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.     

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E acervulina, E maxima, and E. tenella challenge infection. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.     

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.     

A simplified protocol for molecular identification of Eimeria species in field samples

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.     

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.     

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.     

Prevalence of coccidia in pigs in Zimbabwe.

Faecal specimens from 162 piglets, aged 1-5 months, from four farms around Harare were examined for coccidia during 1989-1990. Oocyst count per gram (OPG) of faeces was determined by the McMaster technique. Identification of Eimeria species was done after sporulation of oocysts in 2.5% potassium dichromate solution. Measurements of sporulated oocysts were taken from 20 positive cases by using calibrated eye micrometer. Overall incidence was 25.3% (41/162) varying between 15 and 39.5% at different farms. The OPG ranged between 150 and 11,300. Eight species of Eimeria were identified, namely, Eimeria debliecki, Eimeria perminuta, Eimeria suis, Eimeria polita, Eimeria neodebliecki, Eimeria porci, Eimeria scabra and Eimeria spinosa, shown in the order of their prevalence. Eimeria debliecki was the most predominant, showing highest oocyst counts in 45% of 20 pigs, followed by E. suis (25%) and E. polita (20%). On two occasions only one Eimeria sp. was found while in the other 18 (90%) cases, mixed infections of two to six species were recorded.     

Effects of clopidol on sporulation and infectivity of Eimeria tenella oocysts

Feed additive anticoccidials currently used in Japan were examined for possible effects on oocyst sporulation of Eimeria tenella. Monensin, salinomycin, lasalocid, amprolium plus ethpabate, amporolium plus ethopabate plus sulfaquinoxaline, clopidol, or nicarbazin were given to chickens continuously via the feed at the recommended use level or one-half of that level. Oocysts discharged in feces 7-8 days post inoculation (PI) were collected and aerated for sporulation. Low sporulation rate was noted, when clopidol at 62.5 mg kg-1 was given from 4 to 7 days PI. These oocysts were as infective as oocysts from controls, based on weight gain, feed efficiency, gross lesion score of cecae, and oocyst count 7 days PI. The results of the study indicated that the second schizogony and gametogony are vulnerable to clopidol, as evidenced by oocyst sporulation, but infectivity of these sporulated oocysts was not affected.     

Effects of in ovo vaccination and anticoccidials on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters

The present study reports the effects of various field anticoccidial programs on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters. The programs included in ovo vaccination and various medications with either chemicals, ionophores, or both. In general, serum samples from these chickens showed anticoccidial antibody titers when tested at days 7 and 14 post hatch with the peak response at day 43. Serum anticoccidial titers were highest in birds fed a non-medicated diet compared with those vaccinated or fed medicated diets. Total number of Eimeria oocysts and the composition of Eimeria spp. present in the litter samples from different treatment groups varied depending on the type of anticoccidial program. Oocyst counts in general ranged from 3.7×10(3) to 7.0×10(4) per g of litter. Importantly, both morphological and molecular typing studies revealed four major predominant Eimeria spp., E. acervulina, E. maxima, E. praecox, and E. tenella in the litter samples. Collectively, these results indicate that the field anticoccidial programs influenced the type and abundance of Eimeria spp. present in the litter samples and also modulated host immune response to Eimeria.     

Eimeria infections in litter-based, high stocking density systems for loose-housed laying hens in Sweden

1. Coccidiosis, caused by different Eimeria species, is believed to be a more prominent problem in loose-housed layers kept on litter than in battery cages. In this study, the impact and development of Eimeria infections were investigated in layers kept in litter-based, high stocking density systems for loose-housed hens. 2. Layers from 57 flocks on 26 farms were followed by necropsy of a representative sample of birds that died or had to be culled. Coccidiosis was diagnosed in 11 flocks (19.3%) from 9 (31%) of the farms. The outbreaks occurred when the birds were 19 to 32 weeks old. E. maxima was identified in 6 and E. tenella in 3 of the outbreaks. 3. Sixteen of the flocks were also monitored with faecal and litter samples collected at regular intervals. Oocysts were detected in samples from all these flocks. The pattern of oocyst excretion was similar in most of the flocks, with maximum counts at 4 to 8 weeks after introduction to the laying house. There was no significant correlation between the levels of oocysts in faeces and clinical coccidiosis. 4. Raising pullets without any coccidiostat, to increase their chance to develop immunity against coccidia, was not found to decrease the risk of coccidiosis during the production period when compared to the practice of giving amprolium and ethopabate during the rearing period.     

