Genetic selection of Eimeria parasites in the chicken for improvement of poultry health: implications for drug resistance and live vaccine development.

Apicomplexan parasites of the genus Eimeria are widespread in poultry flocks and can cause the intestinal disease coccidiosis. Early studies, concerned with intraspecific variation in oocyst morphology, indicated that phenotypic changes may be induced by selection experiments conducted in vivo. Genetic selection driven by targeted selection for specific phenotypes has contributed to our understanding of the phenomenon of drug resistance and the development of live attenuated vaccines. Our present knowledge regarding genetics of Eimeria is largely based upon the utilization of such selected strains as genetic markers. Practical advantages of working with Eimeria spp. in the chicken are discussed. The selection of drug-resistant strains by serial propagation has provided useful information regarding the mechanisms of drug resistance and likely longevity of anticoccidial drugs when introduced in the field. Selection experiments to develop precocious strains of Eimeria and growth in chicken embryos have contributed to the development of safe and effective live attenuated vaccines for the control of coccidiosis. Establishment of protocols for genetic complementation by transient or stable transfection of Eimeria is now supporting direct manipulation of parasite genotypes, creating opportunities to expand the range and value of live parasite vaccines. Procedures for developing drug-resistant and precocious lines of Eimeria and/or genetic markers described here are likely to prove useful for researchers investigating the propensity for resistance development to novel compounds and the development of new attenuated vaccines. Such investigations can be helpful in providing a better understanding of biochemical and molecular aspects of the biology of these parasites.

Enhancing the value of meat inspection records for broiler health and welfare surveillance: longitudinal detection of relational patterns.

BACKGROUND: Abattoir data are under-used for surveillance. Nationwide surveillance could benefit from using data on meat inspection findings, but several limitations need to be overcome. At the producer level, interpretation of meat inspection findings is a notable opportunity for surveillance with relevance to animal health and welfare. In this study, we propose that discovery and monitoring of relational patterns between condemnation conditions co-present in broiler batches at meat inspection can provide valuable information for surveillance of farmed animal health and welfare. RESULTS: Great Britain (GB)-based integrator meat inspection records for 14,045 broiler batches slaughtered in nine, four monthly intervals were assessed for the presence of surveillance indicators relevant to broiler health and welfare. K-means and correlation-based hierarchical clustering, and association rules analyses were performed to identify relational patterns in the data. Incidence of condemnation showed seasonal and temporal variation, which was detected by association rules analysis. Syndrome-related and non-specific relational patterns were detected in some months of meat inspection records. A potentially syndromic cluster was identified in May 2016 consisting of infection-related conditions: pericarditis, perihepatitis, peritonitis, and abnormal colour. Non-specific trends were identified in some months as an unusual combination of condemnation reasons in broiler batches. CONCLUSIONS: We conclude that the detection of relational patterns in meat inspection records could provide producer-level surveillance indicators with relevance to broiler chicken health and welfare.

Molecular identification of Sarcocystis wobeseri-like parasites in a new intermediate host species, the white-tailed sea eagle (Haliaeetus albicilla).

A reintroduced white-tailed sea eagle (Haliaeetus albicilla) in moderate body condition was found dead and submitted for post-mortem examination. There were no signs of disease on gross pathological examination. Histopathological examination however revealed the presence of encysted protozoan parasites in pectoral and cardiac muscle sections. Polymerase chain reaction amplification of extracted genomic DNA and sequencing of four regions: the 18S rDNA, 28S rDNA, internal transcribed spacer (ITS) 1, and RNA polymerase B (rpoB) loci, confirmed the presence of a Sarcocystis species in pectoral and cardiac muscle which appeared phylogenetically similar to Sarcocystis wobeseri. This is the first report of S. wobeseri-like infection in a white-tailed sea eagle revealing a new intermediate host species for this parasite.

Identification of production chain risk factors for slaughterhouse condemnation of broiler chickens'.

