Multitissue transcriptomics demonstrates the systemic physiology of methionine deficiency in broiler chickens.

Methionine (Met) supplementation is common practice in broilers to support nutrition, yet there are gaps in the understanding of its role in systemic physiology. Furthermore, several different Met sources are available that may have different physiological effects. This study evaluated the mode of action of Met deficiency (no Met-supplementation) and supplementation (0.25% DL- or L-Met, 0.41% liquid methionine hydroxy analog-free acid (MHA-FA)), and of Met source (DL-, L- or MHA-FA) in broiler chickens, via host transcriptomics. Biological pathway activation modeling was performed to predict the likely phenotypic effects of differentially expressed genes (DEGs) in tissue samples from the jejunum, liver and breast obtained at 10, 21 and 34/35 d of age from three experiments in a combined analysis. Animal performance data showed that Met deficiency reduced BW, daily BW gain, daily feed intake, and breast yield, and increased feed conversion ratio in all experiments (P < 0.05). Effects of Met deficiency on gene expression were least evident in the jejunum and most evident in the liver and breast, as evidenced by the number of DEG and activated pathways. Activated pathways suggested Met deficiency was associated with inhibited protein turnover, gut barrier integrity, and adaptive immunity functions in the jejunum, that predicted reduced breast yield. There was an interaction with age; in Met-deficient birds, there were 333 DEGs in the jejunum of starter vs finisher birds suggesting young birds were more sensitive to Met deficiency than older birds. In the liver, Met deficiency activated pathways associated with lipid turnover, amino acid metabolism, oxidative stress, and the immune system, whereas in breast, it activated pathways involved in metabolic regulation, hemostasis, the neuronal system, and oxidative stress, again predicting a negative impact on breast yield. In the starter phase, supplementation with DL-Met compared to MHA-FA inhibited gamma-aminobutyric acid activity and oxidative stress in breast tissue. When data from all tissues were integrated, increased expression of a liver gene (ENSGALG00000042797) was found to be correlated with the expression of several genes that best explained variation due to the Met deficiency in jejunum and breast muscle. Some of these genes were involved in anti-oxidant systems. Overall, the findings indicate that impaired growth performance due to Met deficiency results from an array of tissue-specific molecular mechanisms in which oxidative stress plays a key systemic role. Young birds are more sensitive to Met-deficiency and DL-Met was a preferential source of Met than L- or MHA-FA during the starter phase.

Evaluation of live-attenuated Histomonas meleagridis isolates as vaccine candidates against wild-type challenge.

Repeated serial in vitro passage of Histomonas meleagridis, the etiological agent of histomoniasis (blackhead) of turkeys, was demonstrated to markedly achieve attenuation and reduction of virulence as compared to the original wild-type isolate. Four experiments were performed to evaluate the route (oral vs. intracloacal) and age (day-of-hatch vs. d 14) for administration of attenuated H. meleagridis isolates as vaccine candidates against homologous or heterologous wild-type challenge. Attenuated H. meleagridis were developed from 2 different strains (Buford strain originating in Georgia; PHL2017 strain originating in Northwest Arkansas). Buford P80a (passage 80, assigned as isolate lineage "a" following repeated passage) was selected as the primary vaccine candidate and was evaluated in Experiments 1-3. Experiment 4 evaluated selected candidates of attenuated PHL2017 (P67, P129) and Buford (P80a, P200a, P138b, P198c) strains against Buford wild-type challenge. As has been demonstrated previously, wild-type H. meleagridis cultures administered orally after 1 day of age were not infective in the current studies, but infection with wild-type cultures could be induced orally at day-of-hatch. Infection was effectively achieved via the intracloacal route at day-of-hatch and in older turkeys (d 21, d 28-29, d 35). Intracloacal inoculation of turkeys with the attenuated passaged isolates as vaccine candidates at d 14 was shown to produce significant (P < 0.05) protection from mortality, reduction in body weight gain, as well as reduction in hepatic and cecal lesions in these experiments following challenge with either the homologous wild-type isolate or from a wild-type strain obtained years later from a geographically disparate area of the United States. Inoculation with the attenuated H. meleagridis isolates at day-of-hatch, either orally or cloacally, did not produce significant protection against subsequent wild-type challenge. While offering significant protection with minimal vaccine-related negative effects, the protection from cloacal vaccine administration was neither significantly robust nor encouraging for industry application using the methods evaluated in the present manuscript since mortalities and lesions were not completely reduced which could thereby potentially allow transmission from residual infection and shedding within a flock.

