No abstract

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subunit rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257bp), Haemonchus placei (176bp), Oesophagostomum radiatum (329bp), Trichostrongylus colubriformis (243bp) and Cooperia oncophora (151bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs.

Summary:The genus Trichinella is currently divided into seven species and at least three additional, unclassified genotypes, Trichinella T6, T8 and T9, where both T8 and T9 have been deemed very similar to T. britovi. Other than for the non-encapsulated species, the absence of distinguishing morphological characters and the overlapping nature of the biological characters within this genus make these traits unsuitable for diagnosis. Consequently, we have developed a simple PCR test for the unequivocal differentiation of all currently recognized species of Trichinella including Trichinella T6. DNA sequence data from each Trichinella genotype were generated from internal transcribed spacers, ITS1 and ITS2, and from expansion segment V (ESV) of the rDNA repeat, from which five different PCR primer sets were chosen. When used simultaneously, this primer mix generates a simple and unique electrophoretic DNA banding pattern for each species and genotype. The ESV-derived primer set contributes at least one band to each agarose gel-derived genotypic pattern and therefore functions as an internal control for PCR integrity. Geographical isolates of each Trichinella genotype were used to verify the reliability and reproducibility of respective DNA banding patterns using single muscle larvae.

Infection with Eimeria acervulina, Eimeria brunetti, or Eimeria tenella affected the composition of the breast, thigh, heart, and liver of 3 or 4-week-old broilers. Liver glycogen was significantly increased at 6 and 8 days postinoculation (PI) with E. acervulina. Conversely, liver glycogen was decreased at 4 and 6 days PI with E. tenella and was unaffected by infection with E. brunetti. The levels of RNA and lipid in the liver were decreased with E. acervulina but unchanged with E. tenella. Both species decreased RNA levels in the breast. None of the three coccidial species had any effect on the moisture, ash, protein, or DNA content of the tissues.

Chickens dying from Eimeria tenella infection revealed four major physiological stresses before death: (1) hypothermia, (2) depletion of carbohydrate stores, (3) metabolic acidosis, and (4) renal tubule-cell dysfunction. These stresses were less pronounced in chickens surviving the infection. Similar stresses could not be demonstrated in pair-feeding trials, in which uninfected chickens were fed only the amount consumed by infected chickens. Prolonged starvation of uninfected chickens only slightly altered the indicators used in assessing the stresses. The variability of previously reported plasma glucose values, in part, may be due to whether the birds tested were those on the verge of death or those that, ultimately, would survive the infection.