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Coccidial Parasites from the Chicken: Their Control by Vaccination and Some new Tools to Examine Their Epidemiology
Martin W. Shirley
(Institute for Animal health Compton Laboratory Compton, Nr Newbury Berks, RG20 7NN, Tel ++44 1635 577275, Fax +44 1635 577263, e-mail shirley@bbsrc.ac.uk)
Coccidiosis, caused by species of intracellular protozoan parasites belonging to the genus Eimeria (phylum Apicomplexa), remains one of the economically most important diseases in modern poultry production. The disease is caused by the replication within the intestine of the asexual and/or sexual stages of seven species of Eimeria (usually several species occur concurrently) and although clinical disease in intensively reared poultry is now relatively uncommon, sub clinical infections are the norm. The total cost of coccidial infections in the UK (circa 780 million broilers) has been estimated to be at least £ 42m per annum, of which 74% is due to sub-clinical effects on weight gain and feed conversion and 24% is the cost of prophylaxis and therapy of commercial birds (Williams, 1999). Infections with Eimeria spp. In intensively reared poultry have always been controlled primarily by prophylactic in-feed medication (especially for broiler production), but vaccination with live, virulent or attenuated parasites has a role to play. For example, within the past few years, use of the safer vaccines has become especially important for the rearing of broiler breeders within Europe. Moreover, it is anticipated that the impact of live attenuated vaccines will increase significantly as they could become a mainstream alternative to the use of anticoccidial drugs in many poultry-producing countries, for example, within those of the European Union (EU). The term coccidiosis actually refers to disease caused by one or more of the seven species of Eimeria that infect the avian avian host. In the chicken the seven recognised species are E. acervulina, E. bruneti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella and they are characterised at the gross level by differences in their pathogenicity and immunogenicity, sizes of oocysts and variation in their prepatent periods, etc. During the course of natural infections, several of the species usually occur together and individual species are generally recognised from examination of gross intestinal lesions the host post mortem or by the morphological appearance of oocysts in the faeces or litter. More specialist (research laboratory) procedures have also been described that allow each of the species to be identified in a definitive manner. Thus studies done in the past five years on the occurrence of the avian Eimeria species have relied either upon a consideration of the traditional characteristics alone, or in combination with methods such as the electrophoretic variation of enzymes. However, approaches that make use of variation within sequences of DNA have been reported recently and in the past year a PCR-based assay has been described that offers a chance for an "Eimeria identification methodology" to bridge the gap between use as a research tool in the laboratory and utility as part of an ongoing programme to monitor infections in the field. CONTROL OF COCCIDIOSIS BY VACCINES 1. General Growth of the Eimeria spp. In the intestine results in the host quickly developing immune responses that will protect it against a subsequent challenge with parasites belonging to the same species (e.g. Rose and Hesketh, 1976; Rose, 1978). In other words, whilst the induced immune responses are potent and effective at controlling challenge infections, they are exquisitely directed only against the species that established the initial immunising infection. The seven species of Eimeria from the chicken vary in absolute terms in their ability to promote protective immune responses, but essentially all are able to induce protection to some degree within a few days of the parasites starting the intracellular phase of their life cycle. In commercial poultry houses in the UK the peak numbers of oocysts are recorded around the third/fourth week of rearing, and one requirement of live vaccines therefore is that they promote protective immune responses that enable a flock to withstand challenge with wild-type parasites within this period of time. A further practical consideration of the induced immune response relates to a potential for strain specificity with the significantly pathogenic species E. maxim. Thus, infection with some strains of E. maxima will protect chickens only partially against challenge with strains that are "immunologically diverse" (e.g. Long, 1974; Norton and Hein, 1976; Fitz-Coy, 1992; Martin et al. 1997, see also below). The ubiquity of immunologically unrelated strains of E. maxima has not been investigated in great detail, but evidence to date suggests that the trait of antigenic diversity if relatively common and that the degree of relatedness may be as little as 10% when the numbers of oocysts that are passed after challenge are counted (e.g. Shirley and Bellatti, 1988). 