The Effect of Different Species of Eimeria with Clostridium perfringens on Performance Parameters and Induction of Clinical Necrotic Enteritis in Broiler Chickens.

Necrotic enteritis (NE) is a common disease that causes great economic loss to the broiler industry due to mortality and reduced performance. Although Clostridium perfringens (CP) is a necessary component of this disease, coccidia species are a well-defined predisposing factor that exacerbates the condition. Different Eimeria species have been reported to influence NE to different degrees. In a pair of experiments, six different Eimeria species were evaluated in the presence and absence of C. perfringens. Male broiler chicks were housed in battery cages for the duration of both experiments. Feed conversion, body weight gain, and NE mortality were reported in both experiments. Experiment 1 challenged birds with E. maxima, E. acervulina, E. tenella, E. necatrix, and E. brunetti at day 13 and subsequently inoculated birds with CP on days 18, 19, and 20. In the second experiment, E. maxima, E. acervulina, E. tenella, and E. praecox were inoculated on day 15 and challenged with CP on days 17, 18, 19, 20, 21, and 22 of the experiment. In the first experiment, E. acervulina, E. brunetti, E. maxima, and E. necatrix with the addition of CP all stimulated necrotic enteritis mortality. In the second experiment, E. praecox had minimal impact on performance during the challenge (14-23 days) while E. maxima + CP decreased body weight gain and increased mortality compared to the CP alone control. Eimeria maxima had the highest mortality (21.9%) in this experiment followed by E. acervulina (6.3%). The remaining Eimeria with added CP in the second experiment did not induce NE mortality. While the challenge with CP alone did not induce mortality, feed conversion was increased compared to the unchallenged control group. When using isolated Eimeria species in these experiments, disturbances created by E. brunetti and E. maxima resulted in the most-severe challenges. These experiments highlight the NE risk of these species of Eimeria and give insight into how other species interact with the host in a controlled CP challenge model.

Artículo regular­Efecto de diferentes especies de Eimeria con Clostridium perfringens sobre los parámetros de rendimiento y la inducción de enteritis necrótica clínica en pollos de engorde. La enteritis necrótica (NE) es una enfermedad común que causa grandes pérdidas económicas a la industria del pollo de engorde debido a la mortalidad y a la reducción del rendimiento. Aunque Clostridium perfringens (CP) es un componente necesario de esta enfermedad, las especies de coccidia son un factor predisponente bien definido que agrava la enfermedad. Se ha informado que diferentes especies de Eimeria influyen en la enteritis necrótica en diferentes grados. En un par de experimentos, se evaluaron seis especies diferentes de Eimeria en presencia y ausencia de C. perfringens. Pollos de engorde machos se alojaron en jaulas en batería durante la duración de ambos experimentos. En ambos experimentos se analizaron la conversión alimenticia, el aumento de peso corporal y la mortalidad por enteritis necrótica. En el Experimento 1 se desafió a las aves con E. maxima, E. acervulina, E. tenella, E. necatrix y E. brunetti en el día 13 y posteriormente se inoculó a las aves con C. perfringens en los días 18, 19 y 20. En el segundo experimento, E. maxima, E. acervulina, E. tenella y E. praecox se inocularon en el día 15 y se desafiaron con C. perfringens en los días 17, 18, 19, 20, 21 y 22 del experimento. En el primer experimento, E. acervulina, E. brunetti, E. maxima y E. necatrix junto con C. perfringens estimularon la mortalidad por enteritis necrótica. En el segundo experimento, E. praecox tuvo un impacto mínimo en el rendimiento durante el desafío (14 a 23 días) mientras que el tratamiento de E. maxima + C. perfringens disminuyó el aumento de peso corporal y aumentó la mortalidad en comparación con el control con solamente C. perfringens. Eimeria maxima tuvo la mayor mortalidad (21.9%) en este experimento seguida por E. acervulina (6.3%). El resto de las especies de Eimeria junto con C. perfringens en el segundo experimento no indujeron mortalidad por enteritis necrótica. Si bien el desafío con C. perfringens no solo no indujo mortalidad, sino que la conversión alimenticia aumentó en comparación con el grupo de control no desafiado. Cuando se utilizaron especies de Eimeria aisladas en estos experimentos, los problemas creados por E. brunetti y E. maxima resultaron en los desafíos más severos. Estos experimentos destacan el riesgo por enteritis necrótica con estas especies de Eimeria y dan una idea de cómo otras especies interactúan con el hospedador en un modelo de desafío con C. perfringens controlado.

