Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2014 Vol. 4 (2) April-June, pp. 355-360/Shojaei Research Article EVALUATION OF EXPERIMENTAL COCCIDIOSIS IN TWO COMMERCIAL STRAINS OF BROILER CHICKEN BY LESION SCORING AND OPG QUANTITATION *S.S.R. Shojaei Department of Pathobiology, Faculty of Veterinary Medicine, Islamic Azad University, Karaj Branch, Karaj, Iran *Author for Correspondence ABSTRACT The study was initiated to compare of two internationally reputed broiler strains in experimental coccidiosis by quantitation of defecated eimerial oocysts and intestinal lesions scoring under the local environmental and management conditions. 40 one-day old male chickens of Arbor Acres strain and Ross308 strain (20 chicks from each strain) used in this study and were divided randomly into four control and challenge groups. Feed and water were provided for ad libitum consumption. At 15 d of age, chickens of challenge groups (from each strain) were individually inoculated with a mixture of 50000 of sporulated oocysts of 4 species including of E. acervulina (20%), E. maxima (40%), E. tenella (25%) and E. necatrix (15%) and from the fourth day after Eimerial challenge, faecal droppings (litter samples) were collected 10 days consecutively for counting oocyst per gram (OPG). Also at day 7 after Eimerial challenge, 5 chicks from each challenge groups were euthanized and intestinal lesions were scored. The results indicated that in both challenge groups, there was an increasing process of OPG in days of 4-7 post challenging and the pick level of OPG was seen at seventh day after inoculation. The average of OPG in the Arbor Acres group was lower than the group Ross in days post inoculation and this difference was significant (P<0.05). Mean lesion scoring of intestine of Arbor acres and Ross challenge groups was 2.0 and 2.6 respectively. Lesions scoring difference was significant (p<0.05). According to the obtained results in this study and since oocyst index and lesions scoring almost always are considered as the most important indicators for coccidiosis evaluation, it can be realized that in the same surveillance condition the, regarding the severity evaluation of coccidiosis, Arbor Acres strain broilers shed less oocysts and had lower lesion scores than Ross308 strain broilers. Keywords: Arbor Acres, Ross308, Broiler, Coccidiosis, OPG, Lesions score INTRODUCTION Coccidiosis of chickens, one of the most costly diseases affecting the poultry industry worldwide, is caused by infection with one or more of 7 species of the intracellular protozoan parasite Eimeria (Lee et al., 2010; Sharman et al., 2010). Eimeria infections occur when susceptible chickens ingest viable sporulated oocysts from contaminated litter. The ingested oocysts invade the intestinal epithelium in a region-specific manner, causing variant pathogenicity in poultry ranging from reduced feed conversion, weight loss,dysentery, enteritis, emaciation, drooping wings, poor growth to, at times, high rate of mortality and morbidity (Shirzad et al., 2011; Lee et al., 2010; Hadipour et al., 2011). After at least 2 generations of asexual reproduction, several hundred thousand Eimeria oocysts can be produced from a single oocyst, and are excreted in feces over several days or weeks (Morris et al., 2006). The oocysts have cyst walls that are highly refractory to environmental extremes and disinfectants. Therefore, the oocysts can be transported mechanically by animals, insects, dust, and contaminated feed, water, and other fomites (Stotish et al., 1987; Morris et al., 2006) Eimeria spp. is omnipresent and can survive in infected birds and the environment for long times (McDougald, 2003). It causes high mortality in young chicks because most of the Eimeria spp affects birds between the age of 3 and 18 weeks (Nematollahi et al., 2009). The tremendous growth of poultry industry in Iran is hampered by various factors and prevalence of various diseases in poultry which is of main concern. Among the various diseases, protozoan parasite of © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 355 Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2014 Vol. 4 (2) April-June, pp. 355-360/Shojaei Research Article the genus Eimeria causing coccidiosis is considered as one of the most common parasites (Hadipour et al., 2011; Shirzad et al., 2011). Due to higher stocking densities and intensive husbandry practices, its incidence is being increased in poultry (Nnadi and George, 2010). The genetic selection of poultry for superior growth rate has arguably been the primary method for increasing productivity. However, many studies have been shown that such selection may be coincidentally accompanied by decreased resistance to diseases or changes in immunological response (Makram et al., 2010; Li et al., 2001; Fathi et al., 2003; Huff et al., 2005). Vaccination programs alone cannot cope adequately with infectious diseases. A combination of vaccination and genetic resistance is essential to maximize the protection from diseases. The endeavor of this work was to compare of response of two modern broiler strains to coccidiosis by OPG counting and lesion scoring during an experimental infection MATERIALS AND METHODS This experiment was carried out at poultry research farm, faculty of Veterinary Medecine, Islamic Azad University, Karaj branch. 