ABSTRACT

Objective

Wild rodents act as important hosts and reservoirs for both zoonotic and non-zoonotic pathogens, playing a key role in maintaining and transmitting infectious agents in nature. Their presence can lead to contamination of food and water sources, affecting both humans and animals.

Methods

This study examined 138 dead rodents from six species (Microtus socialis, Rattus norvegicus, Mus musculus, Meriones libycus, Apodemus witherbyi, and Ellobius lutescens) collected from three regions in Iran. Fecal samples were analyzed for Eimeria spp. and Cryptosporidium spp. using potassium dichromate cultivation and sugar flotation for Eimeria, and modified Ziehl-Neelsen staining for Cryptosporidium.

Results

The infection rate for Eimeria spp. was 5.79%, and the identified species included E. falciformis, E. papillata, E. miyairii, E. musculoidei, and E. hungaryensis. For Cryptosporidium spp., a 4.34% infection rate was observed. While Eimeria infections were limited to three rodent species, Cryptosporidium was detected in all six.

Conclusion

This study presents the first morphological identification of Eimeria species in rodents in Iran, with findings consistent with host-parasite relationships reported globally. Additionally, the widespread presence of Cryptosporidium spp. in multiple rodent species emphasizes the epidemiological importance of these animals as potential reservoirs of zoonotic pathogens. These results contribute to a better understanding of protozoan diversity and distribution in rodent populations of Iran.

INTRODUCTION

Rodents are among the most common reservoirs of zoonotic infections, and they play an important role in the spread of infectious diseases to people and domestic animals. They comprise 42% of the world’s mammalian biodiversity and are the biggest order of extant mammals. They are remarkably diverse. Rodents are thought to be the most successful and adaptable group of mammals (1, 2). Eimeria spp. and Cryptosporidium spp. are protozoan genera of significant concern due to their impact on human and animal health. Cryptosporidium species cause cryptosporidiosis, a gastrointestinal disease that can be particularly severe in immunocompromised individuals. In contrast, the Eimeria species are primarily associated with coccidiosis, which affects a wide range of animal hosts and leads to substantial economic losses in the livestock industry (3, 4).

There are about 2,000 species of Eimeria known to exist in invertebrate hosts as well as mammals, birds, reptiles, and other vertebrates. Eimeria species are found worldwide because their hosts are widely dispersed; over 200 species of coccidia, especially Eimeria spp., have been identified in rodents. Most species of Eimeria are homoxenous, which means they only infect one type of host. Nonetheless, certain avian Eimeria species have been found to have a restricted oligoxenous host range, a trait that could aid in the spread of the parasite by allowing livestock and wildlife to become infected (5, 6). On the other hand, the protozoan genus Cryptosporidium spp. can infect a wide range of hosts, including humans, domestic animals, and wildlife. The oocysts of Cryptosporidium spp. are highly resistant to environmental conditions and can persist for extended periods, enabling transmission through contaminated food and water. Numerous rodent species have been identified as hosts of Cryptosporidium spp., making them significant reservoirs of the parasite. They may actively contribute to the spread of zoonotic Cryptosporidium species and play a role in cryptosporidiosis in both humans and animals (7, 8).

The primary objective of this research is to identify and characterize Eimeria spp. and Cryptosporidium spp. species present in rodents from selected regions. This study aims to expand existing knowledge on the epidemiology of these parasites within rodent populations. The research is conducted in the central and northwestern regions of Iran, specifically in the cities of Arak, Saqqez, and Urmia, where rodent populations are abundant and frequently interact with human habitats.

METHODS

A total of 138 rodents were collected from central and northwestern Iran, specifically from Arak (n=45), Saqqez (n=41), and Urmia (n=52). The rodents were trapped in grain warehouses, cereal pastures, or accidentally captured in homes by rodenticide poisoning (Table 1). All rodents were found dead in the traps before being transported to the laboratory. The samples were securely transported in sealed bags following biosecurity protocols. Following the collection, the taxonomic classification of the rodents was performed and recorded. After the dissection of the gastrointestinal tract, fecal samples were collected from the distal part of the intestine, where the feces had a distinct shape and consistency. These samples were transferred to a 2.5% potassium dichromate solution. Additionally, a portion of the fecal sample was prepared for smear examination and identification of Cryptosporidium.