Prevalence of Isospora suis and Eimeria spp. in suckling piglets and sows in Poland

The aim of this investigation was to determine the prevalence of coccidian infections in suckling piglets and sows in Poland. The research was carried out in 14 out of 16 Polish provinces in the years 2003-2005. The investigation was conducted on three types of farms: large farms (>100 sows), medium farms (25-100 sows) and small farms (<25 sows). Diarrhoea of unweaned piglets was observed on all the examined farms. Overall, 780 litters of suckling piglets from 104 farms and 267 mother sows were examined. The faeces were analyzed with the modified McMaster method. Isopsora suis was found in 217 (27.8%) litters from 70 (66.7%) farms. Eimeria spp. was detected only in 20 (2.6%) litters from 12 (11.5%) farms. On the large farms I. suis infection was detected in 31.7% of litters whereas Eimeria spp. in 1.4% of them. On the medium sized farms I. suis was found in 18.1% of litters and Eimeria spp. in 0.6%. On the small farms I. suis was detected in only 13.2% of litters, whereas Eimeria spp. in as many as 28.9%. I. suis and Eimeria spp. oocysts were found in 18 (6.7%) and 16 (6%) sows respectively. From 72 sows producing I. suis infected piglets only 12 (16.7%) shed I. suis oocysts and as little as 4 (5.6%) shed Eimeria oocysts. In the remaining 56 sows (77.8%) no cases of coccidian infections were detected. The results of this investigation demonstrate the high prevalence of I. suis in suckling piglets on the large swine farms in Poland.     

Oocyst Counts in Crossbred Ewes under Tree-Crop Plantation in the Forest Zone of Ghana

In this paper, the Eimeria oocyst output of two groups, pregnant ewes (group 1) and non-pregnant controls (group 2), which were followed from September 1993 to August 1994, is described. In both groups of animals the level of oocyst output was high during the minor rainy season. However, during the periparturient period the pregnant ewes showed the higher oocyst output. The oocyst output in both groups fell to similar levels after weaning of the lambs in March 1994. The species of Eimeria identified in order of dominance were Eimeria parva, E. pallida, E. faurei, E. ahsata, E. ovina, E. intricata, E. granulosa and E. ninakohlyakimovae. There were no differences in the species composition of oocysts in both groups of animals.     

Eimeria infections of sheep in northwest Germany

To investigate the incidence of ovine Eimeria spp. and seasonal dynamics in oocyst output, fecal samples of sheep from three different management systems were collected monthly over a 1-year period and examined for oocysts. A total of 10 species of Eimeria were observed. The most frequent species were E. bakuensis, E. ovinoidalis, E. weybridgensis/crandallis, E. parva and E. ahsata whereas E. faurei, E. granulosa, E. intricata and E. pallida were found less often. Lambs passed larger numbers of oocysts in their feces than either ewes or yearlings.     

Eimeria brunetti and Eimeria necatrix in chickens of Argentina and confirmation of seven species of Eimeria

Ten poultry farms (broiler breeder pullets, layer pullets, and broilers) in the provinces of Entre Rios and Buenos Aires in Argentina were examined for presence of Eimeria spp. Litter samples obtained from flocks 7-11 wk old were taken to the laboratory for oocyst counting and sporulation, then concentrated for inoculation into coccidia-free chickens. Species were identified by prepatent period, oocyst size, location and appearance of lesions in the intestine, microscopic examination of mucosal smears, and histology (to confirm Eimeria brunetti). On this basis, Eimeria praecox was found in two samples, Eimeria mitis in two, Eimeria acervulina in nine, Eimeria maxima in seven, Eimeria necatrix in three, Eimeria tenella in seven, and E. brunetti in four. These results confirm the presence of all seven recognized species of Eimeria in chickens in the Republic of Argentina.     

Coccidiosis control with toltrazuril in conjunction with anticoccidial medicated or nonmedicated feed

A 42-day broiler floor pen study was conducted comparing the anticoccidial efficacy of toltrazuril (Baycox) as a stand alone treatment and as an additional treatment to in-feed anticoccidial programs. Toltrazuril was administered on days 18 and 19 in the drinking water at 7 mg/kg of body weight. The treatments were 125 ppm nicarbazin (days 0-14) to 66 ppm salinomycin (SAL) (days 15-35) with and without toltrazuril, SAL (days 0-35) with and without toltrazuril, nonmedicated (NM) to SAL with toltrazuril, and NM with and without toltrazuril. The controls were NM noninfected and infected. The treatments were replicated in five blocks of eight pens each in a randomized complete block design. All withdrawal feed was nonmedicated. On day 14, birds, except noninfected, were exposed to coccidial oocysts (Eimeria acervulina, Eimeria maxima, and Eimeria tenella) seeded litter. On days 21, 28, 35, and 42, birds and feed were weighed, four birds per pen were coccidial lesion scored, and litter oocyst counts were performed. The coccidial infection in the NM infected treatment caused a significant (P < 0.05) coccidiosis infection. Coccidiosis was moderately controlled in the anticoccidial treatment birds without toltrazuril. Performance in the NM with toltrazuril was equal to or better (P < 0.05) than the anticoccidial programs without toltrazuril. Toltrazuril was equal to the noninfected birds in performance. Toltrazuril most completely eliminated all coccidial lesions and dramatically reduced oocyst shedding. The performance data, lesion scores, and oocyst counts showed that a 2-day treatment with toltrazuril successfully controlled the coccidiosis with no relapse of infection. Toltrazuril can thus be used for supplemental control with in-feed anticoccidials or as a primary anticoccidial with nonmedicated feed.     

