Abstract
BackgroundAvian coccidiosis is an intracellular intestinal parasitic disease, caused by intracellular intestinal parasites from the genus Eimeria, among which Eimeria tenella is one of the most pathogenic species and causes great economic losses. Frequent applications of anticoccidial drugs have resulted in the development of drug-resistance in E. tenella. In the present study, we sought to determine the genetic diversity of E. tenella isolates prevalent in chicken farms in Hubei Province of China and examine their sensitivity to three anticoccidial drugs. The results provide useful information for the prevention and control of coccidiosis in this region.MethodsEimeria tenella oocysts were isolated from faecal samples collected from different commercial broiler production farms in Hubei Province, China. After oocyst sporulation and animal inoculation for expansion of the field isolates, DNA and RNA were extracted from excysted sporozoites for molecular characterization. Species identification of field isolates were performed by polymerase chain reaction (PCR) amplification of the internal transcribed spacer 1 (ITS1) region of ribosomal DNA. Random amplified polymorphic DNA (RAPD) was used for population genetic analysis. Subsequently, sequences of the major sporozoite surface antigen (SAG), micronemal protein 2 (MIC-2) and cytochrome b (cytb) genes from genomic DNA, and the Eimeria tenella cation-transport ATPase (EtCat ATPase) gene from cDNA were obtained for genotyping using multi-sequence alignments. Finally, sensitivity of the field isolates to three commonly used anticoccidial drugs (diclazuril, decoquinate and maduramycin) were tested to assess the prevalence of drug resistance in E. tenella in Hubei Province of China.ResultsAnalysis of the ITS1 sequences indicated that all the isolates were E. tenella. RAPD analysis and multi-sequence alignments of the SAG, MIC-2, EtCat ATPase and cytb showed genetic diversity among these isolates. Finally, drug sensitivity tests demonstrated that all field isolates were sensitive to diclazuril but resistant to decoquinate (except for the isolates from eastern Hubei) and maduramicin.ConclusionsPopulation genetic analysis indicated that genetic polymorphisms among field isolates were closely related with their regional distributions. Drug sensitivity testing demonstrated that E. tenella isolates in Hubei Province were sensitive to diclazuril, but resistant to maduramycin and decoquinate. The results presented here provide important information for the control and preventions of coccidiosis in the Hubei Province of China.
Highlights
Avian coccidiosis is an intracellular intestinal parasitic disease, caused by intracellular intestinal parasites from the genus Eimeria, among which Eimeria tenella is one of the most pathogenic species and causes great economic losses
Isolation and species identification of Eimeria tenella field isolates from local farms in China Twenty-one Eimeria field samples were isolated from different local commercial broiler production farms in Hubei Province (Fig. 1)
The results indicate that all the field isolates were E. tenella since they produced a specific 278 bp band using the E. tenella-specific primer. This fragment obtained from each strain was subject to DNA sequencing and the results showed that they were 97–100% identical to the internal transcribed spacer 1 (ITS1) sequence of E. tenella (GenBank No AF026388), further confirming that these isolates are E. tenella (GenBank No KY117132–KY117152)
Summary
Avian coccidiosis is an intracellular intestinal parasitic disease, caused by intracellular intestinal parasites from the genus Eimeria, among which Eimeria tenella is one of the most pathogenic species and causes great economic losses. Williams et al developed a RAPD technique based on the amplification of undefined targets by arbitrary primers to detect genetic polymorphisms [8], the technique has been widely used for the analysis of Eimeria genetic diversity [9, 10]. Such analysis can help us to estimate the phylogenetic relationship among different Eimeria isolates [11]
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