Detection of Eimeria Species in Naturally Infected Poultry using Multiplex Polymerase Chain Reaction in Chhattisgarh, India

Among the important diseases of poultry, coccidiosis produces serious problems and heavy economic losses to the poultry industry across the world (Jadhav et al., 2011). The disease is caused by the coccidia parasites of the genus Eimeria. There are seven recognized species viz. E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella which commonly occur in India (Shivaramaiah et al., 2014) and abroad.
       
The prevalence of poultry coccidiosis has been reported from the different countries including India. The favourable climatic condition of India and other factors like poor management is responsible for high rate of coccidial infection in poultry which has been reported time to time by several workers and almost from every part of India (Sharma et al., 2015; Salam and Wani, 2021). The severity of the disease, on the other hand, is determined by the species of Eimeria involved. In a subclinical form, however, it may cause immune-suppression in chicken, which is responsible for secondary infection. Thus, disease management and immune function maintenance for maximum performance, growth and production in the poultry industry are critical requirements for profitable farming. Besides, mixed infection i.e. more than one species is common (Kalita et al., 2021) which will cause different level of pathogenesis depending upon the species present in the birds. Therefore, species identification is required for successful control of the disease which is problematic by using morphological methods. Hence, an alternative diagnostic technique in particular DNA based molecular technique is required to overcome the limitations of traditional methods. Therefore, the objective of this study was to detect the prevalence of Eimeria spp. in poultry farms of four important poultry producing districts of Chhattisgarh.

Sample collection
 
The research work was conducted during 2021-22 at the College of Veterinary Science and Animal Husbandry, Dau Shri Vasudev Chandrakar Kamdhenu Viswavidyalaya, Anjora, Durg, Chhattisgarh. The faecal samples were collected randomly from 3 organized farms with 15 samples from each farm and 15 samples from 6 unorganized poultry farms of each district. A total of 240 fecal samples were collected from 12 organized and 24 unorganized farms of 4 districts of plain region of Chhattisgarh. The samples were collected in sterilized polythene zipper bags and brought to the laboratory by placing in ice jars for further investigations (Manjunatha et al., 2023). Both the direct and Willis techniques were used to diagnose and to separate Eimeria oocysts in faeces as per the method described by Soulsby (1982). The faeces collected from each group were thoroughly mixed using pestle and mortar. Then, an emulsion of faeces was made using equal amount of water which was sieved using wire mesh to remove the coarser particles. Two milliliters of each emulsion were taken in a 10 ml sterile plastic bottle and the saturated sugar solution was added up to the brim of the bottle and a cover slip was placed on top of each with taking care to exclude air bubbles. The bottles were left upright for 15 minutes. The oocysts adhered on cover slip were then collected by means of rinsing.
 
Sporulation of oocysts
 
The Oocysts collected during floatation by Willis method were pooled farm-wise and then washed 3 times in water using centrifuge machine at 2000 rpm for 3 minutes each to remove the sugar. These oocysts were then sporulated by taking in a cleaned Petri dish containing 2.5% potassium dichromate (K₂Cr₂O7) (Munir et al., 2018; Murshed et al., 2023) solution to avoid fungal growth and left for 5 days at room temperature with frequent aeration. The Sporulation was checked under microscope and the number of sporulated oocysts per milliliter of the solution was recorded by using Mc Master technique before storing at 4°C.
 
Extraction of genomic DNA from oocysts
 
The DNA extraction from sporulated oocysts of coccidian parasites was done with DNeasy blood and tissue kit (Qiagen, Germany) as per kit protocol (Kaya et al., 2007) with little modification. A 300 μl of solution containing 1 × 10⁵ oocysts from each sample was centrifuged at 10,000 rpm for 2 minutes to precipitate the oocysts. The supernatant was discarded and the pellet was resuspended in 200 μl nuclease free water after 3 times washing with PBS. Equal volume of glass beads measuring 0.25-0.5 mm in diameter (Sigma-Aldrich, USA) were added and vortex vigorously for 12 minutes to break oocyst and sporocyst walls. Initially, 180 μl of lysis buffer (ATL) was added followed by adding of 20 μl Proteinase K which were mixed by vortexing and incubated at 56°C for 3 hours. Rest part was done as per Manufacturer’s protocol.
 