The Effect of Increased Temperatures on Viability, Morphology, Infectivity, and Development of Eimeria tenella.

Commonly found in backyard and commercial poultry production, coccidiosis, caused by Eimeria species, presents a self-limiting intestinal infection based on the number of ingested oocysts. Heat stress (HS) is one of the major environmental stressors in poultry, predisposing broiler chickens to immunosuppression and rendering them susceptible to diseases. There are suggestions that HS reduces Eimeria oocyst shedding in chickens; however, the relationship between HS and coccidiosis is not well elucidated. The objective of this study was to investigate the effect of temperature on viability, morphology, infectivity, and development of Eimeria tenella in vitro, and merozoite production and oocyst shedding in vivo. In vitro exposure of sporozoites to 55 C for at least 60 min reduced sporozoites viability as shown by morphological changes and rendering them unable to invade Mardin-Darbi bovine kidney (MDBK) cells. Intracellular development of merozoites was significantly reduced by an increase in 2 C in the optimal temperature of incubation in vitro. Most importantly, the induction of HS in the live chickens caused significantly lower lesion scores, reduced merozoite production, and oocyst shedding, resulting in a much less severe disease outcome.

Research Note: Evaluation of deoxycholic acid for antihistomonal activity.

Deoxycholic acid (DCA) is a naturally occurring secondary bile acid that originates from intestinal bacterial metabolic conversion of cholate, a primary bile acid. Deoxycholic acid was shown to have antihistomonal properties in vitro, leading to our hypothesis that DCA inclusion within the feed might prevent histomoniasis. Selected dietary concentrations of DCA were evaluated for effects on body weight gain (BWG), lesions, and mortality of turkeys challenged with wild-type Histomonas meleagridis (WTH). Treatments consisted of non-challenged control (NC; basal diet), 0.25% DCA diet + challenge, 0.5% DCA diet + challenge, 1% DCA diet + challenge, and a positive-challenged control (PC; basal diet). All groups were fed a basal starter diet until day 7, at which time DCA diets were administered to the respective groups. On day 14, 2 × 105 WTH cells/turkey were intracloacally administered. H. meleagridis-related lesions were evaluated on day 13 post-challenge. Pre-challenge day 0 to 14 BWG was higher (P ≤ 0.05) in the 0.25% DCA group than in the 1% DCA group. There were no significant differences in pre-challenge day 0 to 14 BWG between any of the other groups. No significant differences in mortalities from histomoniasis occurred in the DCA groups as compared to the PC group. No H. meleagridis lesions or mortalities were observed at any time in the NC group. Presence of H. meleagridis-related liver lesions was higher (P ≤ 0.05) in the 0.5% DCA group as compared to the PC group. Using the same controls and experimental timeline, an additional group was included to evaluate a biliogenic diet formulated with 20% whole egg powder to encourage endogenous bile acid production. The biliogenic diet had no statistical impact on pre-challenge day 0 to 14 BWG, but did not reduce H. meleagridis-related mortalities or lesions after the challenge. Taken together, these data suggest that DCA inclusion within the feed at these concentrations and under these experimental conditions does not prevent histomoniasis.

