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Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Isolation of Aerobic Bacteria Associated with Fistulous Withers in Donkeys By Shereen Farog Mostafa University of Khartoum B.V.M. 2004 Superviser Dr. Awad Elkarim Abd Elghaffar Ibrahim A thesis submitted to the University of Khartoum in partial fulfillment of the requirements For the Degree of M. Sc. Department of Microbiology Faculty of Veterinary Medicine University of Khartoum ϮϬϭϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. INTRODUCTION The domestic donkey traces its ancestry to the wild asses found in Egypt, the Sudan, Somalia and Ethiopia (Dreyfus, 1976). Packing is one of the most ancient forms of transport which preceded even the invention of the wheel. That it has survived to the present day serves to emphasize its value. The major advantage of pack transport is its effectiveness in the absence of roads. It is particularly effective where access is limited ,this applies not only to remote mountainous areas but also to areas of high population density as in city centres. The steady speed of a donkey’s walk is what makes it so popular as a pack animal or for pulling small carts. Donkeys are usually cheap compared to other draught animals (Clark , 1974). Donkeys are widely used in both rural and urban areas of the Sudan for transporting a wide variety of items such as grain, construction materials, goods and animal feed (Wylam , 1991). Such transport is an important stimulus to trading and also represents a source of income for the donkey owners. A large part of the people and of the economy of Omdurman depends on donkey transport for the movement of goods from wholesale centers to retail outlets and households. The service is cheap, flexible and readily accessible in most parts of the city. It is also an essential source of livelihood and income to many households. Housing for donkeys is rarely constructed as, in most cases, they are simply tied to a pole inside the owner’s compound. Most of the donkeys used in the city are imported from other places. In most countries breast band harnesses are used for donkeys pulling carts or doing field work. The most common causes of saddle sores on donkeys are by and large incorrectly fitting saddles and harness ϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. (Pritchard, 2005). This material causes friction, rubs, sweat and overheating problems. This leaves the animal more prone to withers galls, open back sores and rubs. Conformation faults are often at the root of the problem. If your horse has low withers, or in the other extreme is high withered, or perhaps is a narrowly built horse fitting the saddle can be difficult. A poorly fitted saddle will either put pressure on the donkey where it shouldn't be, or it will slide around, causing friction . Fistulous withers (supraspinous bursitis) is an inflammatory conditions of supraspinous bursa of equine . The bursa is located over the second to fifth thoracic spine of equine horses and donkeys. In the early stage of the disease, a fistula is not present . In more chronic, advanced cases, the ligament and the dorsal vertebral spines are affected, and occasionally these structures undergo necroses . When the bursal sac ruptures or when it is opened for surgical drainage, and secondary infection with pyogenic bacteria occurs, it usually assumes a true fistulous character. The number of donkeys in Sudan is about 7522690 given by (Statistical bulletin for animal resources , 2011). In Sudan, donkeys are mostly used for transport. They pull carts and carry goods and characterized by their body conformation and working speed into three breeds: Mekadi, Atbawi and Reef. Little information is available about donkey type and distribution in Sudan. Their origins are Eddamer and Atbara in the north and Jalabi from Gazira in central Sudan. The scope of the present study is limited to the donkey pack-transport of goods within the city boundary .The main objectives were as follows: 1-To identify and quantify factors associated with the presence of pack wounds in donkeys working in the transport of goods. Ϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2-To isolate and identify aerobic bacteria associated with fistulous wither and other saddle and harness wounds. 3-Assessment of the welfare of working donkeys, using health and behavior parameters. ϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CHAPTER ONE 1. LITERATURE REVIEW 1.1 Donkey 1.1.1 Donkey population and history Donkeys Equus asinus are domestic animals falling under the equine family, which includes horses, zebras and the mules (Wilson, 1990). There are 44.3 million donkeys or Ass (Equus asinus) worldwide. This number has increased by 15.6 per cent since 1981 (FAO, 1992). A small number are kept in the western world as pets. However over 95 per cent live in the developing world where they are kept mainly for work. The precise wild progenitor of the domestic donkey is disputed. Bökönyi (1991) argued that domestication took place in Egypt and Clutton-Brock (1992) notes that the skeletons of three domestic donkeys have been found in an Egyptian tomb dated to 4500–4000 BC. There are comparably early skeletons in the Near East but, whether these are domestic remains uncertain (Eisenmann, 1995).The two recognised races of the ass are: Equus asinus africanus and Equus asinus somaliensis. The Nubian wild ass is one subspecies of the wild ass, Equus africanus which cited in many textbooks as the progenitor of the donkey, although other races may also have contributed to the gene-pool (Epstein, 1984). The dominant color is the mousy grey, although the whole range from black to white are generally rare. The average weight of donkeys (both males and females),was approximately 105 kg (Wilson,1991). Donkeys reach maturity around four years of age .Breeding age for female ϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. donkeys is 4–5 years (Feseha and Yoseph, 1996). Both males (intact males are called jacks and castrated males are geldings) and females (jennies) can be used for work. With good care, donkeys can have a working life of 12-15 years, and they can live even longer. Castration will help to improve the temperament and reliability of males. However, good jacks are important for breeding, and farmers may be able to obtain fees for allowing their jacks to breed. Donkeys are more common in Africa than horses with an exception of Ethiopia and Morocco. Egypt, Nigeria, Mali, Niger and Sudan provide examples of the numerical dominance of donkeys over horses (Payne and Wilson, 1999). Equids used to transport people by cart, to carry goods by pack, or to work in bricks kilns. Rural equids had more problems than urban ones, but urban equids had more lesions. Equids were significantly thinner when climates were warmer. These results should aid the development and targeting of specific welfare interventions. 1.1.2. The place of donkeys in society A number of factors help to explain why donkeys have low status. They are usually the cheapest, often the only affordable, work animal and therefore tend to be associated with the poor. If donkeys are not available, women often have to do the same work (Mohammed, 1991). In contrast with cattle, buffalo and camels which are usually kept for their milk and meat as well as work, whose hides are cured for leather, and whose dung even has a number of uses (Pearson, 1992), donkey bye-products are not generally used. ϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Map 1: Estimates of donkey populations in Africa and the Arabian peninsula in 1996. The figures show the estimated donkey population of each country in thousands. The intensity of shading gives an approximate visual indication of the numbers of donkeys in the different countries. Authors’ estimates are shown in italics. (FAO, 1997). ϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.1.3. General donkey features and Behavior Donkeys are often very inexpensive and have little, or no, disposal value. Although they have sometimes been considered as animals of ridicule or low status, they have excellent reputations as easily trainable and very dependable work animals. Draught animals were used as pack animals for goods and people. In some cases they are the only source of transport. Animals driven vehicles are an important part of the transport network and were prime source of transport used by farmers to bring their agro-products to market and take back their necessities from cities. Such types of vehicles are not only being used in villages but are still on the roads in big cities and are contributing a lot in the saving of petrol energy. donkeys are used as singles or in teams depending on the load and the operations performed. Cattle are hardly used at all for functions other than cultivation except for threshing which is done simply by walking round on top of a heap of crop or by dragging a heavy stone or wooden baulk. Donkeys are the only other species used in this function either loosely attached to each other in pairs or in multiples of up to six animals. Cart transport is much less common than in the past but the importance of this function in urban areas does vary. It is more common in the rural areas of the country . Although the market is the main centre for the donkey pack-transport service most donkey operators reside in the peripheral parts of the city. The majority of the donkey operators are able-bodied males who perform the loading and unloading tasks by ϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. themselves . The donkey pack-transport business, like many other informal activities, has little restriction to entry. Table 1: Main advantages and disadvantages of using donkeys (Jones,1997). Advantages Disadvantages Friendly towards humans Willing to work Can turn in small space Easy to train Need little supervision in work Can utilize poor feed well Not affected much by external parasites Need little water Can survive droughts better than cattle Can survive well in tsetse-infested areas Comparatively cheap to buy Strong relative to size Live and work many years in good care Useful for calming and guarding other kinds of animal Fast walking speed Suffer from being alone Noisy when frustrated or lonely Friends not easily separated Uncastrated males aggressive towards other donkeys Skin easily wounded Wander long distance if not supervised Do not move out of the way of traffic Need shelter from cold and damp Meat not generally eaten Comparatively small in size Mature slowly Breed slowly Manure more fibrous than nutrient-rich 1.1.4. Selection characteristics of a working donkey When selecting an animal for work certain physical characteristics should be observed. These include: a large frame with wide shoulders and a deep chest, a straight back and well-muscled straight legs which have a 90° angle to the ground . The donkey should have good eyesight and agility and an attractive hair coat, without skin ϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. diseases or an abundance of ticks. It is important to observe an animal while it is working, to detect whether it has a physical disability, such as coughing, poor breathing, lameness, sores or wounds. 1.1.5. Economic importance The donkey is one of the man oldest domesticated animals. Donkeys therefore contribute to economic development as they are used in every-day life. They are even used more frequently than cattle and horses and have taken the work that was in the past done by cattle, horses and machinery such as tractors (Starkey, 1994). Most farmers are able to keep donkeys because they do not require technical husbandry practices. They are tolerant to some tropical diseases and parasites. Donkeys are therefore easy to manage and not too demanding in terms of feeding. They can almost survive on poor quality feeds and they can tolerate a considerable heat and dehydration. (Aganga et al., 2000). Most traditional households use their donkeys for transport, fetching water and for gathering firewood . This is shown by the wide spread use of donkeys in urban and rural areas in Africa, as well as parts of the Central America and Asia (Aganga et al., 1994). The major cost of starting a donkey pack-transport business is investment in donkeys. Other start-up costs include straps , packsaddle and whip .The donkeys are usually overloaded and suffer from wounds related to overwork. Therefore it would be a good idea for the country to try to improve the poor animals social standing or social position. Donkeys are multipurpose, as they not only provide transport, but also milk to children as a medicine against whooping cough. ϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.1.6. Fitting Equipment Harnesses and saddles are usually not fitted properly on the donkeys. This causes terrible discomfort and deep wounds that are usually left untreated, since the donkeys are forced to continue working. The face, back, neck, hind legs and under the legs are the areas that are most subjected to deep-seated wounds that can often lead to infection. Breast band harnesses this kind of harness is cheap, and easy to manufacture locally were, however, originally constructed for horses which have a wide and muscular chest, providing enough space for a breast band. This is not the case with donkeys. The breast band on the donkey rests on the shoulder joint and will either have the tendency to glide up, pressing onto the trachea (windpipe), or down getting onto the animal’s forearm. In addition, the donkeys chest being less muscular than the horse’s, increases the risk of bruises. Mostly donkeys were harnessed with homemade breast band harnesses of poor quality .This is not an unusual sight in many parts of Omdurman market places . Donkey users preferred the local round collar harness (traditional) to the threepad collar (improved). The general perception of most farmers on improving harnessing systems underscored the use of quality materials to ensure efficient draught power supply. Saddles sores caused by poor saddle fit usually show up around the withers, shoulder, or back under the cantle. Sometimes saddle sores are caused by a dirty saddle pad or a saddle cinched up with the hair not laying flat underneath. Both of these problems can be big enough irritants to cause open sores or wounds within the space of a short time of work. Saddle sores can be caused by girths and cinches being too ϭϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. tight, too loose, too rough, too dirty, or skin being bunched underneath them- also called girth galls. Fig 1. The arrangement off the breast band, breech strap and saddle for harnessing a donkey to the shafts of a two-wheeled cart (Pritchard, 2005). ϭϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.1.7. General causes of injuries Donkey welfare will be best if donkeys pull well-balanced carts with good, lubricated wheels. They also need good, simple harnesses. This makes pulling easier and the animals stay in good condition. Skin injuries to the back, shoulders or other parts of the body due to saddles, harnesses or collars are brought about by combinations of friction and pressure. If a donkey with a saddle sore must be worked the pressure and friction on the sore must be reduced or preferably eliminated. The ordinary neck collar oscillates from side to side while the animal walks. The breast collar doesn’t oscillate, but has a sawing action. Sawing is obviously a greater source of friction than oscillation, and in consequence the make and fit of the breast collar must be such as to present a perfectly smooth surface next to the donkey’s skin. Neglect of this precaution is soon evident as any small projection in the harness is capable of doing great harm. Harness materials should be from the local environment. Injuries often occur when insufficient attention has been paid to equipment. Many owners consider that the weight of a donkey’s body is equally distributed over its limbs. This is not true, the fore limbs carry more of the body weight than the hind, and the amount which they carry is influenced by the position of the head, which, if held high, relieves the forelegs from weight, and if depressed increases the weight. In regular use the saddle requires attention every day. Saddles should be inspected almost as regularly as the feet. Each weak point in the harness should be known and repaired before trouble occurs (Fahmy, 1992). 