SHORT COMMUNICATION

Single dose of secnidazole treatment against naturally occuring Giardia duodenalis infection in Sakiz lambs

Tratamiento con dosis única con secnidazol en corderos naturalmente infectados con Giardia duodenalis

Kerem Ural,¹* Ph.D, Nuran Aysul,² Ph.D, Huseyin Voyvoda,¹ Ph.D, Bulent Ulutas,¹ Ph.D, Osman S. Aldemir,² Ph.D, Hasan Eren,² Ph.D.

¹University of Adnan Menderes, Department of Internal Medicine, Faculty of Veterinary Medicine, PK: 17, 09016, Isikli, Aydin, Turkey.
²Adnan Menderes University, Faculty of Veterinary Medicine, Department of Parasitology, Işıklı Mevki, 09016, Aydın, Turkey.

*Correspondence: uralkerem@gmail.com

Received: June 2013; Accepted: November 2013.

ABSTRACT

Objetive. The purposes of this study were to determine whether secnidazole administered at a single dose of 10 mg/kg, orally, lessens or eliminates Giardia cyst shedding, and to validate the benefit of secnidazole treatment on clinical signs and faecal consistency in lambs naturally infected with Giardia duodenalis. Materials and methods. To this extent weaned 12 weeks of age lambs were selected and randomly assigned into two groups based on placebo (group C, n=7 untreated control group) or treatment (group S, n=10 lambs treated with a single dose of secnidazole at 10 mg/kg). Results. On days 0 and 10, before and after treatment, respectively, reduction in cyst excretion was determined. The faecal consistency and general health were recorded at each sampling day. Conclusions. During the study there was a high (99.98%) reduction in cyst excretion in the secnidazol treatment group compared to the positive control group on day 10, resulting in a significant (p<0.001) reduction, making secnidazole highly effective treatment option.

Objetivo. El objetivo de este estudio fue determinar si la administración oral de una dosis única de secnidazol de 10mh/kg reduce o elimina la presencia de quistes de Giardia y validar el beneficio del tratamiento con secnidazol en los signos clínicos y la consistencia fecal de corderos infectados naturalmente con Giardia duodenalis. Materiales y métodos. Se seleccionaron corderos destetados de 12 semanas de edad que fueron asignados al azar a dos grupos con base a placebo (grupo C, n=7, grupo de control sin tratar) o tratamiento (grupo S, n=10 corderos con tratamiento de dosis única de secnidazol de 10 mg/kg). Resultados. En los días 0 y 10, antes y después del tratamiento, respectivamente, se determinó una merma en la excreción de quistes. La consistencia de la materia fecal y el estado general de salud fue registrado en cada muestreo diario. Conclusiones. Durante el estudio hubo una alta reducción en la excreción de quistes (99.98%) en el grupo tratado con secnidazol comparado con el grupo control positivo en el día 10, resultando en una disminución significativa (p<0.001), haciendo del tratamiento con secnizadol una opción altamente efectiva.

INTRODUCTION

Giardia duodenalis (G. lamblia and G. intestinalis) is the most commonly diagnosed intestinal parasite of humans and livestock species worldwide. It is suggested that 2.5 million human cases of giardiasis exist annually in the United States (1). Giardiasis also ocur commonly in ruminants. The prevalence of G. duodenalis infection in livestock may present rates from 9 to 38% in adult sheep (2-4) and 68% in lambs (5). Prevalence rates in both humans and animals are often underestimated through the low sensitivity of some parasite detection methods and the intermittent cyst excretion. In Turkey, the real prevalence of giardiasis in sheep remains unclear with relatively few studies indicating 36.6% (6) and 48.48% (7).

Among the morphologically defined Giardia species, Giardia agilis, G. microti, G. muris, G. psicatti and G.duodenalis, only the latter infects humans and most domestic and wild mammals (8). The spectrum of Giardia infection vary from asymptomatic shedding of giardial cysts to symptomatic giardiasis. The infection resulted from G. duodenalis may cause epithelial barrier function loss, villus atrophy and crypt hyperplasia in the small intestine (9, 10). Findings presented that infection and related pathological alterations may result in intermittent and mucous diarrhea (10), within the combination of intestinal malabsorption and hypersecretion (11). Even if overt clinical disease is lacking the pathological alterations in association with the gastro-intestinal system may consequently result in a declined weight gain and to an altered feed efficiency (3,12). Young animals are most likely to develop clinical symptoms especially those stressed or immunocompromised (8-11).

