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Apicomplexan parasites of the genus

The efficacy of the anticoccidial drugs amprolium, clopidol, diclazuril, monensin, monensin + nicarbazin, narasin, narasin + nicarbazin, and salinomycin against field isolates of Eimeria acervulina obtained from a commercial broiler enterprise before and after immunization with a coccidiosis vaccine was investigated. Evaluated by weight gain, feed conversion, and lesion score following challenge, the isolate obtained before vaccination was resistant to all the drugs tested. By contrast, after vaccination the isolate was sensitive to all drugs evaluated by weight gain, and to most drugs judged by feed conversion and lesion score. It is concluded that vaccination had resulted in the restoration of sensitivity to these drugs.

Apicomplexan parasites of the genus Eimeria are widespread in poultry flocks and if present in sufficient numbers can cause the intestinal disease coccidiosis. For many years, the disease has been controlled either by incorporating drugs in the feed (prophylaxis) or by immunization with live parasites (vaccination). Efficacy of anticoccidial drugs has been compromised by the acquisition of resistance which has been documented for all drugs introduced including those most widely used, the ionophorous antibiotics. A consequence of limited efficacy is that these drugs permit some parasite development thus allowing the acquisition of immunity. Combined with improvements in management, this may explain the continued use of drugs for the control of coccidiosis in broiler and turkey production. In recent years vaccination, utilizing live attenuated or non-attenuated parasites has made a significant contribution to the control of coccidiosis in chickens. Vaccines are administered on a single occasion by a variety of methods to newly hatched chicks and initiate an immune response in the bird. Full protective immunity requires secondary exposure to vaccinal oocysts in the litter, an aspect of vaccination over which the producer has little control. Exposure to wild-type parasites already present in the litter may cause clinical coccidiosis, as evident by characteristic intestinal lesions in birds, before protective immunity has time to fully develop. Several vaccines comprise parasites that have never been exposed to anticoccidial drugs and it has been shown that their use in a commercial setting may replace drug-resistant organisms resulting in a restoration of drug-sensitivity. Programs involving alternation of chemotherapy and vaccination have been introduced with the objective of prolonging the life of anticoccidial drugs that have been compromised by drug-resistance. This is the only known example of restoration of drug sensitivity in veterinary parasitology. So-called 'natural' products that include herbs, herbal and fungal extracts, and probiotics are claimed to alleviate Eimeria infections in poultry, but few have been thoroughly investigated. Advances have been made in our knowledge of the structure of proteins released during parasite invasion of host cells and may eventually result in novel vaccines based upon molecular technology. Hens immunized with proteins isolated from gametocyte stages of the life cycle of one species of Eimeria pass antibodies to developing embryos and this may provide short-term passive protection to young chickens' post-hatch. As with other approaches to vaccination, protection is dependent upon exposure to wild-type parasites present in the environment.

This chapter aims to provide an overview of the efficacy and mode of action of the compounds used to control coccidiosis in poultry and livestock and provide a brief outlook of research needs for the future. Satisfactory control of coccidiosis in poultry requires strict attention to hygiene and sanitation, and biosecurity measures that limit human access to poultry facilities. The concept of coccidiosis prevention in chickens by inclusion of drugs in the feed was first described in 1948 and involved the use of sulfaquinoxaline, the first feed additive for poultry. Quinolone coccidiostats inhibit the respiration by blocking electron transport in the parasite mitochondrion reversibly, probably acting at a site near cytochrome b. Clopidol, also known as meticlorpindol or clopindol, is a pyridinol with broad coccidiostatic activity against early development of Eimeria spp. by inhibiting mitochondrial energy production in sporozoites and trophozoites.

Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

An attenuated line of Eimeria meleagrimitis was established by repeated propagation of the parasite in 9-day old turkey poults and subsequent selection for precocious development. Following 20 passages, the prepatent period decreased from 120 to 104h. A series of experiments were conducted to evaluate the pathogenicity, immunogenicity and fecundity of the newly selected line. Judged by body weight gain, feed consumption and feed efficiency following infection, the attenuated line had appreciably reduced pathogenicity. Immunogenicity of the attenuated line was examined by infecting poults successively with incremental doses of 10(2), 10(3) and 10(4) oocysts at 0, 7, and 14 days of age respectively. No oocysts were detected following challenge with 5×10(2) oocysts, indicating that the attenuated line had retained immunogenicity. Fecundity was assessed by infecting two-week old birds with 5×10(2) oocysts of either parent or attenuated line. Oocyst production from 96 to 240h post-infection showed that the patent period of the attenuated line commenced earlier and was of shorter duration than the parent line.

