Impact of fresh or used litter on the posthatch immune system of commercial broilers

This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.     

Efficacy of decoquinate against drug sensitive laboratory strains of Eimeria tenella and field isolates of Eimeria spp. in broiler chickens in China

The efficacy of decoquinate against Eimeria infections in broiler chickens was evaluated using two drug sensitive laboratory strains of Eimeria tenella and 20 field isolates of Eimeria spp. collected from farms in China where various anticoccidials (including maduramicin) had been used. Decoquinate (20-40 ppm in feed) and maduramicin (5 ppm) were efficacious against E. tenella laboratory strains, but decoquinate more so than maduramicin. Body weight gains of E. tenella infected chickens were significantly improved, and caecal lesions were prevented, by feeding either decoquinate or maduramicin. Decoquinate also prevented oocyst production, but maduramicin did not. Most (18/20) Eimeria field isolates were resistant to maduramicin, judged by oocyst production; decoquinate at > or =20 ppm completely controlled all 20 field isolates. Decoquinate has potential value as a broiler anticoccidial in China and other countries where it has not been previously used.     

Role of oregano and Citrus species-based essential oil preparation for the control of coccidiosis in broiler chickens

Background: Due to presence of drug-resistant Eimeria strains and raised public health safety concerns about drug residues in the meat, there is renewed interest in the search for natural alternatives to the coccidiosis control agents. This study was conducted to test the anticoccidial efficacy of oregano and Citrus spp.-based essential oils for broilers.Methods: A total of 280 7-day-old broiler chicks were fed a control diet or diets with salinomycin or essential oils for up to 35 d of age. On d 14, half of the control groups and the treated groups were orally challenged with a coccidiosis vaccine at 25 times higher than the recommended vaccine dose. Control diet-fed chickens that were gavaged with phosphate-buffered saline were considered non-challenged control group.Results: Eimeria challenge or dietary additives failed to affect growth performance during the 7 to 20 d growth period although essential oil-fed chickens exhibited the lowest body wight gain(P = 0.332) and the highest feed conversion ratio(P = 0.062). Oocysts in the litter were detected in the challenged control diet group and the challenged/essential oil-fed groups at 21 and 35 d, respectively. Superoxide dismutase activity in the serum was elevated(P = 0.059) in the salinomycin-fed chickens compared to the challenged controls. Alpha-1-acid glycoprotein was decreased by 28.7% in the salinomycin-fed chickens but increased by 38.1% in the essential oil group compared with the challenged control group. Challenged control group exhibited a significantly higher cooking loss of the thigh meat, compared to the non-challenged control diet group, which was marginally mitigated by dietary supplementation with essential oils. Chickens fed essential oil-added diet had the highest branched-chain fatty acids contents in the cecum.Conclusions: In conclusion, this study shows that oregano and Citrus-based essential oil preparation did not affect growth performance in broiler chickens challenged with the coccidiosis vaccine nor did Eimeria-specific duodenal lesion. However, dietary essential oil preparation lowered oocysts present in litter materials and altered branchedchain fatty acids in cecal digesta. Beneficial findings of the essential oil preparation on volatile fatty acids and oocysts output may warrant further research into assessing its effectiveness and its efficacy in pathogenic fieldisolate Eimeria spp.-induced coccidiosis disease model.     

Higher incidence of Eimeria spp. field isolates sensitive for diclazuril and monensin associated with the use of live coccidiosis vaccination with paracox-5 in broiler farms

