Differential expression of microRNAs in the caecal content and faeces of broiler chickens experimentally infected with Eimeria.

Coccidiosis caused by Eimeria spp. incurs significant morbidity and mortality in chickens, and is thus of great economic importance. Post-mortem intestinal lesion scoring remains one of the most common means of diagnosis; therefore alternative, non-invasive methods of diagnosis and monitoring would be highly desirable. Micro-RNAs (miRNAs) have been shown to be stable in faeces of human and animal species with expression altered in gastrointestinal disease. We hypothesized that miRNA is stable in caecal content of chickens, and that differential miRNA expression patterns would be seen in Eimeria-infected versus uninfected individuals. Initially, RNA was extracted from Eimeria tenella-infected (n = 3; 7 days post infection) and uninfected (n = 3) chicken caecal content to demonstrate miRNA stability. Subsequently, next-generation miRNA sequencing was performed on caecal content from E. tenella-infected chickens with high (lesion score (LS) 3-4; n = 3) or low (LS1; n = 3) levels of pathology, and uninfected controls (n = 3). Comparative analysis identified 19 miRNAs that exhibited significantly altered expression in the caecal content of E. tenella, infected chickens versus uninfected chickens (t-test, False Discovery Rate (FDR) < 0.05). Eight of these miRNAs showed significant up-regulation in infection (fold change of 9.8-105, FDR <0.05). Quantitative PCR was performed using separate biological replicates to confirm differential regulation in eight of these miRNA candidates in caecal and faecal content. This work has identified a panel of miRNA candidates which may be appropriate for use as non-invasive faecal markers of active caecal coccidiosis without the need for culling. RESEARCH HIGHLIGHTSE. tenella induced differential miRNA expression in caecal content and faeces.

Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study.

BACKGROUND: Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study. RESULTS: Of the 47 dogs with ClinL, anti-E. canis/ Ehrlichia ewingii antibodies were detected in 17 (36.2%), anti-Anaplasma phagocytophilum/Anaplasma platys antibodies in 5 (10.6%) and antigen for Dirofilaria immitis in 2 (4.3%). Of the 87 control dogs, anti-E. canis/E. ewingii antibodies were detected in 14 (16.1%) and anti-A. phagocytophilum/A. platys antibodies in 2 (2.3%). No anti-Borrelia burgdorferi antibody tests were positive. No statistical differences between the ClinL dogs and control dogs regarding lifestyle or use of ectoparasitic prevention, were identified. The ClinL was significantly associated with anti-E. canis/E. ewingii antibodies (odds ratio = 2.9, 95% confidence interval: 1.3-6.7, P = 0.010) compared to controls by both multivariable logistic regression and structural equation modelling. CONCLUSIONS: It was demonstrated that an increased risk for E. canis/E. ewingii seropositivity is present in dogs with ClinL compared to clinically healthy control dogs, despite similar ectoparasitic prevention use and lifestyle. Based on these findings it is suggested that dogs with ClinL should not only be tested for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii.

An Experimental Dermal Oedema Model for Apx Toxins of Actinobacillus pleuropneumoniae.

In-vivo models of Actinobacillus pleuropneumoniae (App) infection in pigs are required for the development of vaccines and investigations of pathogenicity. Existing models cause severe respiratory disease with pulmonary oedema, dyspnoea and severe thoracic pain, and careful monitoring and early intervention with euthanasia is, therefore, needed to avoid unnecessary suffering in experimental animals. As a potential replacement for the existing respiratory infection model, an in-vivo protocol was evaluated using intradermal or subcutaneous injection of different App strains and Apx toxins into the abdominal skin of pigs. High concentrations of serovar 1 and serovar 10 App induced diffuse visible dermal oedema and inflammation. Injection of Apx toxins alone did not adequately produce macroscopic lesions, although an influx of inflammatory cells was seen on histopathology. ApxI-producing strains of App induced more inflammation than ApxII- and ApxIII-producing strains. Induction of skin lesions by injection of App or Apx toxins was not sufficiently repeatable or discrete for a robust experimental model that could be used for assessment of novel interventions.