For the purpose of re-describing the Eimeria species that infect the turkey (Meleagris gallopavo) and to establish benchmark biological information linked to genetic markers for each species, a strain of Eimeria meleagrimitis Tyzzer 1929 was obtained from a litter sample from a turkey farm in Minnesota, USA in 2008. Multiple pure lines were derived by infecting turkey poults with a single oocyst; one of these lines was then used to re-describe biological and morphological features of E. meleagrimitis in the turkey and to designate a neotype of E. meleagrimitis in the turkey. Oocyst morphometrics of this line matched those of this species as originally described by Tyzzer (Am J Hyg 10:269-383, 1929). Three asexual generations of merogony (the first generation of meronts large in size and the second and third generations small) were detected in the intestines before the onset of gametogony; no developmental stages were detected in the cecal pouches. No mortality was induced by this line of E. meleagrimitis even when turkey poults were infected with high doses of oocysts (up to 5 × 10(5) oocysts/bird) and despite the ability of E. meleagrimitis to induce severe mucosal damage in the upper and middle duodenum. Macroscopic lesions were characterized to provide a graded lesion scoring guide that should assist assessment of the severity of infections with this species in infected turkeys. The pathogenicity of the strain was investigated, and a significant reduction in weight gain and feed conversion ratio was observed with doses of 10(4) oocysts/bird or more. The maximum yield of oocysts in the feces was obtained when birds were inoculated with 5 × 10(3) oocysts.

Newly hatched turkey poults were infected with 10(2) oocysts of Eimeria adenoeides and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age, respectively. Three peaks in oocyst production were observed in the feces of poults following this series of infections. A second group of poults given the same dosing regimen was challenged with 5 × 10(4) oocysts/poult at different times to evaluate the acquisition of immunity. Judging by weight gain and mortality, no protection had been acquired at 6 d of age, but partial protection was observed by 12 and 18 d of age. A third group of poults were also infected with 10(2) oocysts and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age to evaluate cellular immune responses to infection. Sections of ceca from infected poults showed a significantly higher leukocyte infiltration on d 6, 10, 12, 16, and 18 after infection than uninfected controls. The percent area occupied by CD4+ and CD8+ lymphocytes in the ceca, as assessed by immunohistochemistry, was significantly elevated in infected poults on d 12, 16, and 18. The relative expression of chemokine CXCLi2, and cytokines IL1β, IFNγ, IL10, IL13, IL2, IL12b, and IL18 was measured by real-time reverse-transcription PCR. The expression of CXCLi2 and IL10 was found to be elevated on d 12, and IFNγ on d 10, 12, and 16. Expression of IL13 and IL18 was increased on d 10 and IL2 on d 10 and 16, and that of IL12b on d 16 in infected poults. Increase in the infiltration of leukocytes, percent area occupied by CD4+ and CD8+ lymphocytes, and changes in the relative expression of cytokines in the ceca characterize the dynamics of immune responses in turkey poults infected with E. adenoeides early in life.

The course of natural Eimeria infections in 6 successive broiler flocks at a commercial farm comprising 4 houses, where different anticoccidial drug programs were employed, was studied by counting the number of oocysts in the litter at weekly intervals. The course of infection in all flocks followed a bell shaped curve in which oocyst numbers, initially low, increased to a peak ranging from 36 × 10(3) to 74 × 10(3) oocysts/g (OPG) of litter around 3 to 4 wk of age. Numbers subsequently declined to 3 × 10(3) to 15 × 10(3) OPG. Oocysts could be detected between flocks when birds were not present. Species of Eimeria identified included E. acervulina, E. maxima, and E. tenella Despite the presence of large numbers of oocysts in the litter, coccidial lesions were not observed in the intestines of the birds. The performance of broilers at the study site was comparable to that of other farms in the area where birds from the same settlement were reared to a similar age using the same drug programs. The results indicate the ubiquitous nature of Eimeria spp. infections in commercial broilers despite prophylactic medication.

Thomas K. Jeffers has made many significant contributions to our understanding of the biology of the parasite Eimeria, the cause of coccidiosis in poultry. His work has had direct practical application for the control of this widespread disease. Topics discussed include Jeffers' pioneering work concerned with genetics of the host response to infection, the nature of biological and immunological intraspecific variation, drug resistance and discovery, field surveys of resistance, and his most recognized achievement-the demonstration that the lifecycle of coccidia may be altered by artificial selection. Parasites so modified are attenuated but retain their immunogenicity, a discovery that has led to the development of live vaccines that are inherently non-pathogenic. This article provides a brief biography and describes the contributions that Jeffers has made to our knowledge of coccidiosis.

In July 1971, the polyether ionophorous antibiotic monensin was introduced in the United States for the control of coccidiosis in poultry. At that time, prospects for new anticoccidial agents were not good. Amprolium had enjoyed several years of use, but many other compounds had been abandoned as resistance to them developed. After the introduction of monensin, most commercial broilers were medicated with the drug and it is still widely used for this purpose today. Apart from in poultry, monensin is also used to control coccidiosis in game birds, sheep, and cattle. Indeed, more animals have been medicated with ionophores, such as monensin, for control of disease than any other medicinal agents in the history of veterinary medicine. In this review, we discuss the discovery, mode of action, and efficacy of monensin, together with matters of importance to the poultry industry such as commercial use, drug resistance, toxicity, pharmacology and residues, host immunity to coccidiosis, and effects in other avian species.

