturnal activities are also identified. 2.1 Study area When heavy infestations of lice or mites Samples are collected from 49 are noticed in chicken sheds or nests, the households in 13 villages in the Mnisi area, ectoparasites are aspirated using a hand province Mpumalanga, South Africa. held vacuum cleaner. The parasites are Appendix 2 shows a map of the Mnisi area. then transferred in 70% ethanol. The Mnisi community is located in the north-eastern part of the Bushbuckridge 2.4 Identification of ectoparasites Municipality and covers about 29.500 To determine the different louse, hectare. The study area counts about and mite species, the specimens are 40.000 residents, divided in 8555 mounted on a microscope slide. For households of which many possess determining the tick species, mounting is chickens. not necessary.

2.2 Study animals 2.5 Preparation of mites The research includes chickens For preparation of mites, the (Gallus gallus domesticus) from different specimens are placed (after being age groups and of either sex. conserved in 70% ethanol) in a small

container. The specimens are cleared by 2.3 Parasitological examinations placing a drop of lactic acid on a A thorough examination of the depression slide and transferring the mites chickens is performed for screening for to the acid with a forceps or artist’s ectoparasites. Several regions of the body paintbrush. The mites are carefully heated are examined, including the head, the legs, over a flame, by moving the slide in and the wings, the body and the cloaca. out of the flame. The mites must be Detected ticks, fleas, lice and mites will be continually checked under a removed with a thumb forceps or a brush stereomicroscope to see if they are cleared. and dehydrated in 70% ethanol. Aside When the mites are cleared they are ready from the poultry, also the cracks and to be mounted. A drop of Heinze’s PVA crevices within the sleeping area of the medium is placed in the middle of the glass poultry will be investigated to ensure that slide and the mite is transferred to the middle of the drop. A cover slip is placed

4 on the edge of the drop, touching the After this preparation, the medium and gently lowering onto the specimens can be preserved on a specimen. microscope slide. For preserving specimens for microscopic examination, several types of mounting media are in 2.6 Preparation of fleas and lice vogue.(8) In this study, used mounting For identification of flea and louse media are: species, the specimens first need to be mounted on a microscope slide. Therefore, the specimens that are conserved in 70% 2.6.1 Canada balsam ethanol are now transferred in a 10% After two changes of absolute potassium hydroxide solution for clearing. alcohol, the specimens are placed into When the specimens sink in the potassium clove oil overnight. The next day, the 10% hydroxide solution, the abdomen can specimens are transferred in an small drop be pierced with a fine needle, to allow the of Canada balsam on a microscope slide. potassium hydroxide to enter. The fleas Because the Canada balsam needs to dry now have to stay in the potassium to fixate the specimen, the microscope hydroxide for 24 hours, while the lice slide is placed in a horizontal position in a remain in the potassium hydroxide for 16 dust-free place to overnight. The next day hours. If the body contents are softened, another drop Canada balsam is placed on pressure should be exerted on the abdomen the specimen. A cover slip can be placed with another suitably bent pin. The guts on the microscope slide.(7) should now flow through the hole in the abdomen. This can be repeated several 2.6.2 Entellan times until no opaque matter remains Entellan is a water-free mounting inside the body. After removing the body medium. Because it contains toluene, it contents, the specimens are rinsed in should be used with water-free specimens distilled water and placed in 10% acetic that have been processed with xylene acid for 15 minutes to neutralize the previous mounting.(9) After being in two alkaline potassium hydroxide. The changes of xylene for 15 minutes the specimens are then passed through 40%, specimens can be placed in a drop Entellan 80%, 95% and two changes of absolute on a microscope slide. A cover slip can be alcohol (15 minutes in each different placed. solution).(7)

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2.6.3 Euparal use to prevent ectoparasites infestations. A significant advantage of Euparal Also information regarding the type of is that, in comparison to other mounting housing, feeding and occurring of media, specimens can be directly sick/dying chickens is recorded. transferred from alcohol in to Euparal. The cover slip can directly be placed on the microscope slide.(10) 3. Results