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens.

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).

Impact of Eimeria tenella Oocyst Dose on Parasite Replication, Lesion Score and Cytokine Transcription in the Caeca in Three Breeds of Commercial Layer Chickens.

Eimeria species parasites infect the gastrointestinal tract of chickens, causing disease and impacting on production. The poultry industry relies on anticoccidial drugs and live vaccines to control Eimeria and there is a need for novel, scalable alternatives. Understanding the outcomes of experimental infection in commercial chickens is valuable for assessment of novel interventions. We examined the impact of different infectious doses of Eimeria tenella (one low dose, three high doses) in three commercial layer chicken lines, evaluating lesion score, parasite replication and cytokine response in the caeca. Groups of eight to ten chickens were housed together and infected with 250, 4,000, 8,000 or 12,000 sporulated oocysts at 21 days of age. Five days post-infection caeca were assessed for lesions and to quantify parasite replication by qPCR and cytokine transcription by RT-qPCR. Comparison of the three high doses revealed no significant variation between them in observed lesions or parasite replication with all being significantly higher than the low dose infection. Transcription of IFN-γ and IL-10 increased in all infected chickens relative to unchallenged controls, with no significant differences associated with dose magnitude (p > 0.05). No significant differences were detected in lesion score, parasite replication or caecal cytokine expression between the three lines of chickens. We therefore propose 4,000 E. tenella oocysts is a sufficient dose to reliably induce lesions in commercial layer chickens, and that estimates of parasite replication can be derived by qPCR from these same birds. However, more accurate quantification of Eimeria replication requires a separate low dose challenge group. Optimisation of challenge dose in an appropriate chicken line is essential to maximize the value of in vivo efficacy studies. For coccidiosis, this approach can reduce the numbers of chickens required for statistically significant studies and reduce experimental severity.

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies.

Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. Live coccidiosis vaccines for chickens have been available for almost 70 years, but the requirement to formulate blends of oocysts from multiple Eimeria species makes vaccine production costly and logistically demanding. A multivalent vaccine that does not require chickens for its production and can induce protection against multiple Eimeria species is highly desirable. However, despite the identification and testing of many vaccine candidate antigens, no recombinant coccidiosis vaccine has been developed commercially. Currently, assessment of vaccine efficacy against Eimeria, and the disease coccidiosis, can be done only through in vivo vaccination and challenge experiments but the design of such studies has been highly variable. Lack of a "standard" protocol for assessing vaccine efficacy makes comparative evaluations very difficult, complicating vaccine development, and validation. The formulation and schedule of vaccination, the breed of chicken and choice of husbandry system, the species, strain, magnitude, and timing of delivery of the parasite challenge, and the parameters used to assess vaccine efficacy all influence the outcomes of experimental trials. In natural Eimeria infections, the induction of strong cell mediated immune responses are central to the development of protective immunity against coccidiosis. Antibodies are generally regarded to be of lesser importance. Unfortunately, there are no specific immunological assays that can accurately predict how well a vaccine will protect against coccidiosis (i.e., no "correlates of protection"). Thus, experimental vaccine studies rely on assessing a variety of post-challenge parameters, including assessment of pathognomonic lesions, measurements of parasite replication such as oocyst output or quantification of Eimeria genomes, and/or measurements of productivity such as body weight gain and feed conversion rates. Understanding immune responses to primary and secondary infection can inform on the most appropriate immunological assays. The discovery of new antigens for different Eimeria species and the development of new methods of vaccine antigen delivery necessitates a more considered approach to assessment of novel vaccines with robust, repeatable study design. Careful consideration of performance and welfare factors that are genuinely relevant to chicken producers and vaccine manufacturers is essential.

Vaccination with transgenic Eimeria tenella expressing Eimeria maxima AMA1 and IMP1 confers partial protection against high-level E. maxima challenge in a broiler model of coccidiosis.

