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Microsoft word - 935-940 _9_ pjz-1176-12 1-7-13 final%20pjz%20paper%20sabiqa[2]-1.doc

Pakistan J. Zool., vol. 45(4), pp. 935-940, 2013

Anti-Cryptosporidium Activity of Albendazole, Metronidazole and
Paromomycin in Experimentally Infected Cattle
Sabiqaa Masood,1 Azhar Maqbool,1 Umbreen Javed Khan,2* Zafar Iqbal Chaudhry3 and
Aftab Ahmad Anjum4
1Department of Parasitology, University of Veterinary and Animal Sciences, Lahore
2Department of Zoology,Govt. Post Graduate College (W), Samanabad, Lahore
3Department of Pathology, University of Veterinary and Animal Sciences, Lahore
4Department of Microbiology, University of Veterinary and Animal Sciences, Lahore

Abstract.- Efficacy of albendazole, metronidazole and paromomycin was evaluated against Cryptosporidium in
experimentally infected cattle. Cryptosporidium oocysts harvested from cattle reared at different livestock farms
identified by microscopic morphology and PCR were used for experimental infection. Oocyst per gram (OPG) count
was increased in control (untreated) cattle. A single dose of 10mg/kg body weight of albendazole caused significant
reduction in OPG count and efficacy determined was 43.05, 58.7 and 64.6 percents on 13th, 20th and 27th day post
treatment. At 7.5mg/kg body weight dose efficacy recorded was 34.8, 57.1 and 62.9 percents, respectively. A single
dose of 50mg/kg body weight of metronidazole caused significant decrease in OPG count and efficacy calculated on
days 13, 20 and 27 was 32.8, 53.3 and 56.6 percent, respectively. Similar pattern of reduction in oocyst number and
efficacy was recorded at higher dose of metronidazole. At dose rate of 25mg/kg body weight of paromomycin used
against Cryptosporidiosis under experimental conditions significant reduction in OPG count was observed with
percent efficacy of 55.04, 68.5 and 79.4 on different observational days. At 50mg/kg body weight dose rate of
paromomycin percent efficacy determined was 48.1, 65 and 69, respectively. On comparison the most effective
reduction in OPG was observed by the use of paromomycin at dose rate of 25mg/kg body weight of cattle. Results of
paromomycin were better than other two trialed drugs and may be a choice for therapy of, cryptosporidiosis.
Key words: Cryptosporidiosis, cattle, efficacy, albendazole, metronidazole, paromomycin.
INTRODUCTION
haemorrhagic diarrhoea, lethargy, pyrexia and loss of condition (Xiao et al., 1999). Cryptosporidium, a unicellular intestinal microscopic examination of Cryptosporidium coccidian, infects microvillus of gastrointestinal tract in wide range of animals (Spano et al., 1998). Cryptosporidium in different species ranges from Higher load of oocysts is excreted in the 4.5 to 7.9 µm in length and 4.5 to 6.5 µm in width. environment by infected animals. The oocysts are The shape of C. parvum oocyst is ovoid to elliptical highly resistant to desiccation, disinfectants and depending upon the host of parasite. ELISA has been developed with varying degree of specificity environment surrounding the infected animal herds and sensitivity for antigen detection (Elgun is a constant threat to healthy animals (Chen and and Koltas, 2011). PCR technology is a powerful Kehe, 2012). Warm and humid monsoon months are alternative for the detection of C. parvum in both more amicable for propagation of the disease (Wang environmental and clinical samples (Ware et al., widespread in ruminants for neonatal diarrhea Many vaccines and chemotherapeutic agents (Lorenz et al., 2011) while C. andersoni is have been tested for prophylaxis against the disease responsible for abomasal Cryptosporidiosis in cattle (Hueffer et al., 2013). Paromomycin is most and buffaloes (Putignani and Menichella, 2010). commonly used drug against Cryptosporidiosis. Clinical