Research Note: Evaluation of boric acid as a chemoprophylaxis candidate to prevent histomoniasis.

Histomoniasis, caused by the protozoan parasite Histomonas meleagridis, is a disease to which turkeys are especially susceptible. Currently, no chemoprophylaxis compounds are available to mitigate this disease. Boric acid (BA) exhibits antifungal, antiseptic, and antiviral properties and has been used in the treatment of yeast infections. Based on these characteristics, an experiment was conducted to evaluate whether BA might be an efficacious prophylaxis against challenge with wild-type H. meleagridis (WTH). On day-of-hatch, poults were randomly assigned to either a basal control diet or a BA diet (basal diet + 0.2% BA). Groups consisted of a nonchallenged control (NC; basal diet), 0.2% BA + challenge (BA; 0.2% BA diet), and a positive-challenged control (PC; basal diet). On day 21, challenged groups were intracloacally inoculated with 2 × 105 WTH cells/turkey, and lesions were evaluated on day 14 postchallenge. Individual body weights were recorded on day 0, 21, and 35 to calculate the prechallenge and postchallenge body weight gain (BWG). The BA group resulted in lower prechallenge day 0 to day 21 BWG (P = 0.0001) than the NC group. Postchallenge day 21 to day 35, BWG was also lower (P = 0.0503) in the BA group than the PC group. No differences between the BA and PC groups were detected for mortalities associated with histomoniasis. Moreover, liver and cecal lesions were not statistically different between the BA and PC groups. Taken together, these data suggest that BA was not efficacious in the prevention or reduction of histomoniasis disease severity when provided at 0.2% dietary concentration under these experimental conditions.

Research Note: Evaluation of dietary administration of sodium chlorate and sodium nitrate for Histomonas meleagridis prophylaxis in turkeys.

Histomoniasis is currently a re-emerging disease of major significance for many commercial turkey and broiler breeder production companies because of the unavailability of drugs or vaccines. The protozoa Histomonas meleagridis (HM) requires the presence of enteric microflora to promote the disease. The objectives of this research note were to evaluate the effect of dietary administration of sodium chlorate (SC) and sodium nitrate (SN) in vitro and in vivo for HM prophylaxis in poults. A total of 128 day-of-hatch female poults obtained from a commercial hatchery were wing-tagged and randomly assigned into 1 of 4 experimental groups: negative control (NC), positive control, dietary inclusion of SC (3,200 ppm) and SN (500 ppm). Poults from groups SC and SN started on their respective diets on day 12. All groups, except the NC, were challenged with 2 × 105 HM on day 19. Controls were fed a basal diet, identical to the treatment diets but not supplemented with SC or SN. Body weight gain (BWG) was determined weekly, starting on day 1 until day 28, and postchallenge morbidity and mortality were recorded. On day 28 of age, all surviving poults were lesion scored for hepatic and cecal lesions. Ceca and distal ileum were collected on day 28 for bacterial recovery on selective media for total aerobic, lactic acid bacteria, or gram-negative bacteria. The addition of SC and SN in the in vitro growth of HM greatly reduced the growth of the protozoa after 20 h of incubation when compared with the control nontreated group (P < 0.05). However, dietary supplementation of SC and SN had no effect against HM in vivo, as was demonstrated by BWG, the severity of lesions in the liver and ceca or bacterial recovery of treated poults when compared with the positive control group.

Coccidiosis in the Chukar Partridge ( Alectoris chukar ): A Survey of Coccidiosis Outbreaks and a Test of Anticoccidial Drugs Against Eimeria kofoidi.