40 one-day old male chickens of Arbor Acres strain and Ross strain (20 chicks from each strain) were chosen and divided randomly into four control and challenge groups (10 chicks per group). After leg labeling, the chicks were reared under similar managerial, housed in cages covered plastic floor and according to their strains, fed ad libitum and kept in breeding room with appropriate distance. They were fed a corn-soybean meal commercial diet containing18%-20% crude protein and 2800 kcal ME/kg diet. No antibiotic growth promoter or additives was included in the feed. The temperature was maintained at 30-32 °C for the first week and was reduced by 2°C on weekly basis. Lighting was provided 23 hours light and one hour dark. No vaccine was used during the test period. Ambient ventilation and other environmental conditions fully met the requirements laid down in the technical instructions of each strain. In order to purification and standardization of doses of the inoculum for experimental infection, four selected species including E. acervulina, E. maxima, E. tenella and E. necatrix were propagated in 8-wkold Eimeria-free chickens by oral inoculation and the oocysts were recovered from feces which were collected for up to 8 days post inoculation, sporulated and stored in 2% potassium dichromate at 4°C before inoculation (Conway, 2007). The appropriate dose had been estimated in a preceding test inoculation from the results of these initial tests (Shirley, 1995). Then, at 15 d of age, chickens of challenge groups (from each strain) were inoculated orally with a dosage of 50000 of the mixed sporulated oocysts of 4 species including of E. acervulina (20%), E. maxima (40%), E. tenella (25%) and E. necatrix (15%) From the fourth day after Eimerial challenge, samples of the chickens feces were taken from their cages for 10 consecutive days. With regard to control of probable infection, OPG counting was performed on days 5 and 14 before inoculation. The number of oocysts was quantitated by Mac master method (Conway, 2007). On the other hand, since another current valuable diagnostic method is lesion scoring, an interpretation of subclinical coccidiosis based on macroscopic visible lesions caused by Eimeria (Johnson and Reid, 1970), at day 7 after inoculation, we selected randomly 5 chicks from each challenge group and intestinal lesions were scored after euthanizing. For statistical analysis, data were subjected to a one-way analysis of ANOVA test. When significant differences among means were found, data were analyzed by Post-hoc and compared by Tukey test. RESULTS A comparative study was undertaken on two reputed broiler strains namely, Arbor Acres and Ross308 regarding production and defecation of Eimerial oocysts and intestine lesion scoring, as well. © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 356 Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2014 Vol. 4 (2) April-June, pp. 355-360/Shojaei Research Article Table 1: OPG comparison between Ross308 and Arbor Acres strains in different days post infection Groups Daily oocyst per gram of feces during a 10-day period after Eimerial inoculation 4 5 6 7 8 9 10 11 12 13 0 0 0 0 0 0 0 0 0 Uninoculated 0 group 55200 65000 175000 181000 122000 87000 47500 15000 9800 3500 54000 67000 183000 179000 126000 88000 26500 16000 9600 2500 52500 68000 180000 179000 123000 85000 28000 17000 9750 4000 Group 53500 64000 178000 180500 125000 84000 26000 14000 9650 2000 includes Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Ross308 53000 66000 179000 180000 124000 86000 27000 15500 9700 3000 strain 50000 50000 145000 151000 98000 72000 25000 13500 7900 1800 51000 52000 113000 150500 100000 70000 24000 125000 7700 1600 47000 51500 146000 153000 97000 71500 26000 14000 7500 1650 Group 48000 50500 142000 149000 101000 70500 23000 12000 8100 1750 includes Arbor Acres Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 49000 51000 144000 151000 99000 71000 24500 13000 7800 1700 strain Table 2: Gross Lesion Score for 5 randomly selected chicks from each strain 7 days post inoculation GLS at Day 7 after challenge Selected chickens Arbor Acres strain Ross Strain 2 3 Chick 1 2 3 Chick 2 2 3 Chick 3 1 2 Chick 4 3 2 Chick 5 2 2.6 Mean GLS Figure 1: Oocysts per gram feces in 10 days post experimental infection by Eimeria oocysts in Arbor Acres and Ross308 strains. OPG on vertical axis has been plotted against days post Eimerial inoculation on horizontal axis © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 357 Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2014 Vol. 4 (2) April-June, pp. 355-360/Shojaei Research Article The results of microscopic examination of 90 fecal samples are depicted in the Table 1. The OPG of preinfection period of all groups and control group was zero at all days of sampling such as we expected, which is indicative of the appropriate qualitative control of the experiment process. In the challenge groups, there was an increasing process of OPG in days of 4-7 post challenging and the pick level of OPG was seen at seventh day after challenging. From day8 to 9, decreasing of OPG was happened, this decreasing continues with mild, fast and mild process to day of 13, respectively. In addition, the Mean Gross lesion scoring(GLS) of intestinal damages in Arbor Acres and Ross challenge groups was 2 and 2.6 respectively which were evaluated by Johnson and Reid method (1970) (Table 2). Discussion According to the Table 1, OPG of uninfected control was zero in all experiment days which were representative of proper control process during the study. In both strains on days 4 to 13 post infections, a raising trend, one peak and then a decreasing trend was seen, respectively. Also in both strains the highest OPG was measured in three days 6, 7 and 8 after the challenge and between these days, day 7th dedicated the peak of mean OPG to itself by 180000 in the Ross group and 151000 in the Arbor Acres group. Moreover, the average of OPG in the Arbor Acres group was lower than the group Ross while this difference was significant according to t-test. This significant difference was 95% (p<0.05) in days 4, 9 and 10 post challenge and in the other days except day 13after