disease is characterized by mucous to Azithromycin show partial results against the disease (Gargala, 2008). In vitro and in vivo effect Corresponding author: khanumbreen@yahoo.co.uk of nitazoxanide has been demonstrated using different animal models and finally in clinical trials. Copyright 2013 Zoological Society of Pakistan Repeated doses of nitazoxanide and albendazole are also effective against Cryptosporidiosis. Due to Cattle, 2-3 years of either sex experimentally environmental contamination and severity of infected with Cryptosporidium were kept at four clinical signs (Fayer and Nerad, 1996) protease inhibitors such as saquinavir and ritonavir cause management conditions within the radius of 10 significant decrease in oocyst count. In Pakistan, kilometer throughout the course of treatment. The most of the domesticated livestock is not producing negative control group was kept separate in a shed to their potential (Khan and Maqbool, 2012). Major and no one was allowed to make access there. involvement for this lowered performance and Selected animals (n=150) were divided randomly economic losses is of different parasitic infections. into five groups (A, B, C, D and E) having 30 herds Therapeutic efficacy of albendazole, metronidazole each. Animals of groups A, B and C were further divided into two subgroups having 15 cattle in each. Prior to start therapeutic trials groups A, B, C and D were infected with Cryptosporidium oocyst (n=200) orally whereas group E kept as non-infected control. MATERIALS AND METHODS
On 10th day of infection oocyst/gm of feces counted as already described. The albendazole (ICI, Pakistan Cryptosporidium oocyst load per gram of Ltd.), metronidazole (Sonon aventis, Ltd. Pakistan) collected fecal samples was calculated. Oocysts and paromomycin (Star Laboratory, Ltd. Pakistan) were identified on the basis of microscopic were selected for therapeutic trials against Cryptosporidiosis. Animals in subgroups A, B and polymerase chain reaction (PCR). Efficacy of C were treated with two different doses of different anti-parasitic drugs was evaluated on the albendazole (7.5 and 10 mg/kg), metronidazole (50 basis of log reduction in Cryptosporidium oocysts. and 100 mg/kg) and paromomycin (25 and 50 mg/kg) orally for five consecutive days. Group D was kept as positive control. Fecal samples were Fecal samples (n=720) were collected directly collected from each animal of treated and control from the rectum of each cattle in a sterilized plastic groups on 6, 13, 20 and 27 days post treatment and bag reared at Military dairy farm, Government dairy farm, Gawala colonies and House hold dairies (n=180, each) in and around Lahore city from identification was made by microscopic morphology Cryptosporidium oocysts was calculated as per of Cryptosporidium oocysts following the protocol described by Fayer et al. (2012). Cryptosporidium oocysts were identified at molecular level using PCR as described by Johnson et al. (1995). Cryptosporidium oocyst load per gram of feces was calculated. Known weight by volume suspension of each collected fecal sample was prepared. Side effects of drugs were recorded. Effects Following formula was used for calculation of of treatment on body weight and feed intake were observed including randomly selected animals (n=10) from each group/sub group of animals. All readings were documented in triplicate for each N, number of Oocysts per gram of feces; S, number of oocysts counted on the slide; Vol., volume of the sample examined (20 µl); Wt., weight of the fecal sample (20 g) and pv, pellet volume (1ml). EFFICACY OF DIFFERENT ANTICRYPTOSPORIDIUM DRUGS animal of all groups before Cryptosporidium count from 6th day post treatment and onward infection. After the infection on appearance of (P<0.05). Efficacy of drug calculated