Five successive flocks of broilers were reared in floor-pens and given different drug programs or were vaccinated against coccidiosis. Oocysts of Eimeria were isolated from the litter of pens during the fifth flock and their sensitivity to salinomycin (Sal) investigated by measuring new oocyst production following infection of medicated and unmedicated birds. Parasites obtained following 5 flocks given Sal were not well-controlled and it was concluded that they were partially resistant to the drug. Parasites obtained following 4 unmedicated flocks and one medicated flock were better controlled by Sal and it was concluded that in the absence of continuous medication there had been an improvement in drug efficacy. Sal almost completely suppressed oocyst production of isolates from treatments in which medication was followed by vaccination, indicating that when a drug program is followed by vaccination, restoration of sensitivity to Sal had occurred.

An isolate of Eimeria meleagridis Tyzzer, 1927 was obtained by harvesting oocysts from the ceca of a turkey from northwest Arkansas and a pure line was established by infecting birds with a single oocyst. Oocysts were first produced in the ceca of infected birds from 102 to 108 hr after inoculation and were of similar size (mean length × width, 24.9 × 17.0 μm) to those of Eimeria adenoeides Moore and Brown, 1951 and Eimeria gallopavonis Hawkins, 1952. The line was identified as E. meleagridis based upon the development of large schizonts in the midintestine, and small schizonts in the ceca. Two generations of large schizonts were found 48 and 72 hr after infection, and at least two generations of small schizonts were found from 60 to 108 hr after infection. An inoculum of 2 × 105 oocysts was found to cause a significant reduction in weight gain from days 0–3 and 0–6 after infection, suggesting that the significance of this species of Eimeria as a pathogen of turkeys should be reassessed.

The acquisition of immunity to Eimeria maxima by chicks infected 18 hr after hatch with a single dose of 100 oocysts was investigated. In the first experiment, birds were moved each day to clean cages in order to prevent the possibility of secondary infection resulting from ingestion of oocysts passed in their feces. Immunity was measured at 4 wk of age by calculation of oocyst production following challenge with 500 oocysts or weight gain following challenge with 100,000 oocysts. Large numbers of oocysts were produced by infected birds following challenge, although numbers were significantly less than those from birds that had been reared in the absence of infection (susceptible controls). The weight gain of infected birds following challenge was significantly greater than that of susceptible controls but less than that of unchallenged controls. Thus, only partial protection had been acquired, whether parasite replication or body weight gain was used to assess the extent of immunity development. In a second experiment, acquisition of immunity at 4 wk by chicks infected 18 hr after hatch with 100 oocysts of E. maxima and reared in floor pens in contact with their droppings was investigated. Infected birds produced no oocysts following challenge, and weight gains were not significantly different from the unchallenged controls, which indicates that full immunity had developed by 4 wk. It is concluded that if oocysts of Eimeria species are used to vaccinate day-old chicks, reinfection by oocysts present in the litter is necessary for the establishment of protective immunity.

Walter T. Johnson (1892 to 1937), veterinarian at Western Washington Agricultural Experiment Station (WWAES), Puyallup, Washington, USA, and subsequently poultry pathologist and professor of veterinary medicine at Oregon State University Agricultural Experiment Station (OSAES), Corvallis, Oregon, USA, made many important contributions to our understanding of the disease coccidiosis. His pioneering work included the first description of Eimeria necatrix and Eimeria praecox from the chicken and identification of four other species of Eimeria from the fowl. He demonstrated the relationship between numbers of oocysts ingested and severity of infection, and described the phenomenon of host specificity and the significance of immunity. Contrary to widespread opinion, he considered that coccidia were not involved in blackhead disease and other pathological conditions reported from the fowl. His views on control were ahead of his time and he anticipated the possibility of vaccinating birds by infecting them with ...

The performance of commercial broilers reported by broiler production units in the USA from 1997 to 2001 has been evaluated. There was a linear decrease in calorie conversion (CC) and number of days to produce a 2.27 kg bird (DAYS) but no change in percentage mortality. By contrast, there was a linear increase in final bird weight (FBW) during this period. There was a linear increase in the percentage of production units rearing birds to more than 2.5 kg and a decrease in those rearing birds to less than 2.0 kg or 2.0 to 2.5 kg. There were no differences in CC or DAYS during the first half of the year (January to June), but these traits showed a significant increase in July followed by a steep decline during August, September, and October. FBW was similar from January to June but showed a significant decrease in July. No consistent variation in monthly mortality was noted. It is concluded that improvements in the productivity of broilers have been achieved from 1997 to 2001 but that more attention should be given to the problem of broiler management during the summer months.

