The effect of combining subtherapeutic concentrations of different ionophorous antibiotics on anticoccidial action in chickens

The mode of action of polyether, ionophorous drugs against Eimeria infections in chickens was studied by feeding subeffective doses of drugs separately and in combination, in comparison with the full dose of each drug. Thus, monensin, lasalocid and salinomycin (50, 35 and 30 ppm, respectively) were compared with a full dose of the same drugs. The combinations of ionophorous antibiotics were as effective in controlling Eimeria tenella and E. acervulina infections as the single antibiotic mixed in the feed at recommended prophylactic concentrations. When intracellular sporozoites of E. tenella were treated with the drugs in vitro, the effects on morphology were similar, regardless of which drug was used.The additive effects of these drugs in the chicken, and their similar effects on intracellular sporozoites suggest that they have a similar mode of action on Eimeria sporozoites, despite differences in ion selectivity.     

Resistance to anticoccidial drugs of Dutch avian Eimeria spp. field isolates originating from 1996, 1999 and 2001

Fifteen Eimeria spp. field isolates sampled on Dutch broiler farms were subjected to an Anticoccidial Sensitivity Test (AST) in a battery cage study. Four isolates dated from 1996, another four from 1999 and the last seven isolates from 2001. The selected anticoccidial drugs were monensin, narasin, salinomycin, lasalocid, nicarbazin, diclazuril, halofuginone, maduramicin and meticlorpindol/methylbenzoquate. Maduramicin and halofuginone were not included in the ASTs of 1999 and 2001, while meticlorpindol/methylbenzoquate was not tested in 1996 and 1999. Eimeria acervulina present in each of the four 1996 field isolates showed resistance for almost all products tested except maduramicin (1/4) and salinomycin (1/4), which appeared to be reduced sensitive. In 1999 the same species presented a similar resistance pattern for most products, although reduced sensitivity occurred for salinomycin (1/4), and sensitivity was found for diclazuril (2/4), monensin (1/4) and narasin (1/4). In the year 2001 increased sensitivity to various products was found. Higher sensitivity was found for meticlorpindol/methylbenzoquate (7/7) and salinomycin and narasin (both 4/7), followed by nicarbazin (3/7) and monensin (2/7). Reduced sensitivity was found for monensin (3/7), lasalocid (2/7), salinomycin and narasin (1/7). E. maxima was only found in one field isolate per year. The E. maxima from 1996 was resistant to all products except narasin (sensitive) and halofuginone (reduced sensitive). In 1999 this species was reduced sensitive to narasin and lasalocid, showing resistance for the other products. The strain originating from the 2001 isolate was reduced sensitive to most products except monensin and narasin (resistant). Full sensitivity was found for meticlorpindol/methylbenzoquate. E. tenella was present in one isolate of 1996, two of 1999 and four isolates of 2001. The AST of 1996 showed reduced sensitivity for nicarbazin, and sensitive to narasin, maduramicin and halofuginone. All other products showed resistance. In 1999 both strains showed resistance to all products tested. For the year 2001 full sensitivity was found to meticlorpindol/methylbenzoquate. Sensitivity was also found for salinomycin (1/4), nicarbazin (2/4), diclazuril (2/4) and lasalocid (2/4), monensin (1/4) and narasin (1/4). Reduced sensitivity was found for nicarbazin (1/4), lasalocid (1/4) and narasin (1/4). The different resistance patterns of Dutch coccidiosis isolates and resistance of coccidia in general is discussed.     

Cross-reactivity of anti-Eimeria tenella antibody fragments on merozoites and sporozoites of different chicken Eimeria species

Eimeria tenella-specific antibodies were examined for the cross-reactivity on the sporozoites and merozoites of E. tenella, Eimeria maxima, Eimeria acervulina and Eimeria brunetti in an indirect fluorescence antibody test. Two of nine antibodies showed cross-reactivity with sporozoites of E. maxima, E. acervulina and E. brunetti; however, the localization of specific fluorescence differed between species. No antibody binding was observed on merozoites. The suitability of these antibodies to alter the infectivity of Eimeria sporozoites and/or merozoites must be verified in cell culture models and in vivo experimental infections.     