infection was 99% (p<0.01). On day 13, the difference of counted oocysts declined (p=0.064) (p>0.05), so this difference with the confidence level of 95% is not significant whereas the Arbor acres group had lesser OPG in compare to Ross group. Besides OPG counting, lesion scoring is considered as another common diagnostic method in evaluation of coccidiosis, therefore this index was used in evaluation of two mentioned strains in this study, as well. Total lesion scores of 5 selected chicks from each challenged group indicated that Arbor Acres inoculated group showed mean GLS of 2.6 whereas the average GLS of Ross308 infected group was calculated 2 and this difference was significant among mentioned groups. Regarding both groups were reared under the complete same condition of growing and with consideration of obtained results from both oocysts output and lesion scoring, it could be realized that the Arbor Acres has more partly resistance to coccidiosis than the Ross strain since the Arbor group shed lesser oocysts and had less intestinal lesions than the Ross group in all days post infection. Actually there is no evidence about comparison of OPG and lesion scoring between mentioned strains yet, but in the aforementioned studies, these strains have been compared of other indexes which showed that totally Arbor Acres performance is better than Ross in poultry industry Hussain et al., (1993) conducted a study based on comparison of two stains Ross 308 and Arbor Acres in feed conversion ratio (FCR) and consequently the best FCR was observed for strain Arbor Acres. In another study, performance of these two commercial broiler strains which were reared under local environmental conditions, were examined and compared which each other (Zahid and Hussain, 2002). The observations were recorded on body-weight gain, feed consumption, general health and mortality. Also FRC was computed from the data and dressing percentage was determined for all broilers separately. The average weekly live weight of Arbor Acres was significantly higher than chicks of Ross strain while the statistical analysis revealed no substantial difference in feed consumption and FCR among the mentioned broiler strains. Although statistically there were no significant differences in mortality rate for these strains, apparent basis mortality was substantially higher in Ross as compared to Arbor Acres. Similar findings were reported by Chew (1987) who compared Ross and Arbor Acres strains and found no significant difference in mortality percent. The dressing percentage of Ross strain was considerably higher (P<0.05) as compared to Arbor Acres. Similar results were obtained by Singh et al., (1981) who reported the studies on phenotype correlations among live weight and dressed weight in poultry. Iqbal et al., (2012) compared the performance of these two economic strains and economic traits measured were body weight, feed intake, feed conversion ratio (FCR), mortality, antibody titres, dressing percentage and economic evaluation as cost of broiler per kilogram live weight produced. Although in © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 358 Indian Journal of Fundamental and Applied Life Sciences ISSN: 2231-6345 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jls.htm 2014 Vol. 4 (2) April-June, pp. 355-360/Shojaei Research Article some cases no significant difference was shown between these two strains, overall Arbor Acres indicated better performance as compared to Ross. Evaluation of OPG variation during different days post inoculation was another aim of this study. As can be seen in figure 1, an ascendant and descendant trend occurred in both experimented groups respectively during the study, thus from this aspect no difference was among them. To state the matter differently, the OPG peak in both groups was in days 6 and 7 after challenge (with a negligible difference) since the life cycle accomplishment of the most Eimeria species is occurred at these days (days 6 and 7 post infection) (Velker, 2011) and oocysts are produced and consequently shed by destruction of epithelial cells through poultry feces. In addition, the reduction pattern after this peak is related to natural decrease of oocysts production since the number of merozoites which enter to gametogony phase will decline. Because gametogony starts after the second or third schizogony and after that if no re-infection occurs the number of oocysts will decrease gradually as the intrinsic potential of merozoites declines naturally. As in the present study chicks were reared in chicken cage systems in order to keep the operation clean, efficient and profitable, they did not have the chance of re-infection by feed-pecking of scattered grains on the ground. As a result, the presented survey could be an authentic pattern for future complementary studies. According to the obtained results of this study and since oocyst index and lesion scoring almost always are considered as the most important indicators for coccidiosis evaluation, it can be realized that in the same surveillance condition the severity of coccidiosis in Arbor Acres is less than in Ross. In other words, the Eimerial oocyst shedding potential and the intestine lesions induced by coccidiosis which are substantial factors in continuance procedure of coccidiosis, occurance of this disease and economic losses due to poultry coccidiosis, are lower in Arbor Acres as compared to Ross. Finally, in order to access a more accurate assessment and comprehensive comparison of vital reactions of these two strains against coccidiosis, other important indexes like Carotenoid level and absorption coefficient should be taken in to account in the future complementary studies. 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