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Large-scale management systems and parasite populations: coccidia in rabbits.

Broiler rabbit production has become an important branch of animal protein and fur production, not only in the traditionally rabbit breeding and consuming countries, but recently in many other countries of the world. The profitability of the rabbit industry is dependent primarily on the good feed conversion. Aspects of this include factors such as morbidity and mortality of infections, adequate knowledge and fulfilment of both accommodation and nutritional requirements, appropriate breeding systems, etc. There is much controversy about the role of coccidia in the losses of intensified rabbit breeding enterprises. There is no doubt about the significance of Eimeria stiedai which may cause condemnation of large amounts of liver as a result of infection in rabbit colonies of small holders. However, this parasite is rare in large scale rabbit farms. In the latter, intestinal coccidiosis is most frequently--although not invariably--incriminated either as a primary or more often as a predisposing factor of intestinal enteropathies causing severe mortality, mainly in the early post-weaning period. The ability to isolate and maintain species of Eimeria in specific pathogen-free (SPF) rabbits has made it possible to characterize them, such as the most pathogenic E. intestinalis and E. flavescens: the pathogenic E. magna, E. irresidua and E. piriformis; and the least pathogenic E. perforans, E. neoleporis (syn. of E. coecicola) and E. media species. Prophylactic measures are dealt with briefly, and laboratory and field trials with the currently most promising rabbit anti-coccidial drugs (Lerbek, robenidine and salinomycin) are dealt with in more detail.

Animals↗

Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry.

We present a method based on electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) for determining in muscle and eggs the following nine coccidiostats: halofuginone, diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, monensin, lasalocid, narasin, salinomycin, and maduramicin. Dinitrocarbanilide-d8, nigericin, and diclazuril-bis were used as internal standards. The method uses extraction in acetonitrile followed by a clean-up on an SiOH solid-phase extraction column. High-performance liquid chromatography (HPLC) separation was performed on a Purospher C(18) column (125 mm x 3 mm i.d.) protected by a guard column, the mobile phase being a water-acetonitrile gradient (each gradient component containing 0.1% formic acid) at a flow rate of 1 ml min(-1). For unequivocal identification of each analyte, two ions were detected and chosen for multiple reaction monitoring (MRM). Validation was carried out on spiked muscle and egg samples. The method described meets all the criteria of Decision 2002/657/EC and is easy to use in routine analysis. Validation results are presented with the measured CCalpha and CCbeta values. This whole method allows extraction and analysis of up to 24 samples per day.

Chromatography, Liquid↗

Liquid chromatographic tandem mass spectrometric determination of five coccidiostats in poultry eggs and feed.

A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite 2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC [Official Journal of the European Communities L221 (2002) 8].

Animal Feed↗

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens.

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

Animals↗

Residual activity of anticoccidial drugs in chickens after withdrawal of medicated feeds.

Seven anticoccidial drugs commonly used in poultry (diclazuri), monensin, salinomycin, halofuginone, nicarbazin, robenidine, amprolium, and lasalocid) were tested for residual activity after withdrawal. In each test, the products were given at the recommended level to cages of 10 broiler chickens. Oral inoculation with coccidia was given after withdrawal of medication. Birds pretreated with 1 ppm of diclazuril and inoculated with Eimeria tenella after drug withdrawal had normal weight gain and very low lesion scores. Residual activity depleted gradually over several days, as shown by higher lesion scores when medication was withdrawn for up to 3 days before inoculation. Similar results were observed when young birds were inoculated with a mixture of E. tenella, E. maxima and E. acervulina, and also when birds were given diclazuril to market weight (6 weeks of age) and inoculated with a mixture of six species of Eiméria (The above species plus E. brunetti, E. mitis, and E. necatrix) after withdrawal of medication for 2 days. In contrast, there was no evidence of residual anticoccidial activity with nicarbazin, halofuginone, lasalocid, amprolium, salinomycin or monensin. Overall, the residual activity was unique to diclazuril.

Amprolium↗

Eimeria tenella: experimental studies on the development of resistance to amprolium, clopidol and methyl benzoquate.

