Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.

Efficacy of semduramicin and salinomycin against different stages of Eimeria tenella and E. acervulina in the chicken.

The efficacy of a new ionophore, semduramicin, was compared with salinomycin in a series of in ovo and in vivo trials. Semduramicin was more efficacious than salinomycin against Eimeria tenella sporozoites as judged by oocyst production in embryonated eggs. When the two drugs were given in ovo at 93 h post inoculation (PI), both drugs exerted some effect against late schizogonous stages of E. tenella. In three battery studies, semduramicin (25 ppm) and salinomycin (60 and 66 ppm) were tested against E. tenella and E. acervulina. Medicated feed was withdrawn at 24-h intervals PI to study the stage of action of the anticoccidials. In E. tenella infected chickens, both anticoccidials exerted their maximum effect on weight gain and feed:gain ratio through the first 72 h PI. Semduramicin was more effective than salinomycin in controlling E. tenella lesions and coccidiosis mortality. With E. acervulina, both drugs acted similarly on early life cycle stages and no improvement in performance was recorded when medicated feed was given for longer than 72 h. Semduramicin was more effective than salinomycin in controlling E. acervulina lesions.

In vitro activity of the human neutrophil cathepsin G on Eimeria tenella sporozoites.

The role of human neutrophil cathepsin G (Cat G) on Eimeria tenella sporozoites was studied in vitro. Sporozoites were incubated for 2 hr at 37 C in PO4 buffer, 0.9% NaCl (PBS), pH 7.6 in the presence of Cat G (50 micrograms/ml), diisopropyl fluorophosphate-inhibited Cat G (DFP-Cat G) (50 micrograms/ml) or PBS alone, prior to being inoculated into embryonated eggs. As judged by oocyst production on day 7 postinoculation, embryo mortality and the hemorrhage scores, both Cat G and DFP-Cat G demonstrated anticoccidial activity; greater activity was obtained with the DFP-Cat G. Sporozoites were exposed also to increasing concentrations of native and trypsin-digested DFP-Cat G (0-100 micrograms/ml) under the same conditions. Significant protection (37% and 49% for native and digested DFP-Cat G, respectively) was obtained with a low concentration (5 mu/ml), and higher concentrations resulted in 70% and 84% protection, respectively. The primary bactericidal domain of Cat G, the HPQYNQR peptide, at 3 concentrations (25, 50, and 100 micrograms/ml), reduced the oocyst production by 46%, 16%, and 15%, respectively. The anticoccidial activity of Cat G may involve a peptide fragment different from the antimicrobial domain of the enzyme.

Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken.

Broiler chickens in battery pens were either fed a diet containing 100 ppm lasalocid or no drug for 24 h prior to inoculation with sporulated oocysts of Eimeria tenella or Eimeria acervulina. Different groups of birds remained on medicated feed for 24, 48, 72, 96, 120 or 144 h after inoculation. Conversely, other groups started on an unmedicated diet, were given medicated feed at different times after oocyst inoculation. Starting lasalocid medication 24 h (E. tenella) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain. There was no significant difference in performance of birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. Starting lasalocid medication at 96 or 120 h did not suppress but rather reduced oocyst production.

Effect of lasalocid on the development of Eimeria tenella in chicken embryos.

Sporozoites of Eimeria tenella were treated with lasalocid in vitro and their viability tested by inoculating them into the allantoic cavity of 11-day-old chicken embryos. Concentrations of 0.1, 1.0 and 10.0 micrograms ml-1 reduced sporozoite viability, as judged by oocyst production. Injections into the embryos of 5, 50 or 500 micrograms of lasalocid 92-93 h after infection also reduced oocyst production, indicating activity against the later stages of the life cycle.

Anticoccidial activity of combinations of narasin and nicarbazin.

The efficacy of mixtures of narasin and nicarbazin were evaluated by comparing broiler performance, susceptibility to heat stress, and the mode of action against Eimeria. In a floor pen trial, narasin (70 ppm) alone or in combination with nicarbazin at levels between 10/10 and 50/50 ppm gave significantly better performance than unmedicated birds or birds given nicarbazin at 125 ppm alone. Amelioration of nicarbazin-associated mortality with heat as a stressor was observed in birds given the 50/50 ppm mixture of narasin and nicarbazin: mortality in these birds was similar to that of unmedicated birds and was reduced by 15 to 20% of that occurring in birds in the nicarbazin (125 ppm) treatment. The narasin/nicarbazin mixture (50/50) appears primarily to prevent further development of sporozoites. However, in birds treated with 25/25 ppm of narasin and nicarbazin, both the deleterious action of nicarbazin on merogeny and the antisporozoite activity of narasin were observed.

Eimeria of American chickens: characteristics of six attenuated strains produced by selection for precocious development.

Six attenuated lines of Eimeria species of the chicken of USA origin were produced by selection for precociousness. These lines were less pathogenic, judged by body weight changes and intestinal lesions in infected chickens, than the parent strains from which they were derived. The immunogenicity of the precocious lines and their parent strains was similar. The use of these attenuated Eimeria Unes for immunological control of coccidiosis is discussed.

Parasites of the Christmas turkey.

In the temperate West, the turkey remains popular fare for festive feasts. It is a large bird, amenable to intensive rearing, and now represents one of the cheapest forms of poultry meat available (Box 1). In the USA alone, nearly 100 million birds are raised annually - mainly in Minnesota and North Carolina. But intensive rearing can incur risks of epizootic parasitic diseases, often responsible for severe economic losses. Improved management and medication have reduced the impact of some, such as 'gapezvorm disease', histomoniasis and intestinal coccidiosis; leucocytozoonosis now presents less of a threat than in the past, but some 'newer' diseases such as cryptosporidiosis may yet present severe problems. In this article, Peter Long, William Current and Gayle Noblet review the main parasite challenges faced by the commercial turkey industry.

