Metaproteomics characterization of the alphaproteobacteria microbiome in different developmental and feeding stages of the poultry red mite Dermanyssus gallinae (De Geer, 1778)

ABSTRACT

The poultry red mite (PRM), Dermanyssus gallinae (De Geer, 1778), is a worldwide distributed ectoparasite and considered a major pest affecting the laying hen industry in Europe. Based on available information in other ectoparasites, the mite microbiome might participate in several biological processes and the acquisition, maintenance and transmission of pathogens. However, little is known about the role of PRM as a mechanical carrier or a biological vector in the transmission of pathogenic bacteria. Herein, we used a metaproteomics approach to characterize the alphaproteobacteria in the microbiota of PRM, and variations in its profile with ectoparasite development (nymphs vs. adults) and feeding (unfed vs. fed). The results showed that the bacterial community associated with D. gallinae was mainly composed of environmental and commensal bacteria. Putative symbiotic bacteria of the genera Wolbachia, C. Tokpelaia and Sphingomonas were identified, together with potential pathogenic bacteria of the genera Inquilinus, Neorickettsia and Roseomonas. Significant differences in the composition of alphaproteobacterial microbiota were associated with mite development and feeding, suggesting that bacteