Maung Maung Mya1*, Than Myat Htay2, Aye Win Oo2
1Medical Entomology Research Division, Department of Medical Research, Ministry of Health, Union of Myanmar 2Laboratory Animal Services, Department of Medical Research, Ministry of Health, Union of Myanmar
| ARTICLE INFORMATION | ABSTRACT |
| *Corresponding author: Dr. Maung Maung Mya E-mail: dr.mgmgmya@gmail.com Keywords: Ascariasis Albendazole Deworming drug Fenbendazole Parazivet Pinworm | Pinworms remain most prevalent in laboratory mice and rats. Rats are usually infected with Syphacia muris and mice with Syphacia obvelata and Aspiculuris tetraptera. Therefore, the study was conducted to eliminate worm infection using deworming drugs on laboratory rats in DMR during the study period from March 2021 to February 2022. In of rats the present study, thirty males and thirty females of Wistar rats (Rattus norvegicus) from DMR were randomly selected and 10 each were housed in cages separately according to the treatment of three deworming drugs as fenbendazole, albendazole and parazivet groups of rats. Before the treatment of deworming drugs, all groups of rats were detected worm parasite’s eggs by the method of taping (Graham). Out of 60 rats, 55(91.67%) rats were positive for both Pinworm (Syphacia muris) and Ascariasis (Ascaris suum) eggs. Of these, the highest density of mixed infection was found 40 (72.73%) (Pinworm + Ascariasis) followed by Pinworms only 8(14.55%) of rats and lowest positivity was observed Ascaris suum eggs only 7(12.73%) rats respectively. Among them 28 (93.33%) samples of males and 27 (90%) samples of females were found parasite positive. After treatment of deworming drugs, 100% reduction was observed in all groups of rats. All the deworming drugs were found very effective to control the Pin worm and Ascariasis infection. To reduce reinfection, monthly treatment is needed to control or eliminate the worm infection in laboratory animals. |
INTRODUCTION
Pinworms are most prevalent in laboratory mice and rats (Clifford and Cosentino 006a,b; Livingston and Riley 2003). Rats are usually infected with Syphacia muris and mice are infected with Syphacia obvelata and Aspiculuris tetraptera (Baker, 2007). Rats can be incidental hosts of Syphacia obvelata and mice can be incidental hosts of Syphacia muris (Baker, 2007, Phillipson, 1974). And also rats are infected with Aspiculuris tetraptera (Mathies, 1959).
In the late 1980s and early 1990s, some viruses prevalent in laboratory mice such as Sendai virus, mouse hepatitis virus (MHV), epizootic diarrhea of infant mice (EDIM), and the only parvovirus and helminths are prevalent in laboratory mice and rats. In rats, the picture was similar, with prevalent of viruses including the Sendai virus, coronavirus (also called sialodacryoadenitis virus, SDAV), and the rat parvoviruses. In addition to the viruses, pinworms were prevalent in both mice and rats (Casebolt et al. 1988; Jacoby and Lindsey, 1998; Lussier and Descoteaux, 1986). Pinworms are nematode parasites (Family Oxyuridae) that have simple, direct life cycles and are frequent contaminants of both specific pathogen free (SPF) and conventional colonies of laboratory rats and mice.
Pinworms are transmitted through the ingestion of embryonated eggs. Two species of pinworms Syphacia obvelata and Aspiculuris tetraptera commonly infect laboratory rats and mice. The prevalence of pinworms in an infected rodent population depends on many factors, including parasite environmental load, gender, age, strain, and immune status. Males tend to have higher burdens than females, while young animals tend to have higher worm burdens than older animals.
Mice and rats are the most common laboratory animals used in research and testing of many kinds of traditional and pharmaceutical research. The parasite infections can affect investigations by inducing physiological and immunological alterations in the hosts, increasing or diminishing host susceptibility to experimental stress, inducing tissue damage, stimulating abnormal tissue growth, competing with the host for nutrients, decreasing the volume of the host’s blood and body fluids and by mechanical interference (Baker, 2007). Still little is known about the effects of environmental changes on the biological variation in experimental results (Stahl 1963). In the mouse and rat caecum and colon, Qspicularis tetraptera may be found together with Syphacia muris or Syphacia abvelata (Taffs, 1979).
Syphacia muris is the most prevalent pinworm of rats. The life cycle of Syphacia muris is direct and completed in 7 to 8 days, (Lewis and Silvq, 1986) making this particular pinworm ideal for epidemiologic study. Adult worms of Syphacia muris inhabit the cecum and colon, and female worms migrate to the anus and deposit all their eggs on the perianal region of the host before dying.