Giardia duodenalis has established itself as zoonoses based upon biochemical and morphological similarities among animal and human isolates as well as cross infection surveys. Thus companion and food animals may have the potential to infect humans either through waterborne transmission or direct contact with contaminated feces (13, 14). Albeit the lack of strong evidence connecting contamination of water supplies by sheep with Giardia outbreaks, sheep flocks are considered as a potential threat (14). In this sense, small ruminants are suggested as a reservoir for human infections (2, 14). All animals infected with giardiasis should therefore promptly treated whether presenting clinical signs or not, because of potential source of infection for humans or other animals (9).

The traditional anti-Giardial treatment in humans includes the usage of 5-nitroimidazoles such as metronidazole, tinidazole and ornidazole, albeit effective therapy among populations with high prevalences, may be conformed by the usage of single oral dose drug possessing low toxicity and relatively few side effects (15), for instance might be provived by secnidazole (15-17). Similar to surveys among humans, parasitological eradication rate of 100% was obtained after single-dose treatment with secnidazole in cats infected with G. duodenalis (18). Currently no drug is licensed or FDA approved for the treatment of giardiasis in ruminants. Fenbendazole (9,19) and albendazole (20) resulted in a clinical benefit in treated lambs and sheep. Furthermore, paromomycin was found to be effective in lambs (21). However, fenbendazole and albendazole require multiple doses, and paramomycin is expensive. As data on the efficacy of anti-Giardial treatment in sheep involves limited and aforementioned studies, besides cases refractory to traditional treatment there is a need to evaluate novel and reasonably priced treatment regimens. The purpose of this study was to evaluate the efficacy of a single dose of secnidazole in lambs naturally infected with G. duodenalis.

MATERIALS AND METHODS

Animals, housing and husbandry. A sample size of a total of 17 Sakiz lambs at the age of 12 weeks were purchased from a national commercial vendor, where they were group housed on hay as contact bedding. During the allocation period (10 days) all lambs were screened twice with a 10 days interval to confirm the presence/absence of G. duodenalis cysts and Cryptosporidium parvum oocysts in the faeces. All animals were previously treated prophylactically with toltrazuril (Cevazuril® Ceva-Vet, 20 mg/kg bodyweight) to prevent coccidiosis. Prior to the trial, the housing was cleaned and disinfected with a product containing quaternary ammonium for elimination of probable existing environmental contamination. For 10 days duration straw bedding was added to the pens. In addition strict measures were performed to prevent cross-contamination between both experimental groups. All lambs received commercial lamb flakes, to ensure equivalent diets. Water and hay was provided ad libitum throughout the study period. Ethical guidelines were taken into consideration for enrollment into the groups.

Fecal flotation and microscopic examination of fecal samples. Pretreatment day 0 (D0) was designated as the initial treatment of the trial. Collections from each lamb were obtained on 2 occasions and were designated etiher D0 or day 10 (D10) (after treatment). Fecal samples (approximately 10 g) were collected on D0 manually from the rectum of all lambs involved and were submitted immediately to Department of Parasitology for fecal flotation. Fecal material was mixed with 15 ml of 33% (w/v) ZnSO₄ solution and strained onto centrifuge tubes, which then was spunned in centrifuge at 880 x g for 5 minutes as reported previously (5). Following centrifugation, a small sample of the fecal mixture solution was collected and placed on a microscope slide involving Lugol iodine, which was covered by a slip. The slide was examined 40x power for the presence of Giardia cysts. The latter test was repeated for 2 times from different samples for each lamb collected on day 0 by a single blinded researcher. For enrollment in the present study mono infection with G. doudenalis was proven only by microscopical fecal samples as reported previously by Escobedo et al (17).

Clinical criteria. Data on general health and faecal consistency were recorded before and after treatment. The faecal consistency was scored as previously described by Le Jambre et al (22), and similar to that of Geurden et al (9).

Assessment of efficacy of treatment. Secnidazole treatment efficacy in the present study was assessed by microscopic examination of fecal samples collected on D0 and D10 (after treatment completion), in order to avoid the bias that would be introduced by reinfection, and measured based on the reduction in cyst excretion for treatment group compared to those of control group. The reduction in cyst excretion was calculated using the Henderson-Tilton formula (23), involving geometric mean cyst counts similar to those of Geurden et al (9):

Ta and Tb; represented the geometric mean cyst count in the secnidazole treatment group before and after treatment, respectively; where as Ca and Cb; the geometric mean cyst count in the control animals before and after treatment (24).

The Henderson-Tilton formula is considered as the most appropriate method as described and used previously by Geurden et al (9).