2.7 Identification Ectoparasite collection indicated After being mounted on a that 92% of the households visited are microscope slide, the fleas, lice and mites infected with ectoparasites (table 1). One are ready for identification. Species species from the order Siphonaptera determination is based on microscopic (fleas), six species from the order examination to study the morphological Phtiraptera (lice) and three species from characteristics. Using an identification the order Acarina (ticks and mites) are guide by Walker (1994) and the works of recorded. Emerson (1956), de Meillon (1961), Ledger (1980) and Walker (2013) the Among all ectoparasites found, different species of ectoparasites are fleas were the most prevalent. Fleas determined.(7,11-14) isolated in 100% of the villages were only sticktight fleas (Echidnophaga gallinae). 2.8 Questionnaire The six louse species are isolated in 85% Questionnaires (appendix 1) are of the villages investigated. The tick completed by the persons directly species are Amblyomma hebraeum (found responsible for the management of the in 85% of the investigated villages) and chickens after the chickens are being Haemaphysalis elliptica (found only in examined. By means of this questionnaire Shorty village). Only immature stadia of it will be charted whether ectoparasites are both tick species are recorded. The noticed and what methods the households recorded mite species is Ornithonyssus in the Mnisi area (Mpumalanga) currently bursa.

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Table 1 Ectoparasites isolated from chickens in 13 villages in the Mnisi area

Fleas Lice Ticks Mites Athol P N P N Clare A P P N N Dixie P P P N Gottenburg P P N N Hlala Kahle P P P N Hluvukani P P P N Ludlow P P P P Seville P P P N Share P N P N Shorty P P P P Thlavekisa P P P N Utah P P P N Welverdiend P P P P P = present; N = not found

predilection to attach to bare spots on the 3.1 Fleas skin.(16) In chickens, it mostly feeds on In the Mnisi area, fleas are present the comb and the wattle.(17) In this on chickens in all investigated villages. research, all of the Echidnophaga There are five flea families known to occur gallinacea fleas are found on the head and on chickens i.e. , the neck. Most of the fleas were attached to , , Pygiopyllidae the comb, the wattle, the upper eyelids and and Rhopalopsyllidae.(15) Echidnophaga the chin. Figure 1 shows severe gallinacea, from the family Pulicidae, is accumulation of thousands of the only species found in the Mnisi area. on the neck and

the chin. Fleas are also found on the upper In the Mnisi area, infestation per eyelid, some chickens had difficulties chicken ranged from one to thousands of opening the eyes due to swollen upper fleas. Echidnophaga gallinacea has a eyelids.

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Figure 1 Heavy infestation with Echidnophaga gallinacea on a chicken in Athol village, South Africa

3.2 Lice stramineus, both occurring in 15% of the Lice are found on chickens in 11 of villages investigated (table 2). the 13 investigated villages in the Mnisi area. In South Africa, there are 16 louse Louse infestation per chicken species known to occur on chickens.(7) In ranged from one to dozens of lice. The lice the Mnisi area, six species are found. are mostly found on the ventral part of the Menopon gallinae is found in 77% of all chicken body. Figure 2 shows louse investigated villages and thereby most infestation on a chicken between the fluffy abundant. Followed by Gallacanthus downy feathers. In the villages Athol and cornutus (38%), Goniocotes gallinae Welverdiend accumulation of lice eggs are (31%), Lipeurus caponis (23%) and finally found on the base of feather shafts. Stenocrotaphus gigas and Eomenacanthus

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Table 2 Presence of lice species in 13 villages in the Mnisi area, South Africa

Menopon Gallacanthus Goniocotes Lipeurus Stenocrotaphus Eomenacanthus gallinae cornutus gallinae caponis gigas stramineus Athol P P N P N N Clare A N P N N N N Dixie P N P N N N Gottenburg N N N N N N Hlala Kahle P P P N P N Hluvukani P N N N N N Ludlow P N P P P N Seville P N N N N N Share N N N N N N Shorty P N N N N N Thlavekisa P P P N N N Utah P N N N N P Welverdiend P P N P N P P = present; N = not found

Figure 2 Lice on feather shafts of chicken in the Mnisi area

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3.3 Mites 3.4 Ticks Ornythonyssus bursa, also called Ticks (only immature stadia) are the tropical fowl mite, is found on chickens found on chickens in 11 of the 13 villages in three households. In one household in (85%). No adult ticks were collected and Ludlow village there was a heavy mite none of the investigated flocks bore any infestation. Mites are found on the signs of a tick infestation. Infestation per chickens and chicken nests were heavily chicken ranged from one to dozens of infestated. During inspection, thousands of ticks. The highest amount of ticks in one mites were visible crawling on the chicken household is found in Thlavekisa; 183 nests. ticks are found on only six chickens. The two tick species found are Amblyomma On chickens, mites prefer the hebraeum (11 villages) and Haemaphysalis ventral part of the body. They are elliptica (only found in one household in numerous around the vent and accumulate Shorty village). The ticks are mostly found on a few feathers.(18) This is the region on the head and the neck (figure 3). One where the mites are found on chickens in Amblyomma hebraeum tick is found in the the Mnisi area. cloaca of a chicken in Dixie village (figure 4).