1.1.8. Wounds Wounds are often a result of a direct injury, ill fitting tack, harness or hobbles or being attacked by other animals. Some wounds or injuries may need veterinary attention. Others may simply need to be cleaned. If a wound or injury affects the donkey’s ability to ϭϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. work, it will ultimately affect the livelihood of the owner as well as the welfare of the donkey. If treatment is administered quickly and flies are kept away from the wound, the risk of infection is minimized. Fig 2. Donkey was repeatedly whipped on an open wound, that led to deep infection (Omdurman Market). ϭϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.2. Fistulous withers 1.2.1. Definition of fistulous withers Fistulous withers is a chronic inflammatory disease of the supraspinotus bursa and associated tissues (Gaughan et al., 1988; Rashmir-Raven et al., 1990;Cohen et al., 1992). The supraspinous bursa is located between the funicular portion of the nuchal ligament and the dorsal spinous processes of the second to fifth thoracic vertebrae (Hawkins and Fessler 2000). The bursa is approximately 5 cm wide and ranges in length from 5–11 cm and has a capacity in the normal horse of 30–90 ml. Although infection by Brucella abortus has been associated with the condition (Duff, 1937). Other infectious organisms and trauma can also cause the disease. Indeed organisms commonly isolated from clinical cases in geographical areas with a low prevalence of brucellosis in cattle, B. abortus is rarely isolated from fistulous withers cases (Gaughan et al., 1988; Cohen et al., 1992). Infection by multiple bacteria is often Present. Other than B. abortus, include Streptococcus zooepidemicus ,Streptococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirablis, Actinomyces bovis, Bacteroides fragilis, Escherichia coli, Pasteurella spp. and Corynebacterium spp. (Guard 1932; Gaughan et al., 1988; Cohen et al., 1992; Hawkins and Fessler 2000). Infection by Onchocerca cervicalis has also been incriminated in some cases (Lyons et al., 1988; RashmirRaven et al., 1990; Hawkins and Fessler 2000). ϭϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Fig. 3: Names of the different body parts of a donkey. 1.2.2. Clinical signs Clinical signs of supraspinous bursitis (fistulous withers) include singular or multiple draining tracts or diffuse swelling of the withers without drainage. The onset of clinical signs may be abrupt or insidious. Early signs include localized heat, pain and swelling of the bursa. There may be lethargy and general stiffness. In most cases the bursa ruptures and purulent exudates is discharged from one or more fistulae. These fistulae may heal over, but may subsequently reform .Extensive fibrosis may occur. However, horses with fistulous withers that are seropositive to B. abortus are significantly more likely than seronegative horses with ϭϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. fistulous withers to have radiographic evidence of osteomyelitis of the underlying dorsal spinous processes (Cohen et al., 1992). Poll evil (septic supra-atlantal bursitis) causes similar clinical signs to fistulous withers in the poll region. There is frequently pain and neck stiffness. Swelling of the region occurs which may be followed by discharge of purulent material. 1.2.3. Etiological agent of fistulous withers 1.2.3.1. Brucella abortus Bacteria of the genus Brucella are nonmotile, aerobic, intracellular Gram-negative cocci, cocco-bacilli or short rods. Brucella spp. are transmissible to a wide range of species and among the domesticated animals (Godfroid et al., 2004). B. bortus infections in domestic animals have been reported worldwide, There is no apparent age, gender or breed predisposition to infection in horses, although most cases have been reported in horses aged >3 years and equine infections usually involve the cattle pathogen B. abortus (Nicoletti, 2007). Horses usually become infected by the ingestion of B. abortus-contaminated feed, and most reported cases indicate a history of contact with cattle (Duff 1937; McCaughey and Kerr 1967; Denny 1973; O’Sullivan 1981; Ocholi et al. 2004). Serological surveys in endemic areas indicate that many horses can be exposed and infected by B. abortus without any clinical signs of disease (Denny 1973; Dawson and Durrant 1975; Nicoletti et al., 1982; MacMillan and Cockrem 1986). The commonest clinical diseases associated with Brucella spp. infection in horses are septic supraspinotus bursitis (fistulous withers) and septic supra atlantal bursitis (poll evil). ϭϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Duff (1937) reported 85 cases of fistulous withers and identified B. abortus infection in 80%. 1.2.3.2. Staphylococcus Bacteria of the genus Staphylococcus are strongly Gram-positive cocci (0.5– 1.5 min diameter) that occur singly, in pairs, tetrads, short chains ,spherical bacteria that occur in microscopic clusters resembling grapes (Todar, 2011). Staphylococci grow in clusters because of the special division way of Staphylococci, the cells dividing in both three dimensional axis and the new cells remaining attached to each other followed by each division successively. Since there is no exact point of division, the result is to form an irregular cells cluster (Pinho and Errington, 2003). Colonies are usually large (6-8 mm in diameter), smooth, and translucent. The colonies of most strains are pigmented, ranging from cream-yellow to orange. Staphylococci are nonmotile, non–spore-forming, catalase-positive and oxidase-negative bacteria. Staphylococci are facultative anaerobes that grow by aerobic respiration or by fermentation that yields principally lactic acid. Typical animal strains of S. aureus are important pathogens in veterinary medicine, causing disease in horses, the bacterium may cause dermatitis and cellulitis. Staphylococcus aureus is the most common species of staphylococci to cause Staph. infections. One of the reasons for this is a carotenoid pigment staphyloxanthin that is responsible for the characteristic golden color of S. aureus colonies. This pigment acts as a virulence factor, with an antioxidant action that helps the microbe evade death by reactive oxygen species used by the host immune system(Clauditz et al ., 2006; Liu GY et al., 2005 ). On the basis of the ϭϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. coagulase test, the genus Staphylococcus was originally divided into the coagulase-positive species S. aureus and coagulase-negative staphylococci. 1.2.3.2.1. Bacterial virulence factor Several potential virulence factors have been described as important in staphylococcal infections. Most of these factors have been studied in S. aureus ,but some of them have also been found in other Staphylococcus species. The virulence factors can be divided into cell associated components, exoenzymes and exotoxins. The known virulence factors of staphylococci are described as follows :- 1.2.3.2.2. Cell - Associated Components Which include: - Protein A: is a surface protein of S. aureus that binds IgG molecules by their Fc region. In serum, the bacteria bind IgG molecules in the wrong orientation, which disrupts opsonization and phagocytosis. - Capsular Polysaccharide: These capsular polysaccharides have been proposed to interfere with host defense mechanisms by inhibiting the attachment of antibodies. They have also been described to bind to epithelial and endothelial cells and to monocytes, and they induce the release of cytokines. - Adhesins : Staphylococcal bacteria may express proteins on their surface that promote attachment to host proteins ,such as ϭϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. fibronectin, laminin and collagen which important in promoting bacterial attachment to damaged tissue . 1.2.3.2.3. Exoenzymes Coagulase is an extracellular protein that binds to prothrombin in the host to form a complex called staphylothrombin. The protease activity is activated in the complex, leading to conversion of fibrinogen to fibrin . Probably, the bacteria protect themselves from phagocytic and immune defenses by causing localized clotting. 1.2.3.2.4. Exotoxins Staphylococcus aureus can express several different types of protein toxins which are probably responsible for symptoms during infections: - Superantigens: S. aureus secretes two different toxins that have Superantigens activity. The secreted Superantigens stimulate the nonspecific activity of T cell without a normal antigenic recognition. Cytokines are released in a large amount and lead to disease symptoms (Lowy, 1998). One is enterotoxin which can cause food poisoning. A Second superantigens that S. aureus produced is toxic shock syndronme toxin (TSST-1), which is the cause of toxic shock syndrome (TSS) (Johnson et al., 1991). 1.2.3.2.5. Membrane-damaging toxins Which include: Alpha toxin (alpha-hemolysin), Beta – toxin, Delta – toxin and Gamma – toxin. ϭϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.2.3.3. Streptococcus Streptococci are Gram-positive spherical bacteria less than 2mm that typically grow by cell division in one plane, so that nascent cells form a linear array. Most are facultatively anaerobic and catalase negative. Identification is based on colony characteristics; hemolytic properties; fermentation and other biochemical reactions. The majority of pathogenic streptococci possess a serologically active carbohydrate antigenically different from one species or group of species to another. Some pathogenic streptococci, are identified by features such as fermentation behavior, ability to grow at different temperatures, salt tolerance, optochin sensitivity, bile solubility. 1.2.3.3.1 Virulence factors In general ,streptococcal virulence is based on surface and secreted proteins and on structures that directly or indirectly impede phagocytosis, are involved in adhesion and carbohydrate metabolism, or induce release of pro - inflammatory cytokines. The best understood streptococcal virulence factors are the hyaluronic acid capsule, the antiphagocytic M proteins ,and the pyrogenic exotoxins. However, other molecules including streptolysins, proteases, leukocidal toxins, plasminogen activators (streptokinase), and possibly plasmin receptors found on the surface or secreted, also contribute to pathogenicity. In addition, most pathogenic streptococci have the ability to bind components of the host’s plasma, such as albumin, immunoglobulins, fibrinogen, and to bind to fibronectin, lamini and other components of the host cell. Organisms coated with one or more of these plasma ϮϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. components may be able to evade host defenses either by escaping detection or by blocking deposition of opsonic components of complements . 1.2.3.3.2. Streptococcal toxins Few of the cellular components of group A streptococci appear to be directly toxic for animals or humans. Cell wall fragments produce a chronic Multi nodular inflammatory lesion of dermal connective tissue. The peptidoglycan component of cell walls has many of the biologic features of endotoxins. The exotoxins of group A streptococci include the erythrogenic toxins (pyrogenic exotoxins) and the cytolytic toxins (streptolysins S and O). The pyrogenic exotoxins are associated with the enhancement of endotoxin shock and a wide variety of other biologic properties. Streptolysin S is a non antigenic polypeptide associated with various stabilizing carrier molecules. It lyses a wide range of mammalian cells, influences T lymphocyte functions, and is probably responsible for the leukotoxic property of group A streptococci. Streptolysin O is an oxygen-labile (thiol-activated) cytolysin. It is inhibited by non esterified cholesterol and binds to cholesterol in the membranes of mammalian cells and organelles, an interaction producing ring-like and C-shaped structures demonstrable by electron microscopy. Streptolysin O affects a number of leukocyte functions. It produces profound electrocardiographic changes in experimental animals and toxic effects on pulsating heart cells in tissue culture (Wannamaker,1983). Ϯϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.2.3.3.3. Streptococcus equi Strangles is an acute disease of horses caused by infection with Streptococcus equi. It is characterized by inflammation of the upper respiratory tract and abscessation in the adjacent lymph nodes. The source of infection of strangles is the nasal discharge from infected animals which contaminate the feed and water. The disease is worldwide in distribution. In Sudan it is reported in the list of animal diseases in Statistical Bulletin for Animal Recourses (2000). 1.2.3.4. Corynebacterium Corynebacterium species are non motile, non sporulating, short, pleomorphic, gram - positive rods with a high G + C content in DNA. They belong to the class Actinobacteria and are part of the larger Corynebacterium , Mycobacterium , Nocardia (CMN) grouping. Bacteria of this group are characterized by cell walls containing long – chain fatty acids called mycolic acids; corynebacterial mycolic acids have relatively short chains, typically of 28 – 40 carbons. Corynebacterium species are found in a wide range of ecological niches, such as soil ,sewage, and plant surfaces, and some are important pathogens of humans or animals. Use of partial gene sequences from the RNA polymerase subunit - encoding gene has also proved useful in identification and classification of Corynebacterium species. 