The development of resistance by the Houghton strain of Eimeria tenella to the anticoccidial drugs amprolium, clopidol and methyl benzoquate has been studied. Resistance to amprolium and clopidol developed more readily in experiments where a large number of coccidia were exposed to the drug, either by increasing the number of oocysts in the inoculum or by increasing the number of birds in the group. When 45 birds were given 2.0 X 10(6) oocysts, resistance to amprolium and clopidol appeared after 6 and 7 passages respectively. In previous experiments, under similar conditions, resistance to robenidine developed after 6 passages, suggesting little difference between these three drugs. Resistance to amprolium and clopidol arose gradually as the concentration of drug was increased, but resistance to methyl benzoquate appeared in a single step from sensitivity to high-level resistance. Both amprolium and clopidol-resistant lines showed an 8-fold reduction in drug sensitivity. Attempts to measure the degree of resistance by calculation of the ED50 were unsuccessful.

Amprolium↗

Factors influencing the assessment of anticoccidial activity in cell culture.

A comparative study has been made of the factors influencing the assessment of anticoccidial potency in vitro against Eimeria tenella using established anticoccidials and arprinocid and some of its analogues. Drugs whose potency depended upon medium composition were amprolium, lasalocid and halofuginone. There was a difference in strain sensitivity with robenidine. Host cell type had an important effect on potency of monensin, decoquinate, arprinocid and its analogues. Arprinocid was active in chick liver cell systems but totally inactive in chick kidney cell systems, although its N-oxide was active in both cell types. Arprinocid-containing medium, conditioned by supporting the growth of chick embryo liver cell cultures, had an anticoccidial effect on E. tenella growing in chick kidney cells. It is deduced that the anticoccidial activity of arprinocid in the chick is due to a metabolite.

Adenine↗

Eimeria tenella in chickens: development of resistance to quinolone anticoccidial drugs.

The development of drug resistance by the present Houghton strain of Eimeria tenella to the quinolones, methyl benzoquate and buquinolate, was found to take place after a single experimental passage. The development of resistance was independent of drug selection pressure and showed cross resistance to other quinolones, but not to amprolium and robenidine. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar laboratory conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after 6 passages and the Beltsville after 5. Studies on the response of the Houghton strain to methyl benzoquate and buquinolate revealed that the drugs did not completely control the infection as measured by weight gain and that oocyst production was not suppressed. These observations indicate that the strain had already acquired some resistance to these drugs. This was confirmed by examining the resistance to methyl benzoquate of a culture of the Houghton strain of E. tenella which had been stored frozen in liquid nitrogen since 1969. This showed full sensitivity to the drug and developed resistance after 8 passages. This suggests that drug tolerance has been acquired by the Houghton strain since 1969. Oocyst lines were established from the Houghton strain by infecting single birds with approximately 10 oocysts. Eleven of these lines were found to be sensitive to methyl benzoquate, and nine to give rise to resistant parasites. It is concluded that the Houghton strain is contaminated by a small number of resistant oocysts which can be eliminated from a culture by dilution of the challenge inoculum. One of these Houghton oocyst lines, sensitive to methyl benzoquate, developed resistance after 8 serial passages.

Animals↗

Eimeria tenella, E. acervulina and E. maxima: studies on the development of resistance to diclazuril and other anticoccidial drugs in the chicken.

Resistance to diclazuril was induced by 10 passages of the Houghton strains of Eimeria acervulina and E. tenella in chickens given progressively greater concentrations of the drug. This resistance was, however, not complete since the drug retained some efficacy against the drug-passaged lines. Attempts to passage the Houghton strain of E. maxima in birds medicated with concentrations of diclazuril greater than 0.016 parts per million (ppm) were unsuccessful and after 10 passages at this concentration resistance had not developed. Resistance to methyl benzoquate developed after 6 passages of E. tenella in medicated chickens but resistance to amprolium was only partial after 10 passages. The pathogenicity of diclazuril- and amprolium-resistant lines of E. tenella was lower than that of the parental line. Diclazuril was effective against lines of E. tenella resistant to amprolium, arprinocid, clopidol, dinitolmide, halofuginone, methyl benzoquate, monensin and robenidine.

Amprolium↗

Studies on the growth, chemotherapy and enzyme variation of Eimeria acervulina var. diminuta and E. acervulina var. mivati.

