XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
Lab Anim OnlineFirst, published on December 5, 2014 as doi:10.1177/0023677214562850
l
ab orator y
Original Article
a
n imals
l i m i t e d
Efficacy and security of ivermectin given orally to rats naturally infected with Syphacia spp., Giardia spp. and Hymenolepis nana
Laboratory Animals 0(0) 1–5 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677214562850 la.sagepub.com
VRS Foletto, F Vanz, L Gazarini, CAJ Stern and CR Tonussi
Abstract The results of this study show that the oral administration of ivermectin (48 mg/L) repeatedly for 72 h used in accordance with the present protocol is a safe and highly effective treatment for Giardia spp. and Hymenolepis nana in laboratory rat colonies. The drug can be easily and safely administered using drinking water. This simple regimen should control pinworm infection (Syphacia muris), a problem that can be endemic in laboratory colonies. Experiments using healthy animals are likely to generate more consistent results, thereby requiring a reduced number of animals per group.
Keywords ivermectin, Giardia, Syphacia, Hymenolepis, laboratory animals, faecal examination
Laboratory animals must be kept in appropriate health conditions to ensure the higher efficiency of experimental studies.1–5 Parasitized animals are not suitable for experimental research since they may present nutritional, immunological and haematological differences compared with normal values.6–9 At least three parasites are common threats to rodent colonies: Syphacia spp., Giardia spp. and Hymenolepis nana. A common infection found in rat colonies is due to Syphacia spp., which has also been reported to infect humans.6 In the host, these worms develop into adults from eggs in 4–7 days. New eggs are deposited in the host’s perianal region, which are easily dispersed in the environment, contaminating other animals.10 Such infections are usually asymptomatic, but may be associated with rectal prolapse, intussusception, low weight gain, hypertension, irritability and immunological alteration.11–14 Giardia spp. is another common parasite genus found in rat colonies. The infecting cysts are maximally present in faeces 5–14 days after ingestion by the host.15 Indeed such infections are asymptomatic, but unspecific clinical signals may be present such as weight loss, lower growing rate, abdominal distension and diarrhoea.2 H. nana is a threat to both animals and
humans. It has a direct cycle of between 14 and 16 days duration. Severe infection with H. nana leads to dehydration, abdominal pain, malnutrition, diarrhoea and weight loss. This is the only Taenia that infects humans without needing an intermediate host.9,16,17 Ivermectin is a macrocyclic lactone widely used as an antiparasitic agent either in veterinary or in human medicine. It targets a glutamate-gated chloride channel in some invertebrates as well as the chloride channel of gamma-aminobutyric acid type A (GABAA) receptors, promoting cellular hyperpolarization and worm paralysis.18 Although generally considered secure and welltolerated, ivermectin can cause toxic effects in the host depending on its concentration in the central nervous system (CNS), since it is freely permeable through the blood–brain barrier.19 The aim of the present study was to investigate the occurrence of endoparasites in Department of Pharmacology, Federal University of Santa Catarina, Floriano ´polis, Santa Catarina, Brazil Corresponding author: Vanessa RS Foletto, R Sı´lvio Lopes de Arau ´ jo, 645 ap 205, Floriano ´polis, 88048391, Brazil. Email: [email protected]
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
2
Laboratory Animals 0(0)
the intestinal tract of a conventional Wistar rat colony, and to evaluate the efficacy and security of ivermectin for the control of these natural infections.
Animals Forty male and 50 female Wistar rats (Rattus norvegicus) from a conventional biotery (Floriano´polis, UFSC, Brazil) weighing 200–250 and 150–200 g, respectively, were housed under standard conditions for laboratory animals in groups of five with a controlled 12 h light/ dark cycle, temperature 20 2 C and ad libitum access to standard diet and water. All experimental procedures were conducted according to the European Convention for the Protection of Vertebrate Animals and were approved by the local ethical committee for animal research (CEUA–UFSC).
Materials and methods Sample collections and laboratory techniques Faecal sampling was obtained on three consecutive days before the treatment, and on another three consecutive days a week after the treatment. Faecal material was collected directly from the anus and stored in a thimerosal–iodine–formaldehyde solution at room temperature in a proportion of 1:10. Samples from 10 animals were pooled and submitted to the Hoffman–Pons– Janer (HPJ) technique, Willis technique, and also direct visualization using an optical microscope. HPJ can detect the presence of helminth eggs and larvae, and protozoan cysts. This technique utilized spontaneous faecal sedimentation.20 Briefly, faecal samples were diluted in water and filtered through a gauze strip into a conical sedimentation glass and the sediment was evaluated. The Willis technique is based on the floating effect of helminth eggs in a saturated sodium chloride solution (1.2 g/mL).21 Using this technique, the saturated saline solution with the emulsified faeces was deposited in a round-bottomed flask and a meniscus was formed on the surface. Next, the flask was covered with a glass slide. After several minutes, the slide was removed and examined under a microscope. Finally, a direct examination was performed using an optical microscope and Lugol staining. The results were expressed as number of eggs in the samples.
Drug and method of administration Ivermectin (Hebei Veyong Animal Pharmaceutical Co Ltd, Shijiazhuang, China) was dissolved in water (48 mg/L) and was offered alone ad libitum during a 72 h period. Control groups received tap water only.