on days 13, 20 oocysts in feces blood samples were collected in similar fashion and complete hemogram was respectively. On statistical analysis by Univeriate obtained. The leukocyte count particularly of eosinophil was carried out on blood films stained by (P<0.05) decrease in the OPG count was recorded Giemsa. The comparison of hemoglobin level and on 13th day post treatment whereas on 20th and 27th leukocyte count among different groups was carried days difference was non-significant. Similar pattern out. Data obtained was analyzed by one way of reduction in oocyst number and efficacy was ANOVA using Duncan’s Multiple Range test. recorded at higher dose of metronidazole used in paromomycin used against Cryptosporidiosis under Prevalence of Cryptosporidium oocysts in experimental conditions significant decrease in OPG cattle identified by microscopic oocyst morphology count was observed 6th day post treatment and was 20.55, 12.77, 6.11 and 3.88 percent at onward (P<0.05). Percent efficacy of used drug was Government dairy farm, Gawala colonies, Military 55.04, 68.5 and 79.4 on days 13, 20 and 27 post dairy farm and House hold dairies, respectively. treatment, respectively. On statistical analysis Percent prevalence values confirmed by PCR were significant difference in relation to reduction in oocyst number was observed on different selected days. At 50mg/kg body weight dose rate of paromomycin significant decrease in OPG count Efficacy of different selected drugs against was recorded from 6th day post treatment and Cryptosporidiosis was evaluated on the basis of onward (P<0.05). On days 13, 20 and 27 percent reduction in the oocyst per gram of feces post efficacy of used drug determined was 48.1, 65 and treatment in relation to time. The means of 69, respectively. On comparison the most effective reduction in Cryptosporidium oocysts of treated and reduction in OPG was observed by the use of paromomycin at dose rate of 25mg/kg body weight of cattle. Results for efficacy of selected drugs are metronidazole and paromomycin at different doses against Cryptosporidium oocysts in experimentally Post treatment side effects observed were infected cattle determine on the basis of reduction in sweating and diarrhea. At higher doses three cattle Oocyst per gram of feces (OPG) are presented at Table I. OPG count showed an increasing trend in (metronidazole) and only one in C (paromomycin) control (untreated) animals. A single dose of exhibited the sign of sweating. Diarrhea was noted 10mg/kg body weight of albendazole caused a in the sub group of A and B, which became normal significant decrease in OPG count from 6th day post by day 13. No other side effect was recorded during treatment and onward. The efficacy of albendazole experiments. Increase in body weight of animals in observed was 43.05, 58.7 and 64.6 percents on 13th, non-infected control and treated groups were in 20th and 27th day post treatment. At 7.5mg/kg body same pattern and differed significantly with animals weight dose of albendazole significant reduction in of infected non-treated group. Significant difference OPG count of Cryptosporidium was recorded from was observed in the values of leukocyte and 6th post treatment day and onward. The efficacy of eosinophil in infected cattle on 6th and 13th days post albendazole determined at this dose was 34.8, 57.1 and 62.9 percents on days 13, 20 and 27 post Cryptosporidium non-infected cattle were 58.09, 5.67, 5.33 and 0.59, respectively. Whereas values metronidazole caused a significant decrease in OPG recorded in infected cattle were 59.09, 5.73, 10.29 Table I.-
Cryptosporidium oocyst per gram of feces in cattle treated with selected doses of different drugs in relation to time
Subgroups
Treatment
Oocysts per gram of feces on different days
(mg/kg.b.wt.)