BACKGROUND: Poultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters. METHODS: Populations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts). RESULTS: Vaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites. CONCLUSIONS: This study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.

An investigation of polymorphisms in innate and adaptive immune response genes in canine leishmaniosis.

The outcome of infection with Leishmania infantum in dogs is variable, which is thought to be due to the nature of the immune response mounted by the host. As a consequence, the clinical signs and severity of canine leishmaniosis vary between individual dogs. Host immunogenetic factors might play an important role in determining the outcome of infection. The aim of this study was to examine polymorphisms in innate and adaptive immune response genes, to determine whether any of these were associated with susceptibility or resistance to L. infantum infection. Genomic DNA was obtained from two groups: pet dogs in endemic regions of Europe and a group of Beagles exposed to sand fly infection as part of a vaccine study. Genotyping was performed using a SNP (single nucleotide polymorphism) array for selected immune response genes. The first part of the study compared 62 clinical cases with 101 clinically unaffected dogs that were seronegative for Leishmania antibodies. One SNP in the CIITA gene demonstrated a significantly higher minor allele frequency in the case group, compared with the control group at the individual SNP level after permutation, but was not significant after correction for multiple testing. The second part of the study examined 48 Beagle dogs exposed to L. infantum over two transmission seasons. Twenty-seven dogs with a resistant phenotype (no evidence of clinical disease, seronegative at the end of the study period, negative on lymph node culture and only transiently PCR positive in bone marrow) were compared with 21 dogs demonstrating a susceptible phenotype (clinical disease, seropositive, positive lymph node culture and consistently PCR positive in bone marrow). Three SNPs in TLR3, two SNPs in PTPN22 and one SNP in TLR4 and IL1A were associated with the susceptible phenotype in the Beagle group at the individual SNP level after permutation analysis, but were not significant after correction for multiple testing. Further validation of these SNPs is required in a larger cohort of dogs, ideally with extreme phenotypes to confirm an association with the outcome of L. infantum infection.

Polymorphisms in the canine IL7R 3'UTR are associated with thymic output in Labrador retriever dogs and influence post-transcriptional regulation by microRNA 185.

Interleukin-7 (IL-7) and its receptor (IL-7R) are essential for T cell development in the thymus, and changes in the IL-7/IL-7R pathway have been implicated in age-associated thymic involution which results in a reduction of naïve T cell output. The aim of this study was to investigate the relationship between IL7 and IL7R genetic variation and thymic output in dogs. No single nucleotide polymorphisms (SNPs) were identified in the canine IL7 gene, but a number were present in the canine IL7R gene. Polymorphisms in the IL7R exon 8 and 3'UTR were found to be associated with signal joint T cell receptor excision circle (sj-TREC) values (a biomarker of thymic output) in young and geriatric Labrador retrievers. Additionally, one of the SNPs in the IL7R 3'UTR (SNP 14 c.1371 + 446 A > C) was found to cause a change in the seed-binding site for microRNA 185 which, a luciferase reporter assay demonstrated, caused changes in post-transcriptional regulation, and therefore might be capable of influencing IL-7R expression. The research findings suggest a genetic link between IL7R genotype and thymic output in dogs, which might impact on immune function as these animals age and provide further evidence of the involvement of IL-7/IL-7R pathway in age-associated thymic involution.

Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study.

BACKGROUND: In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. METHODS: We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls. RESULTS: From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or "Candidatus Mycoplasma haematoparvum" DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5-106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis. CONCLUSIONS: Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR.

Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections.

Canine tick-borne pathogens such as Ehrlichia canis and Hepatozoon canis are widespread in the Mediterranean basin but have never been reported or investigated in Cyprus. We describe herein the presence of canine tick-borne pathogens in three dogs with clinical signs compatible with vector-borne diseases from Paphos area of Cyprus. Molecular and phylogenetic analysis revealed the presence of E. canis, Anaplasma platys, H. canis, Babesia vogeli and Mycoplasma haemocanis in Cyprus. One dog co-infected with E. canis, H. canis, B. vogeli and M. haemocanis is, to the best of our knowledge, the first report of this multiple co-infection in dogs. The tick-borne pathogens reported in the current study should be considered in the differential diagnoses in dogs exposed to ticks in Cyprus.