Investigation of ivermectin’s depressive effect on the rats’ CNS To study the potential depressive effect of ivermectin on the CNS, which could cause lasting changes and interfere with behaviour and the dynamics of memory formation, a contextual fear-conditioning protocol22 was performed seven days after ivermectin treatment. Behavioural testing was always carried out under low intensity illumination (70 lux) during the diurnal phase (from 13:00 to 17:00 h). Fear conditioning was performed in a rectangular chamber (35 20 30 cm), with aluminium sidewalls and a front wall and ceiling made of Plexiglas. Its grid floor, made of stainless steel bars (3 mm diameter, spaced 9 mm apart centre-tocentre), was connected to a circuit board and a shock generator (Insight, Ribeira˜o Preto, SP, Brazil) to enable the delivery of controlled electrical foot shocks. In all the experiments, each animal was placed in this environment (Context A), and allowed to explore it freely for 3 min, as an initial familiarization session, and was then returned to its home cage. On the next day, the animal was again placed there for the training session, during which it received, after an initial 30 s delay (preshock period), the unconditioned stimulus (three electrical foot shocks of 0.7 mA, 60 Hz, for 3 s, with a 30 s inter-shock interval). The animal remained in this chamber for an additional 30 s (post-shock period) before returning to its home cage. In the test, the animal was re-exposed to this paired context for 3 min in the absence of unconditioned stimulus presentation. All sessions were spaced 24 h apart. After each behavioural session, both chambers were cleaned with paper tissue soaked with a 10% ethanol–water solution. The experimenter was unaware of the treatment condition in all the studies. Freezing behaviour, a commonly used index of fear in rats,23 and defined as a total absence of body and head movements except those associated with breathing, was continuously recorded during the experimental sessions by a video camera. The freezing time in each period was quantified (in seconds) by a trained observer (inter- and intra-rate reliabilities 90%) blind to the experimental groups, using a stopwatch, and expressed as the percentage of total session time.
Statistical analysis Statistical analysis was carried out using GraphPad Prism 5.0Õ software (Graphpad Software Inc, La Jolla, CA, USA). Egg occurrence was presented as mean number of eggs/mm3 SEM. Comparisons were made using a paired Student’s t-test for simple means. For contextual fear-conditioning protocol the results were expressed as mean SEM. After ensuring the
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
Foletto et al.
3
assumptions of normality and homogeneity of variance, the percentage of freezing time recorded during the test was submitted to an unpaired Student’s t-test. The statistical significance level was set at P < 0.05.
Results In male rats, Syphacia eggs were found in 100% of the samples at 8.7 1.2 eggs/mm3, Giardia eggs in 50% of samples at 2.5 1.4 eggs/mm3 and Hymenolepis in 25% of samples at 1.2 1.2 eggs/mm3. In female rats, Syphacia eggs were found in 50% of samples at 3.7 2.4 eggs/mm3 and Giardia eggs in 25% of samples at 2.5 1.4 eggs/mm3. Hymenolepis was not detected in female rats. Ivermectin treatment eliminated the occurrence of Giardia eggs in males and females, and Hymenolepis in males. The drug reduced the incidence of infection from 100% to 50% in males, which caused a 94% decrease in the number of Syphacia eggs (0.5 0.3 eggs). Similar rates were found in females, where the incidence of infection was reduced from 50% to 25%, with a mean number of eggs of 0.2 0.2 eggs (Figure 1). Ten male rats were randomly allocated to two independent groups (n ¼ 5/group) based on the previous treatment, which consisted of vehicle or ivermectin (48 mg/L in drinking water for 72 h), given a week before fear conditioning. As can be observed in Figure 2, there was no difference between treatment condition during re-exposure to the paired context (t8 ¼ 0.004; P ¼ 0.98), evidenced by the lack of residual or long-lasting effects of the drug on memory processing (Figure 2).
Male
Discussion The endoparasites encountered in our animals were the same as those normally described in laboratory animals.2,24 There are several treatments and protocols adopted for the control and eradication of these infecting parasites, and the efficacy of ivermectin has been demonstrated in several species. In hamsters, a dose of 300 mg/kg subcutaneously produces an eradication rate
Figure 2. The lack of ivermectin-induced impairments in memory encoding or expression in rats. A week after ivermectin treatment (4.8 mg/L in drinking water for 72 h), animals underwent a familiarization session and then entered Context A paired with three foot shocks (US). When re-exposed to the paired context a day later (Test A), groups spent the same amount of time freezing. Bars represent the percentage of total time spent freezing. Values are expressed as mean SEM.
Female
Syphacia ssp
Eggs/mm3
Giardia ssp Hymenolepis nana
10
10
5
5
**
***
***
0
*
*** ***
0
Pre-Ivermectin
Post- Ivermectin
Pre-Ivermectin
Post- Ivermectin
Figure 1. Ivermectin is effective in combating Giardia spp. and Hymenolepis nana. Also, it reduces the prevalence of Syphacia spp. A week before and after ivermectin treatment (4.8 mg/L in drinking water for 72 h), parasitological analysis was performed. Values are expressed as mean SEM of eggs/mm3. Asterisks indicate a significant difference (P < 0.05) from pre-ivermectin treatment (paired Student’s t-test).