Twenty European Eimeria spp. field isolates were subjected to an anticoccidial sensitivity test (AST). The anticoccidial drugs tested were diclazuril (Clinacox) and monensin (Elancoban). The assay was performed in a battery cage trial. Infected medicated birds were compared with an unmedicated control group. Coccidial lesion scores and oocyst shedding were used as parameters. The results of the AST show that resistance is common amongst coccidiosis field isolates, especially Eimeria acervulina (68% and 53% resistance for diclazuril and monensin, respectively). Resistance is less frequent amongst Eimeria maxima (38% and 50% resistance for diclazuril and monensin, respectively) and Eimeria tenella isolates (23% and 38% resistance for diclazuril and monensin, respectively). A highly significant influence of the coccidiosis prevention program (live coccidiosis vaccination with Paracox-5 vs. anticoccidial drugs in feed) on the sensitivity patterns of Eimeria spp. field isolates for both diclazuril (P= 0.000) and monensin (P= 0.001) was found. Further, when looking at the single species and each anticoccidial drug level, significantly more sensitivity of E. acervulina for monensin (P= 0.018), E. maxima for diclazuril (P = 0.009), and E. tenella for diclazuril (P = 0.007) was found in isolates originating from vaccinated flocks. Moreover, for E. acervulina and diclazuril, E. maxima and monensin, and E. tenella and monensin a trend toward higher sensitivity of isolates for these products was found when live coccidiosis vaccination was applied. The present study shows that sensitivity for the anticoccidial drugs diclazuril and monensin is more frequent in Eimeria spp. field isolates originating from broiler farms where a coccidiosis vaccination policy is followed.     

Application of polymerase chain reaction based on ITS1 rDNA to speciate Eimeria

A method was developed to recover Eimeria spp. oocysts directly from poultry litter and determine which species of Eimeria were present using polymerase chain reaction (PCR) based on the ITS1 rDNA sequence. The species composition of Eimeria oocysts was also compared before and after propagation in susceptible chickens to determine if the relative proportion of each species changed after expansion. In samples from two broiler operations, ITS1-PCR was able to detect Eimeria spp. oocysts recovered from litter, with Eimeria acervulina, Eimeria maxima, and Eimeria praecox being the predominant species present therein. Although Eimeria tenella was found in one sample, the other species--Eimeria brunetti, Eimeria necatrix, and Eimeria mitis-were not detected. The species composition as determined by ITS1-PCR did not appear to appreciably alter after expansion in susceptible chickens. The described method represents a rapid means for determining the major Eimeria species in a poultry operation and may be helpful in choosing a particular live oocyst vaccine formulation to protect chickens against coccidiosis.     

An enzyme-linked immunosorbent assay for coccidiosis in chickens: correlation of antibody levels with prior exposure to coccidia in the laboratory and in the field

An enzyme-linked immunosorbent assay was developed for detecting antibody to coccidia to facilitate the survey of laboratory and field infections. Serum antibody levels in chickens were measured against soluble Eimeria tenella oocyst antigen. Sera from breeders aged 10, 23, 37, and 43 weeks were positive with uniformly high antibody titers. Broiler chick sera showed high maternal antibody titer at hatch, decreasing to an almost negligible response at 3 weeks of age. Two-week-old broiler chicks had variable responses to a single infection of E. tenella: titers were elevated at 8 to 10 days postinfection and generally increased through day 24. Weekly reinfection of 2-week-old broiler chickens produced an antibody titer in proportion to the number of oocysts per dose and stimulated protection against challenge with 2 x 10(5) E. tenella. Inbred birds raised in a pathogen-free environment for 6 weeks had no detectable antibody titers.     

Comparison of Eimeria species distribution and salinomycin resistance in commercial broiler operations utilizing different coccidiosis control strategies

The purpose of the present study was to evaluate the species composition and salinomycin sensitivity of Eimeria oocysts isolated from commercial broiler farms that differed by means of coccidiosis control (anticoccidial drugs [ACD] vs. live oocyst vaccines [VAC]). A comparison of Eimeria species composition and salinomycin sensitivity was also made before and after a producer switched from salinomycin to live oocyst vaccines. In general, no significant difference was observed in the concentration of Eimeria spp. oocysts in litter from VAC-utilizing farms compared to litter from ACD-utilizing farms. Application of PCR-based methods to detect coccidia found that Eimeria species distribution in litter from VAC operations more closely resembled the species composition in the live oocyst vaccines. Drug sensitivity testing found that Eimeria oocysts from VAC operations displayed greater salinomycin sensitivity as measured by weight gain and feed conversion efficiency compared to oocysts from ACD farms. These findings provide additional evidence for the usefulness of live oocyst vaccines to restore ionophore sensitivity in poultry operations that contain an ionophore-resistant population of Eimeria spp. oocysts.     