Within a few hours, the eggs developed into embryonic and they are considered infective (Stahl, 1963). Infection is believed to occur via 3 modes: (1) direct ingestion of the eggs; (2) ingestion of food or water contaminated with the eggs; and (3) retro infection (Chan 1952). Ingestion of eggs is considered to be the primary mode of infection, and the eggs are reported to remain viable within the environment for as long as 4 weeks (Cliford and Watson, 2004, Dix et al. 2004).
Antemortem diagnosis traditionally is made by identification of these eggs on a perianal cellophane tape, given the ease of collection and interpretation, although direct examination of the cecum postmortem is considered the most dependable method for Syphacia muris diagnosis (Anya, 1966).
Ascariasis prevalence was found in humans and animals Kindem. Ascaris suum is mostly infected to rats, mice, swine and pigs. Ascaris suum infection is established orally by third stage larvae after their development from embryonated eggs. The third stage larvae invade the small intestine of host migrate into the lever and lung, and finally reach to the cecum and/or proximal colon, where they develop into adult worms (Tsuji et al. 2003).
Ethiopian Study revealed that the prevalence of helminthiasis was higher in mice (28.57% than in rats (7.41%) (Derothe et al. 1997). Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals (Institute for Laboratory Animal Research, 2011). This study reported that the data on the presence of helminth parasites, mainly with regard to pinworm species in laboratory rat colonies.
At the same time, to fine out the effectiveness of deworming drugs on helminth parasites in laboratory rats colonies of DMR. Therefore, the objective of the study was to fine out the effectiveness of deworming drugs for intestinal parasites control on laboratory rats (Rattus norvegicus) breeding in DMR.
MATERIALS AND METHODS
Study Area
The study was conducted on laboratory rats in the Laboratory Animal Service, Department of Medical Research, Ministry of Health, Yangon, Myanmar.
Study period
The study period was one year, from April 2021 to March 2022.
Study Design
Laboratory based descriptive study design was used.
METHODOLOGY
Thirty males and thirty females of Wistar rats (Rattus norvegicus) from DMR were randomly selected. Laboratory rats were housed in cages (250 mm x 170 mm x 100 mm) of their breeding rooms separately. The temperature and humidity of the experimental room was maintained at 22±2°C and humidity 80-90%. An exhaust fan and air-conditioner were provided for good air ventilation in room.
Selected rats were given to free access the diet and tap–water. Before the experiment, 10 Male and 10 female each of rats were separated into 6 different cages (3cages for male and 3 cages for female). Cages were labeled according to three deworming drugs(fenbendazole, albendazole and parazivet) respectively and then all the rats were detected pin worms eggs using taping method (Graham, 1941), after that each group of rats was treated with deworming drugs according to treatment regimens for rats and mice were described in the reference literature. For rats, treatment generally involves a week-off feeding containing fenbendazole.
Pinworm eggs are resistant to desiccation and many common disinfectants, but are susceptible to high temperatures. In this experimental study, different deworming drugs (fenbendazole, albendazole and parazivet) were applied to selected groups of rats. According to the instruction of therapy, it was done by oral administration of 10mg/kg/day for 10 consecutive days. After 7 days of treatment, all groups of rats are checked, and detected the Pinworm and Ascariasis parasite’s eggs again by the method of taping (Graham, 1941).
Analysis of Data
The mean and standard deviation of each parameter were calculated by standard statistical methods. The positive rate was calculated in percent.
RESULTS
Table 1. Shows that before the treatment of deworming drug in the rat population, the positivity rate of eggs was found, in 55(91.67%) out of 60 rats for both Pinworm (Syphacia muris) and Ascaris suum eggs. Of these, the highest density of mixed infection (Pinworm + Ascariasis) was found in 40 rats = (21male +19 females) (72.73 %) in the rat’s population, followed by (14.55 %) of only pinworms (Syphacia muris) positivity 8 (3 males + 5 females), and lowest positivity was observed 7 (4 male + 3 Female) (12.73 %) of only Ascaris suum eggs.