In 1995, an antibiotic (ANT) was used in starter, grower, and withdrawal (WD) feeds by 94.3, 98.2, and 75.1% of broiler production units, but by 2000, ANT use had declined to 64.8, 66.9, and 48.1% respectively. Roxarsone (ROX) was used in the starter and grower feeds by 69.8 and 73.9% of production units. Bacitracin (BAC) was used more frequently than other antibiotics in the starter and grower feed. Virginiamycin (VIR) was used most frequently in the WD feed. Most units (69.4%) reported use of two different antibiotics. The use of programs comprising two ANT decreased, whereas programs with a single ANT increased during the period of study. A combination of ionophore (ION) + ROX + ANT was employed most frequently in the starter and grower feeds, whereas an ANT alone was used most frequently in the WD ration. The use of ION + ROX + ANT declined from 1995 to 2000, but use of ION + ROX increased. There were no significant differences in calorie conversion whether plants used ION + ROX + ANT, ION + ROX, or ION + ANT. The number of days to rear birds to 2.27 kg was significantly greater for production units using ION + ROX. Mortality was lower for units that used ION + ROX + ANT and ION + ROX than for those that used ION + ANT. Production units that used ION + ANT were more likely to rear birds to a weight greater than 2.5 kg than to 2.0 to 2.5 kg. Units in the South and Central states were more likely to use an ION + ROX than those in the Northeast and Atlantic states, whereas for ION + ROX + ANT the reverse was the case. The cost of medicating with ION + ROX + ANT decreased from 1995 to 1998.

This article describes some of the milestones in research concerned with protozoan parasites of the genus Eimeria that infect birds and cause the disease coccidiosis. The time period covered is from 1891, when oocysts were first found in the ceca of diseased chickens, to the present. Progress in our understanding has lagged behind that of other protozoan parasites such as Toxoplasma and Plasmodium despite the enormous importance of Eimeria to animal livestock production. Nevertheless, applied research by universities, government agencies, and private industry has resulted in the successful development of methods of control, research that continues today. The topics covered and the references provided are selective and include life cycles and biology, pathology, ultrastructure, biochemistry, immunity, genetics, host cell invasion, species identification, taxonomy, chemotherapy, vaccination, and literature concerned with avian coccidiosis. This review is primarily concerned with the avian species of Eimeria that infect poultry, but some important advances, principally in immunology, have been made using species that infect rodents and rabbits. These are included where appropriate.

Immunological control is recognised as the only major practical alternative to chemotherapy for the control of coccidiosis. Initial hopes that it would be possible to develop a genetically engineer...

The efficacy of different concentrations of monensin in turkeys exposed to field isolates of Eimeria and the effect of the withdrawal of monensin from their diet were investigated. Results of a battery study indicated that 66 ppm monensin was effective against three species of Eimeria in the turkey. In floor pens in which poults were exposed to infection, use of 59.5, 79.4, and 99.2 ppm monensin resulted in reduced mortality and improved feed conversion at 3 wk of age compared with birds that had received no medication. Poults given 59.5 ppm monensin weighed more and had a better feed conversion at 3 wk than poults given 79.4 or 99.2 ppm monensin, but at 10 wk no differences in the body weight or feed conversion of poults given different concentrations of drug were apparent. At 14 wk (and from 10 to 14 wk), poults that had been given 99.2 ppm monensin had a lower feed intake and weighed less than birds that had been given 59.5 ppm of the drug but there were no significant differences in feed conversion. There was no significant difference in the weight gain or feed conversion of poults from 10 to 14 wk of age whether monensin was present in the feed or had been withdrawn. No evidence of compensatory growth was found in the present study.

Anticoccidial drugs are widely used for the control of coccidiosis in the fowl which has inevitably led to the development of drug resistance. Resistance has developed to all of the compounds that have been introduced and if chemotherapy is to remain the principal method of control of coccidiosis, it will be important to continue the search for new anticoccidial agents. Knowledge of biochemical pathways present in the Eimeria parasite and how they differ from those of the host might help identify novel targets for inhibition. Studies of the mode of action of drugs are required if the biochemical mechanisms of resistance are to be understood. Information on the genetic origins of resistance, the stability of resistance and the factors involved in the spread of resistance throughout parasite populations is required. Since there are no methods at present to prevent resistance, more attention should be given to developing strategies for preserving the efficacy of anticoccidial drugs.

MHC class II alleles clearly contribute a primary genetic component of susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. However, IDDM does not occur in NOD mice made MHC class I-deficient by a functionally inactivated beta2-microglobulin allele (beta2m(null)). In the present study the beta2m(null) mutation was used to examine the relative contributions of MHC class I and class II-dependent T cell responses for initiating autoimmune pancreatic beta cell destruction in NOD mice. Splenocytes from diabetic NOD donors transferred IDDM to both lymphocyte-deficient NOD-scid (class I+) and NOD-scid.beta2m(null) mice (class I-). In contrast, splenocytes from young prediabetic NOD donors only transferred IDDM to class I+, but not class I- NOD-scid recipients. However, splenocytes from prediabetic NOD donors did transfer IDDM to NOD-scid.beta2m(null) recipients previously engrafted with class I+, but not class I-, pancreatic islets. CD4+ T cell lines reactive against some syngeneic class I+ targets could be isolated from NOD.beta2m(null) mice. However, NOD.beta2m(null) T cells underwent activation-driven deletion when transferred into class I+ NOD-scid recipients. Hence, the class I autoreactive T cells present in NOD.beta2m(null) donors did not elicit IDDM when transferred into class I+ NOD-scid recipients. Collectively, these results show that autoimmune IDDM in NOD mice is initiated by MHC class I-dependent T cell responses, but this leads to the subsequent activation of additional T cell populations that can mediate pancreatic beta cell destruction in a MHC class I-independent manner.