Statistical analysis. Statistical analyses were performed using the SPSS statistical software package (version 13; SPSS Inc., Chicago, IL). The results for faecal cyst counts in both control and treatment groups were tested for normality using the Kolmogorov-Smirnov test. The faecal cyst count was not normally distributed; therefore, the data on faecal cyst count were log-transformed to achieve near-normality. An independent-samples t test was conducted to compare faecal cyst counts before (D0) and 10 days after the start of treatment (D10) between the groups. Within-group comparisons with baseline cyst value were made using t test for dependent measures. Probability (p<0.05) was considered to indicate a significant difference. Summarized data were presented as geometric mean and range.

RESULTS

Animal management and treatment applications. No observable and significant adverse reactions to secnidazole treatment were detected in the present study. All involved lambs were in good general health condition. All lambs in both groups had clinical signs compatible with naturally occuring Giardiosis, involving diarrhea. No Cryptosporidium and nor coccidosis infection were found in any of the animals enrolled throughout the study period. Seventeen Sakiz lambs at the age of 12 weeks, of both sexes, that tested microscopically positive for G. duodenalis, were allocated into two groups. Seven lambs were randomly assigned to the positive control group (group C), with taking into account the ethical guidelines and written owners consent. The remaining lambs (n=10) were enrolled in secnidazole group (group S), receiving the latter drug (Flagentyl® Eczacıbaşı, 500 mg tablets) at a single dosage of 10 mg/kg perorally. Due to ethical concerns and commercial value of the lambs, only a limited number of animals served as controls. Albeit at the end of the study all positive control animals were also treated with secnidazol at the same dosage to the previosuly treated animals.

Cyst excretion. The results of the cyst counts were presented in table 1. Throughout the study period lambs in control group C remained positive, besides 5 out of 7 lambs presented an increase in cyst counts on day 10 (ranged 340-47600) compared to the initial values (ranged between 1000-39500), albeit there was no statistical significance. On day 10, after secnidazol treatment, the percentage reduction in cyst excretion calculated based on geometric mean was surprisingly very high as 99.98%. For group S geometric mean for cyst excretion was significantly decreased (p<0.001) after treatment.

Faecal scoring results. The faecal consistency was high in approximately most of the animals and 15 out of 17 lambs had a fecal score of 2 or 3 on a single sampling day 0, whilst on day 10 all treated lambs had normal faecal consistency (score 1).

DISCUSSION

Despite the recognition of giardiasis as a significant human intestinal pathogen, relatively few options, specifically nitroimidazole derivatives are commercially available for therapy (25). For a long while, 5-nitroimidazole compounds, (metronidazole and other ones), are first line anti-Giardial treatment choice and are still been widely using (25,26). Albeit their partial efficacy, cases refractory to therapy applications within the latter compounds are becoming common (27). There is clearly a need for evaluating novel treatment options both in human-being and for small and large animal veterinary fields.

Nitroimidazole derivatives, especially the second generation such as secnidazole, is effective in a single dose, and have proven to be efficacous and inexpensive (25). Secnidazole is related to the commonly used 5-nitroimidazoles. The latter drugs present an activity against anaerobic micro-organisms and are effective for the treatment of giardiasis. (28). Secnidazole is rapidly and completely absorbed after oral administration and has a longer terminal elimination half-life (approximately 17 to 29 hours) than commonly used drugs in this class (28,29). It is commercialized for the treatment of giardiasis in humans. It has the advantage to be administered in a single dose with sound curative effects. Anti-Giardial therapy within secnidazole has been the subject of various research articles in humanbeing (15-17).

An outbreak of giardiasis was detected among sheep in Italy, whereas infected lambs at the age of 30-90 days presented malabsorption syndrome with decreased weight gain and impairment in feed efficiency within the excretion of malodorous and poorly formed faeces. Therapy with fenbendazole at a dose of 10 mg/kg for 3 consecutive days resulted in successfull clearence of the infection (19). In a prior study evaluating the efficacy of fenbendazole at 15 mg/kg/day during 3 consecutive days in lambs experimentally infected with giardiasis, there was a high (≥97.8%) and continuous reduction in cyst excretion in the treatment group compared to the positive control group (placebo) for 12 days, resulting in a significant (p<0.001) reduction of the cumulative cyst excretion (9). Albendazole treatment was the subject of another trial in sheep with Giardia sp. infection at 2 different groups of animals with 10 and 20 mg/kg albendazole for 3 consecutive days, in which the efficacies of 10 mg/kg and 20mg/kg albendazole on sheep were 58.8% and 100%, respectively after 20 days posttreatment (20). In a recent trial lambs with naturally occuring Giardia infection in Turkey, the therapeutic efficacy of 20 mg albendazole, 50 mg and 100 mg paromomycin (kg body weight orally) for 3 consequtive days were compared. According to the results of that study paromomycin apperaed to be more effective than albendozole for the treatment (21).