Figure 3 Chicken with Echidnophaga gallinacea and Amblyomma hebraeum infestation on chin and neck in Hluvukani village, South Africa 10

Figuur 4 Amblyomma hebraeum in cloaca in Dixie village, South Africa

4. Discussion Only 23% of the owners interviewed treat the chickens against ectoparasites. One of Results show that ectoparasites are the causes of high ectoparasite infestations common in rural chickens in the Mnisi in the Mnisi area is possibly that chicken area. In all of the examined villages, owners in rural communities have poor ectoparasties are detected. Due to the fact access to conventional drugs because of that most chickens are free-ranging (and distance and exorbitant prices. This results thereby hard to catch), it was not always in using no or using alternative insecticides possible to examine a standard amount of for controlling ectoparasites, which is less chickens per household. In the examined effective.(19) Besides that, chicken sheds households, ectoparasite collection in the Mnisi area are mostly made of old indicated that 92% of the households is corrugated iron and wire mesh and in the infected with ectoparasites. Data from the sheds there are plenty of cracks and questionnaire indicated that 73% of the crevices, what makes effectively cleaning owners that had been interviewed had the sheds very difficult. Excessive noticed ectoparasites on their chickens. ectoparasite infestation can be reduced by

11 cleaning the complete chicken shed with The fleas have negative effects on insecticides and thereby killing their hosts. Chickens mostly have ectoparasites.(20) In the Mnisi area, every accumulations of Echidnophaga gallinacea chicken owner that owns a shed, cleans it. around the eyes, comb, wattle and other 84% of the sheds are cleaned by just bare spots.(23) The fleas cause irritation removing dirt, without using insecticides. and tissue damage.(19) Ulcerations can be The remaining 16% uses King Kong, Blue caused by both cuts from the mouthparts as Death powder, dip, Jeyes fluid and well as infections due to subcutaneously Karbadust. laid eggs. The resulting infection, blood loss, or both, can compromise growth and even kill young chickens.(17) Older 4.1 Fleas chickens suffer aneamia, reduced egg As described before, the only flea production and may also die from a heavy species found in the Mnisi area is infestation.(21) Besides direct effects from Echidnophaga gallinae. Many of the Echidnophaga gallinacea, flea infestation chickens are heavily infested. is recorded to co-occur with fowl pox

(Avipoxvirus, type Fowlpox virus) in Echidnophaga gallinacea, also poultry and bubonic plague (Yersinia known as the sticktight flea, infests a wide pestis) in burrowing owls (Athene variety of birds and mammals in tropical cunicularia).(17) and subtropical regions worldwide.(16,21)

In those regions it is known as an Besides occurring on chickens and important ectoparasite in chickens.(21) The other animals, Echidnophaga gallinacea is occurrence is probably because of also reported on man.(23,25-27) The flea- relatively poor standards of husbandry and borne rickettsiose murine typhus favourable climatic conditions for ( typhi) is a febrile disease development of the parasites.(24) distributed among humans worldwide.(28) Echidnophaga gallinacea has previously DNA from Rickettsia spp. is previously been recorded in South Africa. In some amplified from Echidnophaga gallinacea regions of the country, Echidnophaga fleas in Egypt (infection rate 100%).(29) gallinacea is reported to be the most Additional studies, using a large sample of ordinary flea on dogs, however the fleas Echidnophaga gallinacea from South are also known to attack chickens and Africa, are needed to determine the other animals.(22,23) infection rate in South Africa, to determine

12 if humans or animals are, in fact, exposed pathogenic for chickens, causing the mass to Rickettsia spp. and to establish the epidemics of fowl cholera, but also for pathogenicity of the agent. man. In man the infection appears mainly as a pleuritis, pneumonia or even encephalitis.(31) 4.2 Lice