1.2.3.4.1. Virulence factor Clear understanding of virulence of this organism, leading to ϮϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. effective, easily delivered means of prevention, will have major impact on many nations but especially on the developing world. Cell wall lipids of C. pseudotuberculosis have been considered virulence factors since their cytotoxicity was demonstrated more than 50 years ago (Carne etal.,1956). They protect the bacteria from intra phagocytic destruction but also induce hemorrhagic necrosis and chronic abscessation (Jolly, 1966; Hard, 1975; Tashjian and Campbell, 1983). An extracellular protein, renalin production by most isolates of C. renale This protein lyses erythrocytes in synergy with S. aureus toxin. The staphylococcal toxin apparently hydrolyzes sphingomyelin, producing ceramide, and then renalin interacts non enzymatically with ceramide to lyse cells (Bernheimer and Avigad, 1982). Others virulence factors, including tissue - damaging toxins and enzymes and attachment and colonization factors. It can cause a variety of diseases in multiple species ,but there is no indication that particular virulence factors are associated with specific disease. 1.2.3.5. Actinomycetes Actinomycetes appear as Gram-positive bacilli (Hall, 2006),which comprise a group of branching unicellular microorganisms. They produce branching mycelium which may be of two kinds substrate mycelium and aerial mycelium. Among actinomycetes, the streptomycetes are the dominant. The non-streptomycetes are called actinomycetes, comprising approximately 100 genera .Members of the actinomycetes, which live in marine environment, are poorly understood Ϯϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. and only few reports are available (Siva, 2001;Vikineswari et al., 1997; Rathana and Chandrika, 1993; Lakshmanaperumalsamy, 1978). actinomyces species require enriched culture and growth is enhanced by an atmosphere with 6-10% added carbon dioxide. The optimum growth temperature is 37°C. Colonies may appear after 3 - 7 days of incubation. Colonies are described often as ‘breadcrumb’ colonies. The reason for different sensitivity between gram-positive and gram-negative bacteria could be described to the morphological differences these microorganisms, gram-negative bacteria having an outer polysaccharide membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to lipophilic solutes, the gram positive should more susceptible having only an outer peptidoglycan layer which is not an effective permeability barrier. 1.2.3.5.1 Virulence factor Actinomyces pyogenes, a gram-positive, normally commensal bacterium, resides on the mucous membranes of cattle, sheep, swine, and other economically important animals (Carter et al., 1991).Despite the versatility of A. pyogenes as an agent of disease in domestic animals . Actinomycetes pyogenes produces virulence factors (pyolysin [PLO], collagen binding protein, neuraminidase, and DNase) are ubiquitous among isolates . The hemolytic exotoxin pyolysin (PLO) (Ding and La¨mmler, 1996), which is cytolytic for the erythrocytes of a number of animal species (Funk et al., 1996) , as well as dermonecrotic and lethal for laboratory animals (Lovell, 1944). The role of this toxin in pathogenesisis unclear. However, it is expressed in vivo and is immunogenic, as anti hemolysin antibodies have been found in the sera Ϯϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. of naturally (Lovell, 1939) and experimentally ( Matthews et al., 1963) . Cytolysin produced by a number of gram-positive bacteria (Alouf , 1980; Boulnois et al., 1991; Cossart et al., 1989) and its potential role in virulence. Also Actinomyces pyogenes produces neuraminidase which cleave terminal sialic acid residues from carbohydrates and glycoproteins. In some bacteria, this allows bacterial adhesion. 1.2.3.6. Bacillus Bacillus species are Gram-positive rods often arranged in pairs or chains with rounded or square ends and usually have a single endospore. The endospores are generally oval and are very resistant to adverse condition sporulation is not repressed by exposure to air (Holt et al., 1994). Bacillus species can be broadly divided in three groups based on the morphology of the spore and sporangium (Berkeley et al., 1997 ) . The genus Bacillus currently comprises in excess of 60 species, commonly found in the environment and as laboratory contaminants (Konemann et al., 1997). They are aerobic or facultatively anaerobic and most species are motile by peritrichous flagella. Most species are oxidase- negative and catalase-positive. 1.2.3.6.1. Virulence factor The many species of the genus exhibit a wide range of physiologic abilities that allow them to live in every natural environment enable them to survive or thrive in harsh environments, ranging from desert sands and hot springs to Arctic soils and from fresh waters to marine sediments. Spores formed under different conditions have Ϯϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. different stabilities and degrees of resistance to heat, radiation, chemicals, desiccation, and other hostile conditions. The genus includes thermophilic, acidophilic, alkaliphilic, pH values and salt concentrations at which few other organisms could survive. Because the spores of many Bacillus species are resistant to heat, radiation, disinfectants, and desiccation, they are exacerbate preexisting infections by producing either tissue-damaging toxins or metabolites such as penicillinase that interfere with treatment. 1.2.4. Epidemiology Very Little data about the epidemiology of fistulous withers in donkeys. They are given less consideration than other species of livestock and their welfare is often neglected. The data concerning the casual agent , transmission and significance of the disease . It is important to look for donkeys , due to their hard working ability and their tough nature. 1.2.5. Disease of equine similar to fistulous withers 1.2.5.1. Granulomatus lesion of the skin A wide range of etiologies have been reported to cause granulomatous inflammatory lesions localized to the deep layers of the skin in equine. Habronema and Onchocerca species of nematode have been shown (Jones et al., 1997). Fungal infections, including sporotrichosis, and histoplasmosis, can produce prolific and often deep granulomatous inflammation (Scott et al., 2003). Similarly, botryomycosis, which has been associated mainly with Staphylococcus Ϯϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. spp., is characterized by a chronic pyogranulomatous dermatitis and formation of micro abscesses and bacterial pseudomycetoma (Pascoe et al., 1999 ; Scott et al., 2003). In addition, a generalized idiopathic granulomatous disease syndrome has been reported in horses(Axon et al., 2004; Heath et al .,1990 ; Sellers et al., 2001) . Although generally thought of as a disseminated disease affecting many organ systems, idiopathic granulomatous disease has been described to produce only cutaneous lesions in some cases (Heath et al.,1990). 1.2.5.2. Saddle sores A saddle sore is an inflammation of the hair follicles of the horse at pressure points where tack comes in contact with the skin. The most common location is the withers. Girth sores are similar friction wounds found in the girth area. Sores can also occur on the horse's face where the bridle contacts the skin. In addition, riding a horse that has not been properly groomed or using dirty tack can result in saddle sores. The pressure of tack grinding against grit or sand on the skin can have to same effect as rubbing the skin with sand paper. Clean tack after every ride, and always groom your horse before every ride. Another cause of saddle sores is poor equitation. An off-balance, bouncing rider can cause the horse a great deal of muscle pain, as well as sores. All that excessive movement causes friction against the horse's skin. The first symptom of a saddle sore is inflammation and sometimes blistering. The condition starts as an acute inflammation of the hair follicles and progresses to a purulent folliculitis. Affected areas show hair loss and are swollen, warm, and painful. The serous or purulent exudates dries and forms Ϯϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. crusts and in the most severe cases infection and necrosis will occur. The best treatment for saddle sores is complete rest until it has healed. Cold water or ice packs can help reduce the swelling. Your veterinary may recommend giving the horse an anti-inflammatory drug. If there are open wounds he/she will probably prescribe antibiotics to ward off infection. A topical antibacterial ointment should be applied to the raw area. In severe cases, where there is a blood blister or necrotic tissue, surgery might be required to remove that tissue. As usual, prevention is easier than the cure. Groom your horse thoroughly before riding, paying particular attention to areas that come in contact with the tack. Make sure your tack is clean, has no rough places, and fits properly. Lift the pad up slightly at the withers before tightening the girth, so that it does not bind in that area. Learn to ride correctly, in balance with your horse, and avoid excessive up and down hill riding. If a donkey has one of the conformation faults it might need extra or specially designed pads under its saddle and/or a breast collar to stabilize the saddle. 1.2.5.3. Capped hocks Capped hock is inflammatory swellings of the subcutaneous bursae (acquired bursitis) located over the olecranon process and tuber calcaneus, respectively, of horses. Trauma from lying on poorly bedded hard floors, kicks, falls , iron shoes projecting beyond the heels, and prolonged recumbency are frequent causes. Edematous swelling develops over and around the affected bursa. Lameness is rare in either case. The affected bursa may be fluctuating and soft at first but, in a short time, a firm fibrous capsule forms, especially if there is a recurrence of an old injury. Initial bursal swellings may be hardly Ϯϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. noticeable or quite sizable. Chronic cases may progress to abscessation. Acute early cases may respond well to applications of cold water, followed in a few days by aseptic aspiration and injection of a corticosteroid. The bursa may also be reduced in size by application of a counterirritant or by ultrasonic or radiation therapy. Older encapsulated bursae are more refractory. Surgical treatment is recommended for advanced chronic cases or for that become infected. 1.2.6. Prevention Since horses usually acquire B. abortus infection from cattle (Denny, 1973; Cramlet and Bernhanu, 1979), they should not be housed or pastured with seropositive cattle. Since trauma is considered to be a predisposing factor for the development of fistulous withers, properly-fitting saddles and tack should always be used. Parasite control measures to reduce the incidence of Onchocerca spp. 1.2.7. Treatment Treatment of fistulous withers and poll evil can be difficult. Medical treatment is directed towards controlling infection and inflammation. Broad spectrum antibiotics are recommended. The wounds generally resulted from poorly fitting pack equipment. Treatment regimens are divided into two categories: 1) conservative topical therapy plus padding . 2) surgical debridement and primary wound closure. Surgical treatment is used primarily in cases refractory to conservative therapy. As a result of surgical intervention, clients tend to comply with Ϯϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. recommendations to stop working the animal. The surgery may be performed in the standing horse or under general anaesthesia. The convalescent time was shorter, and the recurrence rate was lower following surgery than for conservative therapy ( Miguel et al., 1997). Treatment of brucellosis in donkey generally involves a combination of systemic antimicrobials and local surgical drainage of infected tissue. In a survey of veterinarians in Florida concerning the treatment of fistulous withers, treatment with antibiotics was reported to be effective in 37 of 46 horses (80.4%) (Gardner et al., 1983). Lavage of draining tracts with antiseptic solutions and dimethyl sulphoxide may be helpful (Cohen et al., 1992). ϯϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CHAPTER TWO 2. MATERIALS AND METHODS 2.1. Sterilization a-Flaming It was used to sterilize glass slides, cover slip, needles, scalpels points of scissors and mouth of culture tubes by passing them through Bunsen burner flame without allowing becoming red hot . b-Red heat It was used to sterilize loop wires, point of forceps and searing spatulas by holding them over Bunsen burner flame until become red-hot . c-Hot air oven It was used to sterilize glassware such as bottles, flasks, test tubes, Petri dishes, Pasteur pipettes, graduated pipettes and forceps. They were sterilized in hot air oven at 180°C for one hours . d-Steaming at 100°C It was used for sterilization of sugars and media that could not be autoclaved without determent effect to their constituents .It was carried out as described by Cruickshank et al., (1975). e-Moist heat (Autoclave) Autoclaving at 121°C for 15 minutes was used for sterilization of media and ϯϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. plastic wares . Autoclaving at 115°C for 10 minutes was used for sterilization of some media . 2.2. Reagent Most reagents were obtained from British Drug House (BDH). 2.2.1. Sodium Chloride/ normal saline Normal, physiological or isotopic saline was prepared as described in Oxoide manual by dissolving 8.5 g of sodium chloride in 1 liter of distilled water to obtain 0.85% concentration. 2.2.2. Alpha-naphthol solution Alpha-naphthol solution was obtained from BDH Ltd . This solution was prepared as 5% aqueous solution for Voges-Proskaur (VP) test and prepared according to (Hopkins and Williams) . 2.2.3. Potassium hydroxide (Hopkins and Williams): This reagent was prepared as 40% aqueous solution for Voges-Proskaur (VP) test and prepared according to (Hopkins and Williams) . 2.2.4. Hydrogen peroxide (H2O2) This reagent was prepared as 3% aqueous solution and stored in dark and cool place. It was used for catalase test and obtained from BDH Ltd . 2.2.5. Kovac's reagent This reagent composed of 5 g para- dimethyl amino benzaldehyde, 75ml amyl alcohol and 25ml concentrated hydrochloride acid. It was prepared ϯϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. as described by Barrow and Feltham (1993). The aldehyde was dissolved in alcohol gently in water bath at temperature of 50-55C, then cooled and the acid was added with care. The reagent was protected from light and stored at 4°C for indole test . 