A single, multiplex PCR for differentiating all species of Trichinella.

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.

A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle.

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.

A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella.

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichilnella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.

Protection against coccidiosis in outbred chickens elicited by gamma-irradiated Eimeria maxima.

In an effort to develop an attenuated coccidiosis vaccine against coccidiosis, we exposed Eimeria maxima oocysts to an optimum dose of gamma irradiation (17 kRad) that does not affect sporozoite invasion of the intestinal mucosa but does prevent asexual parasite development. Irradiated E. maxima oocysts were suspended in gelatin slabs and placed in battery cages for ingestion by 1-day-old chickens. Separate groups of chickens were given gelatin slabs containing nonirradiated E. maxima oocysts or were inoculated per os with either irradiated or nonirradiated E. maxima oocysts. Chickens infected with irradiated or nonirradiated oocysts by either oral inoculation or gel delivery showed a dose-dependent protection against weight loss associated with E. maxima challenge compared with unimmunized controls. In general, nonirradiated oocysts elicited protective immunity at lower immunization doses compared with irradiated oocysts. These experiments were extended to a floor pen study wherein 1-day-old male and female broiler chickens were given irradiated or nonirradiated E. maxima oocysts in gelatin slabs in hatching boxes and challenged at 4 wk of age. A significant reduction (P < 0.05) in lesion scores was observed for chickens immunized with either irradiated or nonirradiated oocysts compared with unimmunized controls. Although no significant difference (P > 0.05) was observed in weight gain between these groups, both male and female chickens inoculated with irradiated E. maxima oocysts showed about a 10% greater weight gain than unimmunized controls. For both male and female chickens, average weights at challenge were greater in groups that were immunized with 17-kRad-irradiated E. maxima oocysts compared with those animals immunized with nonirradiated oocysts.

Preferential translation of reovirus mRNA by a sigma3-dependent mechanism.

We have characterized reovirus strains that differ in the degree to which they inhibit cellular protein synthesis and used them to investigate mechanisms regulating gene expression in infected cells. A previous genetic study associated distinct effects of reovirus strains on cellular translation with polymorphisms in viral protein sigma3. In cell extracts, sigma3 sequesters double-stranded RNA (dsRNA) and blocks activation of the dsRNA-activated protein kinase (PKR), an interferon-induced enzyme that inhibits translational initiation by phosphorylating elF-2alpha. We found that in infected cells, cellular protein synthesis is translationally regulated in a strain-specific manner. Using immunoprecipitation and indirect immunofluorescence we showed that the effect of a strain on cellular translation is not determined by the level of sigma3, but appears to result from differences in sigma3 localization. In cells infected with a strain that spares cellular translation, sigma3 is present throughout the cytoplasm, whereas in cells infected with inhibitory strains, sigma3 is restricted to perinuclear viral factories. Biochemical studies suggested that diffuse localization of sigma3 is a consequence of low affinity for capsid protein mu1. Our findings are consistent with a model in which the efficiency of cellular translation is determined by the cytoplasmic level of sigma3 that is not complexed with mu1.

Gamma-irradiated and nonirradiated Eimeria tenella sporozoites exhibit differential uracil uptake and expression of a 7- to 10-kDa metabolic antigen.

Eimeria tenella sporozoites were exposed in the oocyst form to either an optimum (15 kRad) or a high (25 kRad) dose of gamma irradiation and used to infect cultured chicken embryo fibroblasts (CEF). The sporozoite-infected CEF monolayer was pulsed at time of infection or 24 hr postinfection with [3H]uracil and harvested 24 hr later to measure sporozoite metabolic activity. Sporozoites exposed to either 0 or 15 kRad gamma irradiation incorporated similar (P > 0.05) amounts of [3H]uracil during the first and second 24-hr periods after infection. However, there was a significant decrease (P < 0.05) in [3H]uracil uptake by 25 kRad-exposed sporozoites compared to nonirradiated and 15 kRad-irradiated sporozoites. Indirect immunofluorescence (IFA) staining of E. tenella sporozoite-infected CEFs using monoclonal antibodies (MAb) specific for somatic or "metabolic" antigens showed that gamma irradiation also affected the release of intracellular metabolites. Regardless of irradiation dose, extracellular sporozoites exhibited similar intensity of immunofluorescence when stained with either somatic antigen- or metabolic antigen-reactive MAb. Also, somatic antigen expression was similar for intracellular parasites irrespective of radiation dose. However, metabolic 7- to 10-kDa antigen expression by 25 kRad-irradiated sporozoites was markedly reduced compared to nonirradiated or 15 kRad-irradiated intracellular sporozoites. These results were corroborated by immunostaining sporozoite/CEF protein-impregnated Immobilon membrane with somatic or metabolic 7- to 10-kDa antigen-reactive MAb. These findings may indicate that the metabolic 7- to 10-kDa antigen is involved in protective immunity elicited by nonirradiated and/or 15 kRad-irradiated E. tenella sporozoites.