2. Vaccines available Four live coccidiosis vaccines (that comprise oocysts) are manufactured within Canada, the USA or Europe. The two produced in the USA or Canada comprise wild-type strains of unaltered virulence, viz. " Coccivac" vaccine (American Scientific Laboratories Inc.) and "Immucox" vaccine (Vetech Laboratories), respectively. Within Europe, the experience of coccidiosis vaccines is formally limited to the use of the second-generation, live-attenuated vaccines, viz. "Paracox" vaccine (manufactured in the UK by Schering-Plough Veterinary Ltd) and "Livacox" vaccine (manufactured in the Czech Republic by Biopharm). Sales of "Paracox" and "Livacox" vaccines have increased steadily since their launch (Fig. 1) and an estimated 240 million doses of " Paracox" vaccine (Williams, 1998) and 200 million doses of "Livacox" vaccine (Bedrnik, personal communication) have been sold. (These figures are well behind the total sales of "Coccivac" vaccine which has now been administered to more than 2,000 million chickens since it was introduced in the USA in the 1950*s [Williams, 1998].) Within Europe, numerically the greatest use of coccidiosis vaccines is for the control of cocidiosis in broiler breeder chickens and the importance of vaccination is now acknowledged by several bodies. For example, a recent report prepared by the Farm Animal Welfare Council (1998) on the "Welfare of Broiler Breeders" points out that it is universal practice to vaccinate these birds against coccidiosis and that " the commercial availability of an effective coccidiosis vaccine in the last 10 years has removed the principal cause of mortality, uneven growth and poor welfare in young breeding birds, which generally have good health status and low mortality. It is now quite reasonable to expect birds to be reared to 18 weeks with no more than 2% losses, either culls or mortality". The four live vaccines manufactured in Europe and the USA all contain oocysts of the species E. acervulina, E. maxima and E. tenella (see Table 1). However, the need for some or all of the four other recognised species to be included within a vaccine is more controversial and n part is determined by both the type of chicken to be vaccinated and a consideration of the epidemiology and virulence of the individual species of Eimeria. Thus, the final composition of a live attenuated vaccine remains a matter of some judgment by the manufacturers. Moreover, in the future the choice of a particular vaccine that is made by the consumer may also rely both upon the products available together with an assessment of the perceived or actual threat posed by some of the species other than E. acervulina, E. maxima and E. tenella. The general trend is that vaccines targeted specifically towards broiler breeders contain more species than vaccines intended for use primarily in broilers. This philosophy may underpin the difference between the two attenuated vaccines available within Europe. "Paracox" vaccine (used more extensively in broiler breeders) is a complete vaccine and comprises eight populations representative of all seven species with two strains of E. maxima, whereas "Livacox" vaccine (used more extensively in broilers) is a trivalent product that comprises oocysts of only one strain each of E. acervulina, E. maxima and E. tenella. 3. composition of live attenuated vaccines The two attenuated vaccines developed in Europe each comprise novel populations of parasites that were selected in the laboratory under controlled conditions of assage. The selected populations of parasites are characterised by some novel features that distinguish them substantially from the normal "wild-type", strains that are included in the first generation live vaccines. So-called "precocious" lines (with a shorter endogenous) life cycle are used exclusively in "Paracox" vaccine (e.g. McDonald and Shirley, 1986; shirley and Millard, 1986; Williams, 1992) and two "precocious" and one "egg-adapted" line are used in "Livacox" vaccine (Bedrnik, 1993). Precocious lines comprise 10 of the 11 parasite populations used in the two attenuated vaccines (see also Tabe 1) and they are characterised by an abbreviated by an abbreviated (shorter) life cycle in which significantly fewer offspring are produced in the intestine. The overall effect is therefore one of marked attenuation of virulence so that attenuated coccidiosis vaccines offer an inherently greater margin of safety than vaccines based on wild-type strains because:
