A cross sectional study was conducted in Woliso from November 2011 to April 2012 to estimate the prevalence of epizootic lymphangitis in 390 cart horses using clinical, direct smear and mycological examination as well as histofarcin test. The overall prevalence of epizootic lymphangitis was 24.9% (97/390) in carthorses in Woliso. The wounds caused by harness were major predisposing factors identified. The majority (38.1%) of the cases were at the moderate stage of the severity of the disease, and the lesions were predominantly (95%) found on the limbs. The fungus was easily demonstrated in the clinical specimens by Gram’s staining technique. The isolation of Histoplasma capsulatum variety farciminosum was attempted on Sabouraud dextrose agar. The sensitivity of histofarcin test was 83.3% while its specificity was 80.6%.The positive and negative predictive values of histofarcin test were 76.9% and 86.2%, respectively. The findings of this study revealed that the disease is prevalent in Woliso and warrants for the launching of a sound control strategy. Furthermore, the potential application of histofarcin is suggested for the diagnosis of epizootic lymphangitis in horses as evidenced from the results of the current study.
The Food and Agriculture Organization estimated that in 2008, there were almost 59 million horses in the world, with around 33,500,000 in the Americas, 13,800,000 in Asia and 6,300,000 in Europe and smaller portions in Africa and Oceania (FAO,2009).. In Ethiopia, there are about 2 million horses, 6.2 million donkeys, 0.38 million mules, and about 1.1 million camels. (Statistical Bulletin, 2011). With respect to age distribution, the majorities of the horses, donkeys and mules are of the age group 3 years and older. Among the horses aged 3 years and older, about 1.2 million were used for transportation, 0.21 million for draught and the remaining 0.21 million for other purposes (Statistical Bulletin, 2011).
Carthorses do provide significant service in transporting people and goods in many rural areas and small town in different parts of Ethiopia. Moreover, carthorse business provides service in generating a large amount of revenue. Many disease situations affect horses inflecting heavy losses and debilitating their working ability. Epizootic lymphangitis (EL) is one of the major endemic diseases in Ethiopia expected to cause a great economic loss especially in carthorses ( Ameni, 2006;Pal, 2012).
EL, a contagious chronic disease of equines, is characterized clinically by a spreading, suppurative, ulcerative pyogranulomatous dermatitis and lymphangitis (OIE, 2000; Pal, 2012). The causative agent of EL is Histoplasma capsulatum variety farciminousum (HCF) that is a dimorphic fungus, having yeast form in the tissue and mycelial (mould) phase in the environment.. The mycelia are relatively resistant to ambient condition, which allows it to persist for many months in warm and moist conditions ( Radostits et al., 2008; Pal,2012).
EL is more common in the tropics and subtropics and is endemic in north, east and north-east Africa, and some parts of Asia, including some countries bordering the Mediterranean sea, India, Pakistan and Japan (OIE, 2000;Pal,2012). The disease is common in Ethiopia, especially in cart horses in warm, humid areas (Ameni, 2006).The incidence of the disease increases with assembling of animals; it is much more common, when large numbers of horses are stabled together for cavalry and other transportation needs .The disease has a high morbidity and low mortality (10-15%) rates (Radostits et al., 2008).The disease may last for several months to a year (Pal,2012).
The disease has three forms, namely the cutaneous, the ocular and the pulmonary forms based on the route of entry of the agent .The pulmonary form of the disease is infrequent and is presumed to occur after inhalation of the organism. The most common form of epizootic lymphangitis is the cutaneous form and affects the skin and lymphatics, particularly on the extremities, chest wall, face, and neck (OIE, 2000; Pal, 2007).
The traumatized skin is either infected directly by infected pus, nasal or ocular excretion or indirectly by the soil or contaminated harnesses, grooming equipments, feeding and watering utensils, wound dressing or flies. It is also believed that ticks may play a role in the transmission of this agent (Ameni and Terefe, 2004; Pal, 2012). The incubation period is usually several weeks, but can be variable (Al-Ani and Al-Delaimi,1996 ; Pal,2012).
Studies conducted in Ethiopia have confirmed that EL is endemic in the country being distributed in humid and hot areas with an altitude ranging from 1600-2400 meter above sea level (Ameni, 2006). Similar studies done in Ethiopia showed that EL possess great threat to the carthorse business (Endebu, 1996 ; SPANA,2003).An average prevalence of 18.8% has been recorded in carthorses at 28 towns in the country (Ameni, 2006).
Woliso is one of the towns in Ethiopia that uses horses for the transportation and thus has large number of carthorses. However, little or no information is available on the epidemiology of EL in Woliso Town. Therefore, the present study was contemplated to estimate the prevalence of EL in carthorses in Woliso Town.
