鸡胚接种柔嫩艾美耳球虫和堆形艾美耳球虫活卵囊的免疫保护试验

给18日龄鸡胚接种一定剂量的柔嫩艾美耳球虫(Eim eria tenella)和/或堆形艾美耳球虫(E.acervulina)孢子化卵囊,出雏后在无球虫环境中笼养,1~10日龄每天收集各组粪便样本,计数克粪便卵囊数(OPG),并于14日龄时以大剂量同源孢子化卵囊攻虫,以相对增重率(RWG)、饲料转化率(FCR)、相对卵囊产量(ROP)评价免疫保护效果。结果显示,以E.tenella或E.acervulina卵囊免疫18日龄鸡胚,其卵囊排出的潜隐期及达到峰值的时间与1日龄雏鸡接种组相一致,有相似的排卵囊曲线,提示其诱导免疫的建立是在出雏后开始建立的。攻虫后各免疫组的RWG由攻虫对照组的31.9%~51.7%提高到了76.5%~83.6%,RCR由攻虫对照组的4.11~4.89改善为2.72~2.96,ROP降至4.7%~23.5%。结果表明以一定剂量E.tenella和E.acervulina卵囊单独或混合经羊膜腔免疫18日龄鸡胚都可以建立起针对出雏后14日龄同源攻虫的良好免疫保护力。比较混合免疫E.tenella和E.acervulina卵囊组与单一接种E.tenella或E.acervulina卵囊组的免疫效果发现,混合免疫组的各项指标均稍优于后者。     

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.     

Prior or concurrent exposure to different species of avian Eimeria: effect on sporozoite invasion and chick growth performance.

The effects of prior (immunity) or concurrent administration of Eimeria acervulina or Eimeria tenella on cellular invasion in vivo and in vitro and on growth performance in white leghorn chickens (WLC) were examined. Weight gains of WLC immunized with E. acervulina and challenged with E. tenella were significantly greater than those of nonimmunized chicks challenged with E. tenella (this occurred despite the increased invasion by E. tenella in E. acervulina-immunized chicks that was reported earlier). The weight gains and modest but consistent improvements in intestinal lesion scores, feed conversion ratios, and oocyst shedding in immunized/challenged WLC indicated that E. acervulina conferred a small measure of protection against E. tenella infection that was independent of the effect on invasion. In contrast, immunization of WLC with E. tenella significantly decreased (41%-51%) invasion by E. acervulina as compared with that in nonimmunized WLC but had little effect on chick growth performance. Concurrent inoculation of chicks with E. tenella and E. acervulina had little effect on invasion by E. tenella sporozoites or on subsequent performance of the chicks. In vitro, prior exposure of cultured cells to either of two isolates of E. tenella also caused a significant decrease in invasion by E. acervulina. No gross changes occurred in the culture morphology between the E. tenella-inoculated and noninoculated cultures. Collectively, the data indicate that prior exposure of WLC and cultured cells to single isolates of avian coccidia markedly influenced invasion by other species but had less effect on the growth performance of the birds.     

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.     

Anticoccidial effects of xanthohumol

Xanthohumol (XN), a prenylated chalcone from the hops flower, was examined for its ability to reduce invasion of Madin-Darby bovine kidney (MDBK) cells by Eimeria tenella sporozoites (SZ), as well as to reduce invasion by E. tenella and E. acervulina SZ in the chick host. Additionally, XN was tested as an anticoccidial feed additive at 20 ppm against challenge infections with E. acervulina, E. maxima, and E. tenella. Cell invasion by E. tenella SZ was inhibited 66% by treatment of SZ with 22 ppm XN. This inhibition was associated with an apparent physical disruption of the apical ends of the SZ. Rectal challenges with E. tenella SZ treated with 5, 10, and 20 ppm XN resulted in significantly reduced gross-lesion scores and normal chick-host weight gains compared with challenge with untreated SZ. Oral challenges with similarly treated E. acervulina SZ, accomplished with prior antacid treatment, resulted in significantly reduced gross lesions and reduced oocyst shedding compared with challenge with untreated SZ and were associated with physical disruption of sporozoite morphology. In a pilot test, provision of feed supplemented with 20 ppm XN for 3 days before challenge to 6 days after challenge did not control challenge infections with E. acervulina, E. maxima, or E. tenella as judged by measurements of weight gain, feed conversion, and gross lesions. Although XN-fed chicks infected with E. acervulina and E. maxima shed fewer oocysts than those on control feed, the differences in numbers were not statistically significant (P > 0.05).     

Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence

Chicken coccidia are protozoan parasites of the genus Eimeria. They cause economical losses in the poultry industry globally. The various species can be distinguished on the basis of the morphology of the oocysts and parasitic site in intestine, but these criteria sometimes are unreliable. Therefore, a species-specific polymerase chain reaction (PCR) was developed. Based on variable sequence regions, specific primers were constructed for the differentiation of five Eimeria species (Eimeria acervulina, E. brunette, E. maxima, E. necatrix, and E. tenella). PCR products were amplified from coccidian vaccine (coccivac-D and coccivac-B) and E. tenella and were subsequently sequenced. Similarities of the five species sequences between the vaccines and Genbank were 94-100%. Analysis of the E. tenella internal transcribed spacer 1 (ITS-1) partial sequence from Taiwan and from Genbank indicated that the similarity was 99.6%. The PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. The five sets of primers will not amplify any non-specific bands of the chicken genome or its intestinal contents. Therefore, the five sets of specifically designed primers are guaranteed to be useful for differential diagnosis of avian coccidiosis caused by Eimeria spp.     

Guar meal ameliorates Eimeria tenella infection in broiler chicks

Guar meal contains relatively high levels of saponins, which are known to have antiprotozoal activity and may be effective against coccidiosis. A 2x2 factorial experiment investigated the impact of guar meal (0 or 5%) corn-soy-based starter broiler diets on chicks unchallenged or challenged with Eimeria tenella. At 1 day of age, 120 unsexed RossxRoss broiler chicks were randomly distributed among four treatment groups. Chicks were challenged with 5x10(3) sporulated oocysts of E. tenella in 0.5ml at 10 days of age by oral gavage. Weekly body weight, body weight gains, feed conversion ratio and mortality rate were recorded for chicks fed from 0 to 21 days of age. Oocysts shed per gram feces were recorded from 6 to 10 days post-challenge. Results showed that challenged chicks fed 0% guar meal had significantly higher oocysts per gram shed in feces than the other groups. No significant differences among treatment groups in mortality rate were observed. Body weights of unchallenged and challenged chicks fed 0% guar meal were significantly higher than those fed 5% guar meal at 2 weeks of age. Results indicated that including 5% guar meal in the diet of chicks challenged with E. tenella decreased oocysts shed per gram feces and prevented bloody diarrhea, but without affects on body weight and feed conversion ratio at 11 days post-challenge.     

福建省鸡球虫病感染情况的调查

为掌握福建省鸡球虫病的发病状况及影响因素,2009年12月至2010年11月,采用粪便漂浮法和卵囊培养法对本省的球虫病情况进行调查,并在实验室对送检的1 500份粪便样品进行了分析.结果发现当地鸡体内有6种球虫,分别是柔嫩艾美耳球虫(Eimeria tenlla)、巨型艾美耳球虫(E.maxima)、堆形艾美耳球虫(E...     

Isolation of five species ofeimeria from chickens in Bangladesh

Five species of Eimeria, namely E. acervulina, E. tenella, E. maxima, E. brunetti and E. necatrix were identified in chickens in Bangladesh on the basis of lesions seen at post-mortem examinations of naturally infected birds, and on the dimensions of oocysts and the lesions seen in chicks experimentally infected with single oocyst derived strains. The use of filter top, polycarbonate cages permitted the isolation of strains without sophisticated animal isolators and is appropriate for use in laboratories throughout the developing world. Responses to a questionnaire sent to all known intensive poultry farms suggested that coccidiosis is a major disease. For control, producers rely mostly on management procedures and the tactical use of sulphonamides; in-feed chemoprophylaxis is not widely used. These control measures are unsatisfactory and recent coccidiosis outbreaks were reported from seven of 16 farms. There was evidence of a seasonal incidence in clinical coccidiosis.     

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.     