Six different louse species are found in 85% of the investigated villages 4.3 Mites in the Mnisi area. During examination of Ornithonyssus bursa is found in the chickens, it was not always possible to three households in three different villages. collect all lice, because the lice crawl The mite is known to be almost entirely through feathers quickly. The six lice restricted to warm and tropical regions and species that are collected all belong to the is previously recorded in South Africa.(18) chewing lice (Phthiraptera: Amblycera, Ischnocera) and are common on domestic The nests and chickens in one chickens worldwide.(12,30) Amblyceran household in Ludlow village were heavily lice are important ectoparasites in infested. In that household, inspection chickens. While mostly living on the skin, revealed thousands of mites crawling on the lice cause irritation, restlessness, the chicken nests. The mites are also found overall weakening, cessation of feeding, on the investigated chickens in that loss of weight and reduced egg production. household. It is unknown why this is the The skin lesions may possibly become only heavily infested household. In the sites of secondary infection. other investigated households in the Eomenacanthus stramineus is one of the Ludlow village, there are no mites found most pathogenic species, because of its and in other villages the infestation was not hematophagous features. It can cause that severe. anaemia, heavy multi-focal skin lesions or even death of infested birds.(30) In chickens, Ornithonyssus bursa leads to crustae, thickening of the skin and Menopon gallinae and cracks and scabs around the cloaca. The Eomenacanthus stramineus both are productivity may be decreased. The mites known to carry Pasteurella multocida in can migrate from chickens to man during the digestive tract, a pathogen of handling or move into homes. importance because it is not only Ornithonyssus bursa can also bite humans.

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The bites are irritating and can be mildly to The hard tick Amblyomma intensely painful. Several lesions can occur hebraeum is also known as the South after being bitten, some individuals react to African bont tick and only occurs in the bite with prolonged itching and painful Southern East Africa. Immature stages dermatitis. Papules most commonly occur feed on several animals and they are also after being bitten. Macules, diffuse known to feed on ground frequenting birds. erythema, vesicles, urticare and It is known that the nymphs and larvae hemorrhagic necrosis may also be mainly attach on the head and the neck. In seen.(18,32) the Mpumalanga province, where the Mnisi area is located, the life cycle of Other then the red mite, Amblyomma hebraeum continues Dermanyssus gallinae, which is thought to throughout the year.(14) transmit diseases by being vector of several pathogenic agents i.e. avian Amblyomma hebraeum is most cholera, avian smallpox, equine important for transmission of Ehrlichia encephalomyelitis and Salmonella ruminantium, the causative agent of enteritidis, Ornythonyssus bursa has not heartwater in cattle, sheep, goats and wild been implicated in vectoring diseases.(33) antilopes. Besides distributing heartwater, The western equine encephalomyelitis Amblyomma hebraeum can also cause tick virus has been isolated from Ornithonyssus typhus in man by transmitting the bacteria bursa mites, however, there is no evidence Rickettsia africae and Rickettsia conorii. In that the virus is actually transmitted by the cattle, benign theileriosis can be caused by mite.(27) the same tick transmitting the prozotoan Theileria mutans. The long mouthparts can 4.4 Ticks cause sores, which often become infected Species of both soft and hard ticks with bacteria leading to acsessation. are known to occur on chickens.(3) In the Besides infection with bacteria, the Mnisi area, only hard ticks are found. The blowfly Chrysomya bezziana, is attracted recorded species are Amblyomma by the wounds and the larvae of the fly can hebraeum and Haemaphysalis elliptica. cause severe myiasis.(14) From both tick species, only immature Haemaphysalis elliptica, the stages are found. Immature ticks of eight southern African yellow dog tick, is found Ixodid tick species are known to occur on on chickens in one household in the Mnisi birds in South Africa.(34)