However, the intrinsically greater safety advantages of the precocious/egg-adapted lines are complemented by the ability of the parasites to induce protective immune responses that are as potent as those generated by naturally occurring wild-type strains. 4. The issue of strain diversity within E. maxima The presence in the field of antigenically diverse strains has some major implications for the use of a "nagging problem" in which a putative immune variant of E. Maxima was causing general morbidity in vaccinated flocks on a farm at which "Immucox" vaccine had previously been used successfully for five years. Protective immunity induced by the strain of E. maxima used in " Immucox" vaccine was thought to be unable to control infections caused by the variant strain. The problem was resolved in a straightforward manner simply by adding small numbers of oocysts of the variant strain of E. maxima to the "Immucox" vaccine. However, this type of "local solution" is not compatible with the large-scale use of coccidiosis vaccines, nor approved within the EU. The approach used for "Paracox" vaccine appears to be the most suitable option and this vaccine is manufactured at the outset to contain two immunologically distinct strains of E. maxima that confer a wider degree of protection than would be achieved by vaccination with a single strain alone. As far as is known the strategy continues to work very ell n practice and the appearance of problems of the type described by Lee (1993) have not been encountered within Europe (or elsewhere). 5. Future developments The recent uptake of attenuated coccidiosis vaccines in Europe and in other parts of the world has given the poulty industry greater confidence that vaccines may be used without any subsequent disease problems that require attendant medication. (Wild-type strains of unaltered virulence used in the first generation coccidiosis vaccines may themselves lead to clinical disease.) Thus the success of the live attenuated vaccines is both likely to prompt the introduction of further similar products into the marketplace and encourage a wider uptake of such products for the vaccination of broilers. Indeed, the future control of coccidiosis in broilers now appears much less straightforward than hitherto, with many factors needing to be taken in to account. Such factors include not only those relating directly to the biology of the parasites (such as the management of drug-resistance in the absence of fewer anticoccidial drugs, etc,) but a changing "political climate", especially within Europe, that may significantly influence the future direction of control methods. For example, the EU is active in looking at the use of antibiotics in poultry production and concerns are being raised about the use of antimicrobial feed additives. In December 1998 the EU countries voted in favour to ban, with effect from January 1st 1999, the use of three digestive enhancers, viz. the antibiotics zinc bacitracin, virginiamycin and spiramycin, that had all been used previously for more than 30 years. Increasingly, the antibiotic ionophorous anticoccidial drugs are being considered along with other antibiotics that have quite different properties and uses, leading to some concerns that within the EU the use of important anticoccidial drugs may be affected in the future. Not surprisingly, the suitability of live attenuated vaccines for use in broilers is being examined further. Shirley and Bedrnik (1997) described the efficacy of "Livacox" vaccine in broilers reared n South America (Table 2) and Williams et al. (1999) examined the efficacy (and economic benefits) of "Paracox" vaccine in broilers as assessed from the results of field trials in the UK involving over 936,000 birds. "Paracox" vaccine comprised all eight precocious populations (see above) and was given of the criteria used to measure the performance of the birds and briefly, the vaccine performed as well as the two drug shuttle control treatments (see Table 3 and Fig. 2). The digestive enhancer virginiamycin was used in all trials and, interstingly, necrotic enteritis occurred only in medicated controls (2/9 flocks). The success of the safe, immunogenic precocious lines as components of two live vaccines has also stimulated further similar studies. Reports unconfirmed by the author indicate that a live attenuated vaccine based on precocious lines is now available in Japan, and a precocious line of E. acervulina been derived in Australia for incorporation into a proposed Australian live vaccine comprising E acervulina, E. maxima, E, necatrix and E. tenella (Stewart and Jorgensen, 1997). Precocious lines that are effective immunogens have also been isolated in China. Huang et al. 1997) described field trials in which 10,000 chickens were vaccinated