Species and strain differentiation ofEimeria spp. of the domestic fowl using DNA polymorphisms amplified by arbitrary primers

Eimeria spp. from the domestic fowl were examined for genetic relatedness by the random amplified polymorphic DNA (RAPD) assay. Nine different oligonucleotide decamers with arbitrary DNA sequences were tested as primers to amplify DNA from sixEimeria species infecting chickens. Two strains each ofE. acervulina andE. tenella were used. Depending on the species/strain-primer combination, between 1 and 12 DNA segments ranging in size from 0.16 to 4.95 kb were amplified. The two strains ofE. acervulina showed minor and major differences in their amplified DNA patterns, giving a similarity coefficient of 61%. The two strains ofE. tenella seemed to be more closely related, yielding a similarity coefficient of 98%. The differences observed between species were greater than those found between strains with every primer used, indicating that the RADP assay could be a useful tool for the study of relationships among these coccidia. The results obtained in this study also indicate the presence of unique, species-specific, amplified DNA segments that could be exploited to identifyEimeria species of the chicken.     

Evaluation of vaccine delivery systems for inducing long-lived antibody responses to Dermanyssus gallinae antigen in laying hens

ABSTRACT

Dermanyssus gallinae, the poultry red mite, is a global threat to the commercial egg-laying industry. Control of D. gallinae is difficult, with only a limited number of effective pesticides and non-chemical treatments available. Here, we characterize the candidate vaccine antigen D. gallinae cathepsin D-1 (Dg-CatD-1) and demonstrate that purified refolded recombinant Dg-Cat-D1 (rDg-CatD-1) is an active aspartyl proteinase which digests haemoglobin with a pH optimum of pH 4. Soluble protein extracts from D. gallinae also have haemoglobinase activity, with a pH optimum comparable to the recombinant protein, and both proteinase activities were inhibited by the aspartyl proteinase inhibitor Pepstatin A. Enzyme activity and the ubiquitous localization of Dg-CatD-1 protein in sections of adult female mites is consistent with Dg-CatD-1 being a lysosomal proteinase. Using Dg-CatD-1 as a model vaccine antigen, we compared vaccine delivery methods in laying hens via vaccination with: (i) purified rDg-CatD-1 with Montanide? ISA 71 VG adjuvant; (ii) recombinant DNA vaccines for expression of rDg-CatD-1 and (iii) transgenic coccidial parasite Eimeria tenella expressing rDg-CatD-1. In two independent trials, only birds vaccinated with rDg-CatD-1 with Montanide? ISA 71 VG produced a strong and long-lasting serum anti-rDg-Cat-D1 IgY response, which was significantly higher than that in control birds vaccinated with adjuvant only. Furthermore, we showed that egg-laying rates of D. gallinae mites fed on birds vaccinated with rDg-CatD-1 in Montanide? ISA 71 VG was reduced significantly compared with mites fed on unvaccinated birds.

RESEARCH HIGHLIGHTS

• Dermanyssus gallinae cathepsin D-1 (Dg-CatD-1) digests haemoglobin

• Vaccination of hens with rDg-CatD-1 in Montanide? ISA 71 VG results in long-lasting IgY levels

• Serum anti-rDg-CatD-1 antibodies reduce egg laying in D. gallinae after a single blood meal

     

Eimeria spp. of the domestic fowl: Analysis of genetic variability between species and strains using DNA polymorphisms amplified by arbitrary primers and denaturing gradient-gel electrophoresis

