A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species.

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as "variation" to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.

Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA.

Coccidiosis of chickens is one of the commonest and economically most important parasitic diseases of poultry worldwide. Given the limitations of traditional approaches, molecular tools have been developed for the specific diagnosis of coccidiosis. Recently, a polymerase chain reaction (PCR)-based capillary electrophoresis (CE) method, employing genetic markers in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, was established for both analytical and diagnostic purposes. The application of this method to investigate the epidemiology of coccidiosis and genetic structures of Eimeria populations on commercial chicken establishments has discovered genetic variants of Eimeria (i.e., new operational taxonomic units OTU-X, OTU-Y and OTU-Z) which were (based on CE analysis) distinct from those of species of Eimeria identified previously in chickens in Australia. The present characterization of these OTUs, based on their ITS-2 sequences and phylogenetic analyses of selected sequence data, provides first evidence to support that OTU-X represents a population variant of Eimeria maxima, and that OTU-Y and OTU-Z represent cryptic species of Eimeria. Further biological and genetic studies are needed to rigorously test these proposals and establish the specific status of these OTUs and their importance as pathogens in chickens. An understanding of the epidemiology of these population variants or cryptic species in Australia is central to designing and implementing effective vaccination and control strategies.

Investigating a persistent coccidiosis problem on a commercial broiler-breeder farm utilising PCR-coupled capillary electrophoresis.

In the present study, we utilised a polymerase-chain-reaction-coupled capillary electrophoresis (CE) approach to investigate the epidemiology of Eimeria species on a broiler-breeder farm in Victoria, Australia. The Eimeria populations of two flocks vaccinated against coccidiosis were followed over an 11-week period. All seven recognised Eimeria species of chickens were detected in both flocks. One flock suffered increased morbidity and mortality in its eighth week and had consistently higher Eimeria oocyst counts, species prevalences and rates of co-infections. Four Eimeria species included in the vaccine administered occurred at higher prevalences before the disease outbreak in the flock. Using the CE approach, two new, previously undescribed Eimeria genotypes were discovered in both chicken flocks, one of which dominated toward the end of the study period. The molecular approach proved versatile and capable of providing useful epidemiological data which could be used to investigate and interpret coccidiosis outbreaks.

The application of a polymerase chain reaction (PCR)-based capillary electrophoretic technique provides detailed insights into Eimeria populations in intensive poultry establishments.

Intestinal coccidiosis, caused by one or multiple species of Eimeria (Protozoa: Apicomplexa), is one of the most important infectious diseases affecting chickens. In this study, we used a polymerase chain reaction (PCR)-based capillary electrophoresis (CE) approach to conduct an epidemiological survey of Eimeria species in seven Australian broiler flocks, varying in age from 18 to 42 days. We confirmed that all seven recognized Eimeria species of poultry were present. Eimeria acervulina and E. maxima were the most common, followed by E. mitis (i.e., 89%, 87% and 64% of chickens, respectively). E. praecox was present in 44% of birds, whereas E. brunetti and E. tenella were uncommon, being found in 36% and 26%, respectively. E. necatrix was rarely detected (10%). Even the least common species were present in more than 70% of sheds. The prevalence of individual species was higher in older than in younger chickens. Most of the chickens sampled were simultaneously infected with multiple Eimeria species (mean=3.6). The number of Eimeria oocysts excreted per gram of faeces reached a peak at 36 days of age, before declining to a considerably lower level by 42 days. As anticoccidial drugs were permanently withdrawn at 36 days, the decreasing Eimeria oocyst excretion rates indicated the development of protective immunity in the chickens. The present study showed that even healthy chickens usually harbour numerous species of Eimeria. The CE technique proved to be a time and cost-effective means of investigating the epidemiology of Eimeria in commercial establishments.