Slaughterhouse condemnation of broiler chickens results from identification of polymorphic pathological conditions during meat inspection from arrival and on the slaughter line. While conditions that result in condemnation are multifactorial, identification of factors that are common for a number of categories could be valuable for developing strategies to reduce total condemnation. This study aimed to identify those condemnation categories that were most common in batches of broiler chickens and to determine and compare associated risk factors. In the first step, retrospective meat inspection records for 55,918 broiler batches from one large broiler integrator for 2015-2017 were used for association rules analysis. Results identified a network of nine associated condemnation categories: whole carcass condemnation for ascites, abnormal colour, perihepatitis, cellulitis, hard breast, tumours and dead on arrival, and liver only and heart only most often associated with hepatitis and pericarditis, respectively. Secondly, a longitudinal study collected data on 109 explanatory variables from broiler parental flocks to slaughterhouse characteristics between January 2015 and December 2017. Condemnation outcome data were obtained from meat inspection records for 539 broiler batches participating in the study. Parental flock-, rearing farm-, shed- and transport-level risk factors were assessed for each outcome using mixed-effects multivariable Poisson regression including shed and farm as random effects. A Poisson regression tree method was used as the first step to identify variables most relevant for analysis and comparison across the outcomes. No single production factor was associated with all nine of the condemnation outcomes investigated in this study, although some were shared across multiple outcomes: age of parental flock at time of lay, flock-level Campylobacter spp. frequency, broiler chick weight at seven days of age, weight at slaughter, type of broiler removal (i.e. thinning, final depopulation), catcher team, number of birds per transport crate, slaughterhouse shift number, and type of slaughterhouse line. Broiler chickens removed during final depopulation were at greatest risk of condemnation. Condemnation rates for cellulitis and tumours were found to be higher in broilers inspected by night shift at the slaughterhouse. Discovery of an apparent protective effect of a higher number of broilers per transport crate was unexpected. These findings provide information for the broiler industry on production chain factors that might be amenable to targeted intervention to improve future efforts for control of condemnation.

Surprisingly long body length of the lungworm Parafilaroides gymnurus from common seals of the Dutch North Sea.

Lungworms of the genera Parafilaroides and Otostrongylus are responsible for parasitic bronchopneumonia, the foremost disease of eastern Atlantic common seals (EACS, Phoca vitulina vitulina) in the Dutch North Sea. Recently, there have been increased reports of lungworm cases and observations of unusually long Parafilaroides sp. adults in this location. The initial aim of this study was to confirm the identity of the Parafilaroides species infecting this population. Parafilaroides are usually small and delicate, making them difficult to extract from host tissue, and there is often difficulty accessing fresh specimens for morphological study. The large size of the Dutch worms and the accessibility of specimens from numerous animals enabled the description and measurement of many intact specimens (N = 64) from multiple host animals (N = 20). Species identity was confirmed by targeted sequencing of ribosomal and mitochondrial DNA amplicons from a subset of worms. Worm morphology was consistent with descriptions for P. gymnurus, but the mature females were 1.9-fold and 3.4-fold longer than those recovered from French EACS (P ≤ 0.001) and Canadian western Atlantic common seals (Phoca vitulina concolor; P ≤ 0.0001). They were also significantly longer than mature female P. gymnurus described from other seal species, with the exception of those from harp seals of Les Escoumins, Quebec. We suggest that intraspecific genetic differences in P. gymnurus and the environment within the host could contribute to the variation reported here. This study is the first to describe P. gymnurus using morphological and molecular methods and should serve as a reference for identification of the species.

The transmission dynamics of Campylobacter jejuni among broilers in semi-commercial farms in Jordan.

