Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

BACKGROUND: Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius.
METHODS: This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively.
RESULTS: About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method.
CONCLUSION: Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.

Introduction

Parasitic infection including protozoa and helminthes in rodents are of special interest because of the role of rodents as reservoirs of many important parasites of man (1). All rodents are susceptible to protozoan infection. Some of these protozoa may be zoonotic. Although Giardia muris has a limited host and its transmission to human from laboratory rodents has not been reported, care should be taken with Giardia and Cryptosporidium spp. (2, 3).

A form of Giardia muris has been observed in the golden hamster, mice, and rats. Infection is usually subclinical but the animal’s exhibit weight loss, hair bristling, bloating with meteorism (4).

Trichomonas muris is common parasite that detected in rodents, as mice. Its pathogenesis in mice is unclear. Although T. muris suspected to be non-pathogenic (5), diarrhea and anorexia have been reported as sign of T. muris infection (6).

Some possible protozoa infection in rat and mice are Chilomastix Bettencourt, Entamoeba muris, Cryptosporidium muris, Giardia muris, Cryptosporidium parvum, Trichomonas muris, Hexamita muris, Eimeria spp., Spironucleus muris that some of them are described as mildly pathogen (2, 3, 7). Eimeria spp. has been reported as common protozoa parasites in rabbits and Balantidium coli identified in guinea pigs (8).

Although helminthic fauna and Leishmania infection in rodents as a host of visceral Leishmaniosis in Meshkin Shahr district were reported (9,10, 11), there is no data on protozoa infection in this area.

This study aimed to determine frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Musmus culus and, Cricetulus migratorius (gray hamster) from Meshkin Shahr district, northwestern Iran.

Materials and Methods

Meshkin Shahr is located in the northwest of Iran in Azerbaijan, It covers an area of approximately 1530 km2 and its population is estimated to be 237585, among whom 29.7% are settled in urban areas and 70.3% live in 323 rural areas. It is the nearest city to the Sabalan high mountain. The weather of this city and the district of Meshkin Shahr is moderate mountainous (12) (Fig.1).

Fig. 1:

Geographical situation of Meshkin Shahr district

Sample collection

From Mar. to Dec. of 2014, intestinal samples from 204 trapped rodents including M. persicus (no.117), M. musculus (no.63) and C. migratorius (no.24) were collected by Sherman method, live animal Traps from northwest of Iran (13). This study was approved by the Research Ethical Review Committee of Tehran University of Medical Sciences, Tehran, Iran with No: 22943.

All of rodents stool samples were preserved in formalin 10%, PVA solution and saline solution in Meshkin Shahr research station and transferred to the Department of Medical Protozoology and Mycology, School of Public Health, Tehran University of Medical Sciences. Formalin-ether concentration method carried out for all of the samples and samples observed with light microscope with 400× magnification. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples that collected in saline solution.

All of suspected cases were stained with trichrome staining method (14). The slide was mounted using Canada balsam and observed under 1000× magnification. Detection of intestinal protozoa was based on morphological characteristic of specific protozoa. Analysis was performed using Excel 2007. Formalin-ether concentration method carried out and smears were prepared from pellet of all samples. The slides were dried at room temperature for 5 min after methanol fixation all of samples were stained with modified acid-fast staining method (15), finally all of slides were observed with under light microscope 1000× magnification. Samples smear were prepared and after drying and methanol fixation, aniline blue staining method carried out according to Ryan method (16). All of samples were observed with 1000× objective and evaluate for detecting microspore spores.

Results

From 204 the caught rodents, 127(62.1%) were male. In general, 121 (59.3%) of rodents were infected with protozoa including M. persicus 88 (75.2%), M. musculus 20 (31.2%) and 13 (54.2%) grey hamster.

Infection rates of protozoa in male M. persicus, M. musculus and grey hamster were 52.3%, 60%, and 84.6%, respectively that they were more than female as 47.7%, 40% and 15.4%, respectively. Generally, the prevalence of protozoa infection in male rodents 69(57%) was more than females 52(43%).

Prevalence of Intestinal protozoa was as below: E. muris 14(6.9%), T.muris 55(27.0%), Chilomastix betencourtti 17(8.3%), G. muris 19(9.3%) and from coccidian group: Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and cryptosporidium spp. 1(0.5%) (Table 1).

Table 1:

Frequency of Intestinal protozoa among 204 trapped rodents from Meshkin Shahr, Ardabil Province in 2014

Parasite	Meriones Infected no	persicus %	Mus Infected no	musculus %	Cricetulus-migratorius Infected no	%	Total Infected no	%
Entamoeba muris	12	5.9	1	0.5	1	0.5	14	6.9
Trichomonas muris	40	19.7	7	3.45	8	3.94	55	27.0
Chilomasix Bettencourt	16	7.9	0	0	1	0.5	17	8.3
Giardia muris	15	7.4	1	0.5	3	1.5	19	9.3
Eimeria spp	37	18.23	7	3.45	2	0.98	46	22.5
Cryptosporidium spp	1	0.5	0	0	0	0	1	0.5
Isospora spp	4	2	0	0	0	0	4	2
Microsporidia Spores	48	23.64	11	5.42	4	1.97	63	31.03

Prevalence of protozoa infection in M. persicus 88 (72.8%) was more than M. musculus 20 (16.5%) and C. migratorius 13(10.7%).

