About 35% of all broiler flocks in the United States receive an anticoccidial vaccine, but it is not possible to easily differentiate Eimeria vaccine strains from Eimeria field isolates. Being able to do that would allow using vaccines in a more targeted way. The objective of this study was to collect Eimeria maxima isolates from broiler flocks that received anticoccidial feed additives and flocks that had been vaccinated against coccidia and then test them with a multilocus sequencing typing (MLST) scheme developed for this study. Fecal samples were obtained from commercial broiler flocks in Alabama and Tennessee. Oocyst counts in samples tended to be lower in flocks receiving anticoccidial feed additives and higher in vaccinated flocks. Selected samples were screened for presence of E. maxima by quantitative PCR, and Eimeria spp. composition was investigated by next-generation amplicon sequencing (NGAS) in 37 E. maxima positive samples. Other detected Eimeria spp. besides E. maxima were Eimeria acervulina in 35 samples, Eimeria praecox in 23 samples, Eimeria mitis or Eimeria mivati in 17 samples, and Eimeria necatrix or Eimeria tenella in 10 samples. Six partial E. maxima genes (dnaJ domain containing protein, 70-kDa heat shock protein, prolyl endopeptidase, regulator of chromosome condensation domain containing protein, serine carboxypeptidase, and vacuolar proton-translocating ATPase subunit) of 46 samples were sequenced. The MLST scheme was able to differentiate two vaccines from each other. Three of 17 samples from vaccinated flocks differed from the vaccine used in the flock, while 16 of 29 samples from unvaccinated flocks differed from the vaccine. However, there was also a large number of low-quality, ambiguous chromatograms and negative PCRs for the selected genes. If and when more advanced, possibly next-generation sequencing-based methods will be developed, the genes should be considered as targets.

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