Campylobacter is the leading cause of foodborne bacterial gastroenteritis in humans worldwide, often associated with the consumption of undercooked poultry. In Jordan, the majority of broiler chicken production occurs in semi-commercial farms, where poor housing conditions and low bio-security are likely to promote campylobacter colonisation. While several studies provided estimates of the key parameters describing the within-flock transmission dynamics of campylobacter in typical high-income countries settings, these data are not available for Jordan and Middle-East in general. A Bayesian model framework was applied to a longitudinal dataset on Campylobacter jejuni infection in a Jordan flock to quantify the transmission rate of C. jejuni in broilers within the farm, the day when the flock first became infected, and the within-flock prevalence (WFP) at clearance. Infection with C. jejuni is most likely to have occurred during the first 8 days of the production cycle, followed by a transmission rate value of 0.13 new infections caused by one infected bird/day (95% CI 0.11-0.17), and a WFP at clearance of 34% (95% CI 0.24-0.47). Our results differ from published studies conducted in intensive poultry production systems in high-income countries but are well aligned with the expectations obtained by means of structured questionnaires submitted to academics with expertise on campylobacter in Jordan. This study provides for the first time the most likely estimates and credible intervals of key epidemiological parameters driving the dynamics of C. jejuni infection in broiler production systems commonly found in Jordan and the Middle-East and could be used to inform Quantitative Microbial Risk Assessment models aimed to assess the risk of human exposure/infection to campylobacter through consumption of poultry meat.

The molecular characterisation of Cryptosporidium species in relinquished dogs in Great Britain: a novel zoonotic risk?

Surveillance was conducted to investigate the occurrence of protozoan parasites of the genus Cryptosporidium in dogs newly admitted to a dog rehoming charity in London, Great Britain. Voided faecal samples were collected from all new admissions between 2011 and 2012 during six separate 4-week sampling periods. Information on host signalment, including age, breed and reason for submission and faecal consistency, was collected. Polymerase Chain Reaction (PCR) targeting the 18S ribosomal RNA gene, confirmed by sequencing, was conducted on the faecal samples to detect Cryptosporidium genomic DNA and determine Cryptosporidium identity. In total, 677 dogs were included in the study. The prevalence of Cryptosporidium-positive faecal samples was 4.6% (31/676). There were positive samples in all of the six sampling periods. Cryptosporidium canis (n = 28), C. parvum (n = 2) and C. andersoni (n = 1) were identified. Sixty KDa glycoprotein (gp60) gene amplicon sequencing of the C. parvum samples identified genotypes IIaA17G1R1 and IIaA15G2R1 for the first time from a dog. There were no significant associations between signalment data and Cryptosporidium status. While this was a study of one rehoming shelter, the presence of the potentially zoonotic C. parvum and C. canis in dogs highlights a public health concern. Further research is needed to better understand the epidemiology and potential impacts of Cryptosporidium infection in dogs.

Knowledge gaps in host-parasite interaction preclude accurate assessment of meat-borne exposure to Toxoplasma gondii.

Toxoplasma gondii is recognized as a widely prevalent zoonotic parasite worldwide. Although several studies clearly identified meat products as an important source of T. gondii infections in humans, quantitative understanding of the risk posed to humans through the food chain is surprisingly scant. While probabilistic risk assessments for pathogens such as Campylobacter jejuni, Listeria monocytogenes or Escherichia coli have been well established, attempts to quantify the probability of human exposure to T. gondii through consumption of food products of animal origin are at early stages. The biological complexity of the life cycle of T. gondii and limited understanding of several fundamental aspects of the host/parasite interaction, require the adoption of numerous critical assumptions and significant simplifications. In this study, we present a hypothetical quantitative model for the assessment of human exposure to T. gondii through meat products. The model has been conceptualized to capture the dynamics leading to the presence of parasite in meat and, for illustrative purposes, used to estimate the probability of at least one viable cyst occurring in 100g of fresh pork meat in England. Available data, including the results of a serological survey in pigs raised in England were used as a starting point to implement a probabilistic model and assess the fate of the parasite along the food chain. Uncertainty distributions were included to describe and account for the lack of knowledge where necessary. To quantify the impact of the key model inputs, sensitivity and scenario analyses were performed. The overall probability of 100g of a hypothetical edible tissue containing at least 1 cyst was 5.54%. Sensitivity analysis indicated that the variables exerting the greater effect on the output mean were the number of cysts and number of bradyzoites per cyst. Under the best and the worst scenarios, the probability of a single portion of fresh pork meat containing at least 1 viable cyst resulted 1.14% and 9.97% indicating that the uncertainty and lack of data surrounding key input parameters of the model preclude accurate estimation of T. gondii exposure through consumption of meat products. The hypothetical model conceptualized here is coherent with current knowledge of the biology of the parasite. Simulation outputs clearly identify the key gaps in our knowledge of the host-parasite interaction that, when filled, will support quantitative assessments and much needed accurate estimates of the risk of human exposure.