Field isolates of coccidia from 20 natural outbreaks in the chukar partridge ( Alectoris chukar ) were received from gamebird farms in 10 U.S. states. These were propagated in the laboratory and identified by microscopy and PCR. Of 20 samples, 18 were Eimeria kofoidi, two were Eimeria legionensis only, and one was a mixture of the two species. One isolate of E. kofoidi also contained an unidentified species detected only by PCR, nucleotide sequencing, and phylogenetic analysis. The efficacy of anticoccidial drugs against chukar coccidia was tested with experimental infections in battery cages. Isolates of E. kofoidi were used to infect 2-wk-old chukars. Anticoccidial products were given in the feed at levels approved for other poultry or for chukars. Tests were terminated at 6 days postinoculation with measurement of weight gains, fecal diarrhea scores, and necropsy to observe for lesion severity. Lasalocid (120 ppm) was moderately effective in one test. When tested against four field isolates, other ionophores (monensin, salinomycin, semduramicin) showed moderate effectiveness in reducing lesions and improving weight gains. Rofenaid (a potentiated sulfa mixture), robenidine (30 ppm), diclazuril (2 ppm), and decoquinate (80 ppm) were highly effective. In a test of nine products against a highly virulent field isolate, only diclazuril (2 ppm) and clopidol (125 ppm) reduced the severity of lesions and improved weight gain relative to infected controls, suggesting the extent to which previous drug usage had selected for drug resistance.

Immunization of chukar partridges against coccidia (Eimeria kofoidi and Eimeria legionensis) with low doses of live oocysts.

Experiments were conducted to determine whether chukar partridge (Alectoris chukar) chicks would develop protective immunity after inoculation with coccidia. Young chukar chicks in battery cages inoculated with 100 or more oocysts of Eimeria kofoidi or Eimeria legionensis had significant protection at challenge 4 wk later, as measured by greatly reduced oocyst shedding and improved weight gain as compared with unvaccinated, challenged controls. However, when birds were housed in litter pens and vaccinated by various regimens (including two species of chukar coccidia at 100/dose), coccidiosis rapidly spread through all treatments and caused significant mortality. Vaccination with Coccivac-T or with 100 oocysts of Eimeria dispersa did not prevent mortality resulting from accidental contamination, and feed treatment with a Lactobacillus competitive-exclusion product had no benefit. Most if not all of the mortality was from E. kofoidi. This study illustrated the natural fecundity of chukar coccidia in a floor-pen environment where multiplication rate and reinfection combine to produce clinical disease from a small original exposure. Further, these results cast doubt on the potential use of low doses of live oocysts as a vaccine in the chukar partridge.

The efficacy of anticoccidial products against Eimeria spp. in northern bobwhites.

To determine whether chemotherapeutic compounds available for use in domestic poultry are effective at controlling coccidiosis in northern bobwhites (Colinus virginianus), we tested 13 chemotherapeutic anticoccidials including amprolium (250 parts per million [ppm]), clopidol (125 ppm), diclazuril (1 ppm and 2 ppm), decoquinate (30 ppm), lasalocid (120 ppm), monensin (90 ppm), narasin/nicarbazin (36/36 ppm), robenidine (33 ppm), roxarsone (50 ppm), sulfadimethoxine/ ormetoprin (125/75 ppm), salinomycin (60 ppm), semduramicin (25 ppm), and zoalene (125 ppm and 150 ppm). Three tests were conducted using two replicates of 10 birds each: Infected, unmedicated controls and medicated birds were challenged with 1 x 10(6) oocysts of a field isolate consisting primarily of Eimeria lettyae. Subsequently, we tested clopidol, lasalocid, salinomycin, diclazuril (1 ppm), and monensin against mixed-species field isolates containing E. lettyae, E. dispersa, E. colini, or all. Weight gain, gross intestinal lesions, severity of diarrhea, and feed conversion ratio (FCR) 6 days postinfection were recorded. Lesion score, as previously reported, was unreliable as a measure of severity of infection in comparison with weight gain, fecal scores, and FCR. Excellent to good efficacy was found in clopidol, decoquinate, diclazuril (1 ppm and 2 ppm), and in lasalocid, narasin and nicarbazin, robenidine, sulfadimethoxine/ormetoprin, and zoalene (150 ppm). Marginal protection was found using monensin, salinomycin, semduramicin, or a roxarsone/semduramicin combination. Amprolium, roxarsone, and zoalene (125 ppm) were ineffective at controlling coccidia. Two of the six isolates tested against diclazuril 1 ppm and clopidol demonstrated a high degree of resistance, but none of the six isolates was resistant to lasalocid. Four of the eight isolates showed mild to moderate, and moderate to high, resistance against monensin and salinomycin, respectively. These findings indicate that several available compounds are effective at controlling coccidiosis in bobwhites.