with early maturing, attenuated eimerian oocysts selected from field strains. The authors reported decreased morbidity rates of coccidiosis (10% vs. 30%), decreased costs for medication (0.02 Yuan vs. 0.10 Yuan) but the same weight gains (2,000 g vs. 1, 800 g) when compared to chickens not vaccinated on the farm. In another experiment, 60 native chickens were vaccinated with early maturing attenuated oocysts of E. acervulina, E. maxima, E. necatrix and E. tenella at 7 days of age. 14 and 26 days after vaccination, half of the vaccinated group and a similar number of control chickens were challenged with field strains (50, 000 and 100, 000 oocysts per bird). After challenge, birds in the vaccinated group passed 90% fewer oocysts; had significantly decreased lesion scores (0.5 vs. 4-4.5); lower bloody caecal scores (0.5 vs. 3-3.5); a lower morbidity rate (0 vs. 10-20%) but improved weight gains (105.6 g vs. 78.0 g) when compared with the controls. A new composition of live oocysts given to broilers was reported by Vermeulen et al. (1998). No details of the types of parasites that comprised the experimental vaccine were given, but the floor pen trial involved the administration of oocysts through nipple drinkers and chickens aged 5 days were vaccinated with a mixture of three species. More than 90% of the chickens took the vaccine and the authors commented that the composition appeared safe as intestinal lesion scores were always less than a score of two and that the vaccinal oocysts were immunogenic as shown by the level of protection conferred to chickens against heterologous challenge. In view of the method of delivery that was used, the authors concluded that the vaccine was compatible with modern broiler management. Other immunological approaches, different to the use of live vaccines, are also being considered. Wallach and colleagues (e.g. Pugatsch et al., 1989; Wallace et al., 1992; Wallace et al., 1995) demonstrated the ability of antigens recovered from the sexual stages of E. maxima (and injected with an adjuvant) to protect the young offspring of vaccinated hens against coccidial infections; i.e. through the mechanism of IgG-mediated maternal immunisation. Targeting the laying breeder bird could be both economically and logistically attractive propositions for preventing coccidiosis in broilers as each female breeding bird produces around 120 broilers - active vaccination of a comparatively small number of birds leads to the passive protection of substantially more. IDENTIFICATION OF EIMERIA SPECIES AND STRAINS; WITH EMPHASIS ON SOME RECENT ADVANCES 1. General The most commonly recognised species of Eimeria in all types of intensively reared chickens worldwide are E. acervulina, E. maxima and E. tenella, but other species may assume greater relative importance in specific types of birds and/or in different regions of the would. For example, E. necatrix is not commonly encountered in broiler flocks but is generaly accepted to pose a special threat to broiler breeders and a similar situation is probably true of E. bruneti. In contrast, the contribution of E. mitis to sub-clinical coccidiosis has almost certainly been underestimated because infections do not result in the appearance of gross intestinal lesions - even when large doses of oocysts are ingested. However, some strains of E. mitis can induce significant negative effects on the body weight of infected chickens (McDonald et al., 1985; Williams, 1998), including actual weight loss, but the lesions are microscopic even though they lead to a dramatic loss of functional surface area of the intestine. Some knowledge of the different species of Eimeria that are present or persist in different countries and regions despite the use of specific control measures, can provide useful information for the planning of anticoccidial drug and vaccination programmes and contribute to an understanding of any problems. Whilst chemotherapy is broadly directed to the control of all species, vaccination strategies are more selective (only "Paracox" vaccine contains all species0 so that methods leading to the unequivocal identification of the seven Eimeria species are expected to become of more value as the reliance of vaccination strategies increases. 