Eimeria species occurrence varies between geographic regions and poultry production systems and may influence parasite genetic diversity.

Coccidiosis is one of the biggest challenges faced by the global poultry industry. Recent studies have highlighted the ubiquitous distribution of all Eimeria species which can cause this disease in chickens, but intriguingly revealed a regional divide in genetic diversity and population structure for at least one species, Eimeria tenella. The drivers associated with such distinct geographic variation are unclear, but may impact on the occurrence and extent of resistance to anticoccidial drugs and future subunit vaccines. India is one of the largest poultry producers in the world and includes a transition between E. tenella populations defined by high and low genetic diversity. The aim of this study was to identify risk factors associated with the prevalence of Eimeria species defined by high and low pathogenicity in northern and southern states of India, and seek to understand factors which vary between the regions as possible drivers for differential genetic variation. Faecal samples and data relating to farm characteristics and management were collected from 107 farms from northern India and 133 farms from southern India. Faecal samples were analysed using microscopy and PCR to identify Eimeria occurrence. Multiple correspondence analysis was applied to transform correlated putative risk factors into a smaller number of synthetic uncorrelated factors. Hierarchical cluster analysis was used to identify poultry farm typologies, revealing three distinct clusters in the studied regions. The association between clusters and presence of Eimeria species was assessed by logistic regression. The study found that large-scale broiler farms in the north were at greatest risk of harbouring any Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. Comparison revealed a more even distribution for E. tenella across production systems in south India, but with a lower overall occurrence. Such a polarised region- and system-specific distribution may contribute to the different levels of genetic diversity observed previously in India and may influence parasite population structure across much of Asia and Africa. The findings of the study can be used to prioritise target farms to launch and optimise appropriate anticoccidial strategies for long-term control.

Real-time PCR-based quantification of Eimeria genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition.

Eimeria species parasites can cause the disease coccidiosis in all livestock species, most notably poultry. Traditional diagnostics such as faecal microscopy have now been supplemented by molecular assays including genus-specific and species-specific quantitative polymerase chain reaction (qPCR), although DNA extracted from faecal samples is commonly affected by PCR inhibition. This was confirmed when genomic DNA extracted from chicken faeces inhibited the threshold cycle value of internal positive control (IPC) DNA amplification by 15.33%. Hence, the objective of the present study was to use IPC qPCR to determine PCR inhibition in a series of experimental samples and use the increase in IPC qPCR threshold cycle value as an individual (sample-specific) correction factor for an established 5S rDNA qPCR used to estimate total Eimeria genome numbers. IPC-corrected genome counts were correlated with conventional oocyst per gram counts and compared with non-corrected counts, revealing a 0.1769 increase in correlation coefficient to outweigh underestimation of oocyst counts. Though the sample size used in this study is small, this limitation would be offset by the sample-specific correction factor determined using the IPC along with each sample.

Two new Eimeria species parasitic in corncrakes (Crex crex) (Gruiformes: Rallidae) in the United Kingdom.