Examination of Eimeria Oocysts

For this purpose, after fecal sample collection, a portion was placed in a 2.5% potassium dichromate preservative solution and stored at 4 °C in a refrigerator for 3 days to allow the oocysts to complete the sporulation process. After that, one slide was prepared from each sample using the direct smear method, and another was prepared using the sugar flotation method (9).

The Eimeria oocysts were identified using a light microscope at magnifications of ×10 and ×40, with each oocyst containing four sporocysts. After observing the target oocysts, further imaging and morphometric measurements of all oocyst samples on each slide were performed using a Dino-Eye digital camera. This camera, which allows simultaneous connection to a light microscope and a computer, facilitated detailed imaging at ×40 and ×100 magnifications using immersion oil.

To identify Eimeria species, images measured with Dino-Eye software were analysed based on key morphological features, including oocyst and sporocyst size, oocyst layers, and the presence of structures like micropyle, polar granules, and Stieda body and large or small refractile globules (6).

Examination and Identification of Cryptosporidium

To identify Cryptosporidium spp. in fecal samples, the modified Ziehl-Neelsen acid-fast staining method was employed. Two fecal smears were prepared for each sample following standard protocols. Under ×100 magnification, Cryptosporidium oocysts were observed as minute red granules within a green-blue background, indicative of the characteristic acid-fast staining properties of the parasite (10).

Statistical Analysis

Descriptive statistical analyses were performed in the study. All data were entered into Microsoft Excel, and descriptive measures, including frequencies and percentages, were calculated using the software’s built-in functions.

RESULTS

A total of 138 rodents were examined, and six species were diagnosed: Microtus socialis, Rattus norvegicus, Mus musculus, Meriones libycus, Apodemus witherbyi, and Ellobius lutescens (11). These species are presented in Table 2.

Out of 138 rodents, 8 (5.79%) were infected with Eimeria spp. Among these, 5 were male and 3 were female. The infected rodents included M. musculus (n=4), R. norvegicus (n=2), and A. witherbyi (n=2). One case of simultaneous infection with Eimeria spp. and Cryptosporidium spp. was observed in A. witherbyi. In the 8 positive samples for Eimeria, 5 species were diagnosed: E. musculoidei (Figure 1) from M. musculus (n=1), E. falciformis (Figure 2) from M. musculus (n=2), E. papillata (Figure 3) from M. musculus (n=1), E. miyairii (Figure 4) from R. norvegicus (n=2), and E. hungaryensis (Figure 5) from A. witherbyi (n=2).

Additionally, 6 (4.34%) out of 138 rodents were infected with Cryptosporidium spp. (Figure 6). Among these, 3 were male and 3 were female. The infected rodent species included M. musculus, R. norvegicus, M. socialis, E. lutescens, and A. witherbyi (Table 3).

DISCUSSION

This study examined the occurrence of Emeria spp. and Cryptosporidium spp. in wild rodents from western, central, and northern Iran, identifying a diverse range of coccidian parasites, including Eimeria species E. musculoidei, E. falciformis, E. papillata, E. miyairii, and E. hungaryensis.

Rodents serve as hosts for a wide range of Eimeria species, most of which exhibit strict host specificity, even among different rodent species. Most in vivo studies on Eimeria spp. have been conducted in rodents, such as the house mouse, due to economic considerations (3).