2. Enzyme electrophoresis Enzyme electrophoresis provided a significant technical advance several years ago and was instrumental in clarifying the taxonomy of Eimeria spp.; especially in establishing the absence of any close relationship between E. acervulina and E. mitis (e.g. Shirley et al. 1983) and confirming as seven the number of species in the chicken. Until very recently, the technique was the only definitive method available and, although relatively cumbersome to set up and use routinely, it roved value both to monitor the purity of laboratory strains and to determine the identity of field isolates. Shirley et al. (1995) used enzyme electrophoresis in combination with an examination of prepatent times, oocyst morphology and growth of parasites in embryonating eggs to study the occurrence in the UK of different Eimeria species in contemporaneous flocks vaccinated with "Paracox" vaccine or medicated with anticoccidial drugs. The predominant species of Eimeria found in the trials in the UK were E. acervulina, E. mitis and E. praecox. Eimeria tenella and E. maxima were more common in medicated flocks and E. brunetti was isolated only from the single flock of unmedicated (unvaccinated) replacement breeders. Eimeria necatrix was not isolated in this study. The detection of two electrophoretic forms of the enzyme glucose phosphate isomerase (GPI) representative of distinct strains of E. acervulina (GPI-7 and GPI-8) and E. tenella (GPI-1 and GPI-9) made possible some differentiation between unequivocal wild-type parasites (GPI-8 and GPI-9) and possible vaccinal lines (GPI-7 and GPI-1). These biochemical differences still remain the only means by which to distinguish between some vaccine parasites and wild-type populations. Similar technical approaches were used by Williams et al.1996) in their survey of Eimeria spp. Occurring in commercially-reared chickens in France during 1994. Six of the seven species were again found, viz. E. acervulina (100% of farms), E. mitis (82%), E. tenella (77%), E. maxima(73%), E. praecox (45%), E. Brunetti (27%)and the study provided the first definite record of the presence of E. praecox and E. mitis in France. Eimeria necatrix was not found during the survey but had been identified previously by other workers in the country. On 21 of 22 farms examined (95%) more than one species was found and on 5 farms (23%) all six most common species were found. Information on the occurrence of strains within a species is scant and most (albeit still very little in absolute terms) is known about E. tenella. Shirley et al. (1989) showed that all isolates recovered from several countries within Europe, Israel and New Zealand comprised only a singly group characterised by GPI-9. No isolates of E. tenella were characterised by GPI-1 which had been found previously in the Houghton laboratory strain (parent strain of the precocious line included within "Paracox"). It is not known why only one "type" of E. tenella occurred in the field at that time and whether the situation remains the same today. The total absence of parasites with CPI-1 suggests that a selection pressure has been imposed on the species and it is possible, therefore, that CPI-9 is linked genetically with an unknown, but very important, trait. Interestingly, of the few laboratory strains examined, those with GPI-9 are innately more pathogenic than those with GPI-1. The epizootiology of E. tenella appears to be less straightforward in the USA where populations characterised by both GPI-9 and GPI-1 were isolated in 1989. One possible reason for the disparity between the situation in the USA and some other countries could have been the constant, although limited, disemination of the live vaccine "Coccivac" that has been shown to contain a strain of E. tenella characterised by GPI-1 (Shirley, 1989). The presence of strains of E. tenella characterised by only one of two known forms of GPI-1 contrasts markedly to the results of similar studies with some of the other species (e.g. E. acervulina and E. mitis) which revealed the concurrent presence in the field of several strains (Shirley 1989). The tenuous link between virulence and GPI-type with E. tenella has a parallel with the lactate dehydrogenase (LDH)-type of E. necatrix (Shirley 1985). Strains characterised by a form of the enzyme coded LDH-7 were highly virulent (almost 100% mortality from a dose of 50,000 oocysts) whereas two strains with LDH-9 were highly attenuated and induced virtually no mortality. Three other enzymes examined were not informative. Unexpectedly, the reproduction of all strains was very similar and these results both suggest that the phenotype of virulence may be associated with specific and predictive markers and that virulence of Eimeria spp. Is not always manifest at the level of parasite reproduction. These initial observations have never been examined further but do warrant further investigation. For example, the availability of DNA markers that were predictive of naturally occurring attenuation would allow the simple identification of strains that might be useful as live vaccines. In the cast of E. necatrix, the detection of polymorphisms in the DNA sequence of LDH might be a useful starting point. The studies by Shirley et al. (1995) of parasites occurring in the UK and by Williams et al.