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
4
Laboratory Animals 0(0)
of 99.1% and 99.5% in the experimental acute and chronic Giardia infections, respectively.25 However, a dose of 200 mg/kg intravenously eliminates a natural Giardia infection.26 Ivermectin doses necessary to control Giardia infection in rats have not yet been described, thus the present study has provided an important reference for a practical treatment in experimental colonies. In humans, ivermectin (0.2 mg/kg) eliminates 50% of the hymenolepiasis.27 On the other hand, in hamsters, this drug is ineffective at an oral dose of 0.5 mg/kg.28 However, an allometric extrapolation29 suggests that the dose used in hamsters should be threefold lower than the effective one used in humans. Ivermectin is effective in eliminating Hymenolepis eggs in rats. Allometric extrapolation indicates that the above human dose would be equivalent to 1.2 mg/kg in rats. Taking into account the daily water ingestion by rats (100 mL/kg), it is conceivable that the total ivermectin dose was about 5 mg/kg, which may explain the effectiveness against hymenolepiasis. The efficacy of ivermectin against pinworm (Syphacia muris) in several studies has been variable, probably due to differences in dose regimen, time of treatment and method of administration. One common factor in such studies that were successful in the elimination of the parasite was the repetition of the treatment at four- to nine-day intervals.30,31 The control of S. muris is difficult, since cross infections occur easily after egg depositions. Although ivermectin is effective against adult and larval forms of Syphacia, it has no effect on eggs. Since eggs are light and resistant, they spread out in the environment, being found intact even in ventilation ducts.10,32 For these reasons, disinfection protocols must be applied concomitantly to pharmacological treatment, until the complete elimination of the eggs is ensured.33 Since ivermectin acts by enhancing GABAAmediated transmission, the main inhibitory system in the nervous system, high doses of this drug can be associated with general neuronal depression.19,34 Despite the high dose of ivermectin administered here (5 mg/kg) and the length of treatment (three days), the rats did not show altered performance in the fearconditioning test. This is in line with a previous report in which it was observed that doses of up to 7.5 mg/kg did not produce visible CNS depression in rats.19 Memory encoding, a process dependent on brain circuitry integrity, is highly modulated by the GABAergic system and, therefore, highly sensitive to interventions in this inhibitory system.35–38 However, as was observed in the present study, the oral treatment with ivermectin did not impair the encoding of this aversive memory, as no differences in freezing behaviour could be observed between groups.
Overall, the present antiparasitic protocol may be considered to be effective against threatening worms in rat colonies, and also secures the subsequent use of such animals for at least some studies in neuroscience. The collective treatment regimen in drinking water is thought to ease its implementation, reducing the sources of animal distress such as physical restraints and invasive administration. Furthermore, we can assume that this prophylactic measure can even target other parasites, beyond those focused on in the present study, since ivermectin is also effective in ectoparasites, thereby providing better sanitary conditions and homeostasis. Such physiological balance is also critical in reducing individual variability in experimental studies and, consequently, in reducing the number of animals required for generating statistically relevant data. Declaration of conflicting interests None declared.
Funding This research was funded by Brazilian government funding agencies CAPES and FAPESC-CNPq/PRONEX.
References 1. Matisz CE, McDougall JJ, Sharkey KA and McKay DM. Helminth parasites and the modulation of joint inflammation. J Parasitol Res 2011; 2011: 942616. 2. Baker DG. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin Microbiol Rev 1998; 11: 231–266. 3. Homberger FR and Thomann PE. Transmission of murine viruses and mycoplasma in laboratory mouse colonies with respect to housing conditions. Lab Anim 1994; 28: 113–120. 4. Pakes SP, Lu YS and Meunier PC. Factors that complicate animal research. In: Fox JG (ed.) Laboratory animal medicine. New York: National Academic Press, 1984, pp.649–665. 5. Pearson DJ and Taylor G. The influence of the nematode Syphacia obvelata on adjuvant arthritis in the rat. Immunology 1975; 29: 391–396. 6. Lindsey J, Boorman G, Colins M, et al. Infectious diseases of mice and rats (Committee of Infectious Diseases of Mice and Rats). Washington: National Academic Press, 1991, pp.275–276. 7. Farrar PL, Wagner JE and Kagiyama N. Syphacia spp. In: Waggie K, et al. (eds) Manual of microbiologic monitoring of laboratory animals, 2nd ed. Washington: National Academic Press, 1994, pp.219–224. 8. Potkay S. Hymenolepis spp. In: Waggie K, et al. (eds) Manual of microbiologic monitoring of laboratory animals, 2nd ed. Washington: National Academic Press, 1994, pp.191–196. 9. Baker DG. Parasites of rats and mice. In: Baker DG (ed.) Flynn’s parasites of laboratory animals, 2nd ed. Iowa: Blackwell Publishing, 2007, pp.303–397.
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
Foletto et al.
