J Arthropod-Borne Dis, December 2018, 12(4): 334–340 S Heidari et al.: Spironucleus muris and … 334 http://jad.tums.ac.ir Published Online: December 25, 2018 Original Article Spironucleus muris and Eperythrozoon coccoides in Rodents from Northwestern Iran: Rare Infections Soudabeh Heidari 1 , Mehdi Mohebali 1, 2 , Zabihollah Zarei 1 , Mehdi Nateghpour 1 , *Afsaneh Motevalli-Haghi 1 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Center for Research of Endemic Parasites of Iran, Tehran University of Medical Sciences, Tehran, Iran (Received 9 Apr 2017; accepted 1 July 2018) Abstract Background: Rodents perform a crucial role in dispersal of zoonosis causes globally. We aimed to investigation about infection levels of parasitic agents in rodents’ population in Meshkinshahr areas, northwest of Iran from Apr to Sep 2014. Methods: Two hundred four rodents were trapped and anaesthetized. A sample of blood was collected via cardi- opuncture from each one. Thin and thick blood smears were prepared and stained with Giemsa. All stained smear were examined under light microscopy with high magnification by two expert microscopists. Every suspected uni- cellular observed were measured microscopically and compared with key references to diagnose. Results: Captured rodents were identified as three genera including Meriones persicus, Mus musculus, Cricetulus migraturius. Protozoa identified in this study were included of Spironucleus muris and Eperythrozoon coccoides, these parasites were observed in blood smear of 0.98% of rodents. S. muris and E. coccoides were seen in M. mus- culus and C. migraturius, respectively. Conclusion: The present study increases awareness about Eperythrozoonosis in rodents and its potential transmis- sion to domestic animals and even to human in rural districts in Iran. Moreover, the attack of Spironucleus on the mucus of colon and its systemic risk was confirmed. Keywords: Spironucleus muris, Eperythrozoon coccoides, Rodents, Iran Introduction Rodentia are the largest group of mammals worldwide. They are found in vast numbers on all continents except Antarctica. Rodents play important roles as reservoirs and carri- ers of diseases agent such as leishmaniasis, plague, leptospirosis, salmonellosis, rat bite fever, dermatophytosis, Sporotrichosis, murine typhus, trichinellosis, cestodes and trematodes infections, toxoplasmosis, relapsing fever (1). Blood parasites in rodent such as Trypa- nosoma lewisi, Leishmania spp., Plasmodium berghei, Babesia microti, Eperythrozoon coc- coides, Haemobartonella muris are important because they are transmitted to humans by ectoparasites of rodents (1). Spironucleus muris (formerly Hexamita muris) is an opportunistic pathogen of sev- eral rodent species including rat, mice, gold- en hamster and European hamster. This flag- ellated protozoan usually inhabits in the crypts of Lieberkuhn in the small intestine after in- gesting parasitic cysts and may cause an acute or choronic form of disease. The organisms invade the lamina propria of the intestinal villi in immunocompromised animals and can dis- seminate systemically through the lymphat- ics or vasculature. Circulating parasite is vis- ible in the peripheral blood of an infected animal (2-4). Eperythrozoon coccoides is blood parasite *Corresponding author: Dr Afsaneh Motevalli- Haghi, E-mail: a-motevalli@tums.ac.ir, amh.mot@gmail.com http://www.ucmp.berkeley.edu/mammal/mammal.html J Arthropod-Borne Dis, December 2018, 12(4): 334–340 S Heidari et al.: Spironucleus muris and … 335 http://jad.tums.ac.ir Published Online: December 25, 2018 in laboratory and wild mice that causes a mild haemolytic anaemia. Eperythrozoon coccoides was first identified in laboratory mice in Ger- many in 1928. Wild mice are natural hosts and rats and rabbits have been experimentally in- fected. Eperythrozoon coccoides was classi- fied into the group of haemotropic myco- plasmas (haemoplasmas) (5-7). Eperythrozo- onosis is a zoonotic disease (transmissible from animals to humans). The first recognized hu- man case of eperythrozoonosis was reported in 1986 worldwide (8). The disease may manifest with fever, he- molytic anemia, lethargy, jaundice, swollen lymph nodes of the neck, leucopenia, neutro- penia, thrombocytopenia, splenomegaly and lymphadenopathy, acidosis (9-11). The most important way for the transmission of E. coc- coides is blood-feeding arthropod vector such as adults and nymphs of the lice Polypax spi- nulosa and P. serrate, this transmission is mechanical (12). We aimed to investigate the diversity and infection levels of parasitaemia in rodent pop- ulation from Meshkinshahr District. Materials and Methods Study area Meshkinshahr City located in Ardebil Pro- vince in the northwest of Iran (38°23′56″N 47°40′55″E) (Fig. 1), is situated at an alti- tude of 1830m above sea level and the aver- age temperature of city is between 22.4 and 2.4 °C. The weather of the city and the dis- trict is moderate mountainous. It is limited from the north to the Moghan City and from the west to the Ahar City