(1996)of the largest field studies to date that have made use of enzyme electrophoresis. However, although useful the methods have generally not found wide favour and will inevitably be replaced by DNA-based technologies that are easier to use and require an input of significantly fewer parasites. 3. DNA-based approaches Surprisingly, comparatively few reports to date have described the analysis of DNA for the identification of Eimeria spp. However, they do include the polymerase chain reaction (PCR) (Stucki et al. 1993), analyses of restriction fragment length polymorphisms (RFLPs) (Shirley 1994a) and the random amplification of polymorphic DNA by PCR (RAPD-PCR) (MacPherson and Gajadhar 1993; Shirley and Bumstead 1994; Cere et al. 1995). Several RFLP markers have been identified in this laboratory that allow discrimination between trains of E. tenella (Shirley 1994a and unpublished). Most of the markers were reiterated sequences of DNA and, for example, using a combination of digestion with EcoRI, Southern blotting and a probe derived from the 5S rRNA gene, it proved possible to discriminate between several strains (and one derived attenuated line) of the species (unpublished data). The PCR amplification of DNA is a very powerful tool and especially sensitive when the targets of amplification are highly reiterated DNA sequences within the genome. Stucki et al. (1993), for example, detected the DNA from a single oocyst of E. tenella when the target of the PCR reaction was the 5S rRNA gene - which appears to be tandemly repeated over 500 times within the genome (unpublished observations). The PAPD-PCR technique may lack the absolute sensitivity achieved by amplification of known highly repetitive sequences with the use of primers of specific sequence, but it does offer some significant advantages. Short, typically nine or 10-mer oligonucleotide primers selected at random are used individually for the amplification reaction and the chance occurrence of sites in the target DNA complementary to the oligonucleotide defines the results. One, or more usually, multiple fragments are amplified and these may be resolved as a series of bands following electrophoresis. Some screening of oligonucleotides is necessary to identify informative ones (those comprising 70% G+C bases work very well with Eimeria spp.) and each of the five oligonucleotides examined in more detail in this laboratory revealed a unique profile of fragments for each of the seven species. Greif et al. (1996) laboratory revealed a unique profile of fragments for each of the seven species. Greif et al (1996) examined the RAPD-PCR profiles of several field isolates of E. brunetti that they also characterised in terms of response to anticoccidial dugs. Fingerprints generated by RAPD-PCR revealed a high degree of similarity between sensitive strains (up to 95%), but polymorphisms, including band shifts, differences in banding intensity and missing bands, gave significantly lower similarities in drug-resistant strains. Greif et al. (1996) also selected a diclazuril-resistant line from a laboratory isolate and amplified from it, by RAPD-PCR, a polymorphic band of around 600 bp. The authors concluded that such polvmorphisms detected by RAPD-PCR would be uselful molecular marker and might lead to the design of diagnostic tests for drug-resistant genotypes. A major drawback of the RAPD-PCR technique is that it is not ideally suited for analyses of mixed populations of parasites - the profiles are far too complex. However, since many of the amplified fragments represent sequences reiterated throughout the genome (a single primer must anneal to the same sequence on opposite strands of the molecule) a virtually unlimited number of oligonucleotide primers could be examined, for example, to identify further DNA targets for a more rational approach to amplifcation. 4. A new PCR-based assay Arguably the most effective procedure described to date to identify species of Eimeria is the PCR amplification of the internal transcribed spacer 1 (ITS1) region between ribosomal DNA (rDNA) genes (Schnitzler et al., 1992), Neospora (Holmdahl & Mattsson, 1996) and Sarcocystis (Jeffries et al., 1996). The ITS1spacer separates the 3* end of the 16S-like ribosomal RNA gene from the 5* end of the 5.8S rRNA gene within individual rDNA transcription units and is typically variable in both sequence length and base composition. Such variation allows for the design of genus- and/or species-specific primers and, as part of the rDNA transcription unit, it belongs to a multiple copy gene family and thus provides a large number of potential PCR targets. Schnitzler et al. (1998; 1999) amplified the iTS1 region from each of the seven species and, using the DNA sequence data, derived unique, species-specific, pairs of primers so that a region