Identification of Eimeria spp. by multiplex PCR
 
The multiplex PCR was used for identification of the Eimeria species of poultry using extracted DNA samples as described by Fernandez et al., (2003) with slight modification. Initially, the PCR amplification was standardized separately for each species using specific primer pairs to have a common reaction for all seven species. Thermo cycling conditions were set with the denaturation step at 96°C for 5 min followed by 30 cycles at 94°C for 1 min, 64°C for 2 min and 72°C for 1 min with a final extension at 72°C for 7 minutes.
       
Once the above conditions were standardized, all the primer pairs were put together in a single 35 μl reaction mixture containing 800 µM dNTPs, 5 U Taq Polymerase, 2.4 mM MgCl₂, 5.6 µl of 1.6X buffer, 0.9 µM E. brunetti, 0.7 µM of E. acervulina, E. praecox and E. necatrix, 0.6 µM of E. tenella, E. maxima and E. mitis, 3.0 µl of genomic DNA and 13.2 µl of nuclease free water for multiplex PCR with the same cycling conditions as described above. The amplification of specific PCR products was checked by gel electrophoresis in 2% agarose gels stained with 0.5 μg/ml ethidium bromide and visualized under gel-Doc UV-transilluminator.

Multiplex PCR was carried out using primer pairs of seven species to know the species present in the poultry of plain region of Chhattisgarh. It revealed that there were only 1-3 species of Eimeria per farm (Table 1) and a total of 4 species were only detected in all 4 districts and these were E. tenella, E. acervulina, E. maxima and E. brunetti with the presence of 43.75%, 50.0%, 37.5% and 12.5% among infected farms, respectively (Fig 1 and 2). The incidence of E. acervulina was found to be the highest and E. brunetti was found to be the lowest in these samples. The E. necatrix, E. mitis and E. praecox were not detected in any farm.
 

 

 

       
As per multiplex PCR, the mixed infection was present in 37.5% farms and single infection was present in 56.25% farms. This molecular test also indicated that highly pathogenic species E. tenella and E. brunetti were present in 9 (56.25%) farms of plain region of Chhattisgarh while moderately pathogenic species E. acervulina and E. maxima were present in 11 (68.75%) farms of which 5 (31.25%) farms were also having highly pathogenic species. The present findings are quite similar with the findings of Lee et al., (2012) who observed E. acervulina and E. tenella as the most prevalent species of Eimeria in chicken. Kaboudi et al., (2016) also reported high prevalence of E. tenella (61.5%) in Sidi Thabet, Tunisia and mixed Eimeria species infection with overall prevalence of 26.5%. However, dissimilar result was revealed by Kumar et al., (2014). They found 5 species with highest prevalence of E. necatrix (43.3%) followed by E. maxima (16.7%), E. tenella (13.3%), E. mitis (3.3%) and E. praecox (3.3%) by multiplex PCR. E. acervulina and E. brunetti were not identified by them. The present test could not detect any species of Eimeria in the sample of oocysts collected from the unorganized farms of Rajnandgaon district. Although the reason was not clear but it might be due to the presence of a smaller number of oocysts.

This multiplex PCR technique demonstrated the presence at least 1 to 3 species of Eimeria in one farm. This technique was significant in detection of multiple species of Eimeria infecting poultry by polymerase chain reaction and the percentage level presence of multiple species of this parasite in the farms of Chhattisgarh.

Present work was carried out in the Department of Veterinary Parasitology for the award of MVSc. Degree. Authors wish to thank to the Dean of the College of Veterinary Science and Animal Husbandry, Anjora, Durg, Chhattisgarh for providing facilities during research work.
 

The authors declare that they have no conflict of interest.