5
10. Taffs LF. Pinworms infections in laboratory rodents: a review. Lab Anim 1976; 10: 1–13. 11. Lubcke R, Hutcheson FAR and Barbezat GO. Impaired intestinal electrolyte transport in rats infested with the common parasite Syphacia muris. Dig Dis Sci 1992; 37: 60–64. 12. Mohn G and Phillip EM. Effects of Syphacia muris and the anthelmintic fenbendazole on the microsomal monooxygenase system in the mouse liver. Lab Anim 1981; 15: 89–95. 13. Wagner M. The effect of infection with the pinworm (Syphacia muris) on rat growth. Lab Anim Sci 1988; 38: 476–478. 14. Flynn BM, Brown PA and Eckstein JM. Treatment of Syphacia obvelata in mice using ivermectin. Lab Anim Sci 1989; 39: 461–463. 15. Roberts-Thompson IC, Stevens DP, Mahmoud AAF and Waren KS. Giardiasis in the mouse: an animal model. Gastroenterology 1976; 71: 57–61. 16. Jueco NL. Hymenolepis diminuta infection. In: Steele JH (ed.) CRC handbook series in zoonoses. Section C: parasitic zoonoses. Vol 1, Florida: CRC Press, 1982, pp.279–282. 17. Jueco NL. Hymenolepis diminuta infection. In: Steele JH (ed.) CRC handbook series in zoonoses. Section C: parasitic zoonoses. Vol 1, Florida: CRC Press, 1982, pp.283–287. 18. Wolstenholme AJ and Rogers AT. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics. Parasitology 2005; 131: 85–95. 19. Trailovic SM and Nedeljkovic JT. Central and peripheral neurotoxic effects of ivermectin in rats. J Vet Med Sci 2011; 73: 591–599. 20. Hoffman WA, Pons JA and Janer JL. Sedimentation concentration method in Schistosomiasis mansoni. Puerto Rico J Publ Hlth 1934; 9: 283–298. 21. Willis HH. A simple levitation method for the detection of hookworm ova. Med J Aust 1921; 2: 375–376. 22. Stern CA, Gazarini L, Takahashi RN, et al. On disruption of fear memory by reconsolidation blockade: evidence from cannabidiol treatment. Neuropsychopharmacology 2012; 37: 2132–2142. 23. Fanselow MS. Conditioned and unconditional components of post-shock freezing. Pavlov J Biol Sci 1980; 15: 177–182. 24. Beyhan YE, Gurler AT, Boluksas CS, et al. Bazi Laboratuvar Hayvanlarinda Nekropsi ve Dis¸ ki Bakisi ile Saptanan Helmintler. Tu¨rkiye Parazitoloji Dergisi 2010; 34: 98–101.
25. Hassan SI, Nessim NG, Mahmoud SS, et al. Effect of a broad spectrum antiparasitic drug ‘ivermectin’ in acute and chronic experimental giardiasis using different dose regimens. J Egypt Soc Parasitol 2001; 31: 419–428. 26. Youssef MY, Essa MM, Sadaka HA, et al. Effect of ivermectin on combined intestinal protozoal infection (giardiasis and cryptosporidiosis). J Egypt Soc Parasitol 1996; 26: 543–553. 27. Heukelbacha J, Wilcke T, Winter B, et al. Efficacy of ivermectin in a patient population concomitantly infected with intestinal helminths and ectoparasites. Arzneimittelforschung 2004; 54: 416–421. 28. Ostlind DA, Mickle WG, Smith SK, et al. The Hymenolepis diminuta–golden hamster (Mesocricetus auratus) model for the evaluation of gastrointestinal anticestode activity. J Parasitol 2004; 90: 898–899. 29. Reagan-Shaw S, Nihal M and Ahmad N. Dose translation from animal to human studies revisited. Faseb J 2008; 22: 659–661. 30. Huerkamp MJ. Ivermectin eradication of pinworms from rats kept in ventilated cages. Lab Anim Sci 1993; 43: 86–90. 31. Klement P, Augustine JM, Delaney KH, et al. An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice. Lab Anim Sci 1996; 46: 286–290. 32. Habermann RT and Williams FP. The identification and control of helminths in laboratory animals. J Natl Cancer Inst 1958; 20: 979–1009. 33. Zenner L. Effective eradication of pinworms (Syphacia muris, Syphacia obvelata and Aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy. Lab Anim 1998; 32: 337–342. 34. Car H, Kuziemka-Leska M and Wis´ niewski K. The NMDA and GABA-A receptors in behavioral activity of rats. Acta Physiol Hung 1996; 84: 269–270. 35. Katz RJ and Liebler L. GABA involvement in memory consolidation: evidence from posttrial amino-oxyacetic acid. Psychopharmacology 1978; 56: 191–193. 36. Izquierdo I, Da Cunha C, Huang CH, et al. Post-training down-regulation of memory consolidation by a GABA-A mechanism in the amygdala modulated by endogenous benzodiazepines. Behav Neural Biol 1990; 54: 105–109. 37. Izquierdo I and Medina JH. GABAa receptor modulation of memory: the role of endogenous benzodiazepines. Trends Pharmacol Sci 1991; 12: 260–265. 38. Bustos SG, Maldonado H and Molina VA. Midazolam disrupts fear memory reconsolidation. Neuroscience 2006; 139: 831–842.