An unusual case of coccidiosis in laboratory-reared pheasants resulting from a breach in biosecurity.

An outbreak of coccidiosis in laboratory-reared Chinese ring-necked pheasants (Phasianus colchicus) resulted in high morbidity and moderate mortality. The outbreak was associated with a breach in biosecurity caused by the cleaning of a sewer line with a mechanical device, resulting in extensive splattering of fecal material throughout the "clean room" where birds were held prior to use in coccidiosis experiments. Mortality and morbidity in the affected birds were seen exactly 5 days after the incident, after birds had been moved to another room for experimental use, corresponding closely with the known prepatent or preclinical period of Eimeria phasiani and Eimeria colchici. Gross lesions in the affected birds varied from dehydration to intestinal and ventricular hemorrhage. Microscopic examination confirmed a diagnosis of severe intestinal coccidiosis. This report underscores the ease of contamination of experimental birds leading to coccidiosis outbreaks during breaches of management and biosecurity.

Evaluation of nifurtimox for potential use in control of histomoniasis in turkeys.

Nifurtimox (NFX), a compound with known antiprotozoal activity, was evaluated for potential use in the prevention or treatment of histomoniasis in turkeys. A test of NFX in vitro showed that the compound was progressively active at concentrations of 12.5-200 ppm. Lower concentrations appeared only to delay growth of histomonads, while a concentration of 200 ppm was completely inhibitory. A series of tests in turkey poults showed that NFX had significant (P < 0.05) efficacy at 300-400 ppm when given in the feed throughout a 14-day experimental infection period. The beneficial effect was most prominent in the reduction of mortality and the suppression of liver lesions. Cecal lesions appeared less affected. Treatment with 400 ppm for a 3-day period after inoculation of turkeys was partially effective. In all tests, liver lesions were suppressed more effectively than cecal lesions, indicating that the concentration of the compound in the liver during metabolic excretion was important in the observed efficacy of this compound. Lack of any effect on growth or feed consumption in uninfected turkeys during a medication period of 16 days indicated that this compound was well tolerated by turkeys at 400 ppm in the feed and might be of benefit in the prevention or treatment of histomoniasis in turkeys.

Low-dose immunization of northern bobwhites (Colinus virginianus) with Eimeria lettyae provides protection against a high-dose challenge.

To determine whether northern bobwhite quail (Colinus virginianus) could be immunized against Eimeria lettyae by a low-dose inoculation of oocysts, we inoculated 30 birds each with either 100 or 1000 oocysts at 2 days of age (given orally by pipette). Four weeks after immunization, the immunized birds and unimmunized controls were challenged with 1 x 10(6) E. lettyae oocysts. Eight days after challenge, birds were killed and weighed, and their intestines examined for gross lesions. Effectiveness of the immunization was measured by analyzing weight gain, intestinal lesions, severity of diarrhea, feed conversion ratio, and oocyst production. After challenge, birds immunized with 100 or 1000 oocysts gained an average of 33.3 g and 28.9 g, respectively, whereas unimmunized challenged birds gained an average of 11.5 g. Immunized quail produced approximately 99.7% fewer oocysts, had minimal gross intestinal and cecal lesions, had minimal diarrhea, and had a 50% lower feed conversion ratio compared to unimmunized challenged controls. These findings indicate that vaccination is a viable option for controlling coccidiosis in quail and that further research into vaccination is warranted.