Materials and Methods
This study was conducted in Woliso which is located at 114 km from Addis Ababa. The town is located at an altitude of 2000 m above sea level, and has an average annual rainfall around 97.2 mm and average annual temperature 19.2 0C.
Information regarding the number of carts in the town was obtained from municipality office. Usually two horses pull a cart with shift, one horse in the morning and the other in afternoon. A total of 237 carts were registered and thus, making the number of cart horses to be 474 out of which 390 horses were included in this study. Sampling was done at cart stations. Each station was visited two times, in morning and afternoon, to examine two horses that were used to pull a single cart in shifts. The remaining 84 carthorses could not be available during the study period mainly because owners were reluctant to stop at cart station and allow their horses examined.
Carthorses were screened for EL by physical examination based on clinical signs of disease. All parts of the body including the nostril, and eyes were examined for the presence of lesions of EL especially for the presence of nodules or ulcers. Emphasis was given to the lymphatic vessels, lymph nodes and skin .Clinical examination of each horse using visual inspection was used as principal method of classification of horses as sick or healthy.
The parts that were difficult for visual inspection and any suspected nodules were examined by palpation. Besides, the driver of the carthorse or the owners were enquired to recall any lesion, or nodules observed in their horses, and the lesions or nodules were considered or rejected from clinical point of EL. The clinical forms of EL were distinguished based on the result of clinical examination. Further confirmation was made by microscopic and mycological examinations of samples collected from the lesions.
Pus sample collected from un-ruptured nodules was used for microscopic and mycological examination. Nodules selected were washed with soap and water, and shaved. Then, disinfected with 70% ethanol to remove surface contaminants .The contents of the nodules were aspirated with sterile syringe and needle and immediately inoculated on to Sabouraud dextrose agar supplemented with 2.5% glycerol and 0.5g/L of chloramphenicol.. The samples were also kept on wet ice and brought to Aklilu Lemma Institute of Pathobiology Laboratory, Addis Ababa as soon as possible. Air–dried smears from swabs of lesions were prepared on clean glass slides for direct detection of yeast form of HCF. Smears from aspirated pus, swabs of fresh lesion, direct impression imprints were fixed with methanol, stained by Gram’s technique for the identification of the yeast form of HCF. The inoculated medium was kept at 25oC (6-8 weeks) for isolation of mycelial form of HCF, while for the isolation of yeast form, incubation was done at 370C.The media were periodically examined for evidence of growth of fungus. Morphology of fungus was studied in lactophenol cotton blue or Narayan stain ( Pal,2004). A total of 105 carthorses were selected in Woliso town for histofarcin test, some clinically affected and others clinically healthy. The remaining horses were not included in this study because of unwillingness of cart owners. The middle cervical area of the lateral side of the neck was washed with soap and water, and shaved. The skin thickness was measured using calipers. Then 0.1 ml of the concentrations (0.2 mg/ml) of antigen was injected into the site intra-dermally. The inoculation site was examined at 24h-48h post-injection. A horse was considered to be positive when the change in size of skin indurations at the site of administration was > 4mm.
Prevalence were defined as the proportion of the numbers of horses positive for EL by clinical and microscopic examinations to the total number of horses examined, and was expressed in percentage. The severity the lesion and distribution the lesions in different body parts were expressed in percentage. Comparison of different proportions was done by chi-square(c2) test.
The prevalence of EL in carthorses in Woliso was 24.9% (97/390) using clinical and microscopic examination. All horses with clinical signs of EL were also positive for the yeast form of HCF by microscopic microscopy.The majority (38.1%) of the cases were at the moderate stage of the severity of the disease; and the lesions in cutaneous form were predominantly (95%) found on the limbs (Figure 1). A few cases of ocular form were also observed (Figure 2).
Figure 3.Ulcer formation on the lymphatic vessels of the neck of a carthorse
Almost all of the carthorses with clinical sings showed one or more nodular ulcer on different parts of their bodies. Up on palpation, recently erupted nodules were found to be firm and freely movable. In some cases, the lesions were observed as a rigid knotted rope following the lymphatic vessels especially involving the limbs and neck (Figure 3).
In severe cases, ruptured nodules arranged in line and discharging white to yellow pus were observed .In such cases, draining lymph nodes were swollen which discharged thick, white, creamy pus upon puncture. In severe cases, ulcerated lesions formed a firm granulation tissue with discharging pus .These lesions appeared on one or more sites of horse with unpleasant odor.In cutaneous form, nodules were commonly observed on the extremities (forelimbs including shoulder and hind limbs), sternal region, neck, face, ventral part of abdomen, and rarely on ear and scrotal area. Based on the location, the lesions were categorized into five groups (Table 1). In majority (94.8%) of the cases, the lesions were noticed on the limbs.
