Intestinal Protozoa in Domestic Cats (Carnivora: Felidae, Felis catus) in Northwestern Iran: A Cross-Sectional Study with Prevalent of Microsporidian and Coccidian Parasites.

Background
In this study, some microsporidial and coccidian parasites were isolated from 103 domestic cats in the Meshkin Shahr area, northwestern Iran during the Jun 2014 to Jun 2015, and their genera were identified using parasitological methods with emphasis on their zoonotic importance.


Methods
One hundred and three fecal samples of domestic cats were collected and preserved in formalin (10%) and conserved in phosphate buffer saline solution, finally examined by microscopy after formalin-ether concentration and specific staining. Preservation in dichromate potassium (2.5%) was performed for all coccidian positive samples and then sporulated coccidian oocysts were investigated.


Results
The detected parasites were Isospora spp. 6/103(5.8%). Microsporidian spores were identified in 46/103 (44.6%) of all samples post-stained by the aniline blue staining method.


Conclusion
Microsporidial infections were more prevalent in domestic cats. Further studies are needed in the identification of microsporidial spores isolated from infected cats.


Introduction
ats as most widespread animals contact directly with humans are reservoir hosts for some of intestinal parasitic infections. They have important role in contamination of environments, humans and animals by excreting eggs, cysts and oocysts of parasites in their stools (1)(2)(3).
A wide range of intestinal protozoa commonly infects cats throughout the world. There are some protozoa infections among feline intestinal parasites including Toxoplasma gondii, Giardia duodenalis, Cryptosporidium spp., Sarcocystis spp. and Isospora felis (I. Felis) and rivolta (2).
Among the coccidial parasites, I. felis and Cryptosporidium spp. infections are the most common (4). Almost all cats can be infected with I. felis. The pathogenicity of I. felis is, however, controversial (4).
Cystoisosporosis cause mild to severe diarrhea that may be bloody in puppies and kittens. The morbidity or mortality rates in severe cases are high (5).
C. parvum infection is a zoonosis that generally infects calves; however, it can also infect cats. C. felis has been reported in cats. Cryptosporidial infections are considered to be clinical importance in immunocompromised cats (4).
Microsporidia have been reported as causative agents of opportunistic infections especially in immunodeficient patients (6)(7)(8); however, are common in immunocompetent people (9). Diagnostic methods based on staining have been described in some previous studies (10,11). Although numerous data associated with the epidemiology of Microsporidia infection exist which describe the zoonotic nature of the parasites, the information about transmission of infection from animals to humans needs more investigations (12).
Although a few studies about intestinal parasites in cat have been completed in different areas in Iran (13,14), there has been no data on cat prevalence of protozoa infection in Meshkin Shahr.
This study aimed at identifying microsporidial and coccidian parasites isolated from domestic cats of Meshkin Shahr area, northwest of Iran using parasitological methods.

Materials and Methods
In this cross-sectional study, 103 cats were caught by trapping (baited cage-traps) from different regions consist of Kojenagh, Ourkandi, Aghbelagh, Sarikhanlou of Meshkin Shahr, Ardabil province north-west of Iran during the Jun 2014 to Jun 2015. The information collected on each cat included age (78 young cats aged between 1-3 yr old and 25 cats aged >3 yr old) and gender (48 males and 55 females).
Two samples were collected from each cat, first sample was preserved in formalin (10%) and second one conserved in phosphate buffer saline (PBS) solution in Meshkin Shahr station of School of Public Health, Tehran University of Medical Sciences, and transferred to the Department of Medical Protozoology and Mycology, School of Public Health, Tehran University of Medical Sciences. The wet mount was prepared with PBS and formalinether concentration method was carried out for all the samples, and consequently observed under a light microscope with a final magnification of 400x.

Acid Fast staining method
Formalin-ether concentration method was finalized, and smears were prepared from pellet of all samples and the slides allowed to dry at room temperature for 5 min following methanol fixation. Subsequently, all the samples were stained by a modified acid-fast staining method (15). Finally, all the slides were observed under the light microscope with a 1000x magnification.

C Preservation in dichromate potassium
Preservation in dichromate potassium (2.5%) was performed for all coccidian positive samples that collected in PBS solution and then sporulated coccidian oocysts were investigated

Aniline Blue Staining Method
Sample smears were prepared, and after drying and methanol fixation, aniline blue staining method was carried out according to Ryan method (11). All the samples were observed with 1000x objective lens and evaluated for the detection of microspora spores.

Ethical approval
This study was reviewed and approved by the Ethics Committee of Tehran University of Medical Sciences (Ethic no. 25287) in accordance with Helsinki Declaration and guidelines.

Data analysis
Descriptive statistical methods relative to absolute and relative frequencies of microsporidial and coccidian intestinal parasites and their distribution in different areas of Meshkin Shahr district were done using SPSS (version 21) (Chicago, IL, USA).