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
(LAN)
[1–5] [PREPRINTER stage]
Lab Anim OnlineFirst, published on December 5, 2014 as doi:10.1177/0023677214562850
l
ab orator y
Original Article
a
n imals
l i m i t e d
Efficacy and security of ivermectin given orally to rats naturally infected with Syphacia spp., Giardia spp. and Hymenolepis nana
Laboratory Animals 0(0) 1–5 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677214562850 la.sagepub.com
VRS Foletto, F Vanz, L Gazarini, CAJ Stern and CR Tonussi
Abstract The results of this study show that the oral administration of ivermectin (48 mg/L) repeatedly for 72 h used in accordance with the present protocol is a safe and highly effective treatment for Giardia spp. and Hymenolepis nana in laboratory rat colonies. The drug can be easily and safely administered using drinking water. This simple regimen should control pinworm infection (Syphacia muris), a problem that can be endemic in laboratory colonies. Experiments using healthy animals are likely to generate more consistent results, thereby requiring a reduced number of animals per group.
Keywords ivermectin, Giardia, Syphacia, Hymenolepis, laboratory animals, faecal examination
Laboratory animals must be kept in appropriate health conditions to ensure the higher efficiency of experimental studies.1–5 Parasitized animals are not suitable for experimental research since they may present nutritional, immunological and haematological differences compared with normal values.6–9 At least three parasites are common threats to rodent colonies: Syphacia spp., Giardia spp. and Hymenolepis nana. A common infection found in rat colonies is due to Syphacia spp., which has also been reported to infect humans.6 In the host, these worms develop into adults from eggs in 4–7 days. New eggs are deposited in the host’s perianal region, which are easily dispersed in the environment, contaminating other animals.10 Such infections are usually asymptomatic, but may be associated with rectal prolapse, intussusception, low weight gain, hypertension, irritability and immunological alteration.11–14 Giardia spp. is another common parasite genus found in rat colonies. The infecting cysts are maximally present in faeces 5–14 days after ingestion by the host.15 Indeed such infections are asymptomatic, but unspecific clinical signals may be present such as weight loss, lower growing rate, abdominal distension and diarrhoea.2 H. nana is a threat to both animals and
humans. It has a direct cycle of between 14 and 16 days duration. Severe infection with H. nana leads to dehydration, abdominal pain, malnutrition, diarrhoea and weight loss. This is the only Taenia that infects humans without needing an intermediate host.9,16,17 Ivermectin is a macrocyclic lactone widely used as an antiparasitic agent either in veterinary or in human medicine. It targets a glutamate-gated chloride channel in some invertebrates as well as the chloride channel of gamma-aminobutyric acid type A (GABAA) receptors, promoting cellular hyperpolarization and worm paralysis.18 Although generally considered secure and welltolerated, ivermectin can cause toxic effects in the host depending on its concentration in the central nervous system (CNS), since it is freely permeable through the blood–brain barrier.19 The aim of the present study was to investigate the occurrence of endoparasites in Department of Pharmacology, Federal University of Santa Catarina, Floriano ´polis, Santa Catarina, Brazil Corresponding author: Vanessa RS Foletto, R Sı´lvio Lopes de Arau ´ jo, 645 ap 205, Floriano ´polis, 88048391, Brazil. Email: [email protected]
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
2
Laboratory Animals 0(0)
the intestinal tract of a conventional Wistar rat colony, and to evaluate the efficacy and security of ivermectin for the control of these natural infections.
Animals Forty male and 50 female Wistar rats (Rattus norvegicus) from a conventional biotery (Floriano´polis, UFSC, Brazil) weighing 200–250 and 150–200 g, respectively, were housed under standard conditions for laboratory animals in groups of five with a controlled 12 h light/ dark cycle, temperature 20 2 C and ad libitum access to standard diet and water. All experimental procedures were conducted according to the European Convention for the Protection of Vertebrate Animals and were approved by the local ethical committee for animal research (CEUA–UFSC).
Materials and methods Sample collections and laboratory techniques Faecal sampling was obtained on three consecutive days before the treatment, and on another three consecutive days a week after the treatment. Faecal material was collected directly from the anus and stored in a thimerosal–iodine–formaldehyde solution at room temperature in a proportion of 1:10. Samples from 10 animals were pooled and submitted to the Hoffman–Pons– Janer (HPJ) technique, Willis technique, and also direct visualization using an optical microscope. HPJ can detect the presence of helminth eggs and larvae, and protozoan cysts. This technique utilized spontaneous faecal sedimentation.20 Briefly, faecal samples were diluted in water and filtered through a gauze strip into a conical sedimentation glass and the sediment was evaluated. The Willis technique is based on the floating effect of helminth eggs in a saturated sodium chloride solution (1.2 g/mL).21 Using this technique, the saturated saline solution with the emulsified faeces was deposited in a round-bottomed flask and a meniscus was formed on the surface. Next, the flask was covered with a glass slide. After several minutes, the slide was removed and examined under a microscope. Finally, a direct examination was performed using an optical microscope and Lugol staining. The results were expressed as number of eggs in the samples.
Drug and method of administration Ivermectin (Hebei Veyong Animal Pharmaceutical Co Ltd, Shijiazhuang, China) was dissolved in water (48 mg/L) and was offered alone ad libitum during a 72 h period. Control groups received tap water only.
