Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction (PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of this important disease of poultry.     

Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.     

DNA technological progress toward advanced diagnostic tools to support human hookworm control

Blood-feeding hookworms are parasitic nematodes of major human health importance. Currently, it is estimated that 740 million people are infected worldwide, and more than 80 million of them are severely affected clinically by hookworm disease. In spite of the health problems caused and the advances toward the development of vaccines against some hookworms, limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of hookworms is central to their effective control. While traditional diagnostic methods have considerable limitations, there has been some progress toward the development of molecular-diagnostic tools. The present article provides a brief background on hookworm disease of humans, reviews the main methods that have been used for diagnosis and describes progress in establishing polymerase chain reaction (PCR)-based methods for the specific diagnosis of hookworm infection and the genetic characterisation of the causative agents. This progress provides a foundation for the rapid development of practical, highly sensitive and specific diagnostic and analytical tools to be used in improved hookworm prevention and control programmes.     

Development of a genetically engineered vaccine against poultry coccidiosis

Coccidiosis is caused by infection with Eimeria spp. The disease is responsible for major economic loss to the poultry industry unless infections are controlled by anticoccidial drugs. John Ellis and Fiona Tomley discuss recent research on the characterization and cloning of antigens from Eimeria spp and advances towards the development of genetically engineered vaccines against poultry coccidiosis.     

Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock--bionomic and biotechnological implications

Parasitic nematodes of livestock have a major economic impact worldwide. In spite of the health problems caused by nematodes and advances toward the development of vaccines and new therapeutic agents against some of them, relatively limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of parasitic nematodes of livestock are central to their effective control, particularly given the current, serious problems with anthelmintic resistance in nematode populations. Traditional diagnostic techniques have considerable limitations, and there have been some advances toward the development of molecular-diagnostic tools. This article provides a brief account of the significance of parasitic nematodes (order Strongylida), reviews the techniques that have been evaluated or used for diagnosis and describes developments in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infection/s and the genetic characterisation of the causative agents. The advances made in recent years provide a solid foundation for the development of practical, highly sensitive and specific diagnostic tools for epidemiological investigations and for use in control programmes.     

Resistance to anticoccidial drugs in fowl

Resistance has been encountered wherever drugs have been used extensively for the control of parasitic infections. The poultry industry is dependent upon drugs for the control of coccidiosis, a major disease of chickens caused by protozoan parasites of the genus Eimeria. In modern poultry production, drugs are used prophylactically for the prevention of coccidiosis by including them in the diet. This has inevitably led to the development of resistance. We have been fortunate in that new drugs have become available to replace those to which resistance has developed, but this situation is unlikely to continue. The problem of drug resistance, discussed here by David Chapman, has provided impetus for the development of new approaches (such as vaccination) for the control of coccidiosis.     

Sustainable coccidiosis control in poultry production: the role of live vaccines

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.     

The effect of cyclosporin A on the development of Eimeria in non-specific hosts.

The role of T lymphocytes on the development of turkey coccidia in chickens was investigated to further document the involvement of immune mechanisms in the reactions of non-specific hosts to infections with species of Eimeria. All chickens treated with the T cell-specific immunosuppressive drug, cyclosporin A (CsA), 1, 2 or 3 days before or for 3 consecutive days immediately before inoculation with sporulated oocysts of Eimeria from the turkey, shed oocysts in their feces 7–12 days after inoculation. Those chickens treated with CsA after inoculation produced no oocysts although in one experiment a small number of oocysts were discharged by birds injected with CsA 1 day after inoculation. High serum levels of CsA were maintained in the CsA-treated chickens for at least 24 h post-inoculation with the oocysts. Immunosuppressive activity of the drug was determined by measuring lymphocyte blastogenesis in response to concanavalin A. These results demonstrate that the suppression of T lymphocyte activity in a non-specific host early during an infection with a heterologous species of Eimeria permits the complete intracellular development of the parasite. Our findings are suggestive of a role for T lymphocytes in preventing the development of Eimeria in non-specific hosts possibly via a lymphokine-mediated mechanism.     