2.2.6. Ttetramethyl-p-phenylene diamine dihydrochloride This reagent was prepared as 1% aqueous solution according to (Hopkins and Williams) and it was used for oxidase test. 2.2.7. Nitrate test reagent Nitrate test reagent was consisted of two solution and they were prepared according to Barrow and Feltham (1993). Solution A was composed of 0.33% sulphanilic acid dissolved by gentle heating in 5N- acetic acid. Solution B was composed of 0.6% dimethyl amine-alpha-naphthylamine dissolved by gentle heating in 5N- acetic acid. 2.3 Indicators 2.3.1. Andrade's indicator This was prepared according to Barrow and Feltham (1993) . It was composed of 5g of acid fuchsin, 1 liter of distilled water and 150ml of NNaOH. The acid fuchsin was dissolved in distilled water, then the alkali solution was added, mixed and was allowed to stand at room temperature for 24 hour with frequent shaking until the color changed from red to brown. It was used in sugar fermentation . ϯϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.3.2. Bromothymol blue It was obtained from BDH Ltd . The solution was prepared by dissolving 0.2g of the bromothymol blue powder in 100ml distilled water. It was used for Oxidation fermentation test . 2.3.3. Plasma The plasma used for coagulase test was human plasma , prepared by centrifugation of citrated human blood . 2.3.4. KOH and HCL KOH and HCL were used for adjusting the pH . 2.4. Collection of blood for enriched media Blood for enriched media was collected aseptically into sterile flask containing glass beads venipuncture of jugular vein of a healthy sheep kept for this purpose. The blood was defibrinated by shaking the sterile flask that containing glass beads while and after collection . 2.5. Preparation of Culture Media Different type of media including solid ,semisolid and liquid media were used for isolation and identification of bacteria in this study . 2. 5.1. Liquid media 2.5.1.1. Nutrient broth This medium contained peptic digest of animal tissue 5g , beef ϯϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. B274 . Thirteen grams of nutrient broth (Oxoid) were added to one litre of distilled water, mixed well and pH adjusted to 7.2 -7.4 ,distributed in 3ml amount into clean test tubes, then sterilized by autoclaving at 121°C for 15 minutes. 2.5.1.2. Peptone water This medium contained peptic digest of animal tissue 10 g and sodium Chloride 5g with code No. M 028 according to (HIMEDIA). To rehydrate the medium, 15g were suspended in 1000 ml distilled water, mixed well and pH adjusted to 7.2 -7.4 , distributed in 3ml amount into clean test tubes and Sterilized by autoclaving at 121°C for 15 minutes. 2.5.1.3. Peptone water sugar This media was prepared according to Barrow and Feltham (1993). 900ml of peptone water were prepared and andrade's indicator 10 ml . Sugar solution was prepared by dissolving 10 grams of appropriate sugar in 90 ml of peptone water . The pH was adjusted to 7.1- 7.3 before the addition of andrade's indicator. The complete medium was mixed, distributed in to 2 ml volumes into test tubes containing inverted Derham's tube (for gas trapping in case of glucose) andsterilized by autoclaving at 115°C for 10 minutes and kept at 4°C until used . 2.5.1.4. Glucose-phosphate (MR-VP test) medium This medium contained peptone (Oxoid L49) 5g , dextrose 5g and phosphate buffer (k2HPO4) 5g with code NO. 43 according to (Oxoid). The powder of 15 g were added to 1 liter of distilled water, dissolved by ϯϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. steaming , mixed well ,the pH was adjusted to 7.5 , distributed into clean test tubes and sterilized by autoclaving at 121°C for 15 minutes. 2.5.1.5. Nitrate broth medium This media was prepared according to Barrow and Feltham (1993). It contained potassium nitrate 0.2g and peptone 5g . Solid ingredients were dissolved into one liter of distilled water. The medium mixture was distributed in 5 ml amount in clean test tubes and then autoclaved for 15 minutes at 121°C. 2.5.1.6. Serum water sugars The medium was consisted of peptone 4g ,sodium phosphate 0.8g ,distilled water 800ml ,sterile serum 200ml and bromcresol purple(0.2%) 10ml. dissolved the peptone and phosphate in the water, steamed at 100°C for 15 minutes. Add the serum ,checked pH to 7.6_7.8 , add the indicator and sterilized at 115°C for 10 minutes. Then aseptically add 1% of the appropriate sugar as sterile solution and distributed into sterile tubes. 2.5.2. Semi solid media 2.5.2.1. Motility medium Motility medium was prepared as described by Barrow and Feltham (1993). It consisted of nutrient broth (Oxoid) 13g and agar (Oxoid agar No.1) 5g. The dehydrated of nutrient broth were added to agar in one liter of distilled water , steamed to dissolved . The pH was adjusted to 7.4 . The medium was dispended in 5 ml volume into 20 ml capacity test tubes containing appropriate Cragie tubes. The medium in test tubes were sterilized by ϯϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. autoclaving at 121°C for 15 minutes. Then the medium was allowed to cool to become semi solid before using. 2.5.2.2. Hugh and Leifson's (O/F) medium This medium was composed of dipotassium hydrogen phosphate ,sodium chloride, peptone, agar and 0.2 % aqueous solution of bromocersol purple. The medium was prepared as described by Barrow and Feltham (1993) . Two grams of peptone powder, 5g of sodium chloride, 0.3g of potassium hypophosphate and 3g of agar were add to 1 liter of distilled water then heated in water path at 55°C to dissolve the solid . The pH was adjusted to 7.1 . Then the indicator bromothymol blue (0.2 % aqueous solution ) was added and mixture was sterilized by autoclaving at 115 °C for 15 minutes. Then sterile glucose solution was added aseptically to give final concentration 1% . Then the medium was mixed and distributed aseptically in 10 ml amount into sterile test tubes . 2.5.3. Solid media 2.5.3.1. Blood agar This medium was composed of dehyderated blood agar base obtained from(Scharlau) and defibrinated sheep blood . The blood agar base with code NO. 01-352 contained meat extract 10g , tryptone 10g , Sodium chloride 5g and agar 15g . It was prepared according to manufacturer’s instruction by dissolved 40g of blood agar base in one liter of distilled water by boiling, mixed and sterilized by autoclaving at 121°C for 15 minutes. Then cooled to about 50°C and defibrinated sheep blood was added aseptically to give final concentration 10% , mixed gently and 20ml of ϯϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. complete medium was poured into each sterile Petri dishes, the plates were allowed to solidify at room temperature on leveled surface . 2.5.3.2. MacConkey agar The dehydrated form obtained from (Scharlau) with code No. 01-118 and it consist of peptone 20g , lactose 10g , bile salt 5g , sodium chloride 5g , neutral red 0.03g , crystal violet 0.001g and agar 15g . Then 51.5 g of the powder were suspended in 1 liter of distilled water, boiling until dissolved the ingredient completely then sterilized by autoclaving at 121°C for 15 minutes. The medium was then cooled to about 50 °C and poured into sterile Petri dishes in 20 ml amount and left to solidify at room temperature on leveled surface . 2.5.3.3. Nutrient agar The medium was obtained from (Scharlau) with code No. 01-144 and it was composed of peptone 5g , meat extract 3g and agar 15g .To rehydrate the medium , 23g of nutrient agar powder and 5 g of sodium chloride were suspended in 1 liter of distilled water, brought to boiling until dissolved completely then sterilizing by autoclaving at 121°C for 15 minutes. The medium was then cooled to about 50°C and distributed in 20 ml amount sterile Petri dishes. 2.5.3.4. Ammonium salt sugars (ASS) The medium was prepared as described by Barrow and Feltham (1993) . One gram of (NH4)2HPO4 , KCL (0.2g) , MgSO4.7H2O (0.2g) and yeast extract (0.2g) were added to 1 liter distilled water .Then indicator bromothymol blue (0.04ml) was added. The solids were dissolved by ϯϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. steaming and sterilized by autoclaving at 115°C for 20 minutes. The mixture was allowed to cool to about 60°C, then appropriate carbohydrate was added as sterilized solution to give final concentration 1% , mixed and distributed aseptically into sterile test tubes and allowed to solidify in slope position. 2.5.3.5. Aesculin – bile azide agar The medium was prepared as described by Barrow and Feltham (1993) . The medium was composed of ox bile (dehydrated) 40g , asculin 1g , ferric citrate 0.5g , sodium azide 0.2g , agar 15g and nutrient broth 1000g . All of the ingredients except aesculin were dissolved in water by heating . Allowed to cool and then aesculin was added . The medium was then suspended in screw capped bottles, sterilized by autoclaving 115°C for 20 minutes and allowed to set in slope position . 2.5.3.6. Milk agar The medium was prepared as described by Barrow and Feltham (1993) . This medium was composed of milk ( skim ) 500ml and nutrient agar (double- strength) 5ooml . Prepared the skim-milk and sterilized by heating at 115°C for 10 minutes. Cool to about 50°C and add to the nutrient agar double- strength , melted and cooled to 50- 55°C. Mixed and distributed in Petri dishes. 2.5.3.7. Nutrient gelatin medium This medium was composed of beef extract 3 g , peptone 15 g , gelatin 120g and Distilled water 1000 ml . One hundred and twenty eight ϯϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. grams of nutrient gelatin (Oxoid) ,were added to one liter of distilled water , steam to dissolved, mixed well, pH was adjusted to 6.8 , the medium distributed in sterile screw capped ( Bijou) bottles in 2ml amount and sterilized by autoclaving at 121°C for 15 minutes. 2.5.3.8. Simmons citrate agar This medium was composed of magnesium sulfate o.2g , manoammonium dihydrogen phosphate 1g , dipotassium phosphate 1g , sodium citrate 2g , sodium chloride 5g , bacto- agar 15g , brom thymol blue 0.08 g . To rehydrate the medium, 24.2g (Scharlau) were suspended in 1000ml distilled water , dissolved by steaming completely and the pH was adjusted to 7.0 . The medium sterilized by autoclaving for 15 minutes at 121°C . Then dispended into Bijou bottles in portions of 5ml and allowed to solidify in slope position . 2.5.3.9. Starch agar It consisted of potato starch 10g , nutrient agar 50g, there were suspended in 1000ml distilled water (Barrow and Feltham, 1993). Triturated the starch with the water to a smooth cream and added to the molten nutrient agar. Mixed and sterilized at 115°C for 10 minutes. Distribute in 20 ml amount sterile Petri dishes . 2.5.3.10. Urea agar Base The base medium contained peptone (Oxoid L37) 1g , dextrose 1g , sodium chloride 5g ,disodium phosphate 1.2g , potassium dihydrogen phosphate 0.8g , phenol red 0.012g and agar No.3 (Oxoid L13) 15g with code No. CM53 . The medium was prepared according to manufacturer’s ϰϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. (Oxoid) by dissolving 2.4g of powder in 95ml distilled water by boiling , pH was adjusted to 6.8 , sterilized at 121°C for 15 minutes and cooled to 50-55°C . Five ml of sterile urea solution was added aseptically .The medium was distributed in 5 ml amount in sterile test tubes and allowed to set in slope position . 2.6. Clinical investigations 2.6.1. Study area A protocol was developed to assess the welfare of a cross-sectional study of randomly selected, male donkeys used for pack and ridden work found in Khartoum State in urban and peri-urban areas namely Elsagana, Elremila, Mayo in Khartoum, Halaib, Elsouk Elshabi Omdurman in Omdurman and shambat in Bahri. clinical examination and a questionnaire survey were both carried out simultaneously between December 2010 and May 2011. 2.6.2. Animals One hundred and fifty of indigenous breeds from regions of selected areas were investigated using direct observation of health and behavior parameters and to isolate and identify aerobic bacteria associated with infection by fistulous wither in donkeys in Khartoum state. Parameters examined were general condition , body temperature and pulse rate. 2.6.3. Clinical signs Clinical signs of fistulous wither include localized heat, pain and swelling of the bursa. The early lesion is a simple thin-walled sac with serous exudates. This exudates arises in the cause of the inflammatory change in inner layer ϰϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. of the bursal sac. The latter stages involve considerable necrosis. 2.6.4. Collection of Samples In this study, sixty out of 150 donkeys, which showed fistulous wither were considered for bacteriological examination. The lesion were open (fistulae) and samples collected after cleaned with cotton pieces soaked in 70% ethyl alcohol. All donkey samples were collected by a sterile cotton swabs , which were then labeled and carried in container with ice to the laboratory and kept at -10°C. 2.7. Bacteriological examination 2.7.1. Primary isolation All samples were cultured onto blood agar and MacConkey agar. a-Blood agar It was used as an enriched , non- inhibiting medium for primary isolation of bacteria and for determination of colonial morphology and hemolytic activity of Streptococci, Staphylococci and other organisms. b-MacConkey agar This medium was used in this study for isolation of enterobacteria . 2.7.2. Isolation Isolation attempts were made for all samples at medical laboratory, University of Khartoum. The swabs were inoculated onto 10% defibrinated sheep blood agar and MacConkey agar .The inoculated plates were then ϰϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. incubated aerobically at 37°C for 24 hours as described by Barrow and Feltham (1993). Further incubation was continued for another 24 h and if no growth was evident ,then the plates were discarded as negative . 2.7.3. Cultural characteristics All cultures on solid media were examined with nacked eye for growth and colonial morphology and any change in medium.The liquid media were also examined with eye for turbidity , color change ,formation of sediments and accumulation of gas in the Durham’s tube in case of carbohydrates media . 2.8. Purification Purification was done by several subculturing from single well separated colony on separate nutrient agar plates or blood agar plates for those which do not grow on nutrient agar. Then examined for purity microscopically as described later for identification. 2.9. Preservation The isolates were preserved for further studies to determine their cultural and biochemical characterizations. Preservation was made by subculturing the purify isolates and store at 4°C. 2.10. Microscopic Examination Smears were made from purified colonies, fixed by heating and stained by Gram stain method of Barrow and Feltham(1993). Then examined microscopically for cell purity , morphology , arrangement characterization of bacteria and staining reaction . ϰϯ , Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.11. Biochemical tests 2.11.1. Sugar fermentation test The test was carried out as described by Barrow and Feltham (1993). The ped isolates on fresh sheep blood agar plates weakly. Cultures were kept. peptone water sugar was inoculated with organism under the test, incubated at 37°C and then examined daily for several days. Acid production was indicated by the appearance of pink color, while gas production was indicated by presence of an empty space in the inverted Durham's tubes. 