The genetic relatedness of 5Eimeria spp. of the domestic fowl, including 11 strains ofE. acervulina, 2 strains ofE. tenella and 1 precocious line ofE. acervulina, was assayed by means of random amplified polymorphic DNA (RAPD) and denaturing gradient-gel electrophoresis (DGGE). Seven different oligonucleotides were used to generate similarity coefficients for the species and strains ofEimeria infecting chickens. Between 1 and 13 DNA segments, depending on the species/strain-primer combination, were amplified with the various primers. Amplification products ranged in size from 0.16 to 3.8 kb.E. acervulina strains demonstrated two to four major common bands unique to the species. These strains also exhibited major and minor differences in their DNA patterns. Band-match analyses from both polyacrylamide and denaturing gradient gels were used to calculate similarity coefficients for theEimeria spp. and strains tested. Species differences, readily detected upon examination of DNA banding patterns, gave similarity coefficients of 4%–38% and 3%–18% when analyzed by polyacrylamide-and denaturing gradient-gel electrophoresis, respectively. A similar analysis ofE. acervulina strains yielded similarity coefficients of 55%–95% and 51%–85%, respectively. The differences observed between both species and strains were greater when the RAPD-assay products were analyzed via DGGE, indicating that a combination of these two techniques may provide a more stringent analysis of the genetic relatedness of these coccidia.     

Highly virulent Marek’s disease virus strains affect T lymphocyte function and viability of splenocytes in commercial meat-type chickens

ABSTRACT

In previous studies, we have demonstrated that very virulent plus Marek’s disease viruses (vv+MDV) are highly immunosuppressive in commercial meat-type chickens. The specific objectives of this work were to evaluate if vv+MDV immunosuppression (MDV-IS) is induced by reduction of lymphocyte responsiveness and/or viability. Three experiments were conducted to (i) compare vv+MDV 686 with a partially attenuated 686-BAC; (ii) compare vv+MDV strains (648A and 686) with vMDV (GA) and vvMDV (Md5); and (iii) compare chickens vaccinated with Md5-BACΔMEQ and with CVI988?+?HVT. In each experiment, spleens were collected at 28–30 days post infection and lymphocytes were isolated and investigated in three ways: their proliferative response to Concanavalin A (ConA) was analysed by MTT proliferation assay; cell death, and expression of CD45 and MHC-I was studied by flow cytometry; and MHC-IA and β-2 microglobulin (B2M) expression was evaluated by real time RT-PCR. Splenocytes of chickens inoculated with vv+MDV were severely impaired to proliferate when exposed to ConA. Furthermore, vv+MDV induced severe splenocyte death that did not occur after infection with v or vvMDV strains. Vaccination with CVI988?+?HVT, and at less level with Md5-BACΔMEQ reduced these negative effects. This is in contrast to our previous results in which Md5-BACΔMEQ but not CVI988?+?HVT protected against MDV-IS suggesting that although cell death and decrease lymphocyte function seem to be related to MDV virulence and certainly will be associated with immunosuppression, they might not fully explain the previously reported MDV-IS.

RESEARCH HIGHLIGHTS

• vv+MDV induces extensive death in splenocytes in meat-type chickens 28–30?dpi.

• vv+MDV impairs lymphocyte function in meat-type chickens 28–30?dpi.

• Vaccination protects against splenocyte death and reduced lymphocyte function.

• Cell lysis and reduced lymphocyte function do not fully explain MDV-IS.

     

Isoenzymes of Eimeria from the domestic fowl: Electrophoretic variants among species, strains and clones

Electrophoretic variation of enzymes in five Eimeria spp. of the domestic fowl, including nine strains, ten single-sporocyst clones and two single-sporozoite clones of E. acervulina, three strains each of E. maxima and E. tenella, two strains of E. praecox and one strain of E. necatrix, were assayed using cellulose acetate electrophoresis. Ten enzymes [aldehyde oxidase (AO), alkaline phosphatase (ALP), amylase (AMY), fumarate hydratase (FUM), glucose-6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase (GPI), glutamate-oxaloacetate transferase (GOT), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) and phosphoglucomutase (PGM)] were analyzed for their ability to distinguish between these species and strains. Enzymatic activity of G6PDH, GPI, IDH, MDH and PGM was detected in all the Eimeria spp. examined. Strains within each species were characterized by the same electrophoretic variant of G6PDH. Electrophoretic variants of GPI and PGM were the most valuable in the identification of inter- and intra-specific variation, particularly in the field strains of E. acervulina and E. tenella.These two enzymes were used to examine single-sporocyst and single-sporozoite clones derived from two strains of E. acervulina. The enzymes in E. maxima appeared to be conserved, showing no variation among strains with the five enzymes detected. Relative mobilities, calculated as described in this paper, were found to be consistent between different electrophoresis runs and may serve as a reference when this medium is used. Received: 5 April 1996 / Accepted: 26 November 1996     