within the ITS1 region in each of the seven species could be amplified by PCR. Sequences of the primer pairs for each of the seven species (given in Table 4) were evaluated in tests using DNA samples prepared from purified oocysts of reference strains, mixed isolates from the field or tissue samples from experimentally infected chickens. In all cases, positive amplification of DNA from a reference strain/isolate was achieved only with the specific primer pair and no cross amplification by the other primer pairs was observed. The results also agreed with previous electrophoretic variation of enzyme analyses of the same samples (Table 5). The level of sensitivity of the PCR assay was found to be around 25 oocysts. In a further refinement to the procedure, Schnizler et al. (1999) described a read-out for the PCR assay that utilised a commercial colourimetric assay based on a paper chromatography assay (PACHA; from Bio-Rad) and the useo of biotinylated oligonucleotides. Simply, the presence within a field or intestinal sample, etc. of DNA from one of the seven species of Eimeria was revealed by the appearance of a blue spot on a PACHA "comb". The commercial kit was easy to use and once the PCR had been completed, the time required to definitively identify a species was reduced to only 30 min in comparison to 2-4 hours for electrophoresis and staining. Furthermore, starting with the extraction of DNA from an intestinal sample at post mortem, a PCR-based assay with one biotinylated primer from each primer pair and an associated colourimetric assay can provide definitive results within little more than half a day. The PACHA read-out proved to be a simple process and in the future this type of assay might provide the basis for the defelopment of a standard technique in which laboratories in different parts of the world can use the same methods for the identification and detection of avian Eimeria species As yet no commonly accepted, definitive and sensitive system exists with which to investigate epidemiological questions concerning poultry coccidia. Although at present the costs of commercially available PACHA kits are high, these are likely to fall in the future. With regard to the epidemiology and identification of avian coccidia, several important questions could be addressed by use of a PACHA or a related type of PCR assay. In addition to more detailed studies on the occurrence of species such as E. brunetti, E. mitis and E. necatrix, a better knowledge of the epidemiology of Eimeria spp. Per se would be helpful to evaluate the efficacy of different chemotherapeutic and immunological strategies and perhaps changes in types of husbandry (such as for example, any consequences arising from the prohibition from 1999 of battery cages in Sweden). Looking further ahead, PCR-based identification approaches should become increasingly more necessary in order to discriminate between cocidial parasites to the level of strains, especially those characterised by different drug-resistant phenotypes. Many suitable DNA targets for amplification by PCR are expected to exist within the large genomes of Eimeria spp. (Shirley 1994b), so that potentially a wide range of strain types/phenotypes, etc. could be identified easily and cheaply and the information used to aid coccidiosis control programmes. SUMMARY In many ways coccidial parasites are ideal targets for immunological control. For example, potent protective immune responses are induced in the host during primary infections and, in the presence of continuing reinfections, these may last for the life time of the host. Until recently only vaccines based on virulent vaccines were available, but their use can be compromised by outbreaks of disease caused directly by the vaccinal oocysts. Following the successful introduction of the attenuated vaccines, it is likely that immunological control measures per se will gain considrable further support as viable alternatives to chemotherapy. Certainly the poultry industry is gaining confidence that vaccination against coccidiosis can work both effectively and safely. Identification of the individual coccidial species remains an ongoing problem for many veterinarians and technical specialists and in the past has relied almost entirely upon traditional approaches. Now for the first time an easy to use, rapid and sensitive procedure (based on the PCR amplification of a repetitive sequence within the genome) has been described that could be used on a much ider-scale. The use of such an objective PCR technique could prove to be of considerable value to the poultry industry as it seeks to define and optimise increasingly more complex and complementary strategies for the control of coccidiosis in the next millennium. |
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