2.11.2. Oxidase test The method of Barrow and Feltham (1993) was followed. Strips of filter paper was soaked in 1% solution of tetramethyl- p-phenylenediamine dihydrochloride and dried in hot air oven and then placed on a clean glass slide by a sterile forceps. A fresh young tested culture on nutrient agar was picked off with sterile glass rod and rubbed on the filter paper strips. If a purple color developed within 5-10 seconds, the reaction was considered positive. 2.11.3. Catalase test The test was carried out as described by Barrow and Feltham (1993). A drop of 3% aqueous solution of hydrogen peroxide was placed in a clean glass slide. A colony of tested culture on nutrient agar was picked off and put on the drop of hydrogen peroxide. Evolution of gas and appearance of bubbles indicated positive test. 2.11.4. Coagulase test ϰϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.11.4.1. Slide coagulase test The test performed on slide by added drops of physiological saline were placed in each end of slide .Two colonies of tested culture were emulsified in each of drops to make two thick suspensions . A drop of human plasma was added to one of the suspension and mixed gently . Clumping of the organisms within 10 seconds was recorded as positive reaction . 2.11.4.2. Tube coagulase test The test was performed as described by Barrow and Feltham (1993). To 0.5ml of 1:10 dilution of human plasma in physiological saline, 0.5ml of an 18-24h old broth culture of the tested organism was added in asterile agglutination test tube then incubated at 37°C and examined after 2-24 h for coagulation. Definite clot formation indicated positive result. 2.11.5. The oxidation- fermentation (o/F) test The test was carried out as described by Barrow and Feltham (1993). Duplicate test tubes of Hugh and Leifeson's medium were inoculated by the tested organism with straight wire. To one of the test tube a layer of sterile melted soft paraffin oil was added to a depth of 3 cm above the medium to seal it from air. The inoculated tubes were incubated at 37°C and examined daily for fourteen days. Yellow color in the open tube only indicated oxidation of glucose, yellow color in both tubes showed fermentation reaction and blue or green color in open tube and green color in the sealed tube indicated production of alkali. 2.11.6. Indole production test ϰϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Indole production test was carried out as described by Barrow and Feltham (1993). The tested organism was inoculated into peptone water and incubated at 37°C for 48h . One milliliter of the Kovac's reagent was added . Production of red color on the upper layer of the reagent indicated positive reaction. 2.11.7. Voges- Proskauer (VP) test The test was performed as described by Barrow and Feltham (1993). The tested culture was inoculated into glucose phosphate medium (MR-VP medium) then incubated at 37°C for 48h. Then 0.6ml of 5% alphanaphthol solution followed by a 0.2ml of 4% potassium hydroxide were added to one ml of the culture, shaked well and examined after 15-30 minutes . When bright pink color developed the reaction was regarded as positive. 2. 11.8. Nitrate reduction The nitrate test was carried out as described by Barrow and Feltham (1993). The tested culture was lightly inoculated into nitrate broth and incubated at 37°C for up to 5 days. Then 0.5ml of solution A followed by 0.5ml of solution B of nitrate test reagent were added. Red color indicated positive reaction that showed nitrate had been reduced. If red did not develop, powdered zinc was added to see whether there was residual nitrate or not. Red color development indicated that nitrate in medium had been reduced to nitrite by zinc but not by organism, whereas unchanged color indicated nitrate in original medium had been reduced completely and nitrite was further broken down by the organism. ϰϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.11.9. Urease activity test The test was carried out as described by Barrow and Feltham (1993). The tested organism was inoculated heavily onto slope urea agar medium and then incubated at 37°C for 7 days. Appearance of pink color indicated positive reaction. 2.11.10. Citrate utilization test The test was performed as described by Barrow and Feltham (1993). The tested culture was inoculated as a single streak over the surface of a slope of Simmon’s citrate medium at 37°C up to 48 hours . Examined daily for 7 days for growth of the organism and changed color to blue indicated positive test. 2.11.11. Aesculin hydrolysis The procedure of Barrow and Feltham (1993) was followed. The tested organism was inoculated into aesculin agar , incubated at 37°C and examined daily for up to 7 days. Black color indicated positive reaction . 2.11.12. Hydrogen sulphide (H2S) production The method of Barrow and Feltham (1993) was followed. The tested culture was inoculated into peptone water, filter paper impregnated with 10% lead acetate solution was inserted between the plug and the tube . The tubes were examined daily for 7 days for black color of paper due to hydrogen sulphide production as positive reaction . 2.11.13. Ammonium salt sugar test ϰϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. The test was performed as described by Barrow and Feltham (1993). The tested organism was inoculated onto a slope of ASS medium and incubated at 37°C for up to 7 days. The medium was examined on alternative days for growth and acid production. 2.11.14. Gelatin hydrolysis Gelatin hydrolysis test was carried out as described by Barrow and Feltham (1993). The tested culture was stabbed into nutrient gelatin and was incubated at 37°C for up to 14 days. The inoculated tube was placed in refrigerator for 2 hours every 2-3 days and was examined. The liquefaction of gelatin indicated positive test. 2.11.15. Motility test The semi-solid motility medium containing Craigi tube prepared as described by Cruickshank et al. (1975) was used to detect motility . A small piece of the colony of the bacterium under the test was picked by the end of the straight wire loop and stabbed in the center of semi-solid agar in the Craigi tube ,then incubated at 37°C.The was considered motile if there was turbidity in the medium in/outside the Craigi tube . ϰϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CHAPTER THREE 3. RESULTS 3.1. Clinical findings The data showed that donkeys demonstrate avoidance or aggressive behavior towards an observer. Fewer than 60% of working donkeys had abnormal mucous membranes, ectoparasites or poor coat condition. Over 30% of donkeys demonstrated limb deformities and abnormalities of gait , overgrown hooves and wounds caused by incorrect tethering or hobbling . In cases of injury seen the animals were usually thinner and the pads under the pack saddle were of a synthetic fiber material such as polypropylene. The results of this study might be used as the initial stage of a long-term strategy to inform priorities for welfare interventions in working donkeys and to establish a welfare benchmark. Therefore, the status and dynamics of donkeys husbandry, as well as a general assessment of the needs of donkeys owners in this area were researched. The prevalence of external injuries in donkeys was observed in different ages with different status from mild, moderate and severe and different causes. The survey which targeted to detect fistulous wither among donkeys, its investigation was focused on etiological agent. Almost all donkeys were under hard working conditions treated by owners. Donkeys were used always exclusively for transport purposes , abused and poorly managed . feeding is mainly green fodder as well as Sorghum grains (Fetarita) (tables 2,3 and 4). ϰϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Table2: Distribution of External Injuries on Various Body Parts in donkeys Location of Injures Donkeys (n) % Wither 60 40 Flank 4 2.7 Back / Shoulder 71 47.3 Under Tail 3 2 Front Leg 5 3.3 Abdomen 3 2 Head / Neck - - Hind Leg 4 2.7 150 100 Total Table 3: Causes of External Injuries in Donkeys in Khartoum State Cause Donkeys n(%) Improper harness and saddle 38 (25.3) Overloading and overworking 55 (36.7) Biting 20 (13.3) Infection disease 14 (9.3) Un known 5 (3.3) Maltifactorial causes 18 (12) Total 150 ϱϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Table 4: Owner’s Responses to the Management of External Injuries in donkeys Owner’s Responses Donkeys (%) (n) Take to nearly health center 32 21. 3 Treat with medication purchased 20 31. 3 Treat with medical plant 28 18. 7 Do nothing 70 46. 7 Total 150 100 from local market 3.2. Bacterial isolation From 150 donkeys, only 60 samples which collected from donkeys infected with fistulous wither and cultured onto blood agar and MacConkey agar media and incubated aerobically at 37°C. Fifty-four (90%) showed bacterial growth, while six samples (10%) did not demonstrated any growth of bacteria. 3.3. Characterization and identification of isolates The bacterial isolates obtained in this investigation were identified to species level on the basis of their cultural Characteristics, cell morphology, Gram stain reaction and their biochemical properties as described by Barrow and Feltham (1993) as showen in (table 5). The isolate consisted of 142 Gram positive isolate (236.7%) as showen in (Table 4). 3.4. Gram- positive Bacteria Total of 142 (236.7%) organism were characterized as Grampositive bacteria. They were further characterized as Staphylococcus spp 55 ϱϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. (91.7%), Corynebacterium spp 8 (13.3%), Streptococcus spp 6 (10%), Actinomycetes 4 (6.6%) and Bacillus spp 69 (115%) as showed in (Table 4). Bacillus spp and Staphylococcus spp represented the highest percentage of the total Gram-positive bacteria isolated (Fig.1). 3.4.1. Staphylococcus The Staphylococcus spp percentage (38.7%) of the total Grampositive isolated. Staphylococcus species were identified according to the scheme of Staphylococcus species identification of Barrow and Feltham (1993). They were identified as twenty-five coagulase –positive staphylococci, this represent (45.5%) of the total Staphylococci species. They were twenty-two (40%) S. aureus and three (5.5%) S. intermedius. Thirty isolates were coagulase –negative Staphylococci, this represent (54.5%) of the total Staphylococci species. They were eighteen (32.7%) S. chromogen, eight (14.5%) S. haemolyticus and four (7.3%) S. epidermidis. 3.4.2. Streptococcus The Streptococcus spp isolated in this study were identified according to their morphology and Gram-stain reaction. They were Grampositive cocci, non-motile, non-sporing and arranged in short or long chain. They were also identified on the basis of their biochemical properties. They were (4.3%) of the total Gram-positive bacterial isolation. 3.4.3. Actinomycetes Four isolates were identified as Actinomycetes spp and these represent (2.8%) of the total Gram-positive isolates. The different species isolated were Actinomycetes bovis and Actinomycetes pyogenes. ϱϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 3.4.4. Corynebacterium Eight isolates were identified as Corynebacterium spp and these represent (5.6%) of the total Gram-positive isolates. The species isolated were Corynebacterium pseudotuberclosis, Corynebacterium aminoniagenes, Corynebacterium kutscheri, Corynebacterium ulcerans. 3.4.5. Bacillus Sixty-nine isolates were identified as Bacillus spp and these represent (48.6%) of the total Gram-positive isolates. The different species isolated were Bacillus mycoides, Bacillussubtilius, Bacillus circulans, Bacillus licheniformis, Bacillus firmus , Bacillus alvei. ϱϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Table 5: Isolation frequency of Gram-positive bacteria isolated from donkeys infected with fistulous withers . No. of Sources of samples Infected donkeys with No. of samples Species of bacteria examined isolated ϲϬ fistulous withers Total isolated Staphylococcus spp ϱϱ Corynebacterium spp ϴ Streptococcus spp ϲ Actinomycetes spp ϰ Bacillus spp ϲϵ ϭϰϮ Table 6: Biochemical properties of some Grame+ve bacteria isolated from ϱϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. donkeys infected with fistulous withers in Khartoum State Staphylococcs Streptococcus Actinomycetes Test spp spp spp Gram reaction + + + Growth + + + Catalase + - + Oxidase - - - Motility - - - Growth +/- + + F F F VP +/- - - Nitrate + - +/- + - - at37°C. anaerobically Oxidation fermentation reduction Coagulase Key: (-) = Negative (+) = Positive F = fermentative Table 7: Gram stain reaction and biochemical properties of Staphylococcus spp isolated from donkeys infected with fistulous withers ϱϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Test Staphylococcus Staphylococcs Staphylococcus Staphylococcus aureus intermedius chromogens haemolyticus St ep Gram-reaction + + + + + Oxidase - - - - - Catalase + + + + + Oxidation F F F F F Coagulase + + - - - VP + - - + + Nitrate + + + + + Urease + + + - + Mannitol + + - + - Maltose + - + + + Sucrose + + + + + fructose + + + + + Xylose - - - - - fermentation Key: (-) = Negative (+) = Positive F =fermentative Table 8: Gram stain reaction and some biochemical properties of Bacillus spp isolated from donkeys infected with fistulous wither ϱϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Test Bacillus Bacillus Bacillus Bacillus Bacillus mycoides subtilius circulans licheniformis firmus B l a Gram-reaction + + + + + + Oxidase - - - - - - Catalase + + + + + + Oxidation F F F F F F Motility - + + + + + VP + + - - - + Nitrate + + - + + + Urease + - - - - - Glucose + + + + + + Gluctose + + + + - + Salicin + - + + - + Xylose - - + + - - Citrate + + - + - - fermentation Key: (-) = Negative (+) = Positive F = fermentative Table 9: Gram stain reaction and some biochemical properties of Corynebacterium spp isolated from donkeys infected with fistulous withers ϱϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Test Corynebacterium Corynebacterium Corynebacterium Pseudotuberclosis aminoniagenes Gram-reaction + + + Oxidase - - - Catalase + + + Oxidation F F F VP - - - Nitrate - + + Urease + + + Mannitol - - - Maltose + - + Sucrose + - + Xylose - - - kutscheri fermentation Key: (-) = Negative (+)= Positive F = fermentative Table 10: Gram stain reaction and some biochemical properties of Streptococcus spp and Actinomycetes spp isolated from donkeys infected with ϱϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. fistulous withers Test Streptococcus Streptococcus