Detection of Cryptosporidium and Giardia doudenalis in equines in Nineveh, Iraq

A total of 107 fecal samples of equines from different regions in Nineveh, were colleted from January 2007 till December 2007 and examined for Cryptosporidium sp., and Giardia doudenalis by using different methods (wet mount, flotation, lugol's iodine, modified Ziehl Nelsecn (hot) and Giemsa stain Just for Giardia doudenalis. The animal age examined ranged from 4 to 10 years. The total prevalence of cryptosporidium sp. was 27.10% (29 out of 107), while the prevalence of Giardia doudenalis was 19.63% (21 out of 107). This study represents the first trial to explore cryptosporidiosis, giardiasis in equines as in Nineveh there is no survey of these intestinal protozoa.


Introduction
Cryptosporidium parvum and Giardia doudenalis are recognized as primary enteric pathogens in animals and human (1).The severity of enteric disease by either of these parasites ranged from self-limiting to a fulminating diarrhoea.These parasites can be transmitted by ingestion of contaminated food or water (2).
Numerous studies have been carried out in our locality concerning Cryptosporidium sp., in farm animals, Baker recorded the infection with cryptosporidiosis in calves (3), Albakry (4) in goats capra hircus in Nineveh governorate in lambs (ovis aries) by khalil (5), while in horses Equus caballus there is no study that deals with this parasite, in comparison to data for bovine and human cryptosporidial infection.
There is a few information concerning equine cryptosporidiosis, although this disease has been reported in various regions of the world, which is connected with diarrhoea in foals (6,7).
Furthermore, there is an important role of equine Cryptosporidium infection as a source of zoonotic disease (8).Some animals may carry Giardia organisms and animals appear healthy with no signs of disease, such hosts have a high potential rate for spreading the parasite.Infected animals can develop bloody diarrhoea or mucous stool often accompanied by gas production (9).
The first occurrence of Giardia sp., was reported in horses in the Czech Republic by (10).Therefore these study deals with those intestinal protozoa (Cryptosporidium sp., and Giardia sp.) in horses it is considered the first trial in our locality in equine family.The aim of this study was to investigate the prevalence of cryptosporidiosis and giardiasis in horses suffering from diarrhoea and those healthy, and also to determine the possible correlation between cryptosporidium and other parasite producing diarrhoea mainly Giardia in horses in Nineveh governorate.

Materials and methods
The study was carried out through a period of 12 months from January 2007 to December 2007.Atotal of 107 faecel samples of horse taken from both sex of equines family were collected from different regions in Nineveh governorate, samples were stored in clean plastic container covered with 2.5% potassium dichromate solution of preservation.Samples were tightly closed and stored at 4 ◌ ْ C till examination.
The following methods were used for detection of Cryptosporidium oocysts and Giardia cysts in faeces samples were stained with the following: wet mount (lugol's Iodine 1% and 5%) (11), and sheather's flotation technique were used for primary diagnosis of oocyst and cyst.Modified Ziehl-Nelseen stain (hot method) for further investigation for cryptosporidium oocysts (12).Giemsa stain was used for distinguishing Giardia cyst (13).The oocysts of cryptosporidium and cyst of Giardia were measured and identified as described by (5,14).
Chi-square X 2 , Fisher test F were used to compare the frequencies of positive and negative specimens (15).