Histomonas meleagridis (Parabasala, Trichomonadea, Monocercomonadidae): presence of natural agglutinins in horse serum.

Cultured Histomonas meleagridis cells were readily agglutinated in vitro by horse serum at concentrations as low as 5%, although clumping was more rapid and prominent at 15% or higher. For observation of clumping, the cultured organisms were washed twice in Hanks balanced solution (HBSS) by centrifugation (1,000 x g for 15 min) and filtered through glass wool. The test sera were added and the mixture incubated in a Petri plate or 24-well culture plates at r.t. for 15-30 min. Formation of clumps was time- and concentration-dependent. Gentle agitation hindered agglutination at low serum concentration and accelerated agglutination at higher concentrations. The agglutinating factor (AF) was detected in several batches of serum from different sources, regardless of whether sera were heat-treated to inactivate complement. Histomonads were not clumped by either fetal horse or bovine serum (5-30%). Neither chicken nor turkey serum agglutinated histomonads to the extent seen with horse serum. Immune turkey serum lysed histomonads, hindering observation of clumping. Complement inactivation of immune serum slightly reduced lysis. AF in horse serum was precipitated with 25-40% ammonium sulfate and was active when cleaned by dialysis and reconstituted in HBSS. Clumping by serum facilitated the cleaning of histomonads for other studies where pure suspensions were needed.

Lectin-binding by sporozoites of Elmeria tenella.

Sporozoites of Eimeria tenella were reacted in vitro with 19 different lectins characterized with a variety of carbohydrate-binding properties. Nine lectins caused sporozoite agglutination, which was inhibited by the specific carbohydrates mannose, sialic acid, melibiose, D-galactose, or D-galNAc. When intact live or fixed whole sporozoites were reacted with fluorescein isothiocyanate-conjugated lectins, another nine lectins bound to sporozoites, giving weak to strong fluorescence but not agglutination. Of these, all nine lectins bound to surface sites, but four also bound to the refractile body. Two of the agglutinating lectins also bound to intracellular organelles of air-dried sporozoites. SDS-PAGE analysis showed that biotinylated lectins bound a wide variety of parasite proteins. Lectins with similar carbohydrate specificities had some similarity in binding patterns of parasite proteins, as well as marked differences. In a few cases lectins with different carbohydrate specificities bound common protein bands. Only one lectin (Dolichos biflorus) showed no evidence of binding to whole sporozoites, organelles, or proteins.

Cell membrane labeling of Eimeria tenella sporozoites with the fluorescent dye PKH-67 GL for tracking parasite-host interactions.

The fluorescent cell linker dye PKH-67 GL was used as a vital stain for sporozoites of Eimeria tenella for tests on viability, invasion of cultured primary chick kidney cells, flow cytometric analysis and fluorescence microscopy. The effect of PKH-67 GL on sporozoites was tested at a range of concentrations of dye and sporozoites. In flow cytometric analysis, 0.5-40x10(-6) M of PKH-67 GL labeled sporozoites to some degree, with the percentage of labeled sporozoites increasing with higher dye concentrations. The optimum concentration was 2x10(-6) M, allowing easy observation by fluorescence microscopy. Morphological changes in the sporozoite at concentrations greater than 5x10(-6) M were accompanied by loss of viability according to a propidium iodide inclusion assay. Sporozoite penetration of primary chick kidney cells was unaffected by the optimal level of 2x10(-6) M, allowing observation of intracellular activities. Overall, the cell linker dye greatly facilitated observation of E. tenella in vitro and in flow cytometric analysis.

Biological effects of gamma-irradiation on laboratory and field isolates of Eimeria tenella (Protozoa; Coccidia).