Actinomycetes equi zooepidemicus bovis Actinomycetes pyogene Gram-reaction + + + + Oxidase - - - - Catalase - - - - Motility - - - - Oxidation F F F F VP - - - - Growth + + + + Mannitol + + - - Sucrose - - + - Lactose + + + + Starch ND ND + + ND ND - + fermentation anaerobically hydrolysis Gelatin liquefaction Key: (-) = Negative (+) = Positive F = fermentative ϱϵ ND=NOT done Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. ϳϬ ϲϬ ϱϬ ϰϬ No. of isolate ϯϬ ϮϬ ϭϬ Ϭ Type of bacteria Figure 4 : Frecquency of Gram Gram-positive positive bacterial species isolated from donkeys with fistulous withers ϲϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Figure 5 : Staphylococcus aureus isolated from donkey with fistulous withers on blood agar medium Figure 6 : Actinomyces spp bacteria isolated from donkey with fistulous withers on blood agar medium ϲϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Figure 7: Smear from blood agar culture of Staphylococcus aureus Figure 8: Smear from blood agar culture of Actinomyces spp ϲϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CHAPTER FOUR Discussion In underdeveloped countries donkeys play an essential role in the agricultural economies, but these animals have not yet been given sufficient care and they are subjected to many diseases which affect their viability and lower their work ability (Khalifa et al., 1988). The use of donkeys in doorto-door transport service also provides urban dwellers with the opportunity of income generation (Kathy et al., 2004). In the Sudan the use of donkeys for transportation will continue for years to come because of the rugged terrain characteristics inaccessible for modern road transportation facilities as well as the absence of well-developed modern transport networks and the prevailing low economic status of the community. This study was carried out in Khartoum state that is heavily populated with donkeys which are considered to be the most important draught animals, in addition to other towns and rural areas of the Sudan. Despite their invaluable contributions, donkeys are the most neglected animals, accorded low social status, particularly the male working donkeys often are involved in more multipurpose activities than horses. They transport goods to and from markets, farms, and shops, traveling long distances. They also pull carts carrying heavy loads 3 to 4 times their body weight. They work from 8 to 12 hours/day, depending on the season and type of work. Unlike horses, donkeys are not provided with feed supplements. They are brutally treated, made to work overtime without adequate feed or health care. The increasing human population, demands for transport of goods to and from far, remote areas, and construction activities around the town are making equines highly demanded animals. ϲϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Though equines provide several advantages, health and welfare is a visible problem. Studies to elucidate the magnitude of this problem are lacking. Such information would be useful for designing strategies that would help improve donkeys health and welfare. This study describes causes and influencing factors of external injuries in a working population of donkeys in Khartoum State. The results of this investigation demonstrated that external injuries were highly prevalent among the working donkeys in the region, showing their inhumane suffering due to inappropriate management and neglect. The study also revealed that donkeys were much more affected than horses contrary to the widely prevailing opinion that they are tolerant to hardship conditions. Donkeys were involved in a wide array of activities, yet very little management was accorded to them. They were made to carry heavy loads over long distances and hours. They travel as far as 70 km/day while carrying an average weight load of 150 kg. This was evident in the present findings as more cases of injuries in donkeys (36.7%) were due to overloading and overweight as reported by Demelash et al. (2006) , a similar situation in Ethiopia where over-weight and type of load/work contributed to high cases of back sores in donkeys. In agreement with the present report, improper harness and saddle were major causes of injuries in equines, these were demonstrated to be commonly distributed on wither and back coinciding with poorly designed and ill-fitted harnesses and saddles. Manufactured by unskilled artisans, equine-drawn carts are often designed unbalanced and too heavy and do not consider load distribution in relation to the body balance and style of movement. Wooden- or iron-made saddles are constantly put on the back/shoulder and strongly tied to the body by plastic rope, which causes persistent irritation and injuries. Donkeys are also ϲϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. rarely bathed by their owners. Sand, dirt and debris from the transported goods and pollution find their way between the equipment and the donkeys skin. The friction caused by these elements rubbing together against the skin cause chaffing and wounds that are left untreated and that can progress to serious injuries and infections: if the owner is actually riding the donkey, there is more weight on the saddle, predisposing for heavier rubbing against the wound. In most cases, harnesses were made of car tire strips, which cut in to the skin of the donkeys and form large open wounds. It can be concluded that whenever an animal works, there is a potential for injuries caused by either absence of or inappropriate equipment and harnessing. This substantiates the report of Pearson, (1992). The high prevalence of infection-related injuries in donkeys suggests the microbial pathogens as either primary or secondary causes, contributing to a decrease in sale value and work performance. A higher number of donkeys with lacerated wounds due to bites was reported in the present study (13.3%) . Damages caused by barbed wire, sharp objects and trauma due to fighting among donkeys were also common causes of lesions in donkeys in Khartoum State. Adequate feeding and proper health care should thus be an integral part of injury prevention. In many areas it is often virtually impossible for the donkey owner to provide fencing around the area in which the donkey lives. In these situations, although far from ideal, a neck tether, leg hobbles, or a combination of both is used to ensure the donkey does not stray. Unsuitable materials, such as wire, and incorrect fitting cause the majority of injuries which can often be very serious. One of the main problems facing donkeys ϲϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. is fistulous withers that being the major cause of ill-health donkeys and owner should have knowledge of these problems for control and prevention. Occurrence of fistulous withers in working donkey and horses were reported previously (Pearson et a1., 1999). In addition , fistulous withers needed surgical removal of necrotic tissue and local systemic prophylactic treatment with antibiotic. This study showed that wound due to fistulous withers which might be due to trauma, inhumane care which constitutes the main problem or continuous irritation of the animal’s back; this is justified by the fact that most of the donkeys in the area examined are used for riding or for pulling carts. Moreover, treatment for most of the infected wounds may prove costly. The isolated bacteria in the present study included Bacillus spp (69) , Staphylococcus spp (55) , Corynebacterium spp (8) , Streptococcus spp (6) and Actinomyces spp (4). In this study Staphylococci isolated from fistulous withers were Staphylococcus Staphylococcus Staphylococcus chromogen, aureus, Staphylococcus Staphylococcus intermedius, haemolyticus epidermidis. Staphylococcus aureus and is the potential pathogen isolated from wounds which in agreement with Ellis et al., (1998). While the isolation of commensal microflora such as Staphylococcus epidermidis is in line with the findings of El Abdeen (2001), who isolated them from contusions of sheep skins. Isolated of Staphylococcus aureus and Staphylococcus epidermidis , substantiated the result of Guard (1932) and Gaughan et al., (1988). The commonest Staphylococcus were Staphylococcus aureus, Staphylococcus chromogen and Staphylococcus haemolyticus. The Staphylococci associated with wound infections were also previously reported by Beers and Berkow (1999) as in the present ϲϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. study, the higher rate of isolation of Staphylococci indicated that these organism were more active in causing putrefaction of the skins as they were isolated alone or in mixed infection with other organisms. The isolation of Staphylococci from putrefied skin was reported by other authors(Unsworth 1946; Esuruoso 1977 and Ruhmann 1987). The species of Corynebacteria were normal flora of the skins (Barrow and Feltham1993). In this study species of Corynebacterium isolated were Corynebacterium Pseudotuberclosis , Corynebacterium aminoniagenes , Corynebacterium kutscheri and Corynebacterium ulcerous.,0 which similar to the bacterial species isolated by Cohen et al., (1992); Hawkins and Fessler (2000). Isolation of Corynebacterium ulcerans confirms what was reported by Asha et al., (2006), whereas isolation of Corynebacterium pseudotuberculosis and Staphylococcus spp. were in agreement with the findings of Stephen (2005). Corynebacterium was isolated from putrefied skins , this confirms what was reported by Ibrahim (1989). The Isolates of Streptococcus, were identified as Streptococcus equi and Streptococcus zooepidemicus. The same organisms were isolated by Gaughan et al., (1988), but Streptococcus salivarius and Streptococcus pyogenes were reported by Asha et al., (2006). The previous report by Woolcock (1975), substantiated the present finding, who isolated Streptococcus zooepidemicus from infected equines. Streptococci spp. caused a wide variety of local and generalized disease conditions in equines such as pneumonia and sinusitis and as commonest secondary invaders in wounds such as fistulous withers and poll evil. Streptococcus spp. was also isolated from damaged skins as reported by Holt et al., (1994). Actinomycetes spp. isolated were Actinomycetes bovis and Actinomycetes ϲϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. pyogenes. Isolation of Actinomycetes bovis, is in agreement with Kimball and Frank, (1945) and Actinomycetes pyogenes is in agreement with Asha et al., (2006). Actinomycetes bovis causes Actinomycosis which is a sub-acute or chronic progressive disease characterized by development of granulomatus, supperative lesion involving bone and soft tissue. The present result demonsterated abscesses discharge through fistulous and sinuses. In cattle the disease involves the mandible or other bony tissue of the head (lumpy jow). The species of Bacillus isolated: Bacillus mycoides, Bacillus subtilius, Bacillus circulans, Bacillus licheniformis, Bacillus firmus and Bacillus alvei. Isolation of Bacillus licheniformis and Bacillus firmus, is in agreement of the previous report by Asha et al., (2006). Bacillus causes putrefaction of skins due to gelatinolytic activity of bacteria (Cooper ,1973). Bacillus spp. were contaminating microorganism and were found in bad hygiene and were main source of contamination. In contrast to Guard (1932) and Asha et al., (2006) in this study Escherichia coli, Pasteurella spp. Enterococcus faecalis, Listeria species and Kurthia species were not isolated. This difference might be due to difference geographical areas . Hawkins and Fessler (2000) , were frequently isolated ß- hemolytic Streptococcus spp. from horses, whereas Staphylococcus aureus was frequently isolated from fistulous withers samples collected from donkeys in the present study, which in agreement with the report of Cohen et al., (1992). Lastly, Equines are important animals to the resource-poor communities in rural and urban areas of Sudan, providing traction power and transport services at low cost. Veterinary clinic service should be upgraded and made ϲϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. available to donkey owners, especially where private and state veterinarians are unavailable or too expensive in such resource-limited community. Bad hygiene and poor general condition are the factor which help the multiplication of bacteria, that lead to offensive skin, hair slopping and putrefaction of skins (Knew, 1952). ϲϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CONCLUSION The present study can be concluded in the followings : 1. The major problems of donkey identified were: • Injuries to the animal. • Inadequate harness materials. • Lack of knowledge of correct adjustment and use. • Different hitching systems for different implements. 2.The major pathogenic bacteria isolated were Staphylococcus spp., Corynebacterium spp., Streptococcus spp. and Actinomycetes spp. 3. The most common health problems of donkeys arise from overuse, misuse and under-nutrition. 4. Donkeys remain to play an important economic and social roles and are still important in transportation and some families depend on donkey as financial source. ϳϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. RECOMMENDATIONS From the findings of the present study it is recommended that : 1. There is a need for greater awareness of donkeys' welfare issues, with improved facilities and services for donkey management, feeding, watering and Veterinary care, supported by possible legislation. 2. There is a need for training and extension programs targeting on donkey owners. Extension staff needs more training related to donkeys . 3. Appropriate padding materials should be used to allow sufficient ventilation on the skin surface hence preventing infections. 4. Local artisans should be given basic training on harnessing theories and working principles of carts to prevent the manufacture of shoddy carts and donkey owners should be adequately trained on how to adjust the improved harnesses. 5. It is important that, with the influx of synthetic material, owners are educated to the benefits of natural materials over synthetics with respect to the long-term health and efficiency of their animals. 6. Bacteriological laboratories should be consulted to determine the organism and most effective treatment to be used. 7. When antimicrobial drugs are administered they should be given in full therapeutic dose for adequate period. 8. Donkeys with fistulous withers or other type of wounds should be prohibited from working by the authorities . 9. Further extensive work should be carried out, to survey the prevalence of fistulous withers in donkeys. ϳϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 10. Possible improvements by development of a new harness for donkeys . 