To the present authors' knowledge the high efficacy of a single dose of an oral treatment with secnidazole at 10 mg/kg against naturally occuring giardiasis in lambs has been demonstrated for the first time. In the present clinical trial, secnidazole was well tolerated, no adverse events were evident within gastrointestinal in nature, thus did not require treatment intervention or withdrawal from therapy. Available evidence suggested that secnidazole may be efficacious as other 5-nitroimidazole drugs in the treatment of giardiasis in lambs.

The efficacy of secnidazole in lambs is comparable to what has been described in cats naturally infected with giardiasis, in which an oral single dose of 30 mg/kg resulted in 100 % efficacy, as no cysts were found in faeces after treatment (18). In the present study secnidazole treatment significantly reduced the cyst excretion calculated based on geometric mean by 99.98% on day 10 after the start of the treatment, resulting in a significant reduction (p<0.001) in cyst excretion in secnidazole treatment group. Similarly in another parasitic species of ciliate protozoan, Balantidium coli infection cattle in Pakistan were treated with secnidazole at a dosage of 10 mg/kg perorally with a final efficacy of 87.5%, making it the most efffective choice in contrast to metronidazole or oxytetracycline in that study (30). The convenience and ease of administration associated with single-dose therapy, combined with a good tolerability profile, make secnidazole a suitable option and an attractive, besides probably superior alternative to multiple dosage regimens with other nitroimidazole derivatives.

Given the efficacy of secnidazole (as it resulted in a significantly decreased cyst excretion) as a single dose for treatment of naturally occuring Giardia infection among Sakiz lambs involved in the present study, the usage of this of drug should be encouraged. The high cyst reducing activity of secnidazol against G. duodenalis may provide an important benefit in a clinical setting where a specific diagnosis is impossible or impractical, likewise livestock field condition. The availability of this inexpensive drug, the cost (approximately 2.2 dollars per a lamb for therapy) and the easily availability for market opportunity have been significant impediments for usage of this antiparasitic drug against giardiasis in sheep.

REFERENCES

1. Furness BW, Beach MJ, Roberts JM. Giardiasis surveillance - United States, 1992-1997. Morbidity and Mortality weekly Report (MMWR) 2000; 49(SS07):1-13.         [ Links ]

2. Geurden T, Vercruysse J, Claerebout E. Is giardia a significant pathogen in production animals?. Exp Parasitol 2009; 124:98-106.         [ Links ]

3. Olson ME, McAllister TA, Deselliers L, Morck DW, Cheng KJ, Buret AG, Ceri H. Effects of Giardiasis on production in a domestic ruminant (lamb) model. Am J Vet Res 1995; 56:1470-1474.         [ Links ]

4. Ryan UM, Bath C, Robertson I, Read C, Elliot A, McInnes L, Traub R, Besier B. Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites. Appl Environ Microbiol 2005; 71:4992-4997.         [ Links ]

5. Wilson JM, Hankenson FC. Evaluation of an inhouse rapid ELISA test for detection of Giardia in domestic sheep (Ovis aries). J Am Assoc Lab Anim Sci 2009; 49(6):809-813.         [ Links ]

6. Ozmen O, Yukari BA, Haligur M, Sahinduran S. Observations and immunohistochemical detection of coronavirus, Cryptosporidium parvum and giardia intestinalis in neonatal diarrhoea in lambs and kids. Schweiz Arch Tierheilkd 2006; 148:357-364.         [ Links ]

7. Ozdal N, Tanritanir P, Goz Y, Deger S, Kozat S. Parasitic protozoans (Eimeria, Giardia, and Cryptosporidium) in lambs with diarrhoea in the Van Province (Turkey). Bull Vet Inst Pulawy 2009; 53:47-51.         [ Links ]

8. van der Giessena JWB, de Vriesa A, Roosa M, Wielingaa P, Kortbeekb LM, Mankc TG. Genotyping of Giardia in Dutch patients and animals: A phylogenetic analysis of human and animal isolates. Int J Parasitol 2006; 36(7):849-858.         [ Links ]

9. Geurden T, Pohleb H, Sarrea C, Dreesena L, Vercruyssea J, Claerebouta E. The efficacy of a treatment with fenbendazole against an experimental Giardia duodenalis infection in lambs. Small Rum Res 2011; 96(2-3):211-215.         [ Links ]

10. Ruest N, Couture Y, Faubert GM, Girard C. Morphological changes in the jejunum of calves naturally infected with giardia spp. and Cryptosporidium spp. Vet Parasitol 1997; 69:177-186.         [ Links ]