Table 1. Positivity rate of Pinworm and Ascariasis eggs, before and after treatment of deworming drugs in laboratory-reared Wistar rats (Rattus norvegicus) from DMR
| Rattus norvegicus | No. of Sample | Treat ment of drugs | Before treatment | Total positi ve | After treatment | |||||
| Pw eggs +ve (Syphacia muris) | Ascaris suums eggs +ve (A. ) | Mixed (Pw+Asca riasis) | Pw Eggs +ve | Ascar eggs +ve | Mixed | % Reducti on | ||||
| Rats (Males) | 10 | FBZ | 2 | 1 | 7 | 10 | 0 | 0 | 0 | 100 |
| 10 | ABZ | 0 | 2 | 6 | 8 | 0 | 0 | 0 | 100 | |
| 10 | PZV | 1 | 1 | 8 | 10 | 0 | 0 | 0 | 100 | |
| Rats (Female) | 10 | FBZ | 2 | 2 | 5 | 9 | 0 | 0 | 0 | 100 |
| 10 | ABZ | 2 | 0 | 8 | 10 | 0 | 0 | 0 | 100 | |
| 10 | PZV | 1 | 1 | 6 | 8 | 0 | 0 | 0 | 100 | |
| Total | 60 | 8 (14.55%) | 7 (12.73%) | 40 (72.73%) | 55 | 0 | 0 | 0 | 100 | |
| Total + ve | 60 | 55 (91.67%) | ||||||||
FBZ= Fenbendazole, ABZ=Albendazole, PZV = Parazivet, Pw=Pin worm, Ascar=Ascariasis, +ve=positivity A total of 30 males and 30 females were tested for intestinal worms parasites before deworming drugs administration, 28 (93.33%) samples of males and 27 (90%) samples of females were found worm parasite positive. Of these 10 each of the rats was positive for pinworm and ascariasis eggs of PZV Parazivet and FBZ Fenbendazole group.
And 8 rats were found positive for both parasite aggs in ABZ Albendazole group. In female groups 10 rats were positive in ABZ Albendazole and 9 and 8 female rats were positive for pinworm and ascariasis in FBS and PZV groups. After treatment of different deworming drugs as Fenbendazole, Albendazole, Parazivet, on rats, all the rats were found parasite eggs negative and a 100% reduction was observed in all groups of rats.
DISCUSSION
Mice and rats are very useful animals in different kinds of research in the laboratory. Some are useful for cancer research, some are useful for snake bite research, some are useful for bone healing research and some are useful for toxicity research. Therefore, healthy laboratory animals are needed to access accurate and good results. In the present study before the treatment deworming drug laboratory rats were found pinworm (Syphacia muris) and Ascaris suum eggs positive by the examination of the tapping method under the compound microscope with 40X lance.
A total of 60 laboratory rats (30 male + 30 Female) were examined 28 male and 27 female rats were positive for pinworm and ascariasis eggs. Of these pinworm and ascariasis mixed positivity was found higher than individual parasite eggs positivity in both male and female rats. In the male group 21 rats were infested with mixed (pin worm + Ascariasis) infection, only 3 were pinworm eggs and 4 were Ascariasis eggs positive. Similar trim of infection results as male has been found in female groups.
In female groups 19 mixed positivity of pinworm and ascariasis eggs and 5 was pinworm and 3 were ascariasis eggs positive individually and finding observed that male rats have a higher burden of infection than female rats. It may be due to pinworm infestations continuing because of prolonged infections, inefficient diagnosis, and the survivability of eggs of some species in the environment.
Other researchers revealed that the prevalence of pinworms in an infected rodent population depends on many factors, including environmental load, gender, age, strain, and immune status. Males tend to have higher parasite burdens than females, while young animals tend to have higher worm burdens than older animals. Laboratory mice tend to be more resistant to experimentally induced infection than wild mice. Athymic mice, as might be expected, have an increased susceptibility to infection (Meade. and Watson, 2014).
In the present study, rats were infected with Ascaris suum in high density, which is morphologically different from Ascaris lumbricoides (Maung, 1973). Recent studies have revealed that Ascaris suum of swine origin can develop in humans, indicating its zoonotic importance (Anderson et al. 1993; Peng et al. 1988). Although numerous studies have been carried out thus far to characterize the two species of parasites on a morphological basis, species discrimination between Ascaris lumbricoides and Ascaris suum has been controversial (38 Abebe et al. 2002; Kurimoto, 1974; Maung, 1973; Nielsen et al. 1997).
After being treated with different deworming drugs (fenbendazole, albendazole and parazivet) in rat groups, all the parasite eggs were disappeared or negative in three tested groups of rats by the diagnosis of the taping method. All the rats were free from pinworm and Ascariasis eggs after treatment of 7 days. Meade and Watson (2014) revealed that egg hatching after treatment with chlorine dioxide was significantly reduced as compared with that of unexposed control eggs (P < 0.01). Eggs exposed to 400 mg/L chlorine dioxide gas hatched at a rate of 0.3%. Biologic indicators supported efficacy of the gaseous treatment.
Furthermore, these eggs showed morphologic differences in the appearance of the capsule, as compared with control eggs. Liquid forms were significantly (P < 0.01) less effective at preventing hatching than the gaseous form of chlorine dioxide. On the basis of his data, he recommended that perianal tape testing should occur as close as possible to the peak egg-shedding time of 1400, to maximize the sensitivity of this particular diagnostic test (Meade and Warson, 2014).