11. Good management, treatment of wounds in addition to use of suitable saddles may decrease chance of bacterial infection of the affected skins in the area of damage. ϳϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. REFERENCES Aganga, A. A. and Maphorisa, K. (1994). Characteristics and uses of donkeys in Botswana. In : Improving animal traction technology. Starkey, P. Mwenya, E. and Stares, J., eds. pp146-149. CTA. Mills Litho, Maitland, Cape Town, South Africa. Aganga, A. A; Letso, M. and Aganga, A. O. (2000). Feeding donkeys. LRRD. http://www.cipav.org.co/Lrrd12/ agan 122.htm. Aganga, A. A; Tsopito, C. M. and Seabo, D. (1994). Donkey power in rural transportation. Botswana case study. Appropriate Tech. J. 21: 32-33. Alouf, J. E. (1980). Streptococcal toxins (streptolysin O, streptolysin S, erythrogenic toxin). Pharmacol. Ther. 11:661–717. Asha, M. E; Salim, M.O; Amel, O. and Ibrahim, A. A. (2006). Bacteria Isolated from Equine Wounds. The Sudan J. Vet. Res. Vol.45 (1,2). Axon, J.E; Robinson, P. and Lucas, J. (2004). Generalized granulomatous disease in a horse. Aust Vet J . 82:48–51. Barrow, G. L. and Feltham, R. K. A. (1993). Cowan and Steel´s manual for identification of medical bacteria. 3rd edit. Cambridge University Press, Cambridge, U .K. Beers, M. H. and Berkow, R. (1999). Merck´s Manual of Diagnosis and Therapy. 17th (ed). pp. 1135-1136. Merck Research Laboratories N.J.P., London, UK. Berche, P. (1989). Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect. Immun. 57:3629–3636. Berkeley, R.C.W. and Logan, N.A. (1997). Bacillus, Alicyclobacillus and ϳϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Paenibacillus. In: Principles and Practice of Clinical Bacteriology. Emmerson, A.M., Hawkey, P.M. and Gillespie, S.H. (eds). pp. 185-207. John Wiley & Sons Ltd. Bernheimer, A. W. and Avigad, L. S. (1982). Mechanism of hemolysis by Renalin, a CAMP – like protein from Corynebacterium renale . Infect. Immun. 36 : 1253 – 1256 . Bökönyi, S. (1991). The earliest occurrence of domestic asses in Italy. In :Equids in the Ancient World Volume II. Meadow, R. H. and Uerpmann, H.P., eds. pp. 178-216. Ludwig Reichert, Wiesbaden, Germany. Carne , H. R; Kater , J. C. and Wickham , N. (1956) . A toxic lipid from the surface of Corynebacterium ovis. Nature. 178 : 701 – 702. Carter, G. R. and Chengappa, M. M. (1991). Essentials of Veterinary Bacteriology and Mycology. 4th ed. Lea and Febiger, Philadelphia, Pa. Clark, B. (1974). Draft proposal to include the African Wild Ass. Ethiopian Wildlife Conservation Organization, Addis Ababa, Ethiopia. p14. Clauditz, A; Resch, A; Wieland, K.P; Peschel, A. and Götz, F. (2006). "Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress". Infect immun. 74(8): 4950–4953. Clutton-Brock, J. (1992). Horse power: a history of the horse and the donkey in human societies. p 192. Harvard University Press, Cambridge, Massachusetts, USA. Cohen, N.D; Carter, G.K. and McMullan, W.C. (1992). Fistulous withers in horses: 24 cases (1984-1990). J. Am. vet. med. Ass. 201:121-124. Cossart, P; Vicente, M. F; Mengaud, J; Baquero, F; Perez-Diaz, J. C. and Cramlet, S.H. and Berhanu, G. (1979). The relationship of Brucella abortus titers to equine fistulous withers in Ethiopia. Vet. Med.Small Anim. ϳϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Clin. 74: 195-199. Cruickshank, R; Duguid, J.P; Marmino, B. P . and Swain, R.H. A. (1975). Medical Microbiology. 12 th (ed). Vol.11. Churchill. Livingstone,Edinburgh. Danny W. S. and Miller, W. H. Jr. (2011). Bacterial Skin Diseases. In: Equine Dermatology. 2nd (ed). pp130-170. Dawson, F.L.M. and Durrant, D.S. (1975). Some serological reactions to Brucella antigen in the horse. Equine vet. J. 7: 137-140. Demelash, B. and Woldemeskel, M. (2006 ). Causes and Factors Associated With Occurrence of External Injuries in Working Equines in Ethiopia. Intern J Appl Res Vet Med . Vol. 4(1). Denny, H.R. (1973). A review of brucellosis in the horse. Equine vet. J. 5: 121-125. Ding, H. and La¨mmler, C. (1996). Purification and further characterization of a haemolysin of Actinomyces pyogenes. J. Vet. Med. Ser. B 43:179–188. Dreyfus, F. (1974). Contribution a l’etude de la zootechine et de la pathologie des equides domestiques en Ethiopie. Ecole Nationale Vétérinaire d’Alfort (ENVA), Paris, France. p.122. Duff, H.M. (1937). Brucella abortus in the horse. J. comp. Path. 50:151. Eisenmann, V. (1995). L’Origine des ânes: questions et réponses paléontologiques. Ethnozootechnie, 56: 5-26. El Abdeen, Maha Z. (2001). Aerobic bacteria associated with spoilage of hides and skins. M. V .Sc. Thesis. Faculty of Veterinary Medicine, University of Khartoum, Sudan. Ellis, H; Christopher, R. C. and Watson, J. E. (1998). Post-operative ϳϱ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. complications of wound infection. Lecture-notes on General Surgery. 9th (ed), Blackwell Science Ltd. Oxford and Edinburgh, UK. Epstein, H. (1984). Ass, mule and onager. In: Evolution of domesticated animals, Mason, I. L., ed. pp. 174-184. Longman, London, UK. Esuruoso, G.O. (1977). Bovine dermodicosis in southern Nigeri. Bull. Anim. Hlth. Prod. Afr. 58:65-72. Fahmy, S.W. (1992). The health and husbandry of donkeys used by Zabbalin rubbish collectors in Cairo, Egypt. In: Are donkeys an overlooked option? Fielding, D. and Pearson, A. pp 238-240. ILEIA Newsletter. Feseha, G. and Yoseph, L. (1996) . Preliminary survey on management aspects and helminth problems of donkeys in Dire Dawa and East Oromia, Ethiopia. Final year paper, Faculty of Veterinary Medicine, Addis Ababa University, Ethiopia. Food and Agriculture Organization (FAO), United Nations (1992). Production yearbook 46. FAO Statistics Series No.112. p. 205. FAO: Rome. Funk, P. G; Staats, J. J; Howe, M; Nagaraja, T. G. and Chengappa, M. M.(1996). Identification and partial characterization of an Actinomyces pyogenes hemolysin. Vet. Microbiol. 50:129–142. Gardner, G.R; Nicolleti, P. and Scarratt, W.K. (1983). Treatment for brucellosis in horses by Florida practitioners. Fla. vet. J. 12: 21-23. Gaughan, E.M; Fubini, S.L. and Dietze, A. (1988). Fistulous withers in horses: 14 cases (1978-1987). J. Am. vet. med. Ass. 193: 964-966. Godfroid, J; Bosman, P.P. and Bishop, G.C. (2004). Bovine brucellosis. In: Infectious Diseases of Livestoc. Coetzer, J.A.W. and Tustin, R.C., eds. 2nd (ed). pp.1510-1527.Oxford University Press, Cape Town. ϳϲ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Guard, W. (1932). Fistula of the withers. North Am. Vet. 13: 19-23. Hall, V. (2006). Anaerobic Actinomycetes and Related Organisms. In: Principles and Practice of Clinical Bacteriology. Hawkey, P.M and Gillespie, S.H., eds. 2nd (ed). pp. 575-86. John Wiley and Sons Ltd. Hard, G. C. (1975). Comparative toxic effect of the surface lipid of Corynebacterium ovis on peritoneal macrophages . Infect. Immun. 12 : 1439 – 1449 . Hawkins, J.F. and Fessler, J.F. (2000). Treatment of supraspinous bursitis by use of debridement in standing horses: 10 cases (1968-1999). J. Am. vet. med. Ass. 217: 74-78. Heath, S.E; Bell, R.J; Clark, E.G. and Haines, D.M. (2004). Idiopathic granulomatous disease involving the skin in a horse. J Am Vet Med Assoc. 197:1033–1036. Holt, J.G; Krieg, N. R; Sneath, P. H. A; Staley, J. T. and Williams, S. T. (1994). Bergey's Manual of Determinative Bacteriology. 9th (ed). Philadeliphia: Williams and Wilkins. Ibrahim, K. E. (1989). Studies on Cattle Hides Meibomiam Gland Dermodicosis Complex. M V Sc, Thesis, University of Khartoum, Sudan. Johnson, W. M; Tyler, S. D; Ewan, E. P; Ashton, F. E; Pollard, D. R. and Rozee, K. R. (1991). Detection of genes for enterotoxins, exfoliative toxins and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J Clin Microbiol. 29:426-430. Jolly , R. D. (1966) . Some observations on surface lipids of virulent and attenuated strains of Corynebacterium ovis . J. Appl. Bacteriol. 29 : 189 – 196 . Jones, P. A. (1997). Donkeys for development. Pretoria: Animal Traction ϳϳ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Network of Eastern and Southern Africa and Agricultural Research Council of South Africa Institute for Agricultural Engineering (ARC/IAE). Jones, T.C; Hunt, R.D. and King, N.W. (1997). Veterinary Pathology. 6th (ed). pp. 601–680. Williams and Wilkins. Kathy, M and Ali, Z. (2004). Gender issues in donkey use in rural Ethiopia. In: Donkeys, people and development. A resource book of the Animal Traction Network for Eastern and Southern Africa(ATNESA). Starkey, P. and Fielding, D. (eds). pp.62-68. ACP-EU Technical Centre for Agricultural and Rural Cooperation (CTA), Wageningen, The Netherlands. Khalifa, R; Mona, M. E. and Mandour, A. M. (1988). A study of parasites infesting equines in Assiut Governorate. Assiut Vet Med J. 20:68. Kimball, A. and Frank, E.R. (1945). The isolation of Actinomyces bovis from fistulous withers and poll evil. The American Journal of Veterinary Research. 6:39-44. Knew. E. (1952 ). Hides damage and defect. In: Sudan cattle hides. Sudan Government, Sudan. Konemann, E.W; Allen, S. D; Janda, W. M; Schreckenberger, P. C. and Winn, W. J. (1997). Color Atlas and Textbook of Diagnostic Microbiology. 5th (ed). pp. 651-708. Philadelphia: Lippincott Williams & Wilkins. Lakshmanaperumalsamy, P. (1978). Studies on actinomycetes with special reference to antagonistic streptomycetes from sediments of Porto Novo coastal zone. Ph.D. thesis, Annamalai University, India. Liu, G.Y; Essex, A; Buchanan, J.T; Datta, V; Hoffman, H.M; Bastian, J.F; Fierer, J. and Nizet, V. (2005). "Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its ϳϴ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. antioxidant activity". J Exp Med 202: 209–215. Lovell, R. (1939). The Corynebacterium pyogenes antitoxin content of animal sera. J. Pathol. Bacteriol. 49:329–338. Lovell, R. (1944). Further studies on the toxin of Corynebacterium pyogenes. J. Pathol. Bacteriol. 56:525–529. Lowy, F.D. (1998). Staphylococcus aureus infections. N Engl J Med. 339(8): 520-532. Lyons, E.T; Drudge, J.H. and Tolliver, S.C. (1988). Verification of ineffectual activity of ivermectin against adult Onchocerca spp. in the ligamentum nuchae of horses. Am. J. vet. Res. 19:16-22. MacMillan, A.P. and Cockrem, D.S. (1986). Observations on the longterm effects of Brucella abortus infection in the horse, including effects during pregnancy and lactation. Equine vet. J. 18: 388- 390. Matthews, P. R. J. and Derbyshire, J. B. (1963). Observations on the mouse lethal power and serology of Corynebacterium pyogenes. Res. Vet. Sci. 4:531–536. McCaughey, W.J. and Kerr, W.R. (1967). Abortion due to brucellosis in a Thoroughbred mare. Vet. Rec. 80: 186-187. Miguel, A; Solá, V. and McClure, J.J. (1997). J Equine Vet Sc . 17 : 43- 45. Mohammed, A. (1991). Management and breeding aspects of donkeys around Awassa, Ethiopia. In: Donkeys, Mules and Horses in Tropical Agricultural Development. Fielding, D. and Pearson, R. A., eds. pp 185188. CTVM: Edinburgh. Nicoletti, P.L. (2007). Brucellosis. In: Equine Infectious Diseases. Sellon, D.C. and Long, M.T., (eds). pp. 348-350. Saunders Elsevier, Philadelphia. ϳϵ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Nicoletti, P.L; Mahler, J.R. and Scarratt, W.K. (1982). Study of agglutinins to Brucella abortus, B. canis and Actinobacillus equuli in horses. Equine vet. J. 14: 302-304. O’Sullivan, B.M. (1981). Brucella abortus titres and bursitis in the horse. Aust. vet. J. 57: 103-104. Ocholi, R.A; Bertu, W.J; Kwaga, J.K; Ajogi, I; Bale, J.O. and Okpara, J. (2004). Carpal bursitis associated with Brucella abortus in a horse in Nigeria. Vet. Rec. 155: 566-567. Pascoe, R.R.R. and Knottenbelt, D.C. (1999). Manual of Equine Dermatology. WB Saunders, Philadelphia, PA. Payne, J. A. and Wilson, T. R. (1999). An Introduction to Animal Husbandry in the Tropics. 5 edition. pp: 546-563. Blackwell science Ltd, oxford. Pearson, R. A. (1992). Management and husbandry of working animals with particular reference to their welfare. In: Proceedings of the Cairo International Meeting on Working Animals. 13-16 April, 1992. Cairo, Egypt. Pearson, R. A; Nengomasha, E. and Krecek, R. (1999). The challenges in using donkeys for work in Africa. In: Improving donkey utilization and management. Starkey, P. H. and Mueller, P. J (eds). DGIS. The Netherlands. Pinho, M.G. and Errington, J. (2003). Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery. Mol Microbiol 50:871-881. Pritchard, J.C; Lindberg, A.C; Main, D. C. and Whay, H.R. (2005 ). Assessment of the welfare of working horses, mules and donkeys, using health and behaviour parameters. Prev Vet Med. 69:265-83. ϴϬ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Rashmir-Raven, A; Gaughan, E.M. and Modransky, P. (1990). Fistulous withers. Comp. cont. Educ. pract. Vet. 12, 1633-1641. Rathna- Kala, R. and Chandrika, V. (1993). Effect of different media for isolation, growth and maintenance of actinomycetes from mangrove sediments. Indian J. mar. sci., 22: 297 - 299. Ruhmann, U. (1987). Microbiological studies on the occurrence of micrococaceae in slaughter cattle. Vet. Rec. 178: p. 17. Scott, D.W. and Miller, W.H. Jr (2003). Equine Dermatology. pp. 245– 320.WB Saunders, St. Louis, MO. Sellers, R.S; Toribio, R.E. and Blomme ,E.A. (2001). Idiopathic systemic granulomatous disease and macrophage expression of PTHrP in a miniature pony. J Comp Pathol . 125:214–218. Siva- Kumar, K. (2001). Actinomycetes of an Indian Mangrove (Pichavaram) environment : An Inventory. Ph.D. thesis, Annamalai University, India. Starkey, P. (1994). A world - wide view of animal traction highlighting some key issues in eastern and southern Africa. In: Improving animal traction technology. Starkey, P. Mwenya , E. and Stares, J., eds. p146-149. CTA. Mills Litho, Maitland, Cape Town, South Africa. Statistical Bulletin for Animal Recourses. (2000). Issue No (10).p.48-49. Ministry of Animal Resource, Sudan. Statistical bulletin for Animal Resources. (2011). Issue No(20). p .18 .Ministry of Animal Resource, Sudan. Stephen, D. W. (2005). Equine Bacterial and Fungal Diseases. Clin Tech Equine Practice. 4 : 302-310. Tashjian , J. J. and Campbell, S. G. (1983) . Interaction between caprine ϴϭ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. macrophages and corynebacterium pseudotuberculosis : an electron microscopic study . Am. J. Vet. Res. 44 : 690 – 693 . Todar, K. (2011). Staphylococcus aureus & Staphylococcal disease. Todar’s Online text book of bacteriology. Available online at: http://www.textbookofbacteriology.net/staph.htmlAccessed 14th March 2011. Unsworth, k. (1946).Studies on clinical and parasitological aspects of canine dermodetic manage. J Comp Patho. 56:114-124. Vikineswary, S; Nadaraj, P; Wong, W.H. and Balabaskaran, S. (1997). Actinomycetes from a tropical mangrove ecosystem - Antifungal activity of selected strains. Asia Pacific J. Mol. Biol. Boitec. 5(2) : 81 - 86. Wannamaker, L.W. (1983). Streptococcal toxins. Rev Infect Dis. 4: S723732. Wilson, R. T. (1990). The donkey. In : An Introduction to Animal husbandry in the tropics. Pakistan Journal of Nutrition. 