Responses of chickens vaccinated with a live attenuated multi-valent ionophore-tolerant Eimeria vaccine

Coccidiosis, caused by Eimeria species, is a serious economic disease of chickens (Gallus gallus) and the search for vaccines to control the disease is intensifying especially with the increasing threat of drug resistance. A live attenuated multi-valent ionophore-tolerant Eimeria vaccine has been developed that contains three ionophore-resistant Eimeria species, E. tenella, E. maxima and E. acervulina. The attenuated lines were derived from virulent field strains resistant to monensin ionophore by selection for early development in chicks. The vaccine was administered by gavage and through drinking water to broiler chickens, Chinese Yellow strain, reared in wire cages. Vaccinated medicated birds performed better than vaccinated unmedicated and medicated unvaccinated groups. The final mean weights of vaccinated medicated birds were significantly higher (P<0.05), and a better vaccine protection index, using both vaccinating methods, was achieved. Results indicated that concomitant use of ionophores and vaccines could be a useful adjunct to planned immunization in the control of coccidiosis.     

Relative value of oocyst counts in evaluating anticoccidial activity.

Birds medicated with roxarsone and in another experiment with zoalene in the feed produced higher oocysts counts than unmedicated control birds receiving the same oocyst dose of Eimeria tenella or a mixture of six species (E. tenella, E. necatrix, E. brunetti, E. maxima, E. acervulina, and E. mitvati). These experiments confirm the conclusion that oocyst counts constitute an unsatisfactory and unreliable parameter for judging effectiveness of an anticoccidial even though such increases are a relatively rare occurrence in anticoccidial evaluation experiments.     

Molecular identification of Eimeria species infecting market-age meat chickens in commercial flocks in Ontario

A previously described multiplex PCR was evaluated for the identification and prevalence of Eimeria species in market-age commercial chicken flocks in Ontario. The multiplex PCR based on species-specific RAPD-SCAR markers was able to distinguish six available laboratory strains of Eimeria species (E. tenella, E. maxima, E. necatrix, E. mitis, E. acervulina, and E. brunetti) and E. tenella, E. maxima and E. acervulina in unknown field samples, including multiple infections in single reactions. No backyard (0/77) and 20/360 market-age commercial chickens were oocyst-positive using standard fecal flotation methods. PCR identified E. tenella alone (9/360, 2.5%), E. maxima alone (5/360, 1.38%), E. maxima plus E. tenella (5/360, 1.38%) and E. acervulina alone (1/360, 0.27%) in market-age commercial broilers. This is probably the first time the multiplex PCR has been evaluated in poultry establishments in Canada and illustrates the value of the tool in coccidiosis epidemiology on commercial farms.     

Prevalence and risk factors for subclinical coccidiosis in broiler chicken farms in Mazandaran province,Iran

Coccidiosis is a disease of almost universal importance in Poultry production. The subclinical form of coccidiosis has most significant economic impact due to impaired growth rate and feed conversion. Some factors such as: age, size of flock, season, etc., may effect on severity of this disease. In this study, the relationships between some major risk factors and prevalence of subclinical coccidiosis were investigated. This study was done in 120 broiler farms in five different cities of Mazandaran province, north of Iran. Five chicks (3–8 weeks of ages) were taken randomly from every 120 farms and post-mortem and parasitological examinations were performed. Five Eimeria spp. were recognized: Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria brunetti, and Eimeria necatrix. The prevalence rate of subclinical coccidiosis among them is 75% (90 farms out of 120). E. acervulina was the most prevalent species (65.5%) followed by E. maxima (17.7%), E. tenella (15.5%), E. brunetti (10%), and E. necatrix (5.5%). According to what the results approve, the occurrence of subclinical coccidiosis is significantly related to the age and size of flock, whereas the other factors such as the season of year, industrial strains, chicken’s keeping system, and anticoccidial drugs do not affect this phenomenon remarkably.     

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.     

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.     

Evaluation of interspecific hybrids of the chicken, guinea fowl, and Japanese quail for innate resistance to coccidia

Experimental chicken/guinea fowl hybrids, guinea fowl, and chickens were orally inoculated with Eimeria acervulina or E. tenella, which are specific for chickens, or with E. grenieri, which is specific for guinea fowl. No intact oocysts were found in feces within 24 hr of inoculation, suggesting that excystation occurred in the normal and abnormal hosts. No oocysts were found in the feces of hybrids during a 9-day postinoculation period. The guinea fowl passed oocysts of guinea fowl coccidia (E. grenieri) but not those of chicken coccidia, and the chickens passed oocysts of chicken coccidia (E. acervulina and E. tenella) but not those of guinea fowl coccidia. Some asexual development (schizogony) occurred in hybrids inoculated with E. tenella, but sexual development (gametogony) did not. In contrast, quail/chicken hybrids became infected with oocysts of chicken coccidia (E. acervulina, E. tenella, and E. maxima) and quail coccidia (E. bateri) and passed a few oocysts during the normal patent period; control chickens and quails became heavily infected with oocysts of chicken and quail coccidia, respectively.     