Anticoccidial effects of Sericea Lespedeza (SL) included in the diet at different levels were evaluated in chickens following oral infection with sporulated oocysts of either Eimeria acervulina, Eimeria maxima or Eimeria tenella. A series of experiments were conducted to determine the effect of SL upon the ability of the parasites to multiply in the intestine, and the effect on bodyweight gain, feed intake, and feed conversion following infection. Chicks infected with a low dose of oocysts (500 oocysts/bird) of E. acervulina or E. maxima did not show differences in the numbers of oocysts produced in the feces whether they were given 0, 1, 2, or 4% SL in the diet. There was no significant difference in the weight gain, feed intake, or FCR of birds infected with high doses of E. acervulina or E. maxima (200,000 or 100,000 oocysts/bird respectively) whether 0, 1, 2, or 4% SL was included in the feed. No significant difference in the numbers of oocysts in the feces, weight gain, feed intake, and FCR of birds infected with E. tenella (low dose of 500 oocysts; high dose of 50,000 oocysts per bird) whether 4% SL was included in the feed. The results of this study indicate that SL has no anticoccidial activity against Eimeria species in the chicken.

The performance of broilers reared in floor pens and given monensin in the feed at 121 ppm was compared with that of birds given no drug. Feed intake and BW gain of medicated birds was significantly lower than that of unmedicated birds from 0 to 22 d of age. Feed intake and feed conversion of medicated birds was significantly reduced, compared with unmedicated birds, from 22 to 53 and 0 to 60 d of age. Total mortality, and mortality due to leg abnormalities from 22 to 53 and 0 to 60 d, was significantly lower in birds given monensin. There was no difference in the incidence of tibial dyschondroplasia (TD) by 60 d. No differences in mortality due to pulmonary hypertension syndrome (PHS) were observed for any age period. Birds removed from pens at 28 d that had received monensin had lower hematocrit and percentage saturation of hemoglobin with oxygen in the blood than unmedicated birds. No differences in these variables were found at 54 d. There were no differences in the right ventricle weight: total ventricular weight ratios or electrocardiogram lead II values at 28 or 54 d. The results indicate that PHS does not occur more frequently in broilers medicated with monensin.

The spectrum of resistance to seven currently used anticoccidial drugs in isolates of Eimeria obtained from farms two broiler complexes was examined. All isolates were resistant to monensin, salinomycin, and narasin. Lasalocid was more effective in controlling coccidiosis than the other ionophores, although most isolates were classified as resistant to the drug. The majority of isolates were sensitive or showed reduced sensitivity to robenidine, clopidol, and decoquinate. It was concluded that for drugs that have been used extensively (e.g., monensin and salinomycin), examination of isolates from one or two farms may give results applicable to the entire complex. For drugs that have been used infrequently, however (such as robenidine, clopidol, and decoquinate), examination of isolates from at least five farms would be desirable to establish the spectrum of drug sensitivity.

The diabetes (db) gene is a recessive obesity mutation in the mouse capable of producing diabetes only through interaction with heretofore undefined modifiers in the genetic background of certain inbred strains. Here we identify the genetic map locations of androgen and estrogen sulfotransferase genes important in maintaining the balance of active sex steroids in the liver. The Std locus encoding dehydroepiandrosterone sulfotransferase was mapped to proximal Chromosome 7, and the Ste locus encoding estrogen sulfotransferase was mapped to Chromosome 5. The db mutation in the diabetes-susceptible C57BL/KsJ strain aberrantly regulated mRNA transcript levels from these two loci. Hepatic Ste mRNA transcripts were increased from undetectable levels in normal males and females to high levels in db/db mice of both sexes. An anomalous suppression of Std transcription was observed in db/db females, but not in normal females. These reciprocal changes in mRNA concentrations in mutant females were reflected by an induction of a high affinity estrogen sulfotransferase activity and a concomitant loss of dehydroepiandrosterone sulfotransferase activity. These db gene-elicited effects were specific for the sex steroid sulfotransferases since other potential sex steroid metabolizing enzymes (phenol sulfotransferase, sex steroid sulfohydrolase, and UDP-glucuronyltransferase) were unaffected. These aberrant changes would virilize hepatic metabolism in females by increasing the ratio of active androgens to estrogens. In human females, non-insulin-dependent diabetes mellitus often develops when visceral obesity and hyperinsulinemia are associated with hyperandrogenization. This study demonstrates that background modifier genes interacting deleteriously with an obesity mutation are not necessarily defective alleles. Rather, some are functional genes whose regulation has been altered by pleiotropic effects of the obesity gene.