A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry

A compartmentalised model is presented for the estimation of the monetary losses suffered by the world's poultry industry resulting from coccidiosis of chickens and costs of its control. The model is designed so that the major elements of loss may be separately quantified for any chicken-producing entity, e.g., a farm, a poultry company, a country, etc. Examples are presented and the sources, reliability and geographical relevance of the data used for each parameter are provided. Loss elements for specific geographical areas should be recalculated at appropriate intervals to take into account local and international fluctuations in costs of chicks feed, labour, financial inflation and world currency exchange rates. Equations are given for relationships among numbers of chickens, liveweights, weights of carcasses, feed consumptions, feed conversion ratio (FCR), prices of feeds, prices of anticoccidial therapeutic and prophylactic drugs, values of chickens, chicken rearing costs; and effects of coccidiosis on mortality, weight gain and FCR. Using these equations, it is theoretically possible for an international team of representatives, each using reliable local data, to calculate simultaneously each relevant loss element for their respective countries. Addition of these elements could give, for the first time, an accurate global estimate of the losses due to chicken coccidiosis. The total cost of coccidiosis in chickens in the United Kingdom in 1995 is estimated to have been at least GB pound silver 38 588 795, of which 98.1% involved broilers (80.6% due to effects on mortality, weight gain and feed conversion, and 17.5% due to the cost of chemoprophylaxis and therapy). The costs of poor performance due to coccidiosis and its chemical control totalled 4.54% of the gross revenue from UK sales of live broilers. This model includes a new method for comparing the profitabilities of different treatments in commercial trials. providing actual costs rather than the arbitrary numerical scores of other methods. Although originally designed for the study of coccidiosis, the model is equally applicable to any disease. It should be of value to agricultural economists, the animal feed and poultry industries, animal health companies, and to research scientists (particularly for preparing grant applications).     

益生菌调控肠道屏障功能预防家禽球虫病的研究进展

球虫病给养禽业带来巨大经济损失,人们对绿色健康食品的迫切需求使球虫病的防控面临新的挑战.伴随世界"禁抗"进程的不断推进,家禽养殖业亟需一种安全有效的新型抗球虫方法.益生菌可竞争性排斥病原菌定殖以防止球虫病继发感染,可刺激宿主抗菌肽、黏蛋白和紧密连接蛋白的分泌以抵御球虫入侵,还可激活免疫反应以增强机体抗球虫感染的能力.本...     

Live attenuated vaccines against avian coccidiosis: Success with precocious and egg-adapted lines of Eimeria

Avian coccidiosis, caused by parasites of the genus Eimeria, is a major disease of intensively reared poultry. Anticoccidial drugs, and vaccines based on live wild-type (virulent) parasites, have aided the management of coccidiosis, but the development of vaccines based on live attenuated parasites has been a significant advance. Here, Martin Shirley and Petr Bedrník explain the basis of these recent vaccines and discuss their use.     

鸡球虫入侵机理、免疫学性质及基因工程在疫苗研制上的应用

鸡球虫病是严重危害养禽业的一种寄生虫病。长期以来主要以化学药物进行防治。但由于球虫抗药性和药物残留等问题日益严重,使得新药开发和应用受到限制。在免疫学中强毒活苗应用广泛但生产费用昂贵,从而促使我们转向对重组亚单位疫苗的研究。不同种类的球虫有较为严格的寄生部位。且不同发育阶段的球虫其免疫原性和抗原构成也有很大差异。在真核寄生生物中,由于受到宿主免疫系统的限制,选用鸡球虫免疫保护性抗原做重组疫苗目前还没有很大突破,因此我们需要采用新方法发现新抗原,有效防治鸡球虫病。     

An assessment of opportunities to dissect host genetic variation in resistance to infectious diseases in livestock

This paper reviews the evidence for host genetic variation in resistance to infectious diseases for a wide variety of diseases of economic importance in poultry, cattle, pig, sheep and Atlantic salmon. Further, it develops a method of ranking each disease in terms of its overall impact, and combines this ranking with published evidence for host genetic variation and information on the current state of genomic tools in each host species. The outcome is an overall ranking of the amenability of each disease to genomic studies that dissect host genetic variation in resistance. Six disease-based assessment criteria were defined: industry concern, economic impact, public concern, threat to food safety or zoonotic potential, impact on animal welfare and threat to international trade barriers. For each category, a subjective score was assigned to each disease according to the relative strength of evidence, impact, concern or threat posed by that particular disease, and the scores were summed across categories. Evidence for host genetic variation in resistance was determined from available published data, including breed comparison, heritability studies, quantitative trait loci (QTL) studies, evidence of candidate genes with significant effects, data on pathogen sequence and on host gene expression analyses. In total, 16 poultry diseases, 13 cattle diseases, nine pig diseases, 11 sheep diseases and three Atlantic salmon diseases were assessed. The top-ranking diseases or pathogens, i.e. those most amenable to studies dissecting host genetic variation, were Salmonella in poultry, bovine mastitis, Marek's disease and coccidiosis, both in poultry. The top-ranking diseases or pathogens in pigs, sheep and Atlantic salmon were Escherichia coli, mastitis and infectious pancreatic necrosis, respectively. These rankings summarise the current state of knowledge for each disease and broadly, although not entirely, reflect current international research efforts. They will alter as more information becomes available and as genome tools become more sophisticated for each species. It is suggested that this approach could be used to rank diseases from other perspectives as well, e.g. in terms of disease control strategies.     

Defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite Eimeria tenella

The apicomplexan pathogen Eimeria causes coccidiosis, an intestinal disease of chickens, which has a major welfare and economic impact on the poultry industry. There is an urgent need to identify molecules that are rational targets for drug design and novel vaccines against coccidiosis. Apicomplexan secretory organelles, including micronemes and rhoptries, are essential for invasion of the host intestinal epithelium and establishment of parasitism. However, relatively little is known about the precise molecular function of these organelles, partly because few organelle proteins have been characterized. In this study, proteomics tools have been harnessed to define the protein repertoire of micronemes. Purified microneme proteins from Eimeria tenella sporozoites were excised from two-dimensional (2-D) gels and analyzed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and chemically assisted fragmentation (CAF)-MALDI with de novo sequencing. Peptide mass profiles were searched against the NCBI non-redundant (nr) database and against Eimeria-specific databases using the Mascot search algorithm, resulting in the identification of 37 of 96 spots excised from the 2-D gels. In addition, we have found CAF-MALDI to be a useful adjunct for identifying proteins, without the need for tandem MS. This global approach to protein characterization will be vital to gain greater understanding of the processes involved in apicomplexan host cell invasion.     

New Biology of Pheochromocytoma and Paraganglioma

Pheochromocytomas and paragangliomas continue to be defined by significant morbidity and mortality despite their several recent advances in diagnosis, localization, and management. These adverse outcomes are largely related to mass effect as well as catecholamine-induced hypertension, tachyarrhythmias and consequent target organ damage, acute coronary syndromes, and strokes (ischemic and hemorrhagic stroke). Thus, a proper understanding of the physiology and pathophysiology of these tumors and recent advances are essential to affording optimal care. These major developments largely include a redefinition of metastatic behavior, a novel clinical categorization of these tumors into 3 genetic clusters, and an enhanced understanding of catecholamine metabolism and consequent specific biochemical phenotypes. Current advances in imaging of these tumors are shifting the paradigm from poorly specific anatomical modalities to more precise characterization of these tumors using the advent and development of functional imaging modalities. Furthermore, recent advances have revealed new molecular events in these tumors that are linked to their genetic landscape and, therefore, provide new therapeutic platforms. A few of these prospective therapies translated into new clinical trials, especially for patients with metastatic or inoperable tumors. Finally, outcomes are ever-improving as patients are cared for at centers with cumulative experience and well-established multidisciplinary tumor boards. In parallel, these centers have supported national and international collaborative efforts and worldwide clinical trials. These concerted efforts have led to improved guidelines collaboratively developed by healthcare professionals with a growing expertise in these tumors and consequently improving detection, prevention, and identification of genetic susceptibility genes in these patients.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Modeling the Genetic Control of HIV-1 Dynamics After Highly Active Antiretroviral Therapy

The progression of HIV disease has been markedly slowed by the use of highly active antiretroviral therapy (HAART). However, substantial genetic variation was observed to occur among different people in the decay rate of viral loads caused by HAART. The characterization of specific genes involved in HIV dynamics is central to design personalized drugs for the prevention of this disease, but usually cannot be addressed by experimental methods alone rather than require the help of mathematical and statistical methods. A novel statistical model has been recently developed to detect genetic variants that are responsible for the shape of HAART-induced viral decay curves. This model was employed to an HIV/AIDS trial, which led to the identification of a major genetic determinant that triggers an effect on HIV dynamics. This detected major genetic determinant also affects several clinically important parameters, such as half-lives of infected cells and HIV eradication times.Key Words: Hardy-weinberg equilibrium, bi-exponential function, quantitative trait loci, HIV dynamics, functional mapping.