Identification and characterization of an Eimeria-conserved protein in Eimeria tenella

The precocious lines of Eimeria spp. have unique phenotypes. However, the genetic basis of the precocious phenotype is still poorly understood. The identification of Eimeria genes controlling the precocious phenotype is of immense importance in the fight against coccidiosis. In the present study, a novel gene of Eimeria maxima was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). Homologous genes were also found in Eimeria tenella and Eimeria acervulina. Alignment of the amino acid sequences from E. tenella, E. maxima, and E. acervulina showed 80–86 % identity, demonstrating a conserved protein in different Eimeria spp. This gene, designated Eimeria-conserved protein (ECP), contained 235 amino acids with a predicted molecular mass of 25.4 kDa and had 100 % identity with one annotated protein from E. maxima (Emax_0517). Real-time PCR and Western blot analysis revealed that the expression of ECP at mRNA and protein level in E. tenella is developmentally regulated. Messenger RNA levels from the ECP gene were higher in sporozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). Expression of ECP protein was detected in unsporulated oocysts, increased in abundance in sporulated oocysts, and was most prominent in sporozoites. Thereafter, the level of the ECP protein decreased, and no ECP-specific protein was detected in second-generation merozoites. Immunostaining with anti-rECP indicated that ECP is highly concentrated in both refractile bodies (RB) of free sporozoites, but is located at the apical end of the sporozoites after invasion of DF-1 cells. The specific staining of the ECP protein becomes more intense in trophozoites and immature first-generation schizonts, but decreases in mature first-generation schizonts. Inhibition of the function of ECP using specific antibodies reduced the ability of E. tenella sporozoites to invade host cells. Compared with the parent strain, both mRNA and protein expression levels in the sporulated oocyst were downregulated in the precocious line of E. tenella. These results suggest that ECP may be involved in invasion and development of the first-generation merogony stage of E. tenella. Findings of downregulation of ECP mRNA and protein expression in the precocious line enrich the study of the precocious phenotype of Eimeria.     

Anatomopathological changes,quantification and distribution of Libyostrongylus spp. in regions of the proventriculus and ventriculus of naturally- and experimentally-infected ostriches

Nematodes of the genus Libyostrongylus parasitize ostriches, causing high mortality rates. These nematodes are found in the proventriculus and ventriculus of ostriches, but little is known about their distribution and the possible anatomopathological changes they cause in the various regions of these organs. This paper describes the distribution and quantification of Libyostrongylus and pathological changes found in regions of the proventriculus and ventriculus of ostriches with high and low levels of both natural and experimental infection. Ostriches were necropsied and tissue samples from the distinct regions of both organs were analysed based on nematode counts and histopathology after staining with haematoxylin and eosin, Masson’s trichrome or Alcian blue/PAS. The cranial and glandular regions of the proventriculus were the most parasitized. The ventriculus contained more nematodes in the caudal region. No macro- or microscopic pathological changes were observed in either of these organs of experimentally-infected birds. However, naturally-infected birds with high levels of infection presented proventriculus with macroscopic lesions and heterophilic infiltrates surrounding nematodes. In the glandular region of this organ, nematodes were located in the adenomeres of the secretory ducts, causing altered architecture and erosions and ulcerative lesions with damaged epithelium. Nematode eggs were found in the koilin layer of the middle and caudal regions of the ventriculus only of these birds. The pH of the regions assessed by Alcian blue/PAS staining changed from acidic in the proventriculus to more alkaline in the caudal region of the ventriculus. These data add knowledge to the biology of Libyostrongylus.

RESEARCH HIGHLIGHTS

• The most parasitized areas were the cranial and glandular regions of the proventriculus.

• Naturally-infected birds with high levels of infection presented macro lesions in the proventriculus and damaged epithelium.