Evaluation of serum and secretory antibody responses to an immunodominant recombinant merozoite surface antigen, p150, using a sensitive enzyme-linked immunosorbent assay

An equation obtained from linear-regression analysis of positive/negative ratios and log of enzyme-linked immunosorbent assay (ELISA) titers of coccidia-immune serum samples was used to accurately predict the antibody titers of chickens immunized with a recombinant merozoite surface protein (p150). Chickens immunized twice intramuscularly with the recombinant p150 antigen emulsified in complete Freund's adjuvant developed a dose-dependent anti-p150 antibody response 14 days after primary immunization. Serum IgG and IgM and secretory biliary IgA antibodies were detected 2 months after primary immunization. Oral challenge with live Eimeria parasites significantly enhanced both the serum and secretory anti-p150 antibody titers. These results indicate that vaccination of chickens with the p150 recombinant merozoite antigen can induce a parasite-specific host immune response.     

Effects of salinomycin and Bacillus subtilis on growth performance and immune responses in broiler chickens

The present study was undertaken to compare the effect of salinomycin and Bacillus subtilis on growth performance, serum antibody levels against Clostridium spp. and Eimeria spp., and cytokine mRNA expression levels in broiler chickens raised in the used litter. Broiler chickens fed a diet containing salinomycin showed lower (P < 0.05) body weights compared with the control diet-fed counterparts. Serum nitric oxide levels were significantly (P < 0.05) elevated in chickens fed the B. subtilis-enriched diet compared with those on either the salinomycin-fed or control diet-fed chickens. None of the dietary treatments affected (P > 0.05) serum antibody levels against Clostridium perfringens toxins. Both salinomycin and B. subtilis significantly lowered (P < 0.05) the serum levels of Eimeria-specific antibodies compared with the control group. Salinomycin, but not B. subtilis, significantly modulated (P < 0.05) the expression of cytokines encoding interferon-γ (IFN-γ), interleukin10 (IL-10) and tumor necrosis factor superfamily 15 (TNFSF15) compared with the control group. In conclusion, dietary salinomycin and B. subtilis affected serum anticoccidial antibody and intestinal cytokine expression, but failed to improve growth performance in broiler chickens. Further study is warranted to investigate the mode of action of salinomycin on host immune response and growth performance in broiler chickens.     

Coccidiosis in poultry: anticoccidial products, vaccines and other prevention strategies

Coccidiosis in chickens is a parasitic disease with great economic significance, which has been controlled successfully for decades using mainly anticoccidial products. However, large-scale and long-term use of anticoccidial drugs has led to the worldwide development of resistance against all these drugs. In order to minimize the occurrence of resistance, the rotation of various anticoccidial drugs in single and/or shuttle programmes is used. Unfortunately, this has not solved the anticoccidial resistance problem. Recently, live anticoccidial vaccines have been incorporated into rotation programmes, resulting in an increasing incidence of anticoccidial drug-sensitive Eimeria spp. field isolates, which may ameliorate the efficacy of anticoccidial drugs. Nevertheless, possible upcoming bans restricting the use of anticoccidials as feed additives, consumer concerns on residues and increasing regulations have prompted the quest for alternative coccidiosis control strategies. Although management and biosecurity measures could halt the introduction of Eimeria spp. to a farm, in practice they do not suffice to prevent coccidiosis outbreaks. Phytotherapy, aromatherapy and pre- and probiotics either show conflicting, non-consistent or non-convincing results, and have therefore not been applied at a large scale in the field. So far, live attenuated and non-attenuated anticoccidial vaccines have proved to be the most solid and successful coccidiosis prevention and control strategy. Despite the drawbacks associated with their production and use, their popularity is increasing. If with time, the immunogenicity of subunit vaccines can be improved, they could represent the next generation of highly efficient and low-cost anticoccidial strategies.     

