An In-vitro Trial on Antifungal Effect of Xanthium strumarium Leaf Extract on the Growth of the Mycelial Form of Histoplasma capsulatum Var farciminosum Isolated from Horse

: Epizootic lymphangitis (EL) has deleterious effect on both welfare and health of the horses and mules so that it has a serious negative impact on the livelihoods of cart-horse owners/drivers in the affected parts of Ethiopia. Unfortunately, antifungal drugs for the treatment of epizootic lymphangitis are costly and mostly unavailable in such areas. The aim of this study was to investigate the effect of xanthium stramorium leaf extract (XSLE) on the growth of the mycelial form of histoplasma capsulatum var farciminosum which has been reported to have a strong antifungal activity in previous studies, in vitro. Histoplasma capsulatum var farciminosum was isolated from a horse with a cutaneous form of EL. Agar dilution assay was performed to evaluate the anti HCF activity of XSLE and measure the minimum inhibitory concentration (MIC). Ketoconazol was used as a positive control. Xanthium stramorium extract inhibited the growth of HCF at concentrations of 0.068 mg/ml. The result indicates that XSLE can potentially be used for the treatment of EL provided that convenient methods of preparation, dose and route of administration are established through rigorous in vitro trials.


INTRODUCTION
North Africa and is also found in Central and Southern Histoplasma capsulatum var farciminosum is the The mode of transmission of the disease is not well causal agent of epizootic lymphangitis, or preferably established [7]. Direct contact with infective materials histoplasmosis, a chronic disease of horses and other through injured skin or through cutaneous abrasions is Equidae [1], generally affecting the subcutaneous lymph the most common mode of infection [8]. In Ethiopia, the nodes and the lymphatics of the neck, chest and legs disease was reported to have an overall prevalence of but it can also be present as an ulcerating conjunctivitis 18.8% in 28 towns being more common in mid altitude of the palpebral conjunctiva, or rarely as a multifocal areas (1500 to 2300 meter above sea level) [9]. pneumonia [2]. In all cases, the lesions are nodular and Recommended treatments for EL include intravenous granulomatous in character and the organism, once sodium iodide or intravenous amphotercin B [4]. Another established, spreads locally by invasion and then via the treatment regimen involves local treatment of skin lesions lymphatics [3]. Epizootic lymphangitis is a contagious by incising nodules, draining the pus and infusing disease which can infect humans [4]. The yeast form of with 4% iodine tincture followed by oral potassium iodide the fungus is found in the infected host and the mycelial (30 g for a horse of 200-250 kg) daily for 5 days and then saprophytic stage is found in the environment [5,3]. every other day for a further 3-4 weeks or longer if Epizootic lymphangitis has a serious negative impact owners comply [10]. This regimen, devised by the Society on the livelihoods of cart horse owners/drivers in the for the Protection of Animals Abroad (SPANA) clinic at affected areas and also compromises the welfare of the College of Veterinary Medicine and Agriculture of working horses [6]. The disease is endemic in countries Addis Ababa University, was reported to result in a bordering the Mediterranean, particularly in Italy and recovery rate of 85% [11].
Africa and in regions of Asia and Russia [4].
Ethnopharmacologists, botanists, microbiologists by OIE (2008). The isolate was then sub cultured into and natural products chemists are combing the Earth for Sabouraud's dextrose agar (SDA) slants (2.5% glycerol phytochemicals and "leads" which could be developed and 0.005% chloramphenicol) and incubated for 21 days for treatment of infectious diseases [12]. Medicinal plants at 27°C. would be the best source to obtain a variety of drugs in developed countries about 80 of plants are used in Agar Dilution Assay: Medicinal plant used for study; traditional medicine [13]. Over 50% of modern clinical Xanthium strumarium / rough cocklebur: is broad leaved, drugs are natural product of medicinal plants and these tap rooted herbaceous annual plant (Fig. 5) which is in natural products play an important role in drug family asteraceae, sub family asteroideae, tribe development programs in the pharmaceutical industry [14] heliantheae, genus xanthium and species xanthium humans are using natural product of animals, plants and strumarium. It grows as weed throughout on waste lands. microbial sources for thousands of years either in the pure Cockleburs are short day plants and they can also flower form or crude extracts [15].
in the tropics where the day length is constant. The herb In Ethiopia, medicinal plants are widely used in the is reputed as medicine in Europe, China, Indo-china, society. This has been examined to assure the plants Malaysia and America also [17]. Stem is erect, ridged, efficacy in treating disease by some researchers. For rough and hairy and frequently branched which results instance, an in-vitro study conducted by [6] indicated that somewhat bushy plants from 30-120cm tall. It has small fresh garlic extract has some degree of growth inhibitory greed unisexual flower occurring in separate cluster at the effect on the mycelial form of histoplasma capsulatum var end of the brunches and main stems. The fruit is brown, farciminosum [16] and has conducted evaluation of hard, woody, bur from 0.4-0.8 inch long and coved with berries of phytolacca dodecandra for growth inhibition of stout, hooked bristle. Its seed are produced in hard, spiny, histoplasma capsulatum var. farciminosum and treatment globes or oval double chambered single seeded bur [18]. of cases of epizootic lymphangitis in Ethiopia and that Beside its medicinal value if a small quantity of parts of was effective.
the mature plants is consumed, the seeds and seedlings The works mentioned above are good indicators that will cause intoxication because extremely toxic chemical xanthium stramorium leaf extract may have a potent carboxyatratyloside is contained in them [19]. inhibitory effect on histoplasma capsulatum var farciminosum (HCF). There was no research that has been Collection of Medicinal Plant: The collection of medicinal done so far in this plant extract to assure whether it has plant was done in Kemissie Amhara rigion of Oromia zone. antifungal effect for the treatment of EL. Therefore, the Fresh plants were collected and shade then powdered. objectives of this study were to investigate the effect of Powder was preserved in zip lock bags at room xanthium stramorium leaf extract on the growth of HCF temperature. and to determine the minimum inhibitory concentration in vitro.

