Epidemiology and identification of Eimeria species affecting poultry in East Gojjam Zone, North West Ethiopia

Abstract Background Despite the expansion of modernized poultry farming in Ethiopia, the presence of high prevalence of Eimeria species is the bottleneck in the sector causing high morbidity and mortality rate in poultry. Objectives The objectives of this study were to estimate the prevalence and identify Eimeria species and investigate the major risk factors. Method A cross‐sectional study was conducted from November 2019 to April 2020 in East Gojjam Zone, North West Ethiopia. A total of 384 chickens were used. Both floatation and McMaster coprological techniques were employed. Univariate and multinomial logistic regression was used to calculate the odds ratio for the associated risk factors. Analysis of variance was used to analyse differences in Eimeria oocyst counts among the groups. Results Overall prevalence of Eimeria species in poultry from the study area was 26.5%. Age (OR = 0.25, p = 0.001), management system (OR = 12.44, p = 0.001) and production system (OR = 0.37, p = 0.001) were found significantly (p < 0.05) associated with the risk of Eimeria species in poultry. The mean Eimeria oocyst count was significantly different by age and management system (F = 6.526, p = 0.002), (F = 5.369, p = 0.005), respectively. The mean Eimeria oocyst count was significantly greater in 6–12 weeks (p = 0.004) and <6 weeks of age (p = 0.025). A total of 6 Eimeria species were identified. Eimeria tenella (46.07%), Eimeria necatrix (24.5%) and Eimeria acervulina (8.82%) were the most common Eimeria species encountered. Conclusion The prevalence of Eimeria species was higher in poultry in North West Ethiopia. Therefore, tailor‐made intervention is required to mitigate risk factors and reduce the prevalence of Eimeria species in poultry from the study area.


INTRODUCTION
Poultry production is one of the key livestock subsectors of Ethiopia.It plays an important roles in terms of generating employment opportunities, improving family nutrition and empowering women.It is a suitable business for low-income households due to the small quantity of land needed and low investment costs required starting up and running the operation (Jordan, 2001).In Ethiopia, total population of chicken is estimated to be 56.38 million.Of the total population in Ethiopia, 94.31%, 3.21% and 2.49% are indigenous, hybrid and exotic breeds, respectively (Central Statistical Agency [CSA], 2017).
Despite the high number of chicken available in Ethiopia, poultry diseases are major constraints to productivity and production causing economic losses to poultry farm sector in Ethiopia.Several infectious agents like viruses, bacteria and protozoan parasites cause poultry morbidity and mortality (Alemu, 2012).Eimeria species are the most important protozoan parasites causing poultry coccidiosis characterized by enteritis and bloody diarrhoea (Adhikari et al., 2008).
Coccidiosis has been a major cause of poor performance and lost of productivity in poultry and other farm animals.The infectious process is rapid 4-7 days in a faecal-oral route and is characterized by parasites replication in host cells with extensive damage to the intestinal mucosa (Taylor et al., 2007).After ingestion of sporulated oocysts, sporozoites are released that enter asexual and sexual cycles of development resulting in the emergence of 1000 of new oocysts in the intestines (Taylor et al., 2007).Soon, they sporulate and become infective for chickens (Bowman, 2009).Affected birds become depressed, have ruffled feathers, the wings droop, have diarrhoea, and tend to huddle (Aarthi et al., 2010).Food and water consumption usually decreased and may become emaciated and dehydrated (Kinung et al., 2004).Laying hens will experience a reduction in the rate of egg production.
In Ethiopia, E. necatrix, E. maxima and E. tenella are endemic in all parts of the country and affect many young growing birds (Hagos et al., 2004).In the past years coccidiosis used to be the most important cause of mortality in backyard and semi-intensive farms.An incidence of the disease was as high as 80% usually occurring in the form of outbreaks (Safari et al., 2004).Although in Ethiopia quantitative losses due to coccidiosis are not well documented, reports from other countries indicate that the disease contributes to 8.4% and 11.86% loss in profit in large-scale farms and small-scale farms, respectively (Lew et al., 2003).
The specific identification of Eimeria species and strains is important for diagnosis and control, as well as for epidemiology and biological studies of the population (Morris & Gasser, 2006).The Eimeria species have been identified by morphology and/or morphometry of their sporocysts and oocysts as well as their modes of development, and assessing the site and extent of the pathological lesions in the intestine of chicken (Aarthi et al., 2010).However, these methods of identifications are costly, time-consuming, require skilled personnel and can be unreliable under the circumstances of mixed-field infections, particularly when the overlap in biological and morphological characteristics make the unambiguous identification and differentiation of Eimeria species impossible (Al-Idreesi et al., 2013;Carvalho et al., 2011).Eimeria oocysts can be isolated according to the method of Davies et al. (1963) and can be identified by sporulation time using potassium dichromate solution for sporulation and preservation of the parasite (Ryley et al., 1976)

Study area
The present study was conducted in three purposely selected districts of Eastern Gojjam Zone, Amhara National Regional State, Ethiopia, which is located at 10

Study design
A cross-sectional study design was conducted from November 2019 to April 2020.Both local and exotic chicken breeds which are kept under the backyard and semi-intensive husbandry system were included in the study.