and from south to the Sabalan high mountain and from the east to Ardebil Province and from the northeast to the Republic of Azerbaijan. Sample collection and Parasitological study Two hundred four rodents (117 Meriones persicus, 63 Mus musculus, 24 Cricetulus mi- graturius or grey hamsters) were trapped alive from Meshkinshahr Ardebil Province, Iran, between Apr to Sep 2014. Trapped rodents were anaesthetized by placing cotton wool soaked with chloroform. Blood was collect- ed from the heart using a needle and syringe. Thin and thick blood smears were prepared with a drop of blood. Thin blood smear was fixed with methanol. Slides were stained with Giemsa stain and examined under light mi- croscopy at 400x magnification for parasites screening and 1000x magnification under oil immersion for identification. Analysis Protozoa were microscopically measured and compared with key references (13, 14). Ethical consideration This study was approved by the Research Ethical Review Committee of Tehran Uni- versity of Medical Sciences, Tehran, Iran (no: 22943). Results Two hundred four rodents include 3 gen- era (117 M. persicus, 63 M. musculus, 24 C. migraturius (grey hamsters) were collected from Meshkinshahr, Ardabil Province. Pro- tozoa identified in this study were included of S. muris and E. coccoides, these parasites were observed in blood smear of 0.98% of rodents. Spironucleus muris was observed in blood smear of one rodent (1.58% of M. mus- culus) (Fig. 2). This organism was ovoid shape approximately 2–3×7–9µm and was bilater- ally symmetrical with two nuclei, 6 anterior and 2 posterior flagella (13, 15). In addition, this organism existed in the feces of this rodent. Moreover, E. coccoides was microscopi- cally detected in one rodent (4.16% of ham- sters). Cocci-shaped objects in large numbers on the surface of red blood cells and red-purple color with size 0.5–3µm (14) were observed in blood smear of this rodent (Fig. 3). https://maps.google.com/maps?hl=en-IR&gbv=2&um=1&ie=UTF-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=Meshgin+Shahr,+Ardabil&sa=X&ei=2aGWVdfxLMG3sQGfnKSICA&ved=0CBUQ8gEoATAA https://en.wikipedia.org/wiki/Ardabil_Province https://en.wikipedia.org/wiki/Ardabil_Province https://tools.wmflabs.org/geohack/geohack.php?pagename=Meshginshahr¶ms=38_23_56_N_47_40_55_E_type:city%2880000%29_region:IR https://tools.wmflabs.org/geohack/geohack.php?pagename=Meshginshahr¶ms=38_23_56_N_47_40_55_E_type:city%2880000%29_region:IR https://maps.google.com/maps?hl=en-IR&gbv=2&um=1&ie=UTF-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=Meshgin+Shahr,+Ardabil&sa=X&ei=2aGWVdfxLMG3sQGfnKSICA&ved=0CBUQ8gEoATAA https://maps.google.com/maps?hl=en-IR&gbv=2&um=1&ie=UTF-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=Meshgin+Shahr,+Ardabil&sa=X&ei=2aGWVdfxLMG3sQGfnKSICA&ved=0CBUQ8gEoATAA J Arthropod-Borne Dis, December 2018, 12(4): 334–340 S Heidari et al.: Spironucleus muris and … 336 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 1. Map of Ardebil Province in Iran (up). The geographical location of collected samples Meshkinshahr in Arde- bil (down) Fig. 2. Spironucleus muris in a blood smear of Mus musculus. Giemsa stain (a and b). Magnification, 1000x (Original) J Arthropod-Borne Dis, December 2018, 12(4): 334–340 S Heidari et al.: Spironucleus muris and … 337 http://jad.tums.ac.ir Published Online: December 25, 2018 Fig. 3. Eperythrozoon coccoides in thick blood smear of hamester. Giemsa stain. Magnification, 1000x (Original) Discussion We aimed to determine the diversity of blood parasitaemia in rodent population from Meshkinshahr Ardabil. Our findings showed presence of two kinds of protozoan parasite including S. muris and E. coccoides. Previ- ously, S. muris has been commonly seen in laboratory rats and mice (16). The current prevalence is unknown and probably is still high. In a study in Brazil con- ducted on 344 mice and 111 laboratory rats, 46.2% colonies of mice and 85.7% of rat’s colonies were infected with S. muris (17). In another study, 15 mice colonies and 10 rat colonies were collected from 18 laboratories and showed that 90% of rats and 80% of mice were infected with S. muris (18). Moreover, in Iraq, 8% of rats were positive for S. muris infection (19). Furthermore, a 6.3% of the in- fection was found in Bandar Abbas, Iran (20). Spironucleus muris were detected in fecal samples, indicating the presence of infection in the intestines. In addition, infection in immunocompetent and adult mice is usually subclinical. How- ever, infection in athymic (nu/nu), young and immunocompromised mice characterized clin- ically by weight loss, enteritis, hunched pos- ture, rough hair coat, hair lacks shine, leth- argy, distention of the abdomen, depression, diarrhea and death (3). Trophozoites can cause degeneration enterocytes and shortening of microvilli on the crypt epithelium and in- crease in crypt depth (2, 21). Histologically, the formation of cysts was lead to dilation Lieberkuhn glands with an in- flammatory reaction in the lamina propria and the sloughing of