Inoculums Preparation:
A pure sub culture of the mycelial colony of HCF on SDA (with 2.5% glycerol and 0.005% chloramphenicol) (Fig. 1) was used for the tests. Fig. 2: A gram-stained smear made from HCF isolate The inoculums suspension was prepared by transferring revealed typical yeast form of the organism, which a portion of the fungal colony to a sterile saline solution appeared as Gram-positive, pleomorphic and ovoid in a sterile test tube using a sterile wire loop. The turbidity to globose structures of the suspension was adjusted by adding fungal colony or saline and vigorously mixing using a Vortex until turbidity approximates that of a 0.5 McFarland standard [21].

Inoculation of the Media:
The inoculums suspension was inoculated into triplicates of the test media and the controls using sterile swabs. After dipping into the suspension, the swab was uniformly streaked all over the agar surface.

Determination of Minimum Inhibitory Concentration:
Minimum inhibitory concentration was defined as the lowest concentration of drug that completely inhibited visible growth [22]. The MIC was determined after incubating the inoculated media at 27°C for 21 days for sub cultured HCF colony.

HCF Isolate:
The Histoplasma capsulatum var farciminosum isolate which was found from Aklilu Lemma Institute of Pathobiology, Addis Ababa University was re chloramphenicol)    [17]. The genus 0.14 mg/ml and 0.068 mg/ml. The MIC of the XSLE against xanthium also possess antibacterial, antiviral, antimalarial, HCF was 0.068 mg/ml and that of the positive control was fungicidal, insecticidal and cytotoxic activities against 0.5 µg/ ml (Fig. 4). The highest concentration of XSLE cancer cell lines [24]. The inhibitory effect of ketoconazole without growth inhibitory effects was 0.034 mg/ml. is 136 times potent than XSLE on the growth of the The results of the agar dilution assays are summarized in mycelial form of HCF in which it is more potent medicinal Table 1. plant.

DISCUSSION
The isolate was checked by gram staining and sub culturing so that it was characteristic HCF both in gram staining and colony morphology as indicated in Weeks et al., (1985), Selim et al., (1985) and OIE (2008). The interesting foundation that was encountered while sub culturing the HCF colony was that well visible growth has been seen in 21 days even if growth is relatively slow and most isolates require from four to eight weeks for development of characteristic colonies [4]. Histoplasma capsulatum var. farciminosum produces moderately this charachterstics on SDA (with 2.5% glycerol and

Antifungal activity was determined by the agar
In-vivo studies must be conducted so that the safety diffusion method. Test samples were diluted in Sabouraud margin, toxicity and cure rates will be known in order dextrose agar followed by solidification in slanting to use them commercially. positions. Test fungal cultures were inoculated on the slant and were incubated at 29°C for 3-7 days [25,26].
ACKNOWLEDGEMENTS Among dermatophytes, the most susceptible strain was microsporum canis which showed 50% and 60% First of all I praise nobody but God and his Mother inhibition against crude extract and ethylacetate fraction, Virgin Merry for helping me being in front of me to cover respectively. Aspergillus flavus was the second most me from evil actions and impediments in each fraction of susceptible fungi, exhibited 50% inhibition against seconds. n-hexane fraction [