Sample size determination and sampling procedures
The sample size used for this study was calculated according to Thrusfield (2005), assuming a 50% expected prevalence, with 95%

East Gojjam Zone
Awoble district (28 peasant associations) D/Markos city (11 peasant associations) Gozamen district (25 peasant associations) 8 peasant associations were selected 3 peasant associations were selected 7 peasant associations were selected 24 villages and 72 household were selected 12 villages and 24 household were selected 21 villages and 63 household were selected 133 poultry were sampled 132 poultry were sampled 119 poultry were sampled

Sample collection
About 3 g of faecal sample were collected from the upper surface of the litter immediately after the dropping of faeces using sterile disposable plastic gloves.During sampling, farm type, date of sampling, age, sex, breed, management system and production system were recorded for each chicken on a recording format.The sample was placed in a labelled clean universal bottle using 2.5% (w/v) potassium dichromate solution and transported in a cool box to Bahir Dar animal diseases diagnostic and investigation laboratory centre on the same day of collection, and preserved at refrigeration temperature until processing within 48 h of arrival.

Coproscopical examination
Qualitative faecal examination was conducted using flotation technique for the detection of the oocysts of Eimeria using concentrated sucrose solution (Sheather's sucrose solution) with specific gravity of 1.27 as described by Hendrix (1998).The slides were examined using a compound microscope (10× objectives).For identification of Eimeria species, positive samples were taken to the National Animal Health Diagnostic and Investigation Center, Addis Ababa, Ethiopia.
Identification of Eimeria oocysts was based on the morphological features of the oocysts (size of oocyst and sporocysts, shape, colour and texture of oocyst wall, presence or absence of micropyle and polar cap) with the aid of taxonomic keys (Kennedy & Kralka, 1987;Sommer, 1998;Soulsby, 2005).Oocysts size was measured under ocular eye piece that is calibrated with a micrometre under a 40× objective of a microscope.Quantitative faecal examination was performed on positive samples using McMaster technique to determine the number of oocysts of Eimeria per gram of faeces (OPG) (Hendrix, 1998;Kaufman, 1996;Carvalho et al., 2011).The severity of the Eimeria infection was evaluated as mild, moderate and severe with OPG level of <1800, 1800-6000 and >6000 OPG, respectively (Carvalho et al., 2011).presence and OPG values of the single species suffer from a high uncertainty that is going to affect the analysis if referred to single species.

Statistical
A statistically significant association between all variables was set at p < 0.05.

TA B L E 1
Prevalence of Eimeria species in poultry in the subgroups of animals identified by the associated risk factors.

Association of Eimeria species prevalence with risk factors
A total of 384 pooled faecal samples were examined for Eimeria species from the 3 selected areas of East Gojjam Zone, Northwest Ethiopia.
The overall prevalence of Eimeria species found in this study was 26.6% (102/384).In the present study, the highest prevalence of Eimeria species in poultry (10.2%) was recorded in Gozamen district.Besides, higher prevalence values were found in exotic breed, in younger animals, in females, and in farms characterised by a poor management and a semi-intensive system (Table 1).
A total of six potential risk factors were tested by using multinomial logistic regression analysis.Of these, three potential risk factors were significantly (p < 0.05) associated with Eimeria species in poultry.These included animals' age, quality of the management and production system (Table 2).Odds ratio of Eimeria positivity in poor management system was 12.44 times higher than good management system (Table 2).

Association of Eimeria oocyst output with risk factors
Mean counts for each subgroup of animals and the significance of the differences are shown in Table 3.The ANOVA indicated that there was the existence of significant differences in the mean Eimeria oocysts counts among the age categories of poultry (F = 6.526, p = 0.002), with significantly greater counts in younger animals (age of <8 weeks), both TA B L E 2 Potential risk factors significantly associated with the prevalence of Eimeria species in poultry using multinomial logistic regression analysis in the study area.

Eimeria species identification
During sampling, 384 pooled faecal samples were collected according to the established protocol and 102 samples were tested positive for Eimeria species.For each sample the coproculture and identification of species was performed.A total of six Eimeria species were identified as displayed Figure 2a-f.E. tenella and E. necatrix were the more prevalent species and accounted for 46.1% and 24.5%, respectively.