Investigation of ivermectin’s depressive effect on the rats’ CNS To study the potential depressive effect of ivermectin on the CNS, which could cause lasting changes and interfere with behaviour and the dynamics of memory formation, a contextual fear-conditioning protocol22 was performed seven days after ivermectin treatment. Behavioural testing was always carried out under low intensity illumination (70 lux) during the diurnal phase (from 13:00 to 17:00 h). Fear conditioning was performed in a rectangular chamber (35 20 30 cm), with aluminium sidewalls and a front wall and ceiling made of Plexiglas. Its grid floor, made of stainless steel bars (3 mm diameter, spaced 9 mm apart centre-tocentre), was connected to a circuit board and a shock generator (Insight, Ribeira˜o Preto, SP, Brazil) to enable the delivery of controlled electrical foot shocks. In all the experiments, each animal was placed in this environment (Context A), and allowed to explore it freely for 3 min, as an initial familiarization session, and was then returned to its home cage. On the next day, the animal was again placed there for the training session, during which it received, after an initial 30 s delay (preshock period), the unconditioned stimulus (three electrical foot shocks of 0.7 mA, 60 Hz, for 3 s, with a 30 s inter-shock interval). The animal remained in this chamber for an additional 30 s (post-shock period) before returning to its home cage. In the test, the animal was re-exposed to this paired context for 3 min in the absence of unconditioned stimulus presentation. All sessions were spaced 24 h apart. After each behavioural session, both chambers were cleaned with paper tissue soaked with a 10% ethanol–water solution. The experimenter was unaware of the treatment condition in all the studies. Freezing behaviour, a commonly used index of fear in rats,23 and defined as a total absence of body and head movements except those associated with breathing, was continuously recorded during the experimental sessions by a video camera. The freezing time in each period was quantified (in seconds) by a trained observer (inter- and intra-rate reliabilities 90%) blind to the experimental groups, using a stopwatch, and expressed as the percentage of total session time.
Statistical analysis Statistical analysis was carried out using GraphPad Prism 5.0Õ software (Graphpad Software Inc, La Jolla, CA, USA). Egg occurrence was presented as mean number of eggs/mm3 SEM. Comparisons were made using a paired Student’s t-test for simple means. For contextual fear-conditioning protocol the results were expressed as mean SEM. After ensuring the
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
Foletto et al.
3
assumptions of normality and homogeneity of variance, the percentage of freezing time recorded during the test was submitted to an unpaired Student’s t-test. The statistical significance level was set at P < 0.05.
Results In male rats, Syphacia eggs were found in 100% of the samples at 8.7 1.2 eggs/mm3, Giardia eggs in 50% of samples at 2.5 1.4 eggs/mm3 and Hymenolepis in 25% of samples at 1.2 1.2 eggs/mm3. In female rats, Syphacia eggs were found in 50% of samples at 3.7 2.4 eggs/mm3 and Giardia eggs in 25% of samples at 2.5 1.4 eggs/mm3. Hymenolepis was not detected in female rats. Ivermectin treatment eliminated the occurrence of Giardia eggs in males and females, and Hymenolepis in males. The drug reduced the incidence of infection from 100% to 50% in males, which caused a 94% decrease in the number of Syphacia eggs (0.5 0.3 eggs). Similar rates were found in females, where the incidence of infection was reduced from 50% to 25%, with a mean number of eggs of 0.2 0.2 eggs (Figure 1). Ten male rats were randomly allocated to two independent groups (n ¼ 5/group) based on the previous treatment, which consisted of vehicle or ivermectin (48 mg/L in drinking water for 72 h), given a week before fear conditioning. As can be observed in Figure 2, there was no difference between treatment condition during re-exposure to the paired context (t8 ¼ 0.004; P ¼ 0.98), evidenced by the lack of residual or long-lasting effects of the drug on memory processing (Figure 2).
Male
Discussion The endoparasites encountered in our animals were the same as those normally described in laboratory animals.2,24 There are several treatments and protocols adopted for the control and eradication of these infecting parasites, and the efficacy of ivermectin has been demonstrated in several species. In hamsters, a dose of 300 mg/kg subcutaneously produces an eradication rate
Figure 2. The lack of ivermectin-induced impairments in memory encoding or expression in rats. A week after ivermectin treatment (4.8 mg/L in drinking water for 72 h), animals underwent a familiarization session and then entered Context A paired with three foot shocks (US). When re-exposed to the paired context a day later (Test A), groups spent the same amount of time freezing. Bars represent the percentage of total time spent freezing. Values are expressed as mean SEM.
Female
Syphacia ssp
Eggs/mm3
Giardia ssp Hymenolepis nana
10
10
5
5
**
***
***
0
*
*** ***
0
Pre-Ivermectin
Post- Ivermectin
Pre-Ivermectin
Post- Ivermectin
Figure 1. Ivermectin is effective in combating Giardia spp. and Hymenolepis nana. Also, it reduces the prevalence of Syphacia spp. A week before and after ivermectin treatment (4.8 mg/L in drinking water for 72 h), parasitological analysis was performed. Values are expressed as mean SEM of eggs/mm3. Asterisks indicate a significant difference (P < 0.05) from pre-ivermectin treatment (paired Student’s t-test).