Results
Twenty nine positive samples out of 107 (27.10%) for cryptosporidium infection, and 19.63% for Giardia infection.
The percentage of infection with Cryptosporidium 27.10%, and 19.63% with Giardia among the total examined specimens.
Also the highest rate Giardia infection was found in March (45.45%), and the lower rate in January and July (0.0%).There was no significant difference in the infection rate between the months of the year for Cryptosporidium and Giardia.
Table (2) shows the distribution regarding to age group, the highest rate of infection was found among 5-6 years (34.38%),followed by the group of 0 < 4 years (31.58%) and (27.59%) in group 7-8 years old, and the lowest rate of infection among the group of 9-10 years old was (14.81%).Statistical analysis shows no significant difference between age groups, for Cryptosporidium, while in Giardia doudenalis the rate of infection among age groups were high at 7-8 (24.13%) was followed by 9-10 years old group (22.22%) then the rate of infection among 0 < 4 age group was (15.79%) and the lowest rate of infection among 5-6 age group was (15.63%) no significant difference between age group for Giardia.
Ocular micrometer calibrated lens was used for measuring the oocyst of Cryptosporidium sp., the results revealed that the average of measurement of oocyst was 4.9-6.2µm which were diagnosed by sugar flotation method and modified Ziehl-Nelseen method the oocysts appears as bright rose -pink (Fig. 1).Cysts of Giardia doudenalis were measured, the average of cysts were 10-11 µm × 8-9 µm, best detected in feces concentrated by sugar flotation method with specific gravity (1.18) (Fig. 2).

Discussion
This is the first report of cryptosporidial infection in equines in Nineveh governorate.The infection was identified in 29 out of 107 animals from different regions in Nineveh.
Equine cryptosporidiosis was previously reported in other parts of the world and it is believed that is neither associated with the age of the horses nor with mode of life (16)(17)(18)(19).In Iraq there are many studies which were conducted in different host such as calves, lambs, dogs, cats and rabbits with recorded different percentages of infection with Cryptosporidium spp.and Giardia spp.(3,5,20,21).While in equines we have no studies in our locality that deals with these parasites.
In this study the percentage of infection with Cryptosporidium was 27.10%, this result was agreement with other studies which were occurred in different countries such as Germany, Poland, Texas, Colorado, USA and Louisiana and these studies were recorded different percentage of infection ranged from 0.33_100% (22)(23)(24)(25)(26)(27)(28)(29) according to the months of the year, there was a variation in the infection rate of Cryptosporidium (10-55.56%)and no significant differences between months of the year, these result were agreement with (3,(30)(31)(32).The differences in the percentage of infection in the months may be related to different factors such as number of samples, environmental condition, age, sex, immunity status, stress factor…etc.
The age distribution among equines in this area covered by this study is relatively even, rang from one to 10 years old in our study over all rate of cryptosporidial infection.According to the age, infection was high in the 5-6 years age group which is higher than those reported by (33) who reported (3.2%) in horses ranging from one to12 years only, and (13.9%) of horses aged 5> years.In Morocco (34)(35)(36) few donkeys with old age over 12 years were infected, the reasons behind this are numerous such as type of life either feral or domestic, or immunity status, or in the sampling which is mostly taken from adult animals, or due to the unsuitable hygienic.Those infected horses with Cryptosporidium could be significant source of the parasite, both for other animals including human and for the environment, in addition horse faeces that used for soil fertilization also enhanced the like hood of contamination of food and water sheds.
In this study the percentage of infection with Giardia doudenalis was19.63%this result was agreement with Kashash (20).Which recorded the infection rate in Baghdad was14.6 % also this result was agreement with other studies occurred in different parts of the world (10,37), while (18) was recorded percentage of infection with Giardia 4.6%, 5% in horses respectively.According to the age high percentage of infection in age 2-4 years 5% in the same region.
While in racing horses two up to four years of age from two studies in the surrounding of Prague Giardia cysts were found in seven horses (35%) out of a total 20 animals (38,39).Diaz (40) was reported high percentage of infection appears in several ages, while Kashash, Olsen, Wade (20,29,31) showed that the infection of Giardia may occurred in all ages.This is study reports for the first time the occurrence of Giardia sp., in equines of the Nineveh.

Table 1 :
Distribution of Cryptosporidium sp., and Giardia doudenalis according to the months of the year.

Table 2 :
Distribution of Cryptosporidium sp., and Giardia doudenalis according to the age.