Prevalence Rate of Isolated Cryptosporidium Spp. of Broiler Chicken in Al- Qadisiyah Province

Submission Track Abstract This study has been carried out to determined the prevalence rate of cryptosporidiosis in broiler flocks. 150 fecal samples from 30 broiler flocks (5 samples for each flock) in different areas of ALQadisiyah province were used. They were collected starting of January till November 2016 with two age groups of chickens, (1015 and 30-35) days old. Samples were examined with Modified Ziel -Neelsen stain to detect the positive samples by microscopic examination. After that, a nested-PCR technique was performed on 60 samples. Results of microscopic examination showed that total infection rate of Cryptosporidium was 29.33% . The highest rates of infection were recorded in 10-15 days group and during spring season, infection rates reached were 35.71% and 34.28% respectively. The lowest rates were observed in 30-35 days group and during summer season were 23.75% and 20% respectively. No significant differences within percentage in both two age group or among percentage in seasons at level (P < 0.05) were recorded. According to Nested Polymerase Chain Reaction test, the results showed that overall percentage of infection is 20%. The highest rate of observed infection is 23.33% in 10-15 days aged group, while the lowest rate of infection is (16.67 %) in the other age. No significant differences was observed between percentage in ages. On the other hand, result of comparing nested-PCR test with microscopic examination shows that there are no significant differences between the percentage of both tests. In conclusion, cryptosporidiosis is widespread in broiler flocks in AL-Qadisiyah province, but there is no significant impact found concerning the relationship between infection rates and age of chickens or seasons of the year. Received : 21/8/2017 Final Revision : 1/3/2018


Introduction
Cryptosporidiosis is one of the essential protozoan infections in birds. It affects a major number of avian species across various continents (1). It is causing either clinical or subclinical infections (2). There are three forms of Avian Cryptosporidiosis: respiratory form, intestinal form and renal form (3). Clinical signs of a respiratory form include cough, nasal discharge, sneezing, dyspnea pneumonia and thickening of air sacs (3). Clinical signs of enteritis form include yellow greenish diarrhea, offensive odor, depression, low feed consumption and high water consumption (4). Transmission of Cryptosporidium parasite is through ingestion of oocysts of the infected individuals by contaminated water and /or food (5,6). The parasite is in Phylum Apicomplexa and part of the group of parasites commonly referred to as Coccidia, which includes cryptosporidium, Eimeria, Cyclospora and Isospora (7). Cryptosporidium infection represents the main public health concern of water utilities in developed nations (8). Identified species of Cryptosporidium in birds are Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium Meleagridis and Cryptosporidium galli (9). The prevalence rate in broiler chickens varies in different countries. In Iraq, 20.71% existed in Karbala (10). In Iran, infection rate is 23.8% (11). In Henan, China prevalence rate was 3.4% (12). In Tunisia, the prevalent rate is 4.5% (13). In Greece, Cryptosporidium oocysts were found in 24.2% of the examined broiler chickens (14). Due to the importance of the cryptosporidiosis on the health of poultry, this study is designed to detect the parasite in the broiler flocks of AL-Qadisiyah province and study the effect of ages and seasons on the prevalence of cryptosporidiosis.

Materials and Methods
Feces samples collection: 150 Fecal samples from 30 broiler chickens flocks (5 samples for each flock) in some regions of AL-Qadisiyah province were used. They have been collected from the beginning of January until November 2016 with two age groups (10-15 and 30-35) days old broiler chickens, the study includes four seasons (60 days for each season) in which they are divided as shown in table (2). The fecal sample has been transferred to a clean, dry plastic container and transported to the laboratory for the analysis. Microscopic examination the oocyst is detected by examining each sample by pigmented the swab by Modified Ziel -Neelsen (MZN) (15) . Subsequently a Nested polymerase chain reaction test has been performed on 60 samples. The data have been analyzed by SPSS program, using Chi-square test(X²).
DNA Extraction DNA has been extracted from feces samples by using (Stool DNA extraction Kit, Bioneer. Korea). The extraction has been done according to company directives by using stool lysis protocol method with Proteinase K. Subsequently, the extracted DNA has been checked by NanoDrop spectrophotometer, Thence stored (-20C) at refrigerator until used in PCR amplification. Nested Polymerase chain reaction PCR technique has been performed for diagnostic of Cryptosporidium parasite. based on 18S rRNA gene by using specific primers are designed by (16). the first round primers forward primer (GACATATCATTC AAGTTTCTGACC) and reverse primer (CTG AAGGAGTAAGGAACAACC) has been amplified (763bp) product size and the nested primers forward primer (CCTATCAGCTTTA GACGGTAGG) and reverse primer (TCTAAGAATTTCACCTCTGACTG) has been amplified (587bp) product size . These primers are provided by (Korea: Bioneer company). The PCR positive samples of first round have been used in nested amplification at the same amplification condition to amplified (587bp) product size.

Result and Discussion
Diagnostic characterization of cryptosporidium spp of microscopically examination by using Modified Ziel -Neelsen stain when examined under high oil emersion (100) lens of microscopic as in figure (1) shows oval-shaped or spherical objects with a color red or dark pink on a blue ground. Table ( 1) shows no significant differences in infection rates between age of chickens (10-15 and 30-35) days old. The results show that the highest rate of infection (35.71%) that is observed in the ages 10-15 days, While the lowest rate ( 23.75%) is in 30-35 days . This is consistent with the results of (12) which are observed in broiler chickens aged from 1 to 20 days have the highest rate of infection more than from 21 to 60 days. But these results do not agree with what (10), who found that the highest rate of infection at the age of five weeks (28.72%) and the lowest at the age of two weeks (13.18%). However, the minimum prevalence rate was in the younger age group (11). Table (2) shows that there are no significant differences among the seasons. the highest rate (34.28%) is seen in spring ,While the lowest rate ( 20%) is in summer and (32.5) (31.42) are seen in winter, autumn respectively. This is partly in line with what (12) have found spring season is the highest rate of infection in chickens and decreases significantly in autumn and summer seasons. While winter season is the lowest rate of infection. Table (3) reveal the results of nested-PCR as showed in figure (2) .The results show that the highest rate of infection (23.33%) is observed in the age group of 10-15 days , while the lowest rate ( 16.67%) is in 30-35 days of age with no significant differences between the percentages in both two age group at level (P < 0.05). The results of examination show that among (150) samples examined microscopically, 44(29.33%) have given positive. While the total infection rate was 20% (12/60) in the nested-PCR test and no significant differences between the percentages in both tests at level (P < 0.05) Table (4).
The different prevalence rates in broiler chickens between different studies such as 3.4% in broilers (12). The overall infection rate of Cryptosporidium was 10% (17). The percentage of infection is 20.71% (10). Total infection rate is 23.75% (11). The difference in prevalence rates observed may due to the animal management differences (18). On the other hand, the use of different diagnostic methods may also be responsible.   Total  60  12 20 Nonsignificant differences at P < 0.05 due to X 2 tab. = 3.84146 > X 2 cul. = 0.27799. 20 A Nonsignificant differences at P < 0.05 due to X 2 tab. = 3.84146 > X 2 cul. = 1.14261.