Study area
Kaduna state is among the seven states of the north-western geopolitical zones of Nigeria. It is situated within the Sudan savannah vegetation zones of Nigeria with distinct dry and wet seasons. The dry season runs from October to April. The wet season begins in most parts of the state in May and lasts up to September or October, with mean annual rainfall of between 510–1140 mm. The samples analysed here were collected from poultry units around Zaria, Nigeria, between April and July 2013.
Farm selection and sample collection
Combined faecal/litter samples representative of a pen, and thus several individual chickens, were collected from 12 commercial poultry farms found during previous routine veterinary monitoring to harbour coccidial oocysts. In each poultry house, samples were collected following an approximate W-shaped path, starting and finishing in the corners of one of the long sides of the house [3]. Along this path, combined faecal/litter samples were collected manually, stopping every three strides to collect one handful which was placed in clean plastic bags and transported on ice pack to the Research Laboratory of the Department of Veterinary Parasitology and Entomology, Ahmadu Bello University-Zaria.
Sample processing and morphometric oocyst identification
In the laboratory, 200 g of each sample was weighed, transferred into a plastic beaker and soaked in approximately 500 ml of distilled water overnight. The soaked samples were then homogenized by thorough stirring using a glass rod and filtered through a metal sieve (mesh size 300-500 μm). The filtrate from each sample was allowed to sediment for one hour on the laboratory bench, after which the supernatant fluid was discarded into a clean beaker. The presence of oocysts was confirmed microscopically by transferring the equivalent of 10 ml of sediment into a centrifuge tube and testing for the presence of coccidial oocysts using the saturated saline flotation technique described elsewhere [26]. Oocysts were assigned putative species identity based upon microscopic morphology [18]. For each positive sample, oocysts were recovered from the remaining sediment using the centrifugal flotation technique [26]. The harvested oocysts were re-suspended in distilled water and washed by centrifugation three to four times to remove the flotation solution (300 g for 5 min). The sediment containing the oocysts was transferred into Petri-dishes, re-suspended in 2.5 % (w/v) potassium dichromate solution and allowed to sporulate at room temperature for 7 days with regular stirring.
After sporulation, oocysts within each sample were cleaned from the residual faecal debris by treatment with sodium hypochlorite (4 % active chlorine) and three successive washes in distilled water as described elsewhere [27]. After cleaning, the oocysts were re-suspended in distilled water and enumerated using a modified-Fuchs Rosenthal counting chamber. The tubes containing the cleaned oocysts were clearly labelled with isolate number, date and amount of oocysts/ml, and stored at 4 °C until required.
Eimeria propagation
Field samples found to contain coccidial oocysts which had sporulated were used for in vivo propagation as a consequence of overall low oocyst recovery and poor sporulation. Individually caged 4 week old specific-pathogen free (SPF) Light Sussex chickens were inoculated orally with 4,000 sporulated mixed oocysts from single field parasite populations. Progeny oocysts were recovered from caecal tissue and contents collected during post-mortem 7 days post infection, sporulated and purified as described elsewhere [25, 27].
Total genomic DNA extraction
Four millilitres of each washed oocyst suspension, containing between 2 and 5 million oocysts after in vivo propagation, were centrifuged (750 g for 10 min) to pellet the oocysts. Each pellet was re-suspended in the minimum volume residual supernatant and transferred to a 1.5 ml screw top plastic tube. Glass beads (0.4-0.6 mm; Sigma, UK) equivalent to the volume of the oocyst pellet were added to the tube and covered with sterile phosphate buffered saline (PBS; pH 8.0). The pelleted oocysts were then disrupted using a Mini Beadbeater-8, (Biospec Products, Bartlesville, USA) for two minutes and total genomic DNA (gDNA) was isolated from the smashed oocyst homogenate using a QIAamp DNA Tissue mini kit (Qiagen, Germany) following the manufacturers protocol.
Molecular identification of Eimeria by nested polymerase chain reaction
A standardized nested PCR assay targeting the internal transcribed spacer (ITS)-1 sequence for identification of Eimeria species of poultry was used to improve detection of minority Eimeria species populations. Primers amplifying the entire ITS-1 sequence based in the flanking 18S and 5.8S rDNA regions of the eimerian genome were used in the first genus-specific PCR phase, while species-specific primers targeting the ITS-1 region were used to amplify the individual Eimeria species in the second nested phase. The primers (as shown in Additional file 1) and the PCR conditions used were as described previously [3, 14]. Genomic DNA purified from the Houghton reference strains of each of the seven recognised Eimeria species were used as positive controls, with molecular grade water (Sigma, UK) used as the negative control starting from the beginning of the nested assay. The amplification products of the specific nested PCR were analysed by gel electrophoresis in 2 % (w/v) agarose gels in 1x Tris Acetate EDTA (TAE; all Sigma, UK) buffer stained with 0.01 % (v/v) SafeView nucleic acid dye (NBS Biologicals, UK).
Molecular identification of new operational taxonomic unit (OTU) cryptic Eimeria genotypes
Primers specific to the OTUx, OTUy and OTUz ITS-2 sequences were used to screen each sample for the occurrence of these novel genotypes (Additional file 1). PCR conditions were as described previously and the PCR products were analysed by gel electrophoresis as described above [5].
PCR amplicon sequencing to confirm and validate species/genotype identification
Two PCR fragments representative of each Eimeria species detected were sequenced to confirm amplicon identity and validate PCR detection, resulting in 14 sequences from 31 positive reactions (45 %). Amplicons were purified using a Qiagen PCR purification kit, cloned using pGEM-T Easy (Promega, Madison, USA) in XL1-Blue MRF Escherichia coli (Stratagene, La Jolla, USA), miniprepped (Qiagen) and sequenced (GATC Biotech, Konstanz, Germany) as described by the respective manufacturers. Sequence assembly, annotation and interrogation were undertaken using CLC Main Workbench v6.0.2 (CLC Bio, Katrinebjerg, Denmark) and sequences were identified using BLASTn against the GenBank non-redundant database with default parameters. The sequences have been submitted to GenBank under the accession numbers LT549029-LT549042.