• Nematode eggs were found in the ventriculus.

• The proventriculus had an acidic pH, which turned alkaline towards the ventriculus.

     

Vaccination of chickens with the 34 kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100?μg (B) or 1?mg (C) protein emulsified with Freund’s incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1?mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P?=?0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections.

RESEARCH HIGHLIGHTS

• Laying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72.

• Immunized birds had a highly significant reduction in the duration of colonization.

• Fewer immunized than control birds had abnormal caecal contents after infection.

• Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

     

CT venography and pathological characterization of crus haemorrhage in Pekin ducks

The crus haemorrhage is one of the main causes of carcass defects in Pekin duck processing houses. However, its pathologic features are currently unclear. In order to examine the injury to the hind limb veins and illustrate the pathologic characteristics of crus haemorrhage in Pekin ducks, a total of 68 Pekin ducks with crus haemorrhage (test group) and 10 unaffected ducks (control group) were collected in this study. Five ducks randomly selected from each group were examined by computed tomographic venography with 2.0?mm thickness, 120?kVp, and 90?mA. Pathological changes were observed macroscopically, and under a microscope and electron microscope. The computed tomographic venography results showed no differences in the main hind limb veins between Pekin ducks with crus haemorrhage and the control. Macroscopic results demonstrated that the haemorrhage only occurred in crural muscles, most frequently in musculus gastrocnemius and musculus tibialis cranialis. In severe cases, muscular rupture and multiple intermuscular blood clots could be observed. Histological analysis showed rupture of myofibers and massive red blood cells between muscle bundles. Besides, infiltration of connective tissues and inflammatory lesions could be seen. However, no differences were observed in other organs between these two groups. The main ultrastructural characteristics were myofibrillar rupture and split, accompanied by mitochondrial membrane disintegration and vacuolization. All these results indicate that the haemorrhage in crus is a focal myopathy with the characteristics of bleeding, rupture, and inflammatory lesions.

• Research highlights

• CTV was a feasible method to evaluate the hind limb veins in Pekin ducks.

• The focal myopathy presented here only affected crural muscles.

• The focal myopathy was characterized by bleeding, rupture and inflammatory lesions.

     

Survey on the prevalence of Dermanyssus gallinae in commercial laying farms in Portugal

ABSTRACT

Dermanyssus gallinae, also known as the poultry red mite (PRM), is a blood-feeding ectoparasite of poultry and sylvatic birds. This mite is endemic in many parts of the globe and poses a threat to the egg industry, while compromising the health and welfare of hens, both directly and as a vector of diseases. In addition, people attacked by D. gallinae may develop gamasoidosis. Despite the high prevalence in several European countries, epidemiological information on D. gallinae in Portugal is scarce. This study aimed to assess the prevalence and infestation levels in laying farms in Portugal and evaluate the perception and attitudes of producers regarding D. gallinae. A survey was performed between August 2016 – November 2017, which included 24 farms in the NUTS2 regions Centro and Norte. Mites were sampled with corrugated cardboard traps and the perception and attitudes of farmers regarding the PRM were evaluated with the European COREMI questionnaire prepared by WG 1 of the COST action FA1404. D. gallinae was detected in 95.8% of farms (95% CI: 79.8–99.3%). The average number of trapped mites among farms was 5200?±?16,522, with a median of 359 mites (interquartile range?=?46–3135). Results from the questionnaire show that insufficient monitoring, under-detection and late and suboptimal treatment may contribute to the maintenance of significant infestation levels. The present data highlight the need for adequate monitoring of D. gallinae, timely action and effective treatment in order to improve poultry productivity and ensure human and animal health and welfare.

RESEARCH HIGHLIGHTS

• A survey on the prevalence of D. gallinae in Portuguese layer farms is presented

• The perceived importance of D. gallinae was assessed with a questionnaire

• D. gallinae was detected in 95.8% of farms

• The results emphasize the need for adequate monitoring and treatment optimization.