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
4
Laboratory Animals 0(0)
of 99.1% and 99.5% in the experimental acute and chronic Giardia infections, respectively.25 However, a dose of 200 mg/kg intravenously eliminates a natural Giardia infection.26 Ivermectin doses necessary to control Giardia infection in rats have not yet been described, thus the present study has provided an important reference for a practical treatment in experimental colonies. In humans, ivermectin (0.2 mg/kg) eliminates 50% of the hymenolepiasis.27 On the other hand, in hamsters, this drug is ineffective at an oral dose of 0.5 mg/kg.28 However, an allometric extrapolation29 suggests that the dose used in hamsters should be threefold lower than the effective one used in humans. Ivermectin is effective in eliminating Hymenolepis eggs in rats. Allometric extrapolation indicates that the above human dose would be equivalent to 1.2 mg/kg in rats. Taking into account the daily water ingestion by rats (100 mL/kg), it is conceivable that the total ivermectin dose was about 5 mg/kg, which may explain the effectiveness against hymenolepiasis. The efficacy of ivermectin against pinworm (Syphacia muris) in several studies has been variable, probably due to differences in dose regimen, time of treatment and method of administration. One common factor in such studies that were successful in the elimination of the parasite was the repetition of the treatment at four- to nine-day intervals.30,31 The control of S. muris is difficult, since cross infections occur easily after egg depositions. Although ivermectin is effective against adult and larval forms of Syphacia, it has no effect on eggs. Since eggs are light and resistant, they spread out in the environment, being found intact even in ventilation ducts.10,32 For these reasons, disinfection protocols must be applied concomitantly to pharmacological treatment, until the complete elimination of the eggs is ensured.33 Since ivermectin acts by enhancing GABAAmediated transmission, the main inhibitory system in the nervous system, high doses of this drug can be associated with general neuronal depression.19,34 Despite the high dose of ivermectin administered here (5 mg/kg) and the length of treatment (three days), the rats did not show altered performance in the fearconditioning test. This is in line with a previous report in which it was observed that doses of up to 7.5 mg/kg did not produce visible CNS depression in rats.19 Memory encoding, a process dependent on brain circuitry integrity, is highly modulated by the GABAergic system and, therefore, highly sensitive to interventions in this inhibitory system.35–38 However, as was observed in the present study, the oral treatment with ivermectin did not impair the encoding of this aversive memory, as no differences in freezing behaviour could be observed between groups.
Overall, the present antiparasitic protocol may be considered to be effective against threatening worms in rat colonies, and also secures the subsequent use of such animals for at least some studies in neuroscience. The collective treatment regimen in drinking water is thought to ease its implementation, reducing the sources of animal distress such as physical restraints and invasive administration. Furthermore, we can assume that this prophylactic measure can even target other parasites, beyond those focused on in the present study, since ivermectin is also effective in ectoparasites, thereby providing better sanitary conditions and homeostasis. Such physiological balance is also critical in reducing individual variability in experimental studies and, consequently, in reducing the number of animals required for generating statistically relevant data. Declaration of conflicting interests None declared.
Funding This research was funded by Brazilian government funding agencies CAPES and FAPESC-CNPq/PRONEX.
References 1. Matisz CE, McDougall JJ, Sharkey KA and McKay DM. Helminth parasites and the modulation of joint inflammation. J Parasitol Res 2011; 2011: 942616. 2. Baker DG. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin Microbiol Rev 1998; 11: 231–266. 3. Homberger FR and Thomann PE. Transmission of murine viruses and mycoplasma in laboratory mouse colonies with respect to housing conditions. Lab Anim 1994; 28: 113–120. 4. Pakes SP, Lu YS and Meunier PC. Factors that complicate animal research. In: Fox JG (ed.) Laboratory animal medicine. New York: National Academic Press, 1984, pp.649–665. 5. Pearson DJ and Taylor G. The influence of the nematode Syphacia obvelata on adjuvant arthritis in the rat. Immunology 1975; 29: 391–396. 6. Lindsey J, Boorman G, Colins M, et al. Infectious diseases of mice and rats (Committee of Infectious Diseases of Mice and Rats). Washington: National Academic Press, 1991, pp.275–276. 7. Farrar PL, Wagner JE and Kagiyama N. Syphacia spp. In: Waggie K, et al. (eds) Manual of microbiologic monitoring of laboratory animals, 2nd ed. Washington: National Academic Press, 1994, pp.219–224. 8. Potkay S. Hymenolepis spp. In: Waggie K, et al. (eds) Manual of microbiologic monitoring of laboratory animals, 2nd ed. Washington: National Academic Press, 1994, pp.191–196. 9. Baker DG. Parasites of rats and mice. In: Baker DG (ed.) Flynn’s parasites of laboratory animals, 2nd ed. Iowa: Blackwell Publishing, 2007, pp.303–397.
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
XML Template (2014) [4.12.2014–12:08pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/LANJ/Vol00000/140046/APPFile/SG-LANJ140046.3d
(LAN)
[1–5] [PREPRINTER stage]
Foletto et al.