We have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotranaferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5′ end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, β1 polypepride (PRKCB1), in the region from the distal portion of 16p11.2 to p12. 1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, we have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers.

In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.

Forty-six broiler houses were examined for the presence of oocysts of Eimeria in the litter before and after withdrawal of lasalocid or salinomycin from the feed. A decrease in number of small oocysts (Eimeria acervulina or Eimeria mitis) was observed following withdrawal of medication Numbers of medium-sized oocysts (probably Eimeria tenella) remained the same, but an increase in large oocysts (Eimeria maxima) was recorded. No lesions attributable to Eimeria tenella were found in the ceca of birds after withdrawal of the drug. More small- and medium-sized oocysts were found at sites with new litter than at sites where the litter had been employed for previous flocks. No difference in the number of oocysts was found, whether birds were reared on oak shavings or a mixture of pine shavings and rice hulls. The number of oocysts was positively correlated with the moisture content of the litter, but there was no correlation between oocysts present and the final BW or feed conversion of the birds. Moisture levels were highest (after withdrawal of drug) for new litter or oak shavings. There was no correlation between moisture content of the litter and BW or feed conversion.

The incidence of oocysts was monitored in the litter at two broiler farms where birds were reared to 8 wk of age using a shuttle program involving nicarbazin (from 0 to 21 days of age) and salinomycin (from 21 to 44 days of age). Some birds from these farms were challenged with three species of Eimeria to see whether immunity had developed as a result of prior exposure to infection. Oocysts of at least three species of Eimeria were present in small numbers in the litter. Birds from both farms were immune to Eimeria acervulina and Eimeria maxima (judged by weight gain) although the immunity was incomplete as evidenced by oocyst production. Birds from one farm were immune to Eimeria tenella (judged by weight gain) but birds from the other farm were susceptible to this species.

The sensitivity of field isolates of turkey coccidia from the United States to the anticoccidial drugs amprolium, clopidol, diclazuril, and monensin was investigated. Clopidol and diclazuril were the most effective, followed by monensin and amprolium. Thirty-one isolates were classified as resistant to amprolium, 23 resistant to monensin, 10 resistant to diclazuril, and 6 resistant to clopidol. Six isolates were partially resistant to monensin, 10 partially resistant to clopidol, and 11 partially resistant to diclazuril. Four isolates were sensitive to monensin, 12 sensitive to diclazuril, and 17 sensitive to clopidol.

Infectious and metabolic disorders are common in poultry and cause stress, poor performance, and mortality that results in considerable economic loss. Identifying the nature of stress in chickens will assist the development of appropriate measures to improve health and welfare. Acute phase proteins are hepatic proteins, the blood concentrations of which change significantly in the event of many health problems including inflammation and physical injuries. Thus, acute phase proteins are used as nonspecific diagnostic markers for various health disorders. Our previous studies showed that serum ovotransferrin (OVT) is an acute phase protein in chickens. Therefore, in the present study, we investigated whether OVT concentration can be a marker of physiological stress using sera from chickens with different infectious and metabolic disorders. A competitive enzyme immunoassay was developed to measure serum OVT concentrations. The results show that with experimentally induced pulmonary hypertension syndrome and tibial dyschondroplasia, there were no significant changes in OVT levels compared with matched controls. In contrast, when chickens were infected with microbes such as the bacterium Escherichia coli, or protozoan parasites such as Eimeria maxima and Eimeria tenella, there was a significant increase in the levels of OVT in the serum. Chickens with spontaneous autoimmune vitiligo also showed a significant increase in blood OVT levels. These studies suggest that blood OVT concentration is modulated under inflammatory and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens.

"Continuous feeding of low concentrations of sulfaquinoxaline for the control of coccidiosis in poultry" by L. C. Grumbles, J. P. Delaplane, and T. C. Higgins [Poult. Sci. (1948) 27:605-608] was the first paper to demonstrate that it was possible to control coccidiosis by the continuous inclusion of a low level of a drug in the feed of chickens. The principle involved (prevention or prophylaxis) has had a profound impact on our ability to grow chickens and turkeys under intensive conditions. Indeed, it is possible that the modern poultry industry could never have developed to its present extent without the advent of drugs used prophylactically to control coccidiosis. One particular insight was that use of a compound in this manner did not necessarily prevent the acquisition of immunity, an important principle that helps explain the continued efficacy of ionophorous antibiotics used today. The significance of this work to the poultry industry and individuals involved in research, whether employed by government, academia, or pharmaceutical companies, cannot be overstated. Economic benefits, in terms of improved productivity, have been demonstrated in numerous studies published in Poultry Science. In addition, the livelihoods of many poultry farmers have been helped by the control of a disease that in the past caused substantial morbidity and mortality in their flocks. The paper is brief and contains no critical science involving novel procedures but has had a profound influence on the health of poultry for the last 6 decades. For this reason, it is nominated as a landmark contribution from the first 100 yr of Poultry Science.