Table.1. Proportion of occurrence lesions of epizootic lymphangitis on different parts of body of carthorses
|Location of lesions||Total animal examined||Prevalence
|Fore and hind limbs||21||21.6||13.3-29.9|
|Limbs and other parts||20||20.6||12.4-28.8|
The classification of severity of lesions of 97 cases into different stages was made according to the method described by Endebu (1996). Table 3 showed the proportion of carthorses number of animals observed with different degrees of severity and their percentages. The highest proportions (38.1%) of horses were found with moderate stage, while the lowest proportion (8%) was observed with most severe stages.
Table2. Proportion of different severity stages of lesions of epizootic lymphangitis in 97 horses
|Location of lesions||Total animal examined||Prevalence
|Confidence interval (95%)|
Since horses used for cart pulling were male, the effect of sex on the prevalence of EL was not considered. On the other hand, the prevalence of EL was affected (c2= 47.7; p<0.001) by body condition of horses. The body condition scoring was made according to the method described by Sevendsen (1997) .The association of prevalence of EL and body condition of carthorses is shown in Table 3.
Table.3. Association of body condition of carthorses and prevalence of epizootic lymphangitis
|Body condition||Total animal examined||Prevalence (%)||Confidence interval (95%)||c2||P-value|
Smears stained with Gram technique from the pus, swabs or impression smears revealed the presence of Gram-positive, globose or pear shaped, double- contoured yeast cells (2-4 milli micron).
After 4-8 weeks of incubation at 37º C on SDA, the yeast colonies appeared as flat, small, white to grey with a pasty consistency. The colonies in lactophenol cotton blue or Narayan stain showed the yeast cells of HCF.
On SDA, the growth of mycelial form was very slow in which the colonies appeared as grey to brown, flaky, raised, dry and wrinkled after 7-8 weeks of incubation at 25ºC. Wet mount preparation in lactophenol cotton blue or Narayan stain revealed branching hyphae, chlamydospoes and macroconidia.
Out of 105 carthorses injected with histofarcin, only 55 could be traced for a complete follow-up and data recording due to various reasons. In majority of clinical cases, injection of histofarcin induced swelling (Figure 4) of various sizes.
Figure 4.Skin swelling at site of injection of histofarcin on aclinically positive carthorse
Out of 24 clinically positive horses, 20 were positive by skin test making the sensitivity 83.3%. The specificity of histofarcin test was 80.64%. The positive and negative predicative values of histifarcin test were 76.9% and 86.2%, respectively.
In the present study, the prevalence of EL was estimated at Woliso Town, using clinical and microscopic examinations. The severity of the lesion was classified into different groups and its distribution on the different body parts was investigated. Both the yeast and mycelial forms of HCF were isolated in the laboratory. Furthermore, histofarcin skin test was evaluated for the diagnosis of EL.
The prevalence of EL recorded in Woliso was 24.9%, and it was higher than that the average prevalence reported from 28 towns (Ameni, 2006). Furthermore, Ameni and Seyoum (2002) recorded an average prevalence of 21.1% in East Shewa. On the other hand, higher prevalence was reported in Mojo, Debre Zeit, and Jimma towns by Ameni, 2006. The prevalence of EL is affected by the altitude, temperature and humidity of the area (OIE, 2000). Furthermore, the size of horse population affects the prevalence of epizootic lymphangitis.
In affected horses, the cutaneous form of disease was most prevailing and the lesions were observed in any part of the body. Nevertheless, it was frequently noticed on the limbs, chest and neck. Similar findings were described in mules by other worker who reported over 90% cutaneous form and a few proportions of ocular and respiratory forms (Ameni et al., 2006). The extremities such as the limbs are prone to trauma and hence, the fungus can easily get access to infect the horses. Diseased horses had poor body conditions as compared to non- infected horses. This is because of the debilitating nature of disease as sick horses become restless and do not feed adequately. The flies feed on the lesions and the horses are always trying to remove the flies and have less time to feed. Furthermore, the disease causes loss of appetite, which also leads to loss of condition.
The sensitivity and the specificity of histofarcin test recorded in the present study were 83.3% and 80.64%, respectively. The ‘’gold standard ‘’ used in this case was primarily a diagnosis based on clinical signs with microscopic examination of smears made from the lesion. Previously, Ameni et al. 2006 recorded slightly higher (90.3%) sensitivity and lower (69%) specificity in endemic area. These authors reported 100% specificity in disease-free area. However, the use of clinical signs as a ‘gold standard’ could lead to false positive since the EL is slow progressing disease, and its pre-clinical stage can cause reaction of the skin to histofarcin (Al-Ani and Al-Delami, 1986; Ameni et al.,2006). Although, EL can be diagnosed easily on the basis of clinical sings, histofarcin test could be important for detecting early infection before the manifestation of clinical signs, and also it can be used as a differential diagnostic tool to differentiate EL from glanders, ulcerative lymphangitis and sporotrichosis.
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