5
10. Taffs LF. Pinworms infections in laboratory rodents: a review. Lab Anim 1976; 10: 1–13. 11. Lubcke R, Hutcheson FAR and Barbezat GO. Impaired intestinal electrolyte transport in rats infested with the common parasite Syphacia muris. Dig Dis Sci 1992; 37: 60–64. 12. Mohn G and Phillip EM. Effects of Syphacia muris and the anthelmintic fenbendazole on the microsomal monooxygenase system in the mouse liver. Lab Anim 1981; 15: 89–95. 13. Wagner M. The effect of infection with the pinworm (Syphacia muris) on rat growth. Lab Anim Sci 1988; 38: 476–478. 14. Flynn BM, Brown PA and Eckstein JM. Treatment of Syphacia obvelata in mice using ivermectin. Lab Anim Sci 1989; 39: 461–463. 15. Roberts-Thompson IC, Stevens DP, Mahmoud AAF and Waren KS. Giardiasis in the mouse: an animal model. Gastroenterology 1976; 71: 57–61. 16. Jueco NL. Hymenolepis diminuta infection. In: Steele JH (ed.) CRC handbook series in zoonoses. Section C: parasitic zoonoses. Vol 1, Florida: CRC Press, 1982, pp.279–282. 17. Jueco NL. Hymenolepis diminuta infection. In: Steele JH (ed.) CRC handbook series in zoonoses. Section C: parasitic zoonoses. Vol 1, Florida: CRC Press, 1982, pp.283–287. 18. Wolstenholme AJ and Rogers AT. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics. Parasitology 2005; 131: 85–95. 19. Trailovic SM and Nedeljkovic JT. Central and peripheral neurotoxic effects of ivermectin in rats. J Vet Med Sci 2011; 73: 591–599. 20. Hoffman WA, Pons JA and Janer JL. Sedimentation concentration method in Schistosomiasis mansoni. Puerto Rico J Publ Hlth 1934; 9: 283–298. 21. Willis HH. A simple levitation method for the detection of hookworm ova. Med J Aust 1921; 2: 375–376. 22. Stern CA, Gazarini L, Takahashi RN, et al. On disruption of fear memory by reconsolidation blockade: evidence from cannabidiol treatment. Neuropsychopharmacology 2012; 37: 2132–2142. 23. Fanselow MS. Conditioned and unconditional components of post-shock freezing. Pavlov J Biol Sci 1980; 15: 177–182. 24. Beyhan YE, Gurler AT, Boluksas CS, et al. Bazi Laboratuvar Hayvanlarinda Nekropsi ve Dis¸ ki Bakisi ile Saptanan Helmintler. Tu¨rkiye Parazitoloji Dergisi 2010; 34: 98–101.
25. Hassan SI, Nessim NG, Mahmoud SS, et al. Effect of a broad spectrum antiparasitic drug ‘ivermectin’ in acute and chronic experimental giardiasis using different dose regimens. J Egypt Soc Parasitol 2001; 31: 419–428. 26. Youssef MY, Essa MM, Sadaka HA, et al. Effect of ivermectin on combined intestinal protozoal infection (giardiasis and cryptosporidiosis). J Egypt Soc Parasitol 1996; 26: 543–553. 27. Heukelbacha J, Wilcke T, Winter B, et al. Efficacy of ivermectin in a patient population concomitantly infected with intestinal helminths and ectoparasites. Arzneimittelforschung 2004; 54: 416–421. 28. Ostlind DA, Mickle WG, Smith SK, et al. The Hymenolepis diminuta–golden hamster (Mesocricetus auratus) model for the evaluation of gastrointestinal anticestode activity. J Parasitol 2004; 90: 898–899. 29. Reagan-Shaw S, Nihal M and Ahmad N. Dose translation from animal to human studies revisited. Faseb J 2008; 22: 659–661. 30. Huerkamp MJ. Ivermectin eradication of pinworms from rats kept in ventilated cages. Lab Anim Sci 1993; 43: 86–90. 31. Klement P, Augustine JM, Delaney KH, et al. An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice. Lab Anim Sci 1996; 46: 286–290. 32. Habermann RT and Williams FP. The identification and control of helminths in laboratory animals. J Natl Cancer Inst 1958; 20: 979–1009. 33. Zenner L. Effective eradication of pinworms (Syphacia muris, Syphacia obvelata and Aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy. Lab Anim 1998; 32: 337–342. 34. Car H, Kuziemka-Leska M and Wis´ niewski K. The NMDA and GABA-A receptors in behavioral activity of rats. Acta Physiol Hung 1996; 84: 269–270. 35. Katz RJ and Liebler L. GABA involvement in memory consolidation: evidence from posttrial amino-oxyacetic acid. Psychopharmacology 1978; 56: 191–193. 36. Izquierdo I, Da Cunha C, Huang CH, et al. Post-training down-regulation of memory consolidation by a GABA-A mechanism in the amygdala modulated by endogenous benzodiazepines. Behav Neural Biol 1990; 54: 105–109. 37. Izquierdo I and Medina JH. GABAa receptor modulation of memory: the role of endogenous benzodiazepines. Trends Pharmacol Sci 1991; 12: 260–265. 38. Bustos SG, Maldonado H and Molina VA. Midazolam disrupts fear memory reconsolidation. Neuroscience 2006; 139: 831–842.
Downloaded from lan.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on January 23, 2015