Coccidiosis is a widespread disease of considerable economic importance to the turkey industry, as is evident by the fact that many millions of dollars are spent annually on its control. Despite this, in recent years little research has been conducted on the causative organisms--protozoan parasites of the genus Eimeria. This is unfortunate because current methods of control are not without problems. It is possible, therefore, that in the future optimal productivity in turkey production will be difficult to achieve. There has been no recent comprehensive literature review of coccidiosis in the turkey. In the present article our knowledge of the biology of the species of Eimeria that infect the turkey is critically reviewed. Other topics considered include host specificity, host resistance, acquired immunity, in vitro cultivation, pathology, pathogenicity, clinical signs, diagnosis, epidemiology, prevalence, significance, chemotherapy, drug resistance, drug toxicity, and vaccination. The review is intended to provide a baseline for future studies of this important disease.

Halofuginone (HAL) and monensin (MON) were effective in controlling coccidiosis in turkey poults when included in the feed at concentrations of 2 and 66 ppm respectively. At 10 and 14 weeks of age, body weight and feed intake were greater in poults given HAL with bambermycins (BAM) than in poults given HAL or MON with bacitracin methylene disalicylate (BAC). Poults given two anticoccidial drugs (MON followed by HAL or HAL followed by MON) weighed more and had a lower feed conversion than poults that received no medication, but no difference was found in the performance of turkeys given one or two anticoccidial drugs. There were no differences in performance whether HAL was used before or after the use of MON. None of the drugs completely suppressed oocyst production. At 11 weeks of age, an increase in the number of oocysts in the droppings was noted following drug withdrawal, but no increase was observed in the number of oocysts in the litter. There was no indication of clinical coccidiosis after drug withdrawal.

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 acquisition of immunity by chickens infected 18 h post-hatch with 100 oocysts of Eimeria maxima and reared in floor-pens in contact with their droppings was investigated. In the first experiment, birds were placed on new litter and immunity was measured at 2, 3, 4, and 5 weeks by calculation of weight gain from days 0 to 7 following challenge with 100000 oocysts or by oocyst production in the faeces from days 5 to 8 following challenge with 500 oocysts. In the second experiment, birds were placed on new litter or reused litter from the first experiment (1 week after birds from the first experiment had been removed when 6 weeks of age), and were challenged at 1, 2, and 3 weeks of age. In the first experiment, immunity had developed in birds challenged at 3, 4, and 5 weeks, judged by weight gain and oocyst production, but immunity was not complete at 2 weeks. In the second experiment, immunity had developed in birds challenged at 1, 2, and 3 weeks measured by either criterion. In both experiments, birds produced small numbers of oocysts in their faeces following challenge. Judged by the weight gain following challenge, no significant difference in the acquisition of immunity was observed whether birds were reared on new or reused litter.

These guidelines are intended to aid those engaged in poultry research in the design, implementation and interpretation of laboratory, floor-pen and field studies for the assessment of the efficacy and safety of live anticoccidial vaccines for immunization of chickens and turkeys against Eimeria species. In addition to efficacy and safety requirements, manufacture, quality control and licensing considerations are discussed. The guidelines do not address subunit vaccines comprising non-viable material, but many of the principles described will be relevant to such vaccines if they are developed in the future. Guidelines are available in some countries for avian vaccines of bacterial or viral origin but specific standards for anticoccidial vaccines in poultry have not, as far as we know, been produced. Information is provided on general requirements of registration authorities (based upon regulations applicable in the European Union and the USA) for obtaining marketing authorizations for vaccines. These guidelines may assist poultry specialists in providing specific information for administrators involved in the decision-making process leading to registration of new vaccines, and are intended to facilitate the worldwide adoption of consistent, standard procedures.

The acquisition of immunity to Eimeria species was studied in broilers reared in floor-pens on new and used litter, and given different anticoccidial drug programmes. Programmes included a single drug (synthetic compound or ionophore) given in the starter and grower feed, or 'shuttle' programmes comprising a synthetic drug followed by an ionophore (or vice versa) given in the starter and grower feeds, respectively. None of the drug programmes prevented parasite multiplication, since oocysts were found in the intestines of birds and in the litter. Birds were challenged at weekly intervals with a mixture of species (E. acervulina, E. maxima and E. tenella) in order to establish whether protective immunity had developed. Birds reared on used litter that received no medication acquired immunity by 5 weeks, whereas birds reared on new litter were not fully protected against a challenge inoculum until 7 weeks of age. Apart from birds given an ionophore followed by a synthetic drug, medicated birds developed immunity by 7 weeks, irrespective of the type of drug programme, or whether they had been reared on new or used litter. Birds reared in commercial houses (where one and seven previous flocks had been raised) and given a synthetic drug followed by ionophores, also developed immunity to Eimeria species. Immunity was acquired more rapidly to E. maxima than E. acervulina or E. tenella.