E. falciformis is reported as the most prevalent Eimeria species in M. musculus, infecting the epithelial cells of the cecum and upper colon. This species can cause acute coccidiosis in rodents. E. papillata is commonly used in laboratory studies on anticoccidial agents, as it specifically infects M. musculus. Eimeria hungaryensis is a host-specific coccidian that primarily infects the genus Apodemus, consistent with our study findings. This species cannot be transmitted to M. musculus, Microtus arvalis, Clethrionomys glareolus, or Cricetus cricetus. Eimeria miyairii is an uncommon species that infects the epithelial cells of the villi and, occasionally, the glands of Lieberkühn in the small intestine of the Norway rat (R. norvegicus), its primary host. This species is host-specific and cannot infect M. musculus, Sylvilagus floridanus, Cavia porcellus, or Spermophilus tridecemlineatus. E. musculoidei has been identified in the upper ileum of Mus (Leggada) musculoides (its primary host) in Africa and is also suspected to infect the house mouse (M. musculus) in Asia (6, 12, 13).

There are few reports on Eimeria spp. infection in rodents in Iran, and most studies have been limited to genus-level identification. A study conducted in 2014 reported an Eimeria spp. infection rate of 22.5% in rodents from Meshkin Shahr, located in northwestern Iran (14). Additionally, a study conducted in Iraq in 2022 reported a 28% infection rate in M. musculus (15). Another study in Egypt reports 19.9% Emeria spp. infection in Psammomys obesus (16). However, Reports on Eimeria infections in rodents from Iran and neighboring countries remain limited. This study provides the first detailed morphological identification of Eimeria species in rodents from Iran.

The first detection of Cryptosporidium spp. in mice dates back to 1907. Since then, more than 40 species and over 120 genotypes of Cryptosporidium spp. have been identified (17).

Many of these species pose a significant threat to human health and economically important animals, with rodents serving as crucial reservoirs for their transmission. Rodents can act as reservoirs for highly prevalent Cryptosporidium species such as C. parvum, which infects both humans and animals, and C. meleagridis, which primarily infects birds (18, 19). Additionally, rodents can facilitate the adaptation of certain rare Cryptosporidium species to humans, including C. ditrichi, C. mortiferum, C. tyzzeri, and C. viatorum (20).

Most studies on rodents focus on their role as reservoirs of zoonotic agents, with Cryptosporidium being one of the most significant. Rodents can serve as reservoirs for Cryptosporidium, easily spreading thick-walled oocysts into food and water sources (8). Reports indicate that approximately 17-20% of rodents worldwide are infected with Cryptosporidium spp., with C. parvum being the most commonly detected species. Among rodent hosts, muskrats are considered the most relevant reservoir (4, 21). Based on the results, all rodent species involved in the study can act as reservoirs for Cryptosporidium spp.

Several studies have reported Cryptosporidium spp. infections in rodents in Iran. A study conducted in 2016 in the Torkaman Sahra Region identified Cryptosporidium spp. in 6.6% of R. norvegicus samples (22). In the same year, another study reported that only 3% of rodent samples tested positive for C. parvum in Ahvaz City (22), while Cryptosporidium spp. was detected in just 1% of R. norvegicus specimens in Tehran (23, 24). However, meta-analyses suggest that the overall prevalence of Cryptosporidium in rodents across Iran ranges from 18% to 20%. These findings indicate that Cryptosporidium spp. infection rates in Iranian rodent populations are consistent with those reported in other regions worldwide, highlighting the potential role of rodents as reservoirs for this parasite (25, 26).

Study Limitations

The results of this study were based on morphometric measurements and taxonomic identification keys. However, molecular methods could not be applied, which represents a limitation since such approaches could have provided more accurate confirmation and deeper insights. Furthermore, although the presence of Eimeria and Cryptosporidium species in rodents was identified, their geographical and regional distribution patterns were not evaluated. Future studies should incorporate molecular techniques and spatial analyses to provide a more comprehensive understanding of the epidemiology of these parasites.

CONCLUSION

In conclusion, the findings of this study provide the first detailed morphological descriptions of Eimeria species infecting rodents in Iran. The rodent host species identified in this study align with the results of similar studies conducted worldwide, further supporting the consistency of rodent species as hosts for various Eimeria species across different geographic locations. The results of Cryptosporidium spp. infection in terms of species were consistent with previous studies conducted in Iran and worldwide. However, the infection rate observed in this study was lower than the average rates reported in Iran, which could be attributed to factors such as the number of cases examined or the lack of molecular diagnostic techniques in this study.