E. Group (negative non infected control). Means carrying same superscript differ non-significantly (P<0.05)

Table II.-

Comparative percent efficacy of different selected drugs used at different doses against Cryptosporidium in cattle
Subgroups
Treatment
Percent efficacy at different days
(mg/kg.b.wt)
and 0.69, respectively. Hemoglobin concentrations caused a significant decrease in OPG count from 6th in infected and non-infected cattle recorded were day post treatment and onward (P<0.05). Similar 11.08 and 11.04 showing non-significant difference findings in relation to the efficacy of albendazole at Cryptosporidiosis were reported by Xiao et al. DISCUSSION
(1999) and Johny et al. (2007). Metronidazole treatment caused a significant decrease in OPG Cryptosporidiosis is an important zoonotic count from 6th day post treatment and onward disease of domestic and wild animals. Infection with Cryptosporidium parvum is common in cattle, Cryptosporidiosis under experimental conditions buffaloes, goats, sheep, horses, cats, human beings showed better results than albendazole and and other vertebrates (Leitch and He, 2011). The infection is endemic in ruminants as well as human Although a number of compounds have been beings in Pakistan. In present study occurrence of tested against Cryptosporidiosis and only limited disease in cattle reared at Government dairy farm, showed effective results. Kelly et al. (1996) Military dairy farm, Household dairy and Gawala colonies was determined. Efficacy of selected drugs Cryptosporidiosis and eradication in four Zambian against Cryptosporidium oocysts was evaluated AIDS patients by albendazole used at dose 80mg under experimental conditions in cattle. twice. However control large scale study was recommended. Only paromomycin has been shown Efficacy of chemotherapeutic agents to have an anti-cryptosporidial activity (Hahn and increasing trend in control (untreated) animals. A documented in relation to efficacy of paromomycin single dose of 10mg/kg body weight of albendazole by Leitch and He (2011). Similar findings were EFFICACY OF DIFFERENT ANTICRYPTOSPORIDIUM DRUGS reported by Sharling et al. (2010), Tzipori et al. ELGUN, G. AND KOLTAS, I.S., 2011. Investigation of (1994) and Verdon et al. (1994) while working on Cryptosporidium spp. antigen by ELISA method in
stool specimens obtained from patients with diarrhea.
animal models regarding efficacy of paromomycin Parasitol. Res., 108:395-7.
against Cryptosporidiosis. Paromomycin in a dose FAYER, R. AND NERAD, T., 1996. Effects of low of 25 to 35 mg/kg/day has a beneficial but limited temperatures on viability of Cryptosporidium parvum effect upon oocyst shedding and stool frequency in oocysts. Appl. environ. Microbiol., 62: 1431–1433.
AIDS patients (Chawla et al., 2011). In conclusion FAYER, R., FARLEY, C.A., LEWIS, E.J., TROUT, J.M. AND paromomycin is probably the most promising GRACZYK, T.K., 1997. The potential role of the oyster compound for treatment of Cryptosporidiosis in Cryptosporidium parvum. Appl. environ. Microbiol., human beings. Paromomycin was suggested to be 63: 2086-2088.
the most valuable drug for the treatment of FAYER, R., SANTIN, M. AND MACARISIN, D., 2012. Cryptosporidium infection based on the clinical Detection of concurrent infection of dairy cattle with Post treatment side effects observed were Enterocytozoon by molecular and microscopic methods. sweating and diarrhea. At higher doses three cattle Parasitol. Res., 111: 1349-55.
GARGALA, G., 2008. Drug treatment and novel drug target against Cryptosporidium. Parasite, 15:275-81.
exhibited the sign of sweating. Diarrhea was noted GRIFFITHS, J.K., BALAKRIHNAN, R., WIDMER, G. AND TZIPORI, S., 1998. Paromomycin and genecitin inhibit in the sub group of A and B, which became normal by day 13. Increase in body weight of animals in trafficking through the host cell cytoplasm: implications non-infected control and treated groups were in for drug delivery. Infect. Immunol., 66: 3874–3883.
same pattern and differed significantly with animals HAHN, N.E. AND CAPUANO, S.V., 2010. Successful of infected non-treated group. Significant difference treatment of cryptosporidiosis in 2 common marmosets (Callithrix jacchus) by using paromomycin. J. Am. was observed in the values of leukocyte and Assoc. Lab. Anim. Sci., 49: 873–875.
eosinophil in infected cattle on 6th and 13th days post HILL, N.J., DEANE, E.M. AND POWER, M.L., 2008. inoculation. The leukocytes/lymphocytes count of Cryptosporidium infected cattle was 58.09 and Cryptosporidium isolates from common brushtail eosinophil 9.69 percent. The difference noted for possums (Trichosurus vulpecula) adapted to urban hemoglobin concentration was non-significant settings. Appl. environ. Microbiol., 74: 5549–5555.
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