In this study we describe 2 new species of coccidia (Apicomplexa: Eimeriidae) parasites isolated from the feces of corncrake (Crex crex) (Gruiformes: Rallidae), bred in captivity in the U.K. Oocysts of Eimeria crecis n. sp. were approximately spherical and measured 15.3 µm (13-18) × 14.3 (12-16), providing an oocyst shape index of 1.1. A micropyle and oocyst residuum were absent, but a polar granule was present. Oocysts of Eimeria nenei n. sp. were ellipsoidal and measured 23.6 (21-26) × 18.1 (17-20), providing an oocyst shape index of 1.3. A micropyle and polar granule were present. Surveying free-living, wild adult corncrakes in Scotland (U.K.) demonstrated both parasite species to be widespread. These are the first species described to infect the corncrake, and they are distinct from those previously found to infect members of the closely related crane family (Gruiformes: Gruidae). Partial amplification and sequencing of the 18S rRNA gene and internal transcribed spacer 2 indicated a close relationship between the 2 proposed new species as a group distinct from the Eimeria species known to infect cranes. These newly identified parasite species have been associated with enteric disease in corncrakes being prepared for reproduction in captivity and reintroduction into England (U.K.).

Eimeria tenella glucose-6-phosphate isomerase: molecular characterization and assessment as a target for anti-coccidial control.

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.

Quantification of Eimeria acervulina in faeces of broilers: comparison of McMaster oocyst counts from 24h faecal collections and single droppings to real-time PCR from cloacal swabs.

Coccidiosis is an economically important disease in chickens, caused by infection with Eimeria species parasites. Diagnosis of coccidiosis is frequently based on oocyst enumeration in pooled faecal samples or litter. In studies on infection dynamics and for monitoring in the field, samples from individual chickens may be more appropriate as these support the determination of infection status of individual birds and more accurately reflect oocyst output at time of sampling. Faecal samples from individual birds can be collected, but the counting procedure limits the number of samples that can be processed and unequivocal microscopic differentiation between Eimeria species is very difficult. A test that overcomes these drawbacks would improve efficiency and quality of the diagnosis. The aim of this study was to compare two methods for Eimeria oocyst quantification in samples from individual birds. A real-time PCR that quantifies oocysts in cloacal swabs (qPCR) and oocyst counts in single droppings were compared to the standard procedure of oocyst counts in bulked 24h faeces. Faecal samples were collected daily from 30 broiler chickens, inoculated with different doses of Eimeria acervulina. The three techniques produced comparable oocyst counts for all inoculation doses. Single dropping counts are applicable for small sample sizes and when a single Eimeria species is used. For larger sample sizes qPCR is preferable as it can be carried out on samples that have been frozen for storage. Furthermore, qPCR can identify and quantify different Eimeria species, which makes it a valuable diagnostic tool for field or experimental work.

Genetic identification of antigens protective against coccidia.

The Eimeria species, causative agents of the disease coccidiosis, are genetically complex protozoan parasites endemic in livestock. Drug resistance remains commonplace among the Eimeria, and alternatives to chemotherapeutic control are being sought. Vaccines based upon live formulations of parasites are effective, but production costs are high, stimulating demand for a recombinant subunit vaccine. The identity of antigens suitable for inclusion in such vaccines remains elusive. Selection of immunoprotective antigens of the Eimeria species as vaccine candidates based upon recognition by the host immune system has been unsuccessful, obscured by the considerable number of molecules that are immunogenic but not immunoprotective. This is a common problem which characterizes work with most eukaryotic parasites. The identification of a selective criterion to directly access genetic loci that encode immunoprotective antigens of Eimeria maxima using a mapping strategy based upon parasite genetics, immune selection and DNA fingerprinting promises to revolutionize the process of antigen discovery. Linkage analyses of DNA markers amplified from populations of recombinant parasites defined by an ability to escape parent-specific deleterious selection by strain-specific immunity and chemotherapy has revealed four discrete regions within the E. maxima genome linked to escape from a protective immune response. These regions now form the basis of detailed study to identify antigens as candidates for inclusion in future vaccination strategies.

Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions.