The long-held view that anticoccidial drugs, to a greater or lesser extent, interfere with the development of immunity to Eimeria species may no longer be correct because few drugs, if any, are capable of preventing some degree of parasite multiplication. Acquisition of immunity is, therefore, a real possibility, providing sufficient parasites are present in the environment. Immunity is an important consideration during the rearing of replacement layers, broiler breeders, turkeys, and gamebirds, but little research has been carried out on the effects of drugs on immunity development in these categories of stock. In recent years, there has been a change in the perception of the significance of immunity in broilers, and some broiler producers have taken this into consideration when designing drug programs for use in the field. Studies in floor pens and the field indicate that broilers given various drug programs can develop immunity when exposed to natural infections but that this process takes time: solid immunity not developing until birds are 6 to 7 weeks of age. This suggests that drugs should not be withdrawn from the feed prematurely since birds may not have had time to develop adequate immunity.

The effects of diclazuril and monensin, when included in the feed of turkeys from 0 to 10 wk, upon performance and development of immunity to Eimeria species was investigated. Birds were initially inoculated with a low dose of oocysts of three species of Eimeria at 3, 5, 7, and 9 days of age in order to simulate a natural infection. Weight gain and feed intake from 0 to 6 wk of age was significantly greater in medicated birds compared with those that received no anticoccidial medication. Weight gain and feed intake from 6 to 10 wk was greater in birds that received diclazuril than in unmedicated birds. No differences in performance were evident after drug withdrawal from 10 to 16 wk. Immunity to Eimeria species developed by 10 wk in birds that received no anticoccidial medication but did not develop in those given diclazuril or monensin.

The timing and magnitude of a coccidian infection, judged by the numbers of oocysts of Eimeria present in the litter, were affected by the duration of medication. In birds medicated for 6 wk and infected at 35 d of age, fewer oocysts were produced than in birds medicated for 4 or 5 wk whether infected at 18 or 35 d of age. Feed conversion at 6, 7, and 8 wk of birds infected at 18 d and medicated for 6 wk was less than that of birds medicated for 4 or 5 wk. Birds infected at 35 d and medicated for 6 wk had a lower feed conversion than birds medicated for 5 wk. Immunity to Eimeria tenella had developed by 8 wk in birds medicated for 4, 5, or 6 wk if infected at 18 d of age. Immunity did not develop in those birds medicated for 6 wk when infected at 35 d.

These guidelines have been written to aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria species in chickens and turkeys. The information provided deals with many aspects of how to conduct controlled studies in battery cages (dose determination), floor pens (dose confirmation), and commercial facilities (field effectiveness studies), the selection of birds, housing, feeding, preparation of medicated rations, record keeping, diagnostic techniques, and methods for the preparation, maintenance and use of parasites. These guidelines are also intended to assist investigators in conducting specific studies, provide specific information for registration authorities involved in the decision-making process, assist in the approval and registration of new anticoccidial drugs, and facilitate the world-wide adoption of standard procedures.

A landmark decision was taken in 2002 by the Biotechnology and Biological Sciences Research Council (BBSRC) in the UK to fund the genome sequencing of an eimerian (apicomplexan) parasite from the fowl. The project is a joint collaboration between the Institute for Animal Health (IAH) at Compton, UK and the Wellcome Trust Sanger Institute, Cambridge, UK (see http://www.sanger.ac.uk/Projects/E_tenella/ and http://www.iah.bbsrc.ac.uk/eimeria/). The species chosen, Eimeria tenella, is the best known member of the genus and the Houghton (H) strain is one of the most widely studied. The H strain was isolated in 1949 in the UK. It was maintained initially at the Houghton Poultry Research Station (HPRS), Houghton, and, following closure of that laboratory in 1992, thereafter at the IAH in Compton. At various times the parasite has been provided to other institutions and research groups carrying out coccidiosis research in the fowl. The H strain has been utilised in many fundamental studies on the eimerian life cycle and its relationship with the host. It has also been used to investigate the nature of drug resistance, and to derive attenuated lines-one of which, a "precocious line", is a component of a multivalent live attenuated coccidiosis vaccine (Paracox vaccine, Schering-Plough Animal Health). In this article, some immunological, epidemiological, genetic, and chemotherapeutic investigations with the H strain are reviewed, a summary is provided of the biological characteristics of the parasite and some of the core methods used to prepare purified extracellular life cycle stages for experimental studies or passage are given.

These guidelines have been prepared to assist in the planning, operation and interpretation of studies designed to assess the effectiveness of drugs against helminth parasites of chickens and turkeys. They are the first to be compiled under the auspices of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for these parasites. The advantages and disadvantages of the widely used critical and controlled tests are discussed. Information is provided on the selection of animals for experiments, animal housing, feed, dose determination studies, confirmatory and field trials, record keeping and necropsy procedures. This document should help investigators and those involved in product approval and registration in conducting and evaluating studies concerned with determining the effectiveness and safety of anthelmintic drugs.