Effect of amprolium on production, sporulation, and infectivity of Eimeria oocysts.

Amprolium reduced the number of oocysts shed by Eimeria acervulina, E. maxima, E. necatrix, and a mixture of susceptible strains of E. tenella. Sporulation of oocysts from mediated chickens was reduced compared with that of oocysts from unmedicated chickens. Sporulation was reduced by levels of 0.0250% amprolium for E. acervulina and by levels of 0.0060% for E. maxima and the susceptible E. tenella. Not enough oocysts were recovered to measure sporulation of E. necatrix. Sporulation reduction was not affected by the method of administration of amprolium (feed or water), except with E. acervulina, for which fewer oocysts sporulated when 0.0120% amprolium was added in the drinking water than when 0.0125% amprolium was added to the feed. Conversely, amprolium medication had no effect on the sporulation of an amprolium-resistant E. tenella. When fed to unmedicated chickens, those oocysts from amprolium-medicated chickens that did sporulate were as infective as oocysts recovered from unmedicated chickens.

Characterization of the toxicity of the mycotoxins aflatoxin, ochratoxin, and T-2 toxin in game birds. II. Ringneck pheasant.

Ringneck pheasants were fed diets containing 1.25, 2.5, or 5 ppm aflatoxin; 1, 2, or 4 ppm ochratoxin A (OA); or 4, 8, or 16 ppm T-2 toxin. Severe toxin-induced mortality was seen during the first to third weeks with 2.50 and 5.00 ppm aflatoxin (92.5% and 97.5%, respectively), compared with the mortality in control pheasants fed no toxin (0%). Slight mortality (less than or equal to 5%) was seen with OA and T-2 toxin. Body weights were significantly decreased by the lowest level (1.25 ppm) of aflatoxin by 2 weeks of age, by the two highest levels of aflatoxin by 1 week of age, and by 16 ppm T-2 toxin by 1 week of age. The feed-conversion ratio was increased by 2.50 and 5.00 ppm aflatoxin compared with the feed-conversion ratio in controls, although high mortality may have influenced the results. Aflatoxin had no effect on liver weight, but OA increased kidney weight in 3-week-old pheasants. Mouth lesions were seen in some of the pheasants fed T-2 toxin.

Supplemental use of liquid amprolium in skip-a-day feeding of replacement pullets.

The addition of liquid amprolium to the drinking water on days when medicated (amprolium) ration was not fed in a restricted feeding (skip-a-day) program improved protection against a primary exposure to Eimeria acervulina and Eimeria tenella, yet still allowed for the development of protective immunity to subsequent challenge. With E. tenella, the best protection, as measured by reduction of lesion score, was provided by amprolium given in the drinking water on alternate days to feed medication when compared with the use of amprolium only in the feed or liquid amprolium at less frequent intervals (every second or third nonfeeding day). With Eimeria maxima, amprolium in the feed did not significantly lower lesion score compared with the score in unmedicated pullets; however, the further addition of amprolium to the drinking water did. When pullets were reared in floor pens previously seeded with coccidia, amprolium medication in the feed alone reduced the E. tenella-induced mortality rate from 28 to 8%. The addition of amprolium in the drinking water on nonfeeding days eliminated all deaths. Floor-reared pullets were caged after 3 wk and challenged 1 wk later with the same species of coccidial oocysts used to immunize on the floor. Coccidial lesion scores following challenge were eliminated or markedly lower than in pen-reared (unimmunized) pullets similarly challenged. This indicated that protective immunity developed despite the use of amprolium in the drinking water.

Prevention of coccidiosis in bobwhites by medication.