The extraintestinal stages of Eimeria tenella and E. maxima in the chicken.

Donor chickens given feed medicated with one or two levels of decoquinate or given non-medicated feed were infected with oocysts of Eimeria tenella or E. maxima per os. Twelve hours after inoculation with oocysts liver, mid-intestine or ceca homogenates were fed to previously uninfected recipient chickens. The results showed that continuous medication with decoquinate was effective in preventing the transfer of sporozoites from the intestine to the liver. Oocysts were detected in the feces of all recipients of tissue from non-medicated donors, showing that some sporozoites of E. maxima and E. tenella are normally transferred to liver. Young broiler chickens were immunized by oral inoculation of E. maxima oocysts. The immune status of similar chickens inoculated with sporozoites of the same species directly into the liver or spleen were assessed. During the experimental period half of the chicks were provided with non-medicated food and the remainder were given feed supplemented with decoquinate; decoquinate was effective in arresting the development of the sporozoites. Two weeks after initial infection the birds were challenged with oocysts of E. maxima per os. Injection of sporozoites into the spleen did not protect against challenge. Birds inoculated with sporozoites into the liver were unable to develop a significant level of immunity. When the drug pressure was removed from these birds, parasitism of the intestine occurred and immunity developed.

Gut stasis in chickens infected with Eimeria.

The duration and locations of gut stasis were examined in chickens infected with either Eimeria acervulina or E. maxima. Gut passage time (GPT) was used to determine gut stasis. The location of feed retention was determined qualitatively and quantitatively. Infections with both species were associated with increased GPT from Days 5 to 13 postinoculation. Feed appeared to be retained in the crop and gizzard of infected birds when judged visually. However, measurements of total dry matter retained in various regions of the gastrointestinal tract did not differ significantly from each other.

Control of chicken coccidiosis.

Coccidiosis could potentially cause enormous economic loss to the poultry industry, especially in the production of broiler chickens (see Box 1). Losses are currently minimized by chemotherapeutic treatment but the effectiveness of many drugs seems to be declining. In this article, Peter Long and Tom Jeffers discuss the future for coccidial chemotherapy, and the potential for immunological control methods.

Immunization of chickens against coccidiosis with extracts of Eimeria-infected tissues.

Immunization of chickens with extracts prepared from Eimeria-infected tissues was attempted. Significant protection against weight depression was conferred when chickens were inoculated intra-abdominally with tissue extracts prepared from ceca infected with two different strains of E. tenella. When extracts were given intravenously, with or without the adjuvant, polyandenylic-polyuridylic acid, weight depression was not ameliorated after challenge. Experiments using extracts from two strains of E. acervulina- and E. maxima- and one strain of E. brunetti-infected tissues failed to protect against weight gain depression after challenge.

Immunisation of young broiler chickens with low level infections of Eimeria tenella, E. acervulina or E. maxima.

Chickens given 200 oocysts of Eimeria tenella at day-old followed by a dose of between 300 to 500 oocysts at 8 days of age were afforded substantial protection against challenge at 15 and 22 days of age. Chickens given 2,000 oocysts of E. acervulina at day-old were partially protected against challenge infection given at 15 or 22 days of age. When chickens were given doses of 2,000 and 10,000 oocysts at 1 and 8 days, respectively, significant protection against challenge at 15 and 22 days was obtained. Chickens given five oocysts of E. maxima at day-old were partially protected against challenge at 15 or 21 days of age. A dose of 50 oocysts at day-old gave substantial protection judged by body weight changes and lesion scores. The protection was slightly greater when the immunising dose was given at 8 days of age. The results indicate that with chickens kept on wire floors where the conditions for reinfection were minimal, substantial immunity to challenge infection could be achieved by giving small numbers of oocysts to chickens 1 to 8 days of age.

Coccidial oocysts in the liver of a turkey poult.

The liver of a 5-week-old turkey poult submitted for examination was found to have multiple focal lesions which contained unsporulated coccidial oocysts. These oocysts sporulated after incubation and were subsequently identified as oocysts of E. adenoeides. The source of the oocysts was not determined. This appears to be the first time a spontaneous occurrence of a species of Eimeria has been recorded in the liver of an avian host.

The pathogenicity, immunogenicity and endogenous development of a precocious line of Eimeria brunetti.

A precocious line of Eimeria brunetti with a prepatent period of 75 hours was compared with the parent strain (normal prepatent period of 120 hours) in a battery study. The precocious line was less pathogenic than the parent, and caused less weight depression and lower lesion scores in chickens given any dose of oocysts (1,000, 5,000, 25,000 or 125,000). No chickens receiving the precocious line died, whilst 19% and 25% mortality occurred in groups receiving 25,000 and 125,000 oocysts of the parent strain. The precocious line was also highly immunogenic. Chickens given 1,000 or 5,000 oocysts of the precocious line or parent strain developed immunity sufficient to protect against a challenge of the parent strain which caused 83% weight depression and 13% mortality in non-immunised, challenged controls. Histological observations showed that the endogenous development of the parent strain and precocious line was identical up to 48 hours. However, by 72 hours macrogametocytes and microgametes were seen only with the precocious line. The abbreviated asexual cycle of the precocious line would account for its lower reproductive potential.