DISCUSSION
In the present study, the overall prevalence of  Fikre et al. (2005) in central Ethiopia (11.0%) and by Firmaye et al. (2015) in the same area (19.5%).The variation of these findings may be due to the differences in agro-ecology and climatic conditions in the study areas, although most of these studies were conducted in the highland part of Ethiopia.
Most probably, many confounding factors influenced the outcome of the different studies, such as different management system, seasonal variation, study design and different target animal.
Concerning risk factors, there was a significant (p < 0.05) association of age of chicken with the risk of Eimeria species infection in the present investigation.This finding is in agreement with most of the international literature, which consider the young chickens at major risk for Eimeria species infection (Taylor et al., 2007), and with other reports in Ethiopia.In particular, both Firmaye et al. (2015)  weeks of age (Allen & Fetterer, 2002).However, it might be associated also with the presence of another immunosuppressive disease, such as Gumboro diseases (Hachimi et al., 2008).
The more important risk factor for Eimeria species infection found in this study was the management system that resulted highly influencing the presence of the infection (animals kept with a poor management are 12.44 times at risk compared to the ones with good management) and the oocyst output (p < 0.01), resulting in a higher environmental contamination.This finding is in line with other reports in Ethiopia (Anteneh et al., 2019;Belaynew et al., 2016;Tekalign & Teshome, 2018) that found poor management systems at high risk of acquiring Eimeria species infection than good and medium management systems.This might be due to high exposure to environmental oocysts due to many managerial factors, such as lack of clean litter, poor ventilation, absence of isolation pen or defective feeders and waterers that increase the risk of oocyst ingestion.Besides, in poultry farm with poor management, the lack of vaccination for other immunosuppressive diseases like Marek's disease may reduce resistance to Eimeria species by interference with coccidial immunity.Consequently, coccidiosis may not respond to either preventive or curative chemotherapy in a normal way because the normal self-limiting immunity to Eimeria species fails to become established.
Finally, there was difference of prevalence of Eimeria species in poultry also between sex, with a slightly high prevalence of coccidiosis were recorded in male than female chicken.This finding is in line with previous reports in Ethiopia, which reported higher prevalence in male than female (Anteneh et al., 2019;Kefyalew & Hailegebrael, 2018), although other studies disagree with our results (Eshetu & Nigussu, 2019) reporting a higher prevalence of Eimeria species in female than in male.The absence of a statistically significant difference between males and females might be due to an equal chance of exposure for the parasite infection.This indicates there is no significant biological resistance variation with the sex (Pinard- Van Der Laan et al., 1998).
In the present study, positive samples were further investigated for Eimeria species identification based on morphological features of different Eimeria oocysts.Worldwide, more than 13 Eimeria species are reported in chicken.However, in the present study, six Eimeria species were isolated, with E. tenella (47%) followed by E. necatrix (25%) and E. acervulina (9%) as the most prevalent species.Except E. mitis, this result is in line with Solomon (2006), who reported the other five Eimeria species.We may consider that the six species of Eimeria, namely
Package for the Social Sciences (SPSS) statistical software version 20 was used to run logistic regression.Initially, the association of eight individual risk factors with an outcome variable was screened by univariate logistic regression.Those variables significantly associated with the outcome variable at p < 0.05 significance level in the univariate analysis were recruited for multiple logistic regression to see their independent effect.In the multinomial logistic regression analysis, a model was fitted for each outcome variable by stepwise backward elimination of insignificant variables (p > 0.05).ANOVA was used to analyse differences in mean oocyst count.It is not possible to perform any factor analysis for each Eimeria species, since the estimation of the relative percentage of each species, since only a limited number of oocyst were identified for each sample.Therefore, both compared to the ones with 8-20 weeks (p = 0.025) and to the oldest ones with >20 weeks (p = 0.004), as indicated by the Bonferroni pair-wise comparison.Similarly, different management systems were significantly different in the mean count of Eimeria oocysts (F = 5.369, p = 0.005), with the poor management system showing higher outputs, both compared to the medium (p = 0.001) and to good (p = 0.0001) ones.However, chicken under good and medium management system did not show a significant difference (p > 0.05).Instead, there were no significant difference in the mean Eimeria oocysts count in poultry between sexes (F = 0.062, p = 0.8040), breeds (F = 3.108, p = 0.079) and production systems (F = 3.173, p = 0.076).

E
. tenella together with E. maxima (2.94%), or E. necatrix (3.92%).The distributions of Eimeria species as a single and mixed infection are shown Figure 3.
Potential risk factors associated with the Eimeria oocyst output (OPG) using ANOVA test.
TA B L E 3 Distribution of Eimeria species as a single and mixed infection.in central Ethiopia (25.8%) by Hagos et al. (2004).Generally, these prevalence values are halfway in the Eimeria species prevalence rates encountered in poultry in Ethiopia, with values ranging from about 10% to 80%.Higher prevalence values were reported by Dinka and Yacob Eimeria species in poultry was 26.6% which is comparable to what reported in a study done in Arsi zone (22.6%) by Getachew et al. (2008) and in another study performed F I G U R E 3