The efficacy of amprolium, monensin, and salinomycin in preventing coccidiosis in bobwhite quail was studied using a mixed inoculum of equal numbers of Eimeria dispersa and E. lettyae. A total dosage per quail of 10(6) sporulated oocysts was chosen because this dosage gave a good (77%) depression of weight gain from Day 18 to Day 24. Levels of .008% monensin or .0055% salinomycin were the most effective for prevention of coccidiosis as evaluated by body weight gains. These levels significantly reduced parasite numbers in the duodenum with monensin administration and in both the duodenum and ileum with salinomycin administration. Monensin reduced parasite numbers in the illeum significantly in one experiment and in a second. Amprolium was ineffective for prevention of coccidiosis, as evaluated by body weight gains. Amprolium was also ineffective in consistently reducing parasite numbers in the duodenum and ileum. Both monensin and salinomycin had a reasonable safety margin in quail. Levels of monensin of .016%, twice the proposed level, significantly reduced body weight at 14 days of age compared with unmedicated controls or quail given .008% monensin. By 28 days, however, this effect was no longer significant. Levels of salinomycin at the proposed level of .0055% significantly reduced body weight at 14 days of age compared with unmedicated controls. By 28 days, however, this effect was no longer significant in quail given .0055% or .00825% salinomycin, although in quail fed .011% salinomycin body weights remained significantly lower (16.5%) at that date. There were no detectable monensin residues in the liver of quail fed a ration containing .008% monensin for 8 wk.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition of heart, liver, and skeletal muscle from broilers with coccidiosis.

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.

Physiological basis of Eimeria tenella-induced mortality in individual chickens.

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.

Relationship of restricted feeding and medication to coccidiosis control.

Hubbard breeder pullets were fed a complete pullet developer ration on an ad libitum (AL) or restricted feeding (RF) regimen. The ration either was unmedicated or contained .0125% amprolium, .0125% clopidol, or .0110% monensin. The relationship between the feeding schedule and coccidial infection was determined on the basis of 1) efficacy of the medication in controlling a single infection in susceptible pullets and 2) the development of immunity to subsequent challenge inoculation after a series of immunizing inoculations. Cage-reared, susceptible pullets were inoculated with sporulated oocysts of either. Eimeria tenella, 2 strains of E. acervulina, or E. maxima. With all three medications, the infection with a least one species was more severe, as measured by intestinal lesion score, in the RF pullets than in the corresponding AL pullets. Other pullets were kept for three weeks in floor pens that contained coccidial oocysts to allow natural infection and immunity to develop. The pullets were then transferred to suspended cages to prevent reinfection and fed an unmedicated ration. After one week, the pullets were challenged with the same coccidial strain as that used for immunizing. All pullets initially exposed to coccidia and given no medication were resistant to challenge inoculation. Control pullets are exposed to coccidia during rearing were susceptible to challenge. The administration of anticoccidial drugs (especially monensin) by ad libitum feeding interfered with development of immunity under these conditions, but, when the same drugs were given with a restricted feeding regimen, they did not interfere with development of immunity.

Robenidine resistance in Eimeria tenella.

Robenidine protected chickens against cecal coccidiosis infections initiated by a strain of the parasite that had no previous exposure to drugs. No cross resistance was found with 13 strains resistant to other anticoccidials. A strain of Eimeria tenella that was serially propagated in chickens fed mash containing robenidine became resistant to the chemical. No cross resistance was detected when this experimental strain was tested against 12 other anticoccidials.

Effect of dimetridazole on transmission of Histomonas meleagridis by Heterakis gallinarum.

The administration of an antihistomonal drug, dimetridazole, at a dose of 0.08% in feed, controlled experimental infections with Histomonas meleagridis in chickens. The treated birds developed no lesions and the duration of infection with H. meleagridis was reduced. This drug regimen, however, did not always prevent incorporation of H. meleagridis into eggs of Heterakis gallinarium; heterakid eggs pooled from medicated chickens in which H. meleagridis had never been detected transmitted the protozoan to 1 of 10 turkeys fed the eggs. Thus, therapeutic treatment of chickens with dimetridazole may reduce, but not eliminate, transmission of H. meleagridis by eggs of H. gallinarum from medicated birds.

Sequential use of coccidiostats: effect on development by Eimeria tenella of resistance to amprolium, nicarbazin, Unistat, and zoalene.

A strain of the cecal coccidian of chickens, Eimeria tenella, was propagated serially in chickens fed mash containing amprolium, nicarbazin, Unistat, or zoalene. Each group of chickens received a different coccidiostat on a rotating basis. The strain was propagated through 40 groups of chickens; thus, the strain was intermittently exposed 10 times to each coccidiostat. The end product of this simulated shuttle program of prophylactic anticoccidial medication was a strain resistant to three of the four coccidiostats involved. Resistance to nicarbazin was not evident.