14 area. The tick is common on domestic dogs Ornithodorus spp. could have possibly in South Africa, especially in rural been found if the parasite is searched for in areas.(35) Immature stages of the chicken sheds at night.(39) Another Haemaphysalis elliptica are not yet reason is because, during this research recorded infesting chickens, though project, it was winter time in the Mnisi immature stages of other Haemaphysalis area. It could be that the temperature was species are known to occur on other birds not optimal for development of the soft in South Africa.(34) Haemaphysalis ticks. For example, the optimal elliptica is the only proven vector of temperature for development of Argas Babesia canis rossi, the cause of virulent spp. is 22-28°C.(3) babesiosis in dogs.(36) Because chickens in the Mnisi area While known to occur on chickens are commonly heavily infested with in South Africa, there are no soft ticks ectoparasites and the ectoparasites found found on chickens in the Mnisi area. The on chickens in the Mnisi area are not only soft tick Argas spp. is thought to be negatively affecting chickens but may also causing many deaths because of the blood- affect human health, improving sucking habit and also acting as a vector of ectoparasite control is recommended. Aegyptionella spp. and Borrelia spp.. The There is need for an enlightenment larvae can cause tick paralysis in campaign for chicken owners on the chickens.(37,38) Ornithodorus spp., consequences and hazards of ectoparasite another soft tick, also occurs on chickens infestation. Proper sanitation and the use of in Africa. The ticks are nocturnal. Bites certain chemicals in the approved manner from this tick are painful and the ticks are also help in the control of ectoparasites. known as the major vector of Relapsing Veterinary assistance to the chicken fever in man.(6,39) There are some reasons owners needs to be extended. Further that soft ticks are not found during this research to access the ectoparasites impact study in the Mnisi area. The nymphs and on health and production performance of adults of Argas spp. for example, are the chickens in the Mnisi area is suggested, temporary obligate parasites and only visit cost effectiveness of control strategies chickens when feeding.(37) The sheds, if included. Studies on seasonal variation on available, are examined, however, different ectoparasite species infesting sometimes the sheds were hard to examine poultry should be carried out to determine due to size and height. Argas spp. and

15 the appropriate time of the year for control (5) Bala AY, Anka SA, Waziri A, Shehu strategies. A. Preliminary Survey of Ectoparasites As noticed before, additional study Infesting Chickens (Gallus domesticus) on Echidnophaga gallinacea and its in Four Areas of Sokoto Metropolis. infection with Rickettsia spp. is Nigerian Journal of Basic and Applied recommended. Science 2011;19(2):173-180.

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(31) Derylo A. Mallophaga as a reservoir (36) Fourie JJ, Fourie LJ, Horak IG, of Pasteurella multocida. Acta Snyman MG. The efficacy of a topically Parasitologica Polonica; 1970 17:301-313. applied combination of cyphenothrin and pyriproxyfen against the southern African (32) The Center for Food Security & yellow dog tick, Haemaphysalis elliptica, Public Health, Iowa State University. and the , felis, on Acariasis, Mange and Other Mite dogs. J S Afr Vet Assoc 2010;81(1):33-36. Infestations. 2012; Available at: 20 ref. http://www.cfsph.iastate.edu/Factsheets/pd fs/acariasis.pdf. Accessed October 4, 2013. (37) Mukaratirwa S, Hove T. A survey of ectoparasites, cestodes and management of (33) Wang FF, Wang M, Xu FR, Liang free-range indigenous chickens in rural DM, Pan BL. Survey of prevalence and Zimbabwe. J S Afr Vet Assoc 2009 control of ectoparasites in caged poultry in Sep;80(3):188-191. China. Vet Rec 2010 Dec 11;167(24):934- 937. (38) Taylor MA, Coop RL, Wall RL. Veterinary Parasitology. Oxford: (34) Van Niekerk DJ, Fourie LJ, Horak IG. Blackwell; 2007. Birds as hosts of immature ixodid ticks in Free State Province, South Africa. (39) Wall R,Ph.D. Veterinary ectoparasites Onderstepoort J Vet Res 2006 : biology, pathology, and control. 2nd ed. Jun;73(2):123-130. Malden, MA: Blackwell Science; 2001.

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6. Appendices

6.1 Appendix 1: Questionnaire used in the baseline survey of determining the prevalence and control of ectoparasites in the Mnisi area

Date:………… House number:………… Village:…………

Amount of chickens examined:…………

What poultry do you keep (chickens, ducks, geese)? .…………………………………………

How many chickens do you have? ……………………………………………………………...

What do the chickens eat? ………… ……………………………………………………………

Where do they sleep (sheds, on top of trees)? …………………………………………………

Do you see parasites on your chickens? Yes / No

 What do you see? ……………………………………………………………………

Do you see sick chickens? Yes / No

 What do you see? ……………………………………………………………………

Do you see chickens lying on their sides and unable to get up? Yes / No

Do you treat chickens against ectoparasites? Yes / No

 What do you use? …………………………………………………………………...

 How often? …………………………………………………………………………

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Do you clean the poultry sheds? Yes / No  What do you use? ………………………………………………………………….

 How often? ………………………………………………………………………….

Comments: ……………………………………………………………………………………………….…. ……………………………………………………………………………………………….… …………………………………………………………………………………………………. ….…………………………………………………………………………………...………… …………………………………………………………………………………………………...

6.2 Appendix 2: Map of the Mnisi area with its villages

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