11. Buret AG. Pathophysiology of enteric infections with Giardia duodenalius. Parasite 2008; 15(3):261-265.         [ Links ]

12. Sweeny JP, Jacobson C, Robertson I, Ryan UM. Carcass productivity consequences of trichostrongylid and protozoan parasites in Merino x Suffolk prime lambs in the South West of Western Australia. Conference proceedings: Melbourne, Australia: The International Congress of parasitology (ICOPA); 2010.         [ Links ]

13. Gaydos J. Giardia and Wildlife South Coop Wild Dis St Br 1998; 14:2.         [ Links ]

14. Robertson LJ. Giardia and Cryptosporidium infections in sheep and goats: A review of the potential for transmission to humans via environmental contamination. Epidemiol Infect 2009; 137:913-921.         [ Links ]

15. Di Prisco MC, Jiménez JC. Rodríguez N, Costa V, Villamizar J, Silvera A, Carrillo M, Lira C, Zerpa E, López Y. Clinical trial with secnidazole in a single dose in venezuelan children infected by Giardia intestinalis. Invest Clin 2000; 41:179-188.         [ Links ]

16. Almirall P, Escobedo AA, Ayala I, Alfonso M, Salazar Y, Cañete R, et al. Mebendazole compared with secnidazole in the treatment of adult giardiasis: A randomised, no-inferiority, open clinical trial. J Parasitol Res 2011; 1-6. doi:10.1155/2011/636857.         [ Links ]

17. Escobedo AA, Canete R, Gonzalez ME, Pareja A, Cimerman S, Almirall P. Randomized trial comparing mebendazole and secnidazole for the treatment of giardiasis. Ann Trop Med Parasitol 2003; 97(5): 499-504.         [ Links ]

18. DaSilva AS, Castro VSP, Tonin AA, Brendler S, Costa MM, Jaques JA, Bertoletti B, Zanette RA, Raiser AG, Mazzanti CM, Lopes STA, Monteiro SG. Secnidazole for the treatment of giardiasis in naturally infected cats. Parasitol Int 2011; 60(4):429-432.         [ Links ]

19. Aloisio F, Filippini G, Antenucci P, Lepri E, Pezzotti G, Cacciò M S, Pozio E. Severe weight loss in lambs infected with Giardia duodenalis assemblage B. Vet Parasitol 2006; 142(1-2):154-158.         [ Links ]

20. Mohammed BA. Efficacy of albendazole with two different doses for the treatment of giardiasis in domestic ruminants. Iraq J Vet Sci 2006; 20(2):265-282.         [ Links ]

21. Albay MK, Sahinduran S, Adanir R, Yukari BA, Kose O. Efficacy of albendazole and two different doses of paromomycin for treatment of naturally occurring giardia infection in lambs. Kafkas Univ Vet Fak Derg 2011;17:1021-1024.         [ Links ]

22. Le Jambre LF, Dominika S, Eadya SJ, Henshalla JM, Colditza IG. Adjusting worm egg counts for feacal moiture in sheep. Vet Parasitol 2007; 145:108-115.         [ Links ]

23. Henderson CF, Tilton EW. Tests with acaricides against the brown wheat mite J Econ Entomol 1955; 48:157-161.         [ Links ]

24. Presidente PJA. Resistance in nematodes to anthelmintic drugs. Australia: CSIRO Division of Animal Health Australian Wool Corporation; 1985.         [ Links ]

25. Rossignol JF. Cryptosporidium and Giardia: Treatment options and prospects for new drugs. Exp Parasitol 2010; 124:45-53.         [ Links ]

26. Busatti HGNO, Santos JFG, Gomes MA. The old and new therapeutic approaches to the treatment of giardiasis: Where are we? Biologics 2009; 3:273-287.         [ Links ]

27. Nash TE, Ohl CA, Thomas E, Subramanian G, Keiser P, Moore TA. Treatment of Patients with Refractory Giardiasis. Clin Infect Dis 2001; 33:22-28.         [ Links ]

28. Gillis JC, Wiseman LR. Secnidazole. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic use in the management of protozoal infections and bacterial vaginosis. Drugs 1996; 51:621-638.         [ Links ]

29. Videau D, Niel G, Catalan F. Secnidazole A 5-nitroimidazole derivative with a long half-life. Brit J Vener Dis 1978; 54:77-80.         [ Links ]

30. Bilal CQ, Khan MS, Avais M, Ijaz M, Khan JA. Prevalence and chemotherapy of Balantidium coli in cattle in the River Ravi region, Lahore (Pakistan). Vet Parasitol 2009; 163(1-2):15-17.         [ Links ]