AIM: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae. METHODS AND RESULTS: Through the use of a validated in vitro simulation of the porcine ileum, the transmission of antibiotic resistance was detected between commensal Escherichia coli, E. coli O157 and Salmonella spp. Countable transconjugant populations arose readily and, in one example, proved capable of indefinite persistence. CONCLUSIONS: Genetic material conferring antibiotic resistance is readily transmissible between members of the Enterobacteriaceae under ileal conditions. Recipient phenotype influences the persistence of multi-resistant transconjugants. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that the conjugal transmission of antibiotic resistance is commonplace under ileal conditions impacts primarily on the risk of food contamination by multi-resistant bacteria. The establishment of a multi-resistant transconjugant population as a dominant member of the microflora maintains a genetic reservoir of antimicrobial resistance.

Amplified fragment length polymorphism analyses of Eimeria spp.: an improved process for genetic studies of recombinant parasites.

Application of the amplified fragment length polymorphism (AFLP) technique to genetic mapping studies requires high quality DNA as a template. In the case of Eimeria spp., this has previously been in the form of chromosomal DNA obtained from purified sporozoites recovered from large numbers of oocysts (generally up to 2 x 10(8)). In order for the AFLP technique to be more easily applied to studies on the genetics of Eimeria maxima, for which only smaller numbers of oocysts are available, a simplified, more efficient method for the recovery of genomic DNA from small numbers of oocysts was developed. Our new method should also be useful for genetic analyses of other coccidial parasites and for the recovery of AFLP-quality DNA from other pathogens.

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations.

AIMS: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli. METHODS AND RESULTS: Through the adoption of a novel combination of multiple breakpoint selection, phenotypic characterization and the application of a polymerase chain reaction based gene identification system it proved possible to monitor the influence of antibiotic exposure on resistance gene possession. Using tetracycline as a case study a clear hierarchy was revealed between tet genes, strongly influenced by host antimicrobial exposure history. CONCLUSIONS: The antimicrobial exposure regime under which an animal is produced affects both the identity and magnitude of resistance gene possession of a selected bacterial population within its enteric microflora. Among the ramifications associated with such resistance gene selection is the degree of resistance conferred and the carriage of linked resistance determinants. This selection is applied by exposure to antibiotic concentrations well below recognized minimum inhibitory tetracycline concentration breakpoints widely adopted to characterize bacterial 'susceptibility'. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the ability of minimal antibiotic exposure to select for the continued persistence of resistance genes within the enteric microflora. It is clearly demonstrated that different antimicrobial regimes select for different resistance genes, the implications of which are discussed.

Adrenocortical neoplasm disguised as a post-traumatic perinephric hematoma.

Trauma and cancer are entities that are encountered frequently by surgeons and occasionally may exist simultaneously in certain patients. Illustrated here is the case of a man with a perinephric mass, discovered during evaluation for blunt abdominal trauma, which was originally thought to be a perinephric hematoma and later proved to be an adrenocortical carcinoma.

Improved detection of cardiac contusion with cardiac troponin I.

Detecting cardiac injury in patients with chest trauma is difficult because the level of the MB isoenzyme of creatine kinase (MBCK) can be elevated from skeletal muscle injury alone. However, the level of cardiac troponin I (cTnl) is not elevated by skeletal muscle injury. To determine whether its measurement would improve the ability to detect cardiac injury in patients with blunt chest trauma, 44 patients were studied. Serial echocardiograms and serial blood samples were obtained. Six patients had evidence of cardiac injury by echocardiography; all had elevations of MBCK and cTnl. One patient had elevations of both MBCK and cTnl with only a pericardial effusion. Twenty-six of the 37 patients without contusion had elevations of MBCK; none had elevations of cTnl. The ratio of MBCK to total creatine kinase improved specificity at the expense of sensitivity. Measurement of cTnl accurately detects cardiac injury in patients with blunt chest trauma and should facilitate the diagnosis and management of such patients.