In the present study, all the infected rats were found free from pinworm and ascariasis eggs after treatment with deworming drugs, although other researchers informed that Eggs may contaminate ventilation ducts (Hoag, 1961) or shared equipment or procedure areas (Huerkamp, 1993) and can recontaminate a colony after the completion of treatment. Knowledge of egg longevity in the environment is important to determine the need for environmental decontamination, but specific data are unavailable.
Aspiculuris tetraptera eggs are thought to be long-lived in the environment, remaining dormant for several months at 4°C Stahl 1966). Anya (1966a) reported, however, that culturing newly shed eggs at 37°C accelerated embryonation, decreased the number of viable eggs, and reduced their longevity. In a study to determine methods to inactivate viable Syphacia muris eggs, 100% inactivation occurred only with temperatures of 100°C for 30 minutes and ethylene oxide, although high killing rates with formaldehyde and chlorine dioxide suggested that these chemicals could be successful with adjustments to the protocol (Dix et al. 2004). Huerkamp and colleagues (2000) reported the eradication of S.
muris without environmental decontamination, suggesting that the eggs in the environment may not have outlived the treatment period (fenbendazole in feed every other week for five treatments). S. obvelata eggs appear to be unstable, they are reported to survive only 42 hours under ideal conditions, and may be inactivated by drying or immersion in liquids (Chan 1952; Grice and Prociv, 1993). As noted above, Syphacia muris eggs are resistant to the most common disinfectants (Dix et al. 2004), and it is assumed that Aspiculuris tetraptera eggs have similar properties.
Physical methods (e.g., scrubbing with detergent, steam cleaning, or painting) are thus most likely to be effective for environmental decontamination. Biosafety cabinets used to protect mice from aerosolized pathogens may actually be a route to widespread egg dissemination given that eggs shed in the cabinet are resistant to the routine disinfectants used to prevent transmission of other pathogens between cages. Tsuji and associates suggest the possibility of developing a mucosal vaccine for human and pig Ascariasis prevention. One of the current golds in the field of human and veterinary vaccines is the development of a noninvasive and practical route for administration via the mucosal surfaces (Tsuji et al. 2003).
The same author previously developed a nasal immunization technique with rAs14 that involvers protective immune responses against Ascaris suum infection (Tsiji et al. 2001).
Clifford and Watson (2008) revealed that, Long recognized agents that remain in research facilities in the 21st century include parvoviruses of rats and mice, mouse rotavirus, Theiler’s murine encephalomyelitis virus (TMEV), mouse hepatitis virus (MHV), and pinworms.
The reasons for their persistence vary with the agent. The resilience of parvoviruses, for example, is due to their resistance to inactivation, their prolonged shedding, and difficulties with detection, especially in C57BL/6 mice. Rotavirus also has marked environmental resistance, but periodic reintroduction into facilities, possibly on bags of feed, bedding, or other supplies or equipment, also seems likely. TMEV is characterized by resistance to inactivation, periodic reintroduction, and relatively long shedding periods.
Although MHV remains active in the environment for at most a few days, currently prevalent strains are shed in massive quantities and likely transmitted by fomites. (Clifford and Watson 2008). In the present study after treatment with Fenbendazole, Albendazole, Parazivet, and deworming drugs, all the rats were found parasite eggs negative, and a 100 % reduction was observed in all groups of rats.
All the deworming drugs were found very effective to control both pinworm and ascariasis infections. Monthly treatment is needed to control or eliminate pinworm and Ascariasis reinfection in laboratory animals.
CONCLUSION
Pinworms and ascariasis remain prevalent in laboratory mice and rats, swine, and pigs. Rats are usually infected with pinworm Syphacia muris and mice with Syphacia obvelata and Aspiculuris tetraptera as well as Ascaris suum in both rats and mice in laboratory. Therefore, it was needed to eliminate worm infection in laboratory rats. For this purpose, thirty males and thirty females of Wistar rats (Rattus norvegicus) from DMR were randomly selected and 10 each were housed in cages separately for three deworming drugs fenbendazole, albendazole, and parazivet) were applied to selected groups of rats.
Results found that 55 (91.67%) out of 60 were positive for Pin worm and Ascariasis eggs. Of these the highest density of mixed infection (Pinworm + Ascariasis) was found in 40 (72.73 %) followed by 8 (14.55 %) was pin worms positive and the lowest positivity was observed in 7 (12.73 %) of Ascariasis eggs. A total of 30 males and 30 females were tested, 28 (93.33 %) samples of males and 27 (90 %) samples of females were found parasite positive.
After treatment with deworming drugs, all the rats were free from parasite eggs and a 100 % reduction was observed in all groups of rats and very effective to control both pinworm and ascariasis infection. And need to prevent from pinworms and Ascariasis reinfection in laboratory animals by monthly treatment of deworming drugs.
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