Sporulated oocysts of a field strain (FS-111) and a laboratory strain (WIS) of Eimeria tenella were exposed to 0, 50, 100, 150, or 200 Gy of gamma-radiation from a 60Co source. Irradiated oocysts of WIS and FS-111 were not significantly more fragile after irradiation as shown by the release of sporocysts after 5-105 s of vortex agitation with glass beads. Excystation was normal in both strains after treatment of the sporocysts with trypsin and sodium taurodeoxycholate, even in groups exposed to 200 Gy of radiation. Sporozoite release from irradiated sporocysts was more rapid than that from unirradiated sporocysts, primarily because of a shorter lag phase during the first 30 min. Irradiated sporozoites were slower to parasitize cultured chick kidney cells than were control sporozoites (4 h postinoculation), but after 24 h there was no significant difference (P < 0.05) between irradiated and control groups except for the WIS treated with 200 Gy. After 48 h, developing schizonts were reduced by 77-94% on exposure to 50-200 Gy. Strain FS-111 did not develop as well as WIS in vitro, but the effect of irradiation was similar. When irradiated oocysts of WIS or FS-111 were inoculated into chickens the prepatent period was unaffected, but fewer oocysts were produced, lesion scores were lower, and the weight gain was less strongly affected in proportion to the doses of radiation. These results suggest that the effects of radiation damage were largely confined to the mechanism of nuclear and cellular reproduction rather than other physiological processes.

Flow cytometric analysis of the response of Eimeria tenella (Coccidia) sporozoites to coccidiocidal effects of ionophores.

Fluorescein diacetate (FDA) and propidium iodide (PI) were used as indicators of membrane integrity after Eimeria tenella sporozoites were treated with polyether ionophores. Flow cytometry was used to quantitate the structural and functional effects based on red or green fluorescence and shape index of the sporozoites. Two field isolates (FS119 and FS139) were essentially resistant to polyether ionophores administered under practical conditions, whereas a laboratory strain was considered sensitive. The shape of sporozoites changed after treatment with ionophores, and this could be detected by flow cytometry. Green-fluorescing cells declined in number as the membranes were compromised by ionophore treatment. Red-fluorescing cells increased as the compromised membranes allowed entry of PI to bind with the nucleic acids. These effects were generally slower to develop in ionophore-tolerant field isolates compared with the sensitive laboratory strain. The effect of lasalocid on FDA and PI uptake, change in shape of the sporozoites, and lysis of sporozoites was more rapid than that of monensin or salinomycin. The 2 field isolates responded at different rates to salinomycin and monensin. Flow cytometry was a sensitive and accurate instrument for analysis of the effects of ionophores on sensitive and resistant lines of coccidia.

Effect of dietary raw soybeans on coccidiosis in chickens.

Six battery experiments determined the effect of feeding raw soybeans (RSB) in corn-soybean diets on coccidiosis in broiler chickens. The experimental design represents a 2 x 3 factorial arrangement of treatments with three dietary levels of incorporation of RSB (0, 50, and 100%), and chickens either infected or uninfected. The coccidial species tested singularly were Eimeria acervulina, Eimeria maxima, Eimeria necatrix, Eimeria brunetti, and Eimeria tenella. With all species of Eimeria a significant diet by infection interaction was demonstrated with a stepwise increase in weight gain and reduction in lesion scores with increasing levels of raw soybeans. To cause the pancreas to become hypertrophied and hyperfunctional, the 100% RSB diet was fed for an extended period. The experimental design represents a 2 x 3 factorial arrangement of treatments with three feeding regimens [Treatment A: 0% RSB (0 d to termination), Treatment B: (0% RSB: 0 to 12 d then 100% RSB 12 d to termination), and Treatment C: (100% 0 d to termination)] and chickens either infected (E. tenella) or uninfected. There was a significant difference for weight gain between uninfected and infected chicks of Treatments A and C but this difference was absent for Treatment B. Coccidiosis was not reduced by continuously feeding 100% RSB (Treatment C).

Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens.

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.