Differences between the anticoccidial potencies of monensin in maize-based or wheat-based chicken diets

Experiments were carried out to investigate why the anticoccidial ionophore monensin is more potent against the coccidiumEimeria tenella in chickens fed on a maize-based diet (as in the USA) than in chickens fed on a wheat-based diet (as in the UK). The explanation seems to be that the pathogenicity ofE. tenella is lower in maize-fed chickens than in wheat-fed chickens, whether monensin is present in the diet or not. Possible reasons for this are suggested. The better survival of maize-fed birds may be partly due to protective effects of the higher concentrations of vitamins A and E derived from their diet. Furthermore, the higher concentrations of niacin and riboflavin in wheat than in maize may enhance coccidial pathogenicity. These opposing factors might combine to cause equivalent infection levels to produce more severe coccidiosis in chickens fed on a wheat-based diet than on a maize-based diet.     

Anticoccidial effect of green tea-based diets against Eimeria maxima

Anticoccidial effects of green tea (GT)-based diets were evaluated in chickens following oral infection with Eimeria maxima an ubiquitous intestinal parasite of poultry that impairs the growth and feed efficiency of infected birds. Five-week-old chickens were assigned to four groups (GT 0.5%, GT 2.0%, untreated/infected and non-infected control) and each group consisted of 15 chickens. Chickens were fed a standard diet supplemented with ground green tea for 2 weeks prior to infection with E. maxima (10,000 sporulated oocysts per bird). The effects of green tea on E. maxima infection were assessed by two parameters, fecal oocyst shedding and body weight gain. The green tea-fed chickens produced significantly reduced fecal oocysts (P<0.05) when compared to the E. maxima-infected group fed standard diet. The green tea-based diet, however, did not improve body weight loss caused by E. maxima infection. This study is the first to demonstrate anticoccidial effect of green tea on Eimeria parasites.     

Avian eimeria: invasion in foreign host birds and generation of partial immunity against coccidiosis

Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.     

Sensitivity of field isolates of Eimeria to monensin in the turkey

A method previously described by Jeffers and Bentley, involving calculation of a growth and survival ratio and optimal anticoccidial activity index, was used to investigate the sensitivity of 23 field isolates of Eimeria obtained from turkey flocks to the ionophorous antibiotic monensin. Isolates were obtained from litter and intestinal samples from several major turkey-growing regions of the United States, and in most cases contained at least two species of Eimeria. A mixture of strains that had been maintained in the laboratory for many years in the absence of exposure to anticoccidial drugs was found to be sensitive to monensin. Six of the field isolates were judged sensitive, seven partially resistant, and ten resistant to the drug, judged by the Jeffers and Bentley criteria. This is the first report of the acquisition of resistance to monensin in isolates of Eimeria from turkey flocks in the United States.     

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine in a commercial trial in broiler chickens in China

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine were examined and compared with an anticoccidial drug in a trial in broiler chickens under modern commercial conditions in China. In total, 40,660 chickens were used in the present study, half of which were vaccinated with the Supercox vaccine comprising a precocious line of Eimeria tenella and non-attenuated lines of Eimeria maxima and Eimeria acervulina, and the other half were medicated with Diclazuril delivered as feed additive at the dosage of 1mg/kg of feed. The vaccine was administered orally to 7-day-old chickens. No clinical diseases were diagnosed in any of the vaccinated birds. However, clinical coccidiosis occurred in a large proportion of medicated control birds and these chickens had to be treated with anticoccidial drugs (Diclazuril and Toltrazuril). Comparison of production performance between vaccinated birds and medicated control birds revealed that the vaccine Supercox performed better than anticoccidial drugs in terms of mortalities, costs and overall economic benefits (profits). These findings demonstrated that the use of the Supercox vaccine could control clinical coccidiosis in broilers and achieve production performance superior to that using anticoccidial drugs, particularly where drug resistance might result in failure to control clinical diseases.     

柔嫩艾美耳球虫地克珠利抗药株的实验室诱导

采用药物浓度递增法,以0．07mg／L地克珠利为起始诱导浓度连续传代,以最适抗球虫指数（POAA）、病变记分减少率（RLS）和相对卵囊产量（ROP）3项指标综合判定抗药性。经12次传代,该诱导虫株对1．5mg／L地克珠利具有完全抵抗力。试验结果表明,用实验室方法诱导柔嫩艾美耳球虫抗药株完全可行。     

Assessment of dietary supplementation with probiotics on performance, intestinal morphology and microflora of chickens infected with Eimeria tenella