Eimeria acervulina var. diminuta was serially passaged 12 times in chicken embryos, but growth in cultured chick kidney cells was limited to 2 generations of schizonts. After 7 embryo passages the sensitivities of E. acervulina var. diminuta and an embryo-adapted strain of E. acervulina var. mivati to the anticoccidial drugs amprolium, methyl benzyquate, robenidine and sulphaquinoxaline were compared. Both parasites were sensitive to all the anticoccidials but E. acervulina var. diminuta was more sensitive to sulphaquinoxaline and amprolium. The chicken-maintained strain of E. acervulina var. diminuta was extremely sensitive to clopidol, sulphaquinoxaline and decoquinate. Electrophoretic analyses of several enzymes from E. acervulina var. diminuta revealed enzyme profiles with similarities and differences to the embryo-adapted strain of E. acervulina var. mivati.

Animals↗

Integrating genetics and genomics to identify new leads for the control of Eimeria spp.

Eimerian parasites display a biologically interesting range of phenotypic variation. In addition to a wide spectrum of drug-resistance phenotypes that are expressed similarly by many other parasites, the Eimeria spp. present some unique phenotypes. For example, unique lines of Eimeria spp. include those selected for growth in the chorioallantoic membrane of the embryonating hens egg or for faster growth (precocious development) in the mature host. The many laboratory-derived egg-adapted or precocious lines also share a phenotype of a marked attenuation of virulence, the basis of which is different as a consequence of the in ovo or in vivo selection procedures used. Of current interest is the fact that some wild-type populations of Eimeria maxima are characterized by an ability to induce protective immunity that is strain-specific. The molecular basis of phenotypes that define Eimeria spp. is now increasingly amenable to investigation, both through technical improvements in genetic linkage studies and the availability of a comprehensive genome sequence for the caecal parasite E. tenella. The most exciting phenotype in the context of vaccination and the development of new vaccines is the trait of strain-specific immunity associated with E. maxima. Recent work in this laboratory has shown that infection of two inbred lines of White Leghorn chickens with the W strain of E. maxima leads to complete protection to challenge with the homologous parasite, but to complete escape of the heterologous H strain, i.e. the W strain induces an exquisitely strain-specific protective immune response with respect to the H strain. This dichotomy of survival in the face of immune-mediated killing has been examined further and, notably, mating between a drug-resistant W strain and a drug-sensitive H strain leads to recombination between the genetic loci responsible for the specificity of protective immunity and resistance to the anticoccidial drug robenidine. Such a finding opens the way forward for genetic mapping of the loci responsible for the induction of protective immunity and integration with the genome sequencing efforts.

Animals↗

Deposition and depletion of five anticoccidials in eggs.

Anticoccidials are compounds that are widely used as feed additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a certain time interval for broilers and pullets but not for laying hens. It was shown in the past that carry-over at the feeding mill is found to be the main reason for the presence of residues in eggs. An animal experiment was set up to investigate the effect of carry-over at the feeding mill on the presence of residues of anticoccidials in eggs. For the compounds diclazuril, robenidine, halofuginone and nicarbazin in combination with narasin, two concentration levels were tested: the maximum allowed concentration for broilers (100%) and a concentration corresponding to 5% carry-over during feed preparation. Also dimetridazole was included in the experiment but only at one concentration level. Eggs were sampled during treatment (14 days) and for a period of 30 days after withdrawal of the anticoccidial-containing feed. Residues were determined, and deposition and depletion curves were generated. Analyses were performed by ELISA and LC-MS/MS. For all compounds, substantial residues could be found in the 5% groups, which points out the risk of carry-over at the feeding mill. The distribution of the residues between egg yolk and white was determined by analyzing both fractions.

Animal Feed↗

Incidence of residues of nine anticoccidials in eggs.

A survey of the presence of residues of anticoccidials was performed. Three hundred and twenty egg samples, purchased in eight different European countries, were analysed for the presence of nine different compounds: dimetridazole, diclazuril, halofuginone, robenidine, nicarbazin, narasin, salinomycin, lasalocid and monensin. Analyses were performed by LC-MS/MS. Of the samples analysed, 114 (35.6%) contained one or more of the nine anticoccidials in concentrations ranging from 0.1 to 63 microg kg-1. Salinomycin and lasalocid account for more than 60% of all positive samples. Almost 90% of all positive samples contained less than 2 microg kg-1. Results were put into perspective of the farming method and country of origin.