Single nucleotide polymorphisms in major histocompatibility class II haplotypes are associated with potential resistance to inflammatory bowel disease in German shepherd dogs.

German shepherd dogs (GSD) in the UK are at increased risk of developing the Inflammatory Bowel Disaese (IBD). IBD is believed to be a multifactorial immune mediated disease affecting genetically predisposed dogs. The aim of the current study was to investigate whether susceptibility to IBD in GSD is associated with the major histocompatibility complex (MHC) class II locus (Dog Leukocyte Antigen, DLA). Sequence-based genotyping of the three polymorphic DLA genes DLA-DRB1, -DQA1 and -DQB1 was performed in 56 GSDs affected by IBD and in 50 breed-matched controls without any history of gastrointestinal signs. The haplotype DLA-DRB1*015:02-DQA1*006:01-DQB1*023:01 was found to be present only in the control population and was associated with a reduced risk of IBD (P<0.001). In contrast, the haplotype DLA-DRB1*015:01-DQA1*006:01-DQB1*003:01 was associated with IBD (Odds ratio [OR]=1.93, confidence interval [CI]=1.02-3.67, P=0.05). This study has identified an association between DLA-type and canine IBD, supporting the immunogenetic aetiology and immunopathogenesis of this disease.

Analysis of DLA-DQB1 and polymorphisms in CTLA4 in Cocker spaniels affected with immune-mediated haemolytic anaemia.

BACKGROUND: Cocker spaniels are predisposed to immune-mediated haemolytic anaemia (IMHA), suggesting that genetic factors influence disease susceptibility. Dog leukocyte antigen (DLA) class II genes encode major histocompatibility complex (MHC) molecules that are involved in antigen presentation to CD4(+) T cells. Several DLA haplotypes have been associated with autoimmune disease, including IMHA, in dogs, and breed specific differences have been identified. Cytotoxic T lymphocyte antigen 4 (CTLA4) is a critical molecule involved in the regulation of T-cell responses. Single nucleotide polymorphisms (SNPs) in the CTLA4 promoter have been shown to be associated with several autoimmune diseases in humans and more recently with diabetes mellitus and hypoadrenocorticism in dogs. The aim of the present study was to investigate whether DLA-DQB1 alleles or CTLA4 promoter variability are associated with risk of IMHA in Cocker spaniels. RESULTS: There were a restricted number of DLA-DQB1 alleles identified, with a high prevalence of DLA-DQB1*007:01 in both groups. A high prevalence of DLA-DQB1 homozygosity was identified, although there was no significant difference between IMHA cases and controls. CTLA4 promoter haplotype diversity was limited in Cocker spaniels, with all dogs expressing at least one copy of haplotype 8. There was no significant difference comparing haplotypes in the IMHA affected group versus control group (p = 0.23). Homozygosity for haplotype 8 was common in Cocker spaniels with IMHA (27/29; 93 %) and in controls (52/63; 83 %), with no statistically significant difference in prevalence between the two groups (p = 0.22). CONCLUSIONS: DLA-DQB1 allele and CTLA4 promoter haplotype were not found to be significantly associated with IMHA in Cocker spaniels. Homozygosity for DLA-DQB1*007:01 and the presence of CTLA4 haplotype 8 in Cocker spaniels might increase overall susceptibility to IMHA in this breed, with other genetic and environmental factors involved in disease expression and progression.

Restricted dog leucocyte antigen (DLA) class II haplotypes and genotypes in Beagles.

Beagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restriction within this breed and the impact this might have on vaccine responses are rarely considered. This study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were determined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles had significantly different DLA haplotype frequencies in comparison with pet Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in selective antigen presentation and vaccine responses that are not necessarily representative of those seen in other dog breeds.