Prevalence and age-dependent occurrence of intestinal protozoan infections in suckling piglets

A cross-sectional survey was performed on 20 pig breeding farms in southern Hesse, central Germany, to evaluate the prevalence and age-dependent occurrence of intestinal protozoan parasites in unweaned piglets. Faecal samples of 514 clinically unaffected piglets of different age (< 1 to 5-7 weeks) were examined using the sodium acetate-acetic acid-formalin (SAF) concentration technique. Infections with the following protozoan species were detected: Balantidium coli (16 of 20 farms), Entamoeba sp. (15), Jodamoeba sp. (14), Isospora (I.) suis (9), Chilomastix sp. (6) and Eimeria spp. (6). The protozoan species differed in the start and course of (oo)cyst excretion. I. suis oocysts and Jodamoeba cysts were detected already in the first week of life whereas shedding of the other parasites started later on. The prevalence of Isospora oocyst excretion increased to a maximum (18%) in 2-3 weeks old animals followed by a sharp decline. The proportion of Balantidium, Entamoeba or Jodamoeba positive suckling piglets continously increased until the age of 5-7 weeks to 60%, 52% and 22%, respectively, whereas that of Chilomastix positive animals remained on a low level of 8-12% independent of the age. Eimeria oocysts were found transiently in the faeces of 1-4 weeks old piglets.     

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.     

Coccidial infections in housed lambs: oocyst excretion, antibody levels and genetic influences on the infection

Faecal Eimeria oocyst excretion, body weights, humoral antibodies against E. ovinoidalis sporozoite antigen and related heritabilities were determined in housed Merinoland sheep lambs throughout a period of 100 days after birth in Germany. Altogether 10-11 Eimeria spp. were found. Cumulative incidences of E. ovinoidalis and E. weybridgensis/crandallis increased rapidly resulting in almost 100% incidence in 8 weeks old lambs. In the other species, the cumulative incidence increased more continuously. Except for E. granulosa oocysts of all species had been excreted at last once until day 30. By far the highest oocyst counts (OpG) were observed with E. ovinoidalis, followed by E. weybridgensis/crandallis. High counts were limited to the period of 5-8 weeks after birth. In the other Eimeria species oocyst counts persisted at comparatively low levels until the end of the observation period although their proportion of the total counts increased with age of the lambs. Time courses of oocyst excretion suggest an early onset of effective immunity to the major Eimeria spp., which differed for the minor species. Mean and maximum oocyst counts and body weights of the lambs were inversely correlated suggesting negative effects of the infection on the lamb's performance. High mean antibody levels on day 7 after birth dropped until day 40 and increased subsequently again. There were no indications that maternal antibodies were protective. Antibody levels on day 40 after birth were positively correlated with oocyst counts in the faeces whereas those determined on day 80 were independent of infection parameters. Heritabilities of log(10)OpG were not significantly different from 0 up to an age of 60 days. Later estimated values were between 0.54 and 0.79 suggesting that immune protective effects rather than innate effects determining disease susceptibility are under genetic influence.     

巨型艾美耳球虫和柔嫩艾美耳球虫早熟株对8种抗球虫药的敏感试验

为检测巨型艾美耳球虫和柔嫩艾美耳球虫早熟株对常用抗球虫药的敏感性,选用地克珠利、氯苯胍、二硝托胺、癸氧喹酯、尼卡巴嗪、马杜米星、盐霉素和拉沙洛西等8种抗球虫药进行鸡体试验,通过抗球虫活性百分比（percent of optimum anticoccidial aetivity,POAA）、病变记分减少率（reduction of lesion scores,RLS）、相对卵囊产量（relative oocyst production,ROP）和抗球虫指数（anticoccidial index,ACI）4项指标进行综合评价。结果表明,巨型艾美耳球虫早熟株对8种药物敏感;柔嫩艾美耳球虫早熟株对盐霉素轻度敏感,对地克珠利、氯苯胍、二硝托胺、癸氧喹酯、尼卡巴嗪、马杜米星和拉沙洛西敏感。本研究结果可为对球虫早熟株疫苗的开发与应用提供实验依据。     

Subclinical coccidiosis in broilers and pullets

In the period from 1985 to 1987, 24 broiler crops (12 houses; one integration and 3 farms) and 9 pullet flocks (9 houses, 4 farms) were examined for parasites. Intestinal lesion scores and the number of parasites in the intestinal lumen (semiquantitative estimation) were recorded, and the Eimeria species determined when possible (broilers crops: 3rd and 5th week, pullet flocks 4th, 8th, 12th and 18th week). Additionally, the quantity of parasites in litter or faecal samples was examined in regular intervals. Clues for economic damage were only found in broiler crops with increased numbers of coccidial oocysts per gram litter in the 5th week of the fattening period. Eimeria tenella and E. acervulina were the dominating species in broiler chickens and also pullets, E. maxima oocysts however were only casual findings. No ectoparasites, helminths or other protozoa than Eimeria were observed. With regard to the intestinal lesions and the quantities of parasites in the intestine no significant differences were seen when comparing selected broiler chicks and birds taken at random. In pullet flocks the examination of random samples was the superior method, because only freshly dead bodies, collected in insufficient numbers, were suitable for diagnostics. In 3 broiler crops and one flock of replacement pullets kept on a wired floor no Eimeria were diagnosed.