Results
One hundred and three stool samples from cats including 48 males and 55 females were collected. Generally, protozoa infections were attributed to Isospora spp. 6/103(5.8%), involving 4 female and 2 male cats. They were of different sizes and shapes. Large oocyst with a bulge on one side and a size of 38-51 by 27-39 μm that was similar to I. felis (Fig. 1A, B, C), and one medium oocyst that was more rounded and smaller than the previous ones and having a size of 18-28 μm by 16 -23 ( Fig. 1 E). Finally Isospora spp. has been identified because of lack of confirmation by molecular method. Three Isospora positive cases belonged to cats collected from Ourkandi and 3 cats collected from Kojenagh. The most positive cases of Isospora spp. (4/6) were seen in cats more than 3 yr old.
Result of Isospora preservation in dichromate potassium 2.5% has been shown in (Fig. 1D). The sporulated Isospora spp. oocyst contains two sporocysts and 4 sporozoites after preservation in dichromate potassium. The result of acid-fast staining method showed no Cryptosporidium spp. oocysts in this study.
In aniline blue staining method for detecting microspora, ovoid, transluminant spores with a belt-like strip in the middle were observed, with a size of spores ranging between 0.7-2.0 µm. Against the microspora spores characterization, bacteria portray blue color and fungi is displayed red, but with different sizes and shapes (Fig. 1F).
Microsporidian spores were identified in 46 (44.6%) among all samples stained by the aniline blue staining method. Parasites spores were found in 29 female cat fecal samples and 17 male ones. The most positive cases were prominent in Kojenagh [22] and then Ourkandi [15] followed by Aghbelagh [7] and Sarikhanlou [2] (Table 1).

Discussion
Animals, especially dogs and cats, play an important role in communities all over the world. Cats are the reservoir hosts of parasites including protozoa and helminthes because of their provision of nutritional and biological conditions (13).
Although parasitic infections have been reported in rodents from Meshkin Shahr areas (3,16). There are limited investigations regarding gastrointestinal protozoa, especially coccidial and microsporidial infection in cat in this area. In this study, coccidia infections in 103 cats caught from Meshkin Shahr were found as follow: Isospora spp. 6/103(5.8%). Contrary to our study, high levels of infections have been reported in stray cats in Zanjan Province.  I. rivolta (80%), I. felis (70%) and T. gondii (42%) were reported in stray cats (13). The prevalence of intestinal parasites may be different with respect to geographical regions, climate and soil conditions of the areas, the type of cat population and seasons (2).
The prevalence of intestinal parasite depending on rural or urban areas may vary. Protozoan infections are more common in urban areas than rural area (2).
In this study, considering the situation of the regions, different pattern was recorded where Isospora infection was less common than previous study in Zanjan (13). Notably, the high levels protozoan infection rates have been reported in some previous studies (17,18). In a fecal examination survey, among 217 cats obtained from Illinois, I. felis oocysts (23%), I. rivolta oocysts in (24%), and Toxoplasma or Besnoitia oocysts (1%) were identified (17).
Prevalence rates of T. gondii (18.2%), I. felis (15.1%) and G. duodenalis (17.0%) have been reported from Aboriginal communities in the west Kimberley region of Western Australia (19). A survey of pups for sale at pet stores in Atlanta showed that 34% were infected with Giardia, even though none had diarrhea or clinical illness (20).
Conversely, low levels of protozoa infection have been reported in some studies (2,21,22), where these findings are consistent with that of our study. In a study, overall fecal prevalence of coccidia (1.4%) and Giardia species (0.58%) were found in USA (22).
The prevalence Isospora spp. recognized in this study is in agreement with the data obtained by other researchers in different countries. Thus, in cats exhibiting clinical signs, Isospora was reported in 12% of stool samples in Chile (23) and 3% in England (24). In cats without clinical signs, the prevalence of Isospora spp. infection has been reported as 6.3% in Spain (25), 5.6% in Australia (26) and between 0.2% and 9.7% in USA (27,28).
Microspora spores were identified in 46/103 (44.6%) of all samples stained by the aniline blue staining method. There have been few studies concerning microsporidia in cats in Iran. E. bieneusi was detected using molecular method in 3/26 (11.53%) fecal samples collected from cats in a study in Tehran, Iran (29), and (3/40) 7.5% of the specimens obtained from cats in Iran (30).
Microsporidial spores were detected in 29.4% (10/34) of cat fecal samples by MT stain in Portugal (31). Our result was somewhat higher than that of Lobo et al, and this might be attributed to differences in geographic criteria of the two countries.
Taking into account the limitation of this study, PCR was unavailable and only parasitological method was used in the identification of the parasites.

Conclusion
Microsporidia with a prevalence of 44.6% were more prevalent than coccidia such as Isospora (5.8%) due to zoonotic characteristics of microsporidia, hence this must be considered.