The first unequivocal report of Encephalitozoon cuniculi in rabbits and Encephalitozoon intestinalis in cattle in Mosul city-Iraq

The aim of present study was to report spontaneous Encephalitozoon cuniculi (E. cuniculi) infection in rabbits and Encephalitozoon intestinalis (E. intestinalis) infection in cattle in Mosul city Iraq. Fecal samples were collected randomly from 142 rabbits and 115 cattle during the period from June, 2012 to June, 2013. Thin smears were prepared from the fecal samples and stained with different fecal stains, as well as the indirect immunofluorescent antibody technique. Infected rabbits were necropsied and pathology of the infection was described. The result showed that microsporidiosis was detected in 41 (26.9%) rabbits and in 12 (10.4%) cattle. E. cuniculi spores were identified in 31 out of the 41 rabbits (73.2%) and E. intestinalis in 7 out of the 12 cattle (58.3%). Infected rabbits showed vasculitis and granulomatous encephalitis, chronic interstitial nephritis, granulomatous hepatitis and cholangitis, vasculitis and non – suppurative pneumonia, infiltration of mononuclear cells in most layers of the intestines, and hyperplasia of lymphoid tissues. E. cuniculispores were seen in the liver, kidneys and lungs. This study considered the first one that reportmicrosporidiosis (E. cuniculi, E. intestinalis) in Mosul city Iraq.


Introduction
Microsporidia are obligate intracellular microorganisms that infect a wide range of vertebrate and invertebrate hosts (1).They have been traditionally considered as protozoa but they were recently reclassified as fungi (2)(3)(4).Approximately 143 genera and more than 1,200 species are known, and at least 14 species are known to infect humans being (5).Spores are the infecting form of microsporidia, and in humansinterhuman, water -borne, food -borne, transplacental, sexual, and vector routes of transmission have been described (6).Encephalitozoon cuniculi and E. intestinalis are the most wide spread microsporidia species in animals and humans and is the cause of a disease known as encephalitozoonosis (7).The zoonotic potential of Encephalitozoon spp.has been confirmed and microsporidia became of considerable interest of human and veterinary medicine (8).Natural variety of hosts including small and large animals and their lists were given by ( 9) and (10).Despite the zoonotic and scientific importance of microsporidiosis, the disease has never been studied in humans and animals in Iraq.Therefore, the aim of the present study was to report the occurrence of E. cuniculi in rabbits and E. intestinalis in cattle with emphasis on pathology of natural infection in rabbits in Mosul city/ Iraq.

Fecal samples
152 rabbits fecal samples (collected randomly from local markets of Mosul city) and 115 cattle fecal samples (collected randomly from cattle bring to Teaching Hospital of College of Veterinary Medicine, University of Mosul) have been collected in period of one year (from June, 2012 to June 2013).Fecal samples were collected directly from the rectum into containers and held at 4°C in large container contained dry ice (CO 2 ice) until further assay.In the laboratory, a preservative (5-10% neutral buffered formalin -sodium acetate and 4% sodium permanganate) were added to feces at the rate of 10 ml of the preservative to each gram of the fecal specimens, and then the mixture was kept in the refrigerator at 4°C till examination (11).

Processing of fecal samples
The fecal samples were sieved through gauze and cotton to get rid of the large objects or sediments, placed in 15 ml test tubes, and centrifuged at 7500 rpm for 40 minutes.Resuspension of the sediment was made using phosphate buffered saline (PBS) and the test tubes were centrifuged at 7500 rpm for 40 minutes.The filtrate was discarded and the sediment (1 ml) from each tube was resuspended in a test tube and thus became ready for examination (12).

Staining of fecal samples for presence of microsporidia
A drop of the precipitate was placed on a glass slide and was spread by a second slide and the smear was left to dry at room temperature.The slides were then dipped in absolute methyl alcohol for fixation and left to dry at room temperature.Smears were stained quick -hot Gramchromotrope, Weber -green modified trichrome, Ryanblue modified trichrome (13)(14)(15), for microscopic examination.

Detection and morphometric of spores
The criteria used to define microsporidia -positive animals was the presence of one or more pinkish or pinkish -violet ovoid structure with a spore wall and a belt -like stripe, over an examination of at least 100 field (at lens 100x).Measurements of 50 spores per slide were done using optical micrometer as described by (16).

Concentration and purification of microsporidial spores
Collections of microsporidia spores from fecal samples were done according to the method described by (11).Resuspension of the sediment using PBS pH 7.2 was done and smear was prepared using a drop of the solution, and stained with Ryan -blue modified trichrome stain and examined for presence of spores (12).

Indirect immuno-fluorescent antibody technique (IIFA)
Kit manufactured by Bordier Affinity products (SA Chatanerie 2, CH-1023 Crissier, Switzerland) were used to identify E. cuniculi in rabbit fecal samples and E. intestinalisin cattle fecal.Procedure for this technique was done as mentioned by (17).

Histopathological examination
Rabbits that were positive for presence of E. cuniculi spores in their fecal samples were euthanized using chloroform (18).Following gross pathological examination, tissue specimens were collected from internal organs and preserved in 10% formalin for 48 -72 hours (15).Specimens were processed for staining by the conventional methods, and stained with hematoxylin and eosin (15).

Statistical analysis
Data concerning the measurements of the spores in various experiments were analyzed using statistical software (SPSS), version 16.0 and the values were presented as mean, standard error, and range in micrometers (19).produced by injured tissue from the replication of spores as mentioned by (38).

Prevalence of microsporidial infection in rabbit and cattle in this study is shown in
In conclusion, the result of current study showed that natural microsporidiosis is very common in both rabbits and cattle, also natural infection by E. cuniculi was more common in rabbits and E. intestinalis was mostly reported in cattle in Iraq.In addition to that pathology caused by natural infection with E. cuniculi in rabbits in Iraq was typically similar to pathology caused by same pathogens all over the world.
Figu of li prese liver