the epithelium. The main pathological changes had happened in the du- odenum, and then in the jejunum and ileum. In acute infections, the lumen of the small intestine has a large number of flagellated or- ganisms that tend to invade the lamina pro- pria of intestinal villi and filling glands. In chronic infection, dilated lymph glands were seen which were similar to the cyst and were filled with inflammatory cells, cellular de- bris, mucoid material and cystic organisms. Such clinical symptoms has been previously explained (22, 23). Associated pathological findings with the presence of parasite such as pyogranulomatous pneumonia, colitis, lym- phadenitis and multifocal abdominal abscess has been previously reported in two immu- nosuppressed monkeys suffering from sys- tematic Spironucleus infection (24). S. muris is an opportunistic pathogen that feeds the intestinal bacteria. Some stress or debilitating factors such as lack of the thymus is necessary for disease (13, 25). Spironucle- us muris increase the mortality of treated mice with cadmium, as well as is associated with changes in immune function of macro- phages in mice and immune responses. Young animals are more sensitive, older animals are not at risk of disease and spontaneously re- cover mice with history of disease may be resistant to re-infection (26-28). Co-infection with B. microti, P. berghei, and P. yoelii, has been reported with reducing of the tropho- zoite production in S. muris (29). https://maps.google.com/maps?hl=en-IR&gbv=2&um=1&ie=UTF-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=Meshgin+Shahr,+Ardabil&sa=X&ei=2aGWVdfxLMG3sQGfnKSICA&ved=0CBUQ8gEoATAA https://maps.google.com/maps?hl=en-IR&gbv=2&um=1&ie=UTF-8&fb=1&gl=ir&ll=38.398889,47.681944&z=12&ftid=0x40185ccc47f21603:0xf37817dad315726a&q=Meshgin+Shahr,+Ardabil&sa=X&ei=2aGWVdfxLMG3sQGfnKSICA&ved=0CBUQ8gEoATAA J Arthropod-Borne Dis, December 2018, 12(4): 334–340 S Heidari et al.: Spironucleus muris and … 338 http://jad.tums.ac.ir Published Online: December 25, 2018 Furthermore, microvilli damage, loss of height of microvilli, increase in crypt depth, hyperplasia of crypt, abscesses in crypts and atrophy of villis have been reported in mice with S. muris (21, 28). This organism is like- ly to be systemic through the attack to the mucus of colon. Large lesions in the lymph nodes are most likely to indicate the diffu- sion of parasites from the liver through lym- phatic vessels as compared to blood circu- lation. However, the vessels diffusion cannot be disproved. After enterocytes destruction and necrosis, trophozoites of S. muris pass from the intestinal barrier and enter the blood stream in these areas (24, 30). In the present study, one out of 204 (0.49%) rodents was in- fected with S. muris in the intestinal contents and blood. It may be interpreted that the par- asites had passed from the intestinal barrier and entered to the bloodstream. Numerous parasites were observed in peripheral blood. In this study, E. coccoides was another organism detected and is a parasitic bacteria that attach to the surface of erythrocytes in mouse which can induce erythrocytic dam- age. This organism was previously classified as protozoa but in 2005, based on phyloge- netic evidence and 16S rRNA gene sequenc- es was classified in haemotropic mycoplas- mas and family of mycoplasmataceae (7). Eperythrozoon coccoides is mechanically transmitted to humans by arthropods and it is important because human infection have been reported. Infection is more common in farm- ers and veterinarians who are in close con- tact with domestic animals. Congenital trans- mission has also been reported (31). Infected people may be asymptomatic or symptoms, including fever, severe hemolytic anemia and jaundice, especially in infants. Pregnant wom- en and infants are more vulnerable than oth- ers and show more severe symptoms. In Iran, there is no report of E. coccoides in animals and humans. In this study, one out of 204 rodents (0.49%) was infected with E. coccoides. A molecular and microscopic study of blood in China indicated that E. coccoides were causative agent of anemia in a person who has a long history of anemia (9). In a meta-analysis and systematic study, the rate of infection with Eperythrozoon species in the population of Chinese has been investi- gated. Overall, 14951 out of 52433 cases were infected. According to the seasons and the geographical areas, the infection rate var- ied from 0% to 97.29% (32). Conclusion The present study increases awareness about Eperythrozoonosis in rodents and its poten- tial transmission to domestic animals and even to human in rural districts in Iran. Moreover, information obtained from this study confirmed the attack of Spironucleus on the mucus of colon and its systemic risk. Acknowledgements The authors would like to thanks Prof MJ Gharagozlou from Faculty of Veterinary Med- icine University of Tehran. This study was financially supported by Tehran University of Medical Sciences, Iran. 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