Microspora spores were identified in 63(31.03%) of all samples were stained by the aniline blue staining method. In aniline blue staining method was used for detecting Microspora, ovoid, transluminant spores were observed with 0.7–1.2 μm size. The spores had a belt-like strip in the middle or at the end of body. Trophozoite of E. muris and T. muris stained with trichrome staining method with1000× magnification are shown in (Fig. 2, A) and an unsporulated oocyst of coccidia in a wet mount sample with 400× magnification are shown in (Fig. 2, B)

Fig. 2:

A: Trophozoite of E. muris and T. muris in trichorom staining method (1000×); B: An unsporulated oocyst of coccidia in a wet mount sample (400×); C: appearance of Eimeria spp after cultivation in dichromate potassium (400×); D: Cryptosporidium spp in acid-fast staining method (1000×), in trapped rodents from Meshkin Shahr in 2014.

The result of coccidia cultivation in dichromate potassium 2.5% is shown in Fig. 2, C. In each oocyst four sporocysts and in each sporocyst two sporozoites exist, and finally Eimeria spp. diagnosis was made.

The result of acid-fast staining method showed partial acid-fast positive cases with the size of almost 4 μm, in a sample that belongs to a male M. persicus and finally, Cryptosporidium spp diagnosis was made (Fig. 2, D).

In general 121(59.3%) were positive that 63(52.1%) have just one parasite, 32(26.4%) two parasite, 15(12.4%) three, 8(6.6%) four, 2(1.6%) five, 1(0.8%) six, parasites.

The most common single protozoa were Microspora 24/63 and T. muris 19/63 and then Eimeria 13/63 and double infection were between Eimeria and Microspora (8/32), T. muris and Microspora spp. (8/32), Trichomonas and Eimeria (5/32).

Discussion

This study was conducted to determine the prevalence of intestinal protozoa infection of rodents from Meshkin Shahr district, northwestern Iran.

In general, 121 (59.3%) of rodents were infected with protozoa. The most common protozoa were T. muris 27.0%, followed by G. muris (9.3%) and E. muris (6.9%). T. muris was the most common protozoa (8, 17, 18). Overall prevalence of intestinal parasites in rat in Arbil was 76% including T. muris with the higher incidence of 56%, G. muris 12%, H. muris 8% and the least infection percentage was 4% for E. muris (17). The result of our study is consistent with that.

Prevalence of intestinal protozoa was reported in the mice as follows: S. muris (46.2%); G. muris (46.2%); T. muris (53.8%); T. minuta (61.5%) and E. muris (84.6%), while in the rat colonies the prevalence of infection was higher: S. muris (85.7%); T. muris (85.7%); T. minuta (85.7%) and E. muris (85.7%) (7). The high prevalence of parasitic infections was found in an animal house in Brazil. The prevalence rate of protozoa were: T. muris (80.0%), G. muris (66.0%), E. muris (20.0%), and Eimeria sp. (13.3%) (18).

Prevalence of protozoa infections have been reported in mice and rat, Entamoeba sp. (8.08%, 3.18%), Giardia sp. (0%, 0%), Trichomona sp. (8.88%, 1.58%), Chilomastix sp. (3.74%, 1.65%) Spironucleus sp. (0.08%, 0.19%) respectively in North America and Europe (19). Prevalence rate of protozoa infection in our study was higher than those have been reported from North America and Europe.

Overall, 37 mice (74%) from 50 Swiss-Webster mice were infected with at least one parasite. The highest prevalence was related to S. muris (64.8%) then follow by G. muris (27.01%), T. muris (21.6%) and the lowest prevalence rate was related to Blastocystis spp. (2.7%) (20).

Encephalitozoon sp. has been reported from rabbits, mice, guinea pigs and rats. The organisms are small, bipolar and rod-like. They occur singly or in clumps (4). Three strains (I, II, and III) are recognized in E. cuniculi, which, according to the host of the originally characterized isolates, are also designated “rabbit strain,” “mouse strain” and “dog strain” (21).

In the present study, microspora spores were identified in 63(31.03%) of all samples. Encephalitozoon sp. was found in (55%) of mice samples by parasitological method (22).

In our study from coccidia group: with the higher incidence of Eimeria spp 46(22.7%), followed by Isospora spp 4(2%) and 1(0.5%) Cryptosporidium spp were found. In concentrated pellets, some unsporulated oocysts were found and after cultivation in dichromate potassium and observation of pattern of four sporocysts and two sporozoites, Eimeria spp diagnosis was made and about Isospora unsporulated and sporulated oocysts were seen. Eimeria is the most parasites from coccidian group in rodents in our study and it was also described in some previous study (4, 23, 24).The overall prevalence of Eimeria spp in rabbits from pet shops and farms were 46.2% and 41.7%, respectively in Taiwan that is similar our result (23, 24).

C. muris has been reported from mice and the guinea pig. They are true coccidia whose developmental stages appear to take place on the surface of the host cell but not within the cell proper. In the case of C. muris, the parasite may be seen in large numbers in sections of the stomach and is a parasite of the peptic glands, but C. parvum has been reported in rat and found in the glandular structures of the small intestine of the mouse (4, 25). In this study, one Cryptosporidium spp positive was detected in a sample belongs to a M. persicus. Molecular investigation is needed for confirmation of Cryptosporidium species.

Protozoa infection in male rodents 69(57%) was more than female 52(43%). In the present study, there was no significant difference between male and female rodents in consistent with another others (26).

In a study, single parasitic infection was the highest (52%), followed by double infection (16%), and triple infection (8%) (17). In the present study, 63(52.1%) of rodents stool samples had just one parasite, 32(26.4%) two parasite, 15(12.4%) three, 8(6.6%) four, 2(1.6%) five, 1(0.8%) six parasites. Prevalence of single parasite was similar but double and triple infection was more than similar study in Arbil (17).

Conclusion

Rodents as reservoirs of some important parasites in this area infected with some zoonotic parasites, hence control of these animals has an important role on prevention of public health problems.