2 : 138-147. Wilson, R. T. (1991). Equines in Ethiopia. In: Donkeys, mules and horses in tropical agricultural development. Fielding, D. and Pearson R. A., eds. pp. 3347. ACP-EU Technical Centre for Agricultural and Rural Cooperation (CTA), Wageningen, The Netherlands. Woolcock, J. B. (1975). Epidemiology of equine streptococci. Res Vet Sci; 18:113-4. Wylam, C. B. (1991). Experiences in donkey draft from West Sudan. In: Donkeys, Mules and Horses in Tropical Agricultural Development. Fielding, D. and Pearson, R. A. (eds). pp 286-292. CTVM: Edinburgh. ϴϮ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. Appendix Age : Sex : Type of breed : Feeding : Activity : Behavior : Posture and gait : Coat condition : Infected by injury : Site of injuries : Site of injuries : Causes of injuries : Treat of injuries : Fate of injuries : Clinical sign : Specimen collection : ϴϯ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. ϴϰ Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. DEDICATION To the soul of my father, to my lovely mother, supportive Husband, unique sweet son and daughters, wonderful sisters and brother and Friends for their unlimited assistance. With my deep love. i Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. PREFACE This work was carried out at the Department of Microbiology, Faculty of Veterinary Medicine, University of Khartoum, Under supervision of Dr. Awad Elkarim Abd Elghaffar Ibrahim. ii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. ACKNOWLEDGEMENTS First and foremost I would like to thank my Merciful Allah for giving me strength and health to do this work. I would like to express my sincere thankfulness, indebtedness and appreciation to my Supervisor Dr. Awad Elkarim Abd Elghaffar Ibrahim for his guidance, advice, help, encouragement and patience throughout the period of this work. My gratitude is also extended to all staff of the Bacteriology laboratory for the technical assistance during the laboratory work. My thanks also extended to my friends, and collages who help me. iii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. ABSTRACT Working animals provide an essential transport resource in developing countries worldwide. Many of these animals are owned by poor people and work in harsh environments, so their welfare is a cause for concern. The present study was carried out to assess the welfare of working donkeys in Khartoum State, using clinical examination and a questionnaire survey , to identify and quantify factors associated with the presence of wounds in donkeys working in the transport of goods and to isolate and identify aerobic bacteria associated with fistulous withers. One hundred fifty male donkey were randomly chosen. They were used for work and found in markets and along the roads in Elsagana, Elremila, Mayo, Halaib, Elsouk Elshabi Omdurman and Shambat. The study was carried out between December 2010 and May 2011.The results showed that improper harness and saddle were the major causes of injuries in donkeys. The prevalence of injuries was influenced by the type of work carried out, and the body lesions occurred predominantly in the areas of the breast/shoulder, withers and girth, while Lesions on the head, neck, flank and tail base were seen in less than 10% of the animals. From 150 donkeys examined, only 60 of them were had fistulous withers, and their collected samples were cultured on blood agar , nutrient agar and MacConkey’s agar. Bacterial isolates were identified to the species level, according to their cultural characteristics and biochemical tests. A total of one hundred and forty two isolates were obtained and identified as Gram positive bacteria. These isolates were belonging to Bacillus spp. (48.6%), Staphylococcus spp. (38.7%), Corynebacterium spp. (5.6%), Streptococcus spp.(4.3%) and Actinomyces spp. (2.8%),which represents potential pathogens that induced fistulous withers. The following iv Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. species were isolated: pseudotuberclosis, Staphylococcus aureus, Corynebacterium Streptococcus zooepidemicus, Actinomyces bovis and Actinomyces pyogenes, which might lead to the chronicity of the disease. v Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. ϢϴΣήϟϦͤήϟͿϢδΑ Δ Σϭ ή σϷ κ ΨϠ ϣ ϚϠ Θ Ϥ ϳ ϭ Ϣ ϟ Ύ ό ϟ ˯ Ύ Τϧ ϊ ϴ Ϥ Ο ϲϓ Δ ϴ ϣ Ύ Ϩ ϟ ϥ Ϊ Ϡ Β ϟ ϲϓ Ϟ Ϙ Ϩ Ϡ ϟ Ύ ˱ Ϥ Ϭ ϣ Ω έ Ϯ ϣ Δ Ϡ ϣ Ύ ό ϟ ΕΎ ϧ Ϯ ϴ Τϟ ϞΜ Ϥ Η ϩ ά ϫ Ζϳ ή Ο ϖϠ Ϙ Ϡ ϟ Γ Ύ ϋΪ ϣ ϲϫ Ϣ Ϭ Θ ΤϠ μϣ Ϛϟ ά ϟ ˬ Δ ϴ γΎ ϗ ΕΎ Ό ϴ Α ϲϓ Ϟ Ϥ ό Η ϭ ΕΎ ϧ Ϯ ϴ Τϟ ϩ ά ϫ Ϧϣ Ϊ ϳ Ϊ ό ϟ ˯ ή Ϙ ϔ ϟ ϱή ϳ ή δϟ κΤϔ ϟ ϡ Ϊ ΨΘ γΈ Α Ϛϟ Ϋ ϭ ˬ ϡ Ϯ σή Ψϟ Δ ϳ ϻϭ ϲϓ Ϟ Ϥ ό Η ϲΘ ϟ ή ϴ Ϥ Τϟ Δ ϟ Ύ ΣϢ ϴ ϴ Ϙ Θ ϟ Δ γ έ Ϊ ϟ ϊ Ύ πΒ ϟ Ϟ Ϙ ϧ ϲϓ ϞϤ ό Η ϲΘ ϟ ή ϴ Ϥ Τϟ ϲϓ Ρϭ ή Πϟ Ι Ϊ ΣΈ Α Δ τΒ Η ή Ϥ ϟ Ϟϣ Ϯ ό ϟ αΎ ϴ ϗ ϭ Ϊ ϳ Ϊ ΤΘ ϟ ˬ ϥΎ ϴ Β Θ γϹ ϭ Ύ ˱ ϴ Ϯ θϋ έ Ϯ ϛ ά ϟ ή ϴ Ϥ Τϟ Ϧϣ Εή ϴ Θ Χ .ϙέ Ύ Τϟ έ Ϯ γΎ Ϩ Α Δ τΒ Η ή Ϥ ϟ Δ ϴ Ϯ Ϭ ϟ Ύ ϳ ή ϴ Θ Ϝ Β ϟ Ϊ ϳ Ϊ ΤΗ ϭ ϝΰ ϋϭ ˬ ΐϳ ϼΣ ˬ Ϯ ϳ Ύ ϣ ˬ Δ Ϡ ϴ ϣ ή ϟ ˬΔ ϧ Ύ Πδϟ ϲϓ ϕή τϟ ϰΒ ϧ Ύ Ο ϰϠ ϋϭ ϕ Ϯ γϷ ϲϓ Γ Ω Ϯ ΟϮ ϣ ϰϫ ϭ ϞϤ ό Ϡ ϟ ϡ Ϊ ΨΘ δΗ ϭ . Ϯ ϳ Ύ ϣ ϭ ή Β Ϥ δϳ Ω Ϧϴ Α Γ ή Θ ϔ ϟ ϲϓ Δ γ έ Ϊ ϟ Ζϳ ή Ο ϭ ΕΎ Β Ϥ η ϭ ϥΎ ϣ έ Ω ϡ ϲΒ ό θϟ ϕϮ δϟ ϲϓ ΕΎ Α Ύ λϺϟ Δ ϴ δϴ ή ϟ ΏΎ Β γϷ Ζϧ Ύ ϛ ΐγΎ Ϩ Ϥ ϟ ή ϴ Ϗαή ϔ ϟ Ϣ Ϙ σϭ Νή δϟ ϥ Ξ Ύ Θ Ϩ ϟ Εή Ϭ χ έ Ϊ μϟ ϖσΎ Ϩ ϣ ϲϓ ΕΎ ϓ ϵ Ϣ ψό ϣ ΕΪ Οϭ ϭ ˬ Ϫ Α ϡ Ϯ Ϙ ϳ ϱά ϟ ϞϤ ό ϟ ωϮ Ϩ Α ΕΎ Α Ύ λϹ έ Ύ θΘ ϧ · ή Λ ΄ Η Ϊ ϗ ϭ ή ϴ Ϥ Τϟ Ϧϣ Ϟ ϗ ϲϓ Ϟϳ ά ϟ Γ Ϊ ϋΎ ϗ ϭ Γ ή λΎ Ψϟ ϭ Δ Β ϗ ή ϟ ϭ α ή ϟ ϰϠ ϋ ΕΎ ϓ ϵ Εή Ϭ χ Ύ Ϥ Ϩ ϴ Α ϭ ˬϙέ Ύ Τϟ ϭ ϒΘ Ϝ ϟ ϭ Ζϋέ ί ϭ ϙέ Ύ Τϟ έ Ϯ γΎ Ϩ Α Ύ ˱ Α Ύ μϣ ϥΎ ϛ ςϘ ϓ ˬ ΖμΤϓ ˱ έ Ύ Ϥ Σ Ϧϴ Α Ϧϣ ΕΎ ϧ Ϯ ϴ Τϟ Ϧϣ ̃10 ϰΘ Σ Δ ϳ ή ϴ Θ Ϝ Α ϝ Εϻΰ ϋ Ζϔ Ϩ λ ϯά ϐ ϣ έ Ύ Ο ϭ ϲϜ ϧ Ϯ ϛ Ύ ϣ έ Ύ Ο ϭ ϡ Ϊ ϟ έ Ύ Ο ϰϠ ϋ Ύ Ϭ ό Ϥ Ο Ϣ Η ϲΘ ϟ ΕΎ Ϩ ϴ ό ϟ ˬ Ϫ ϟ ΰ ϋ ϰϠ ϋ ϝϮ μΤϟ Ϣ Η.Δ ϴ Ύ ϴ Ϥ ϴ ϛ Ϯ ϴ Β ϟ Ε έ Ύ Β Θ ΧϹ ϭ Δ ϴ ϋ έ ΰ Θ γϹ Ύ Ϭ μ Ύ μΧϝ Ύ ˱ Ϙ ϓ ϭ ωϮ Ϩ ϟ ϯϮ Θ δϣ ϰϟ · Εϻΰ ό ϟ ϩ ά ϫ ϲϤ Θ Ϩ Η ˬ ϡ ή Πϟ Δ Β ΟϮ ϣ Ύ Ϭ ϧ ϰϠ ϋ Ζϓ ή ϋϭ Staphylococcus spp. (38.7%), Bacillus spp (48.6%), Corynebacterium spp. (5.6%), Streptococcus spp. (4.3%) and Actinomyces spp. (2.8%). Δ ϴ Η Ϸ ω Ϯ ϧ Ϸ Ζϟ ΰ ϋϭ ϙέ Ύ Τϟ έ Ϯ γΎ ϧ Ι Ϊ ΣϹ Δ Ϡ Ϥ Θ ΤϤ ϟ ΕΎ Β Β δϤ ϟ Ϟ Ϝ θΗ ϰϫ ϭ Staphylococcus aureus, Corynebacterium pseudotuberclosis, Streptococcus zooepidemicus, Actinomyces bovis and Actinomyces pyogenes. . νή Ϥ ϟ ϥΎ ϣ ί · ϰϟ · ϱΩ Ά ϳ Ϊ ϗ ϱά ϟ ή ϣ Ϸ vi Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. TABLE OF CONTENTS DEDICATION…………………………………………………………………i PREFACE……………………………………………………………………...ii ACKNOWLEDGEMENTS……………………………………………………iii ABSTRACT……………………………………………………………………iv ARABIC ABSTRACT…………………………………………………….......vi LIST OF CONTENTS…………………………………………………………vii LIST OF TABLES…………………………………………………………….xv INTRODUCTION…………………………………………………………......1 1. CHAPTER ONE: LITERATURE REVIEW ……………………………….4 1.1. Donkey…………………………………………………………………….4 1.1.1. Donkey population and history …………………………………………4 1.1.2. The place of donkeys in society………………………………………...5 1.1.3. General donkey features and behavior…………………………………7 1.1.4. Selection characteristics of a working donkey………………………….8 1.1.5. Economic importance……………………………………………… 9 1.1.6. Fitting equipment………………………………………………… 10 1.1.7. General causes of injuries ………………………………………… 12 1.1.8. Wounds…………………………………………………………… 12 vii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.2. Fistulous withers…………………………………………………… 14 1.2.1. Definition of fistulous withers…………………………………… 14 1.2.2. Clinical signs…………………………………………………….. 15 1.2.3. Etiological agent of fistulous withers………………..................... 16 1.2.3.1. Bruccella abortus…………………………………..................... 16 1.2.3.2.Staphylococcus spp………………………………………………. 17 1.2.3.2.1. Bacterial virulence factor…………………………………… 18 1.2.3.2.2. Cell - Associated Components……………………………… 18 1.2.3.2.3. Exoenzymes………………………………………………… 19 1.2.3.2.4. Exotoxins………………………………………....................... 19 1.2.3.2.5. Membrane-damaging toxins ………………………………… 19 1.2.3.3. Streptococcus………………………………………………… 20 1.2.3.3.1.Virulence factors…………………………………………… 20 1.2.3.3.2. Streptococcal toxins………………………………………… 21 1.2.3.3.3. Streptococcus equi…………………………………………. 22 1.2.3.4. Corynebacterium……………………………………………… 22 1.2.3.4.1.Virulence factor…………………………………………….. 22 1.2.3.5.Actinomycetes……………………………………………........ 23 1.2.3.5.1. Virulence factor……………………………………………. 24 viii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 1.2.3.6. Bacillus………………………………………………............. 25 1.2.3.6.1. Virulence factor …………………………………………… 25 1.2.4. Epidemiology…………………………………………………… 26 1.2.5. Disease of equine similar to fistulous withers………………… 26 1.2.5.1. Granulomatus lesion of the skin……………………………… 26 1.2.5.2. Saddle sores…………………………………………………... 27 1.2.5.3. Capped hocks…………………………………………............. 28 1.2.6. Prevention………………………………………………………. 29 1.2.7. Treatment ………………………………………………………. 29 2. CHAPTER TWO: MATERIALS AND METHODS……………… 31 2.1. Sterilization……………………………………………………… 31 a-Flaming……………………………………………………………… 31 b-Red heat……………………………………………………………... 31 c-Hot air oven………………………………………………………… 31 d-Steaming at 100°C………………………………………………….. 31 e-Moist heat (Autoclave)…………………………………………….. 31 2.2. Reagent………………………………………………………….. 32 2.2.1. Sodium Chloride/ normal saline……………………………… 32 2.2.2. Alpha-naphthol solution …………………………………….. 32 ix Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.2.3. Potassium hydroxide ………………………………………… 32 2.2.4. Hydrogen peroxide ………………………………………… 32 2.2.5. Kovac's reagent………………………………………………. 32 2.2.6. Ttetramethyl-p-phenylene diamine dihydrochloride ………… 33 2.2.7.Nitrate test reagent…………………………………………… 33 2.3. Indicators ……………………………………………………… 33 2.3.1. Andrade's indicator…………………………………………… 33 2.3.2. Bromothymol blue……………………………………………. 34 2.3.3. Plasma………………………………………………………… 34 2.3.4. KOH and HCL………………………………………………. 34 2.4. Collection of blood for enriched media…………………………. 34 2.5. Preparation of Culture Media…………………………………… 34 2. 5.1. Liquid media………………………………………………… 34 2.5.1.1. Nutrient broth ……………………………………………. 34 2.5.1.2. Peptone water……………………………………………. 35 2.5.1.3. Peptone water sugar……………………………………… 35 2.5.1.4. Glucose-phosphate (MR-VP test) medium………………. 35 2.5.1.5. Nitrate broth medium…………………………………….. 36 2.5.1.6. Serum water sugars………………………………………. 36 x Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.5.2. Semi solid media…………………………………………… 36 2.5.2.1. Motility medium…………………………………………. 36 2.5.2.2. Hugh and Leifson's (O/F) medium………………………. 37 2.5.3. Solid media………………………………………………… 37 2.5.3.1. Blood agar ……………………………………………… 37 2.5.3.2. MacConkey agar………………………………………… 38 2.5.3.3. Nutrient agar…………………………………………….. 38 2.5.3.4. Ammonium salt sugars (ASS) ………………………….. 38 2.5.3.5. Aesculin – bile azide agar ………………………………. 39 2.5.3.6. Milk agar ……………………………………………….. 39 2.5.3.7. Nutrient gelatin medium ………………………………… 39 2.5.3.8. Simmons citrate agar ………………………………….. 40 2.5.3.9. Starch agar………………………………………………. 40 2.5.3.10. Urea agar Base ………………………………………… 40 2.6. Clinical investigations………………………………………. 41 2.6.1. Study area………………………………………………… 41 2.6.2. Animals…………………………………………………… 41 2.6.3. Clinical signs……………………………………………… 41 2.6.4. Collection of Samples …………………………………… 42 xi Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2.7. Bacteriological examination………………………………… 42 2.7.1. Primary isolation………………………………………….. 42 a- Blood agar ……………………………………………………. 42 b- MacConkey agar……………………………………………… 42 2.7.2. Isolation…………………………………………………… 43 2.7.3. Cultural characteristics …………………………………… 43 2.8. Purification………………………………………………….. 43 2.9. Preservation………………………………………………..... 43 2.10. Microscopic Examination…………………………………. 44 2.11. Biochemical tests…………………………………………... 44 2.11.1. Sugar fermentation test…………………………………… 44 2.11.2. Oxidase test ……………………………………………... 44 2.11.3. Catalase test ……………………………………………… 45 2.11.4. Coagulase test…………………………………………… 45 2.11.4.1. Slide coagulase test …………………………………..... 45 2.11.4.2. Tube coagulase test ……………………………………. 45 2.11.5. The oxidation- fermentation (o/F) test…………………… 45 2.11.6. Indole production test……………………………………. 46 2.11.7. Voges- Proskauer (VP) test …………………………….. 46 xii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. 2. 11.8. Nitrate reduction………………………………………… 46 2.11.9. Urease activity test ……………………………………… 47 2.11.10. Citrate utilization test…………………………………… 47 2.11.11. Aesculin hydrolysis……………………………………… 47 2.11.12. Hydrogen sulphide (H2S) production…………………… 48 2.11.13. Ammonium salt sugar test ……………………………… 48 2.11.14. Gelatin hydrolysis……………………………………….. 48 2.11.15. Motility test……………………………………………… 48 3. CHAPTER THREE: RESULTS………………………………. 50 3.1. Clinical findings……………………………………………... 50 3.2. Bacterial isolation …………………………………………… 52 3.3. Characterization and identification of isolates……………… 52 3.4. Gram- positive Bacteria…………………………………….. 52 3.4.1. Staphylococcus ………………………………………….. 53 3.4.2 Streptococcus …………………………………………….. 53 3.4.3. Actinomycetes …………………………………………… 53 3.4.4. Corynebacterium ………………………………………… 54 3.4.5. Bacillus …………………………………………………… 54 xiii Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. CHAPTER FOUR: DISCUSSION…………………………........ 64 CONCLUSION………………………………………………….. 71 RECOMMENDATIONS………………………………………... 72 REFERENCES………………………………………………….. 74 APPENDIX…………………………………………………….. 84 xiv Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. LIST OF TABLES Page Table 1. Main advantages and disadvantages of using donkeys............. 2. Distribution of External Injuries on Various Body Parts in 8 donkeys………………………………………………………… 51 3. Causes of External Injuries in donkeys in Khartoum State…… 51 4. Owner’s Responses to the Management of External Injuries in donkeys………………………………………………… 52 5. Isolation frequency of Gram-positive bacteria isolated from donkeys infected with fistulous withers …………………………. 6. Biochemical properties of some bacteria isolated from donkeys infected with fistulous withers in Khartoum State……………… 7. 55 56 Gram stain reaction and biochemical properties of Staphylococcus spp isolated from donkeys infected with fistulous withers……… 57 8. Gram stain reaction and biochemical properties of Bacillus spp isolated from donkeys infected with fistulous withers……… 58 9. Gram stain reaction and biochemical properties of Bacillus spp isolated from donkeys infected with fistulous withers………. 59 10. Gram stain reaction and some biochemical properties of Streptococcus spp and Actinomycetes spp isolated from donkeys infected with fistulous withers……………………………………. 60 xv Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark. LIST OF FIGURES Figure 1. Page The arrangement of the breast band, breech strap and saddle for harnessing a donkey to the shafts of a two-wheeled cart …. 11 2. Donkey repeatedly whipped on an open wound, that led to deep infection (Omdurman Market)…………………… 13 3. Names of the different body parts of a donkey………. 15 4. Frecquency of Gram-positive bacterial species isolated From donkeys with fistulous withers………………………. 5. Staphylococcus aureus isolated from donkey with Fistulous withers on blood agar medium…………………….. 6. 61 62 Actinomyces spp bacteria isolated from donkey with fistulous withers on blood agar medium…………………… . 62 7. Smear from blood agar culture of Staphylococcus aureus…… 63 8. Smear from blood agar culture of Actinomycetes spp………… 63 xvi