     

Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil Nadu, India

Coccidosis is one of the most commonly prevalent and economically important parasitic diseases of poultry worldwide. Chicken coccidia are protozoan parasites of the genus Eimeria. This study aimed at analysing the molecular prevalence of seven species of Eimeria infecting chickens in Tamil Nadu, India. Tissue samples (caecum, rectum and upper and mid intestines) collected from chickens exhibiting symptoms of coccidiosis were used for DNA extraction, followed by amplification of the internal transcribed spacer (ITS) region of Eimeria genome with genus-specific primers and speciation in nested polymerase chain reaction (PCR) with species-specific primers. Of 43 tissue samples examined, 25 were positive in ITS PCR and all the seven species could be identified. However, the prevalence of each species varied. In broilers, Eimeria necatrix was present in all infected chickens with Eimeria brunetti, Eimeria tenella, Eimeria maxima and Eimeria acervulina present in more than 50% of infected chickens, while Eimeria praecox and Eimeria mitis were only present in 11% to 16%. Although only 7 samples were positive among layers, the prevalence was largely similar, but with a higher prevalence of E. praecox and E. mitis and a lower prevalence of E. tenella. Multiple infections were most common, with 2–6 Eimeria species infecting the same chickens. In order to estimate the preponderance of each infecting species of Eimeria, a random cloning technique was adopted. The genus-specific ITS PCR product was cloned in a TA vector and ten clones were randomly picked and used as template for amplification of all the seven genera of Eimeria. If the specific species of Eimeria is preponderant, then the frequency of the clones showing that species-specific PCR amplification would be higher. Using this method, the most preponderant species present in the rectum, mid and upper intestines of layers was assessed to be E. acervulina, E. brunetti and E. necatrix. E. acervulina was present in 60–90%, E. necatrix in 10–30% and E. brunetti in 10–20% of the clones screened, indicating that these species could be the most preponderant Eimeria species. Intervention strategies should aim at these species. This new method of estimating preponderance of infecting Eimeria species could be used to assess the relative importance of each species at the farm or region level instead of relying only on prevalence estimates.     

Fatal necrotic tracheitis by Aviadenovirus in captive Alagoas curassows (Pauxi mitu) extinct from the wild

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises.

RESEARCH HIGHLIGHTS

• Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A.

• Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis.

• Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows.

• Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).

     

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek’s disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC.

• RESEARCH HIGHLIGHTS

• Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host–pathogen interactions.

• pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons.

• pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

     

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR?<?0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR?<?0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community.

• HIGHLIGHTS

• Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds.

• Differences were found between infected and healthy animals in gut microbiota.

• Candidatus Arthromitus was closely associated with infected birds.

• M. ornithogaster can affect intestinal microbiota composition of canaries.

     

Pathology of clade 2.3.2.1 avian influenza virus (H5N1) infection in quails and ducks in Bangladesh

We performed pathological and molecular virological investigation of three outbreaks of highly pathogenic avian influenza (HPAI) in a quail farm and two duck farms of Mymensingh and Netrokona districts of Bangladesh in 2011. HPAI viruses of subtype H5N1 were detected from all three outbreaks and phylogenetic analysis of HA gene sequence placed the viruses into clade 2.3.2.1. The outbreak in the quail farm was characterized by acute death with 100% mortality within two days. Marked haemorrhages and congestion with necrotic and inflammatory lesions in the respiratory tract, liver, pancreas and kidneys were the major gross and histopathological lesions. In the case of ducks, nervous signs were the remarkable clinical manifestations and the mortality was around 10%. No significant gross lesions were observed at necropsy. Non-purulent encephalitis with gliosis and neuronal degeneration was observed on histopathological examination. By immunohistochemistry, viral antigen could be detected in different organs of both quails and ducks. This study records varying clinical and pathological manifestations of HPAI in ducks and quails following natural infection with the same strain of the virus.

RESEARCH HIGHLIGHTS

• HPAIV of clade 2.3.2.1 was detected from clinical outbreaks in quails and ducks

• Sudden death with severe haemorrhages in various organs was found in quails

• Pronounced nervous signs with non-purulent encephalitis were observed in ducks

• Viral antigen could be localized in different organs by immunohistochemistry