Effect of ibuprofen on coccidiosis in broiler chickens

Ibuprofen (IBU)-a nonsteroidal anti-inflammatory drug-inhibits the biosynthesis of prostaglandins with pro-inflammatory and immunosuppressive properties and is therefore proposed as a candidate molecule for the treatment of coccidiosis in broiler chickens. In all experiments, IBU was administered via drinking water. In a first experiment, chickens were infected at 10 or 21 days of age with oocysts of Eimeria acervulina (5 X 10(4)), Eimeria maxima (3 X 10(4)), and Eimeria tenella (7.5 X 10(3)) and medicated with IBU at a dose of 15 mg/kg body weight (BW). In a second experiment, chickens were infected at 6 days of age with 10(4) oocysts of E. acervulina and medicated with IBU at a dose of 100 mg/kg BW. In the third experiment, an inoculum consisting of 5 x 10(4) or 10(5) E. acervulina oocysts was administered at 6 days of age to chickens medicated with IBU at a dose of 100 mg/kg BW. In a fourth experiment, the effect of IBU on sporulation and infectivity of E. acervulina oocysts was studied. Coccidial lesion scores (CLSs), oocyst shedding, and weight gain were used as evaluation parameters in all experiments except the fourth, where weight gain was not taken into account. In addition, the sporulation percentage was determined in the last experiment. No influence of IBU on the indicated parameters was observed after providing the drug at a dose of 15 mg/kg BW, whereas CLSs and oocyst shedding were reduced when IBU was provided at a dose of 100 mg/kg BW. However, IBU did not significantly show any effect on the degree of sporulation and infectivity of E. acervulina oocysts at a dose of 100 mg/kg BW.     

Diagnosis of Eimeria species using traditional and molecular methods in field studies

The objective of this study was to identify and characterize species of Eimeria in broiler chickens using traditional morphological and pathological plus molecular (DNA amplification) diagnostic methodologies. Using a combination of those techniques it was possible to identify the presence of multiple circulating species in the flock as well as higher frequencies for some of them, especially Eimeria praecox and Eimeria maxima, which were identified in 100% of the flocks. The frequencies of the other species were Eimeria mitis and Eimeria necatrix (93.3%), Eimeria tenella (76,7%), Eimeria acervulina (56.7%) and Eimeria brunetti (16.7%). However using the lesion score, the most common species were E. maxima (46.7%), E. acervulina (30%), E. tenella (23.3%), and E. necatrix (10%). E. brunetti and E. praecox were not identified by using lesion score. DNA amplification had detection sensitivity for Eimeria species in the field samples of at least 20 oocysts. The implementation of DNA amplification as a routine diagnostic technique in aviaries can assist Eimeria population.     

Comparison of disease susceptibility and subclass-specific antibody response in SC and FP chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima

Susceptibility to disease and the subclass-specific antibody response to Eimeria tenella, E. acervulina, and E. maxima were compared in two inbred strains of chickens, FP (B15B21) and SC (B2B2). FP strain was more susceptible to coccidiosis than SC chickens based on oocyst production, lesion score, and clinical signs. FP chickens infected with E. tenella had more severe cecal lesions and a significantly lower hematocrit level than SC chickens. FP chickens infected with E. acervulina excreted five times as many oocysts at 6 days postinfection as SC and showed a 71% reduction in plasma carotenoid level compared with controls (56% reduction in SC chickens). Body-weight change did not correlate with other signs of disease. Both SC and FP chickens produced high levels of serum IgM and IgG and biliary IgA. Although SC chickens had a slightly higher antibody response than FP chickens at 7 days postinoculation, both strains maintained high levels of IgM, IgG, and IgA for a prolonged period post primary inoculation. Although SC and FP chickens show different disease susceptibility to coccidiosis, they demonstrate similar antibody response.