Animal Husbandry↗

Single and low-level oocyst infections of drug-resistant field strains of Eimeria tenella in medicated birds.

Five out of ten birds infected with a single oocyst of strain Gt2 of Eimeria tenella and medicated with the recommended level of robenidine were found positive in the first experiment and four in the second in comparison with seven and six respectively in nonmedicated birds. Six birds out of ten were found positive in the two groups of similarly medicated birds infected with two or four oocysts each. Although single oocyst infections of strain Lilly 155 were unsuccessful, six out of ten birds were found positive in birds infected with ten oocysts each.All single and low-level oocyst infections were accomplished with oocysts previously treated with beta-glucuronidase and broken into sporocysts prior to infections. The overall results suggested that when a coccidium became resistant to an anticoccidial drug, only one or a few occysts were needed to start an infection if the drug was continued. The results also showed that, perhaps, successful single or low-level oocyst infections can also be used as a criterion for demonstrating drug resistance in coccidia.

Animals↗

Development of immunity to coccidiosis in chickens administered anticoccidials in feed.

New combinations of techniques were employed for comparing the development of immunity of Eimeria tenella in chickens being medicated with 12 different anticoccidials. Broiler-type birds in batteries received a daily measured dose of E. tenella oocysts for 15 consecutive days while the drug was administered at the manufacturer's recommended level. Two or more tests of each drug gave the following ratings: strong suppression, monensin (121 ppm), salinomycin (80 ppm), lasalocid (75 ppm); moderate suppression, monensin (100 ppm), decoquinate (30 ppm), clopidol (125 ppm), and narasin (80 ppm); slight suppression, arprinocid (70 ppm), nicarbazin (125 ppm), and amprolium (125 ppm + ethopabate (4 ppm); no effect, robenidine (33 ppm), zoalene (125 ppm), and aklomide (250 ppm).

Amprolium↗

The anticoccidial efficacy of arprinocid in broiler chickens under floor pen conditions.

The efficacy of arprinocid was tested against artificial infections of mixtures of Eimeria spp. in broiler chickens under floor pen conditions in 3 experiments. Treatment with arprinocid at 60 ppm over a 56 d period significantly increased the live mass gain and feed efficiency of broiler chickens. This increase compared favourably with that obtained by treatment with lasalocid, robenidine and halofuginone. Birds treated with arprinocid had substantially reduced numbers of sporulated oocysts in their litter, and less severe lesion scores than non-mediated birds.

Adenine↗

Coccidiosis in rabbits: a field study.

During a survey 1052 faecal samples were examined in commercial and domestic rabbitries in Belgium. Nine different species of Eimeria were recognised. E magna, E media and E perforans were very common in both production methods. E flavescens, E intestinalis and E stiedai occurred more commonly in traditionally reared rabbits. E stiedai was not recorded in commercial rabbitries. E coecicola, E irresidua and E piriformis were less common. Oocyst output was suppressed in increasing efficacy by clopidol, sulphaquinoxaline/pyrimethamine (10:1), sulphadimidine and robenidine (P less than 0.01). The use of a droppings pit reduced the level of coccidial infection significantly compared with a sledge type of cleaning mechanism (P less than 0.001).

Animal Husbandry↗

Resistance and cross-resistance studies with narasin, a new polyether antibiotic anticoccidial drug.

The Wisconsin (Wis) strain and a field strain (FS-196) of Eimeria tenella were propagated in the presence of 80 ppm narasin in an attempt to select for narasin resistance. Comparisons of the narasin sensitivity of the selected strains (Wis-NR and FS-196-NR) and of the parent strains (Wis and FS-196) from which they were derived revealed no resistance development. These findings agree with the results of similar studies with the polyether antibiotic anticoccidial drugs monensin and lasalocid and support the conclusion that chicken coccidia do not readily develop resistance to this class of anticoccidial drugs. In a separate series of experiments, the efficacy of narasin was evaluated against five E. tenella field strains, one each resistant to amprolium, clopidol, decoquinate, nicarbazin, and robenidine. Narasin effectively controlled infections that were inadequately controlled by the anticoccidial drugs to which the strains were resistant. There was no evidence of cross-resistance to narasin.

Animals↗