We evaluated the effects of dietary supplementation with different preparations of probiotics on the performance of broiler chickens experimentally infected with 2 × 10(4) sporulated oocysts of Eimeria tenella at 14 days of age. Three hundred, day-old, Cobb-500 chicks, as hatched, were separated into 10 equal groups with three replicates. Two of the groups, one challenged with E. tenella oocysts and the other not, were given a basal diet and served as controls without medication. The other challenged groups were given the anticoccidial lasalocid (60 mg/kg) or Enterococcus faecium (5 × 10(8) or 5 × 10(9)cfu/kg feed), Bifidobacterium animalis (5 × 10(8)cfu/kg feed), Lactobacillus reuteri (5 × 10(8)cfu/kg feed), Bacillus subtilis (5 × 10(8)cfu/kg feed), or a multi-species probiotic mix at 5 × 10(8) or 5 × 10(9)cfu/kg feed, respectively. The trial lasted 6 weeks. Individual body weight, feed intake per pen and feed conversion ratio values were recorded weekly, along with the extent of bloody diarrhea, excreta oocyst numbers and bird mortality. Caecal lesions were assessed and intestinal samples were taken for histopathological and bacteriological evaluation from ileum and caecum. Overall growth performance of chickens fed the multi-species probiotic mix at both levels was higher (P<0.05) compared to the infected control. Overall oocyst shedding was lowest (P<0.05) in the lasalocid supplemented group. Villous height was higher (P<0.05) in Bacillus supplemented groups compared to infected controls. The Lactobacillus supplemented group had the highest (P<0.05) numbers of both Lactobacillus and Bifidobacterium in ileum and caecum. In conclusion, dietary probiotics are promising for further investigation on improving intestinal health and growth performance of broiler chickens experimentally challenged with E. tenella.     

Isolation and selection of ionophore-tolerant Eimeria precocious lines: E. tenella, E. maxima and E. acervulina

Eimeria parasites were isolated from Nanhai Guangdong province (southern China) and studied in chickens in wire cages to evaluate their drug resistance against commonly used ionophores: monensin (100 mg/kg of feed), lasolacid (90 mg/kg), salinomycin (60 mg/kg), maduramicin (5 mg/kg) and semduramicin (25 mg/kg). Chinese Yellow Broiler Chickens were infected with 40,000 crude sporulated Eimeria oocysts at 15 days of age and prophylactic medication commenced a day prior to infection. Drug resistance was assessed for each ionophore drug by calculating the anticoccidial index (ACI) and percentage optimum anticoccidial activity (POAA) based on relative weight gain, rate of oocyst production and lesion values. Results revealed that Nanhai Eimeria oocysts comprising of E. tenella, E. maxima and E. acervulina, were resistant to monensin, sensitive to both salinomycin and lasolacid and partially sensitive to maduramicin and semduramicin. By selection for early development of oocysts during passage through chickens, the prepatent time of E. tenella, E. maxima and E. acervulina were reduced by 49, 36 and 22 h, respectively. The precocious lines are less pathogenic than the parent strains from which they were selected and conferred a satisfactory protection for chickens against coccidiosis. These ionophore-tolerant precocious lines could have wider applications in the development of anticoccidial vaccines for sustainable control of coccidiosis.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

Field basis evaluation of Eimeria necatrix-specific enzyme-linked immunosorbent assay (ELISA) for its utility in detecting antibodies elicited by vaccination in chickens

Eimeria necatrix-specific ELISA, using a recombinant antigen (the cDNA-clone NP19 expressing protein), was utilized to detect antibodies against E. necatrix in breeder pullet flocks that had previously received an attenuated live vaccine to E. necatrix. Vaccinated flocks were discriminated significantly from non-vaccinated flocks by their antibody titers and antibody positive rates at 30-55 days post-vaccination. In addition, E. necatrix-oocysts were confirmed in fecal samples of vaccinated flocks using PCR in the case where the antibody positive rates rose. These findings implied that the vaccination prompted repeated infections, and consequently the chickens generated antibodies and secured their protection against virulent field-E. necatrix. Therefore, the ELISA was suggested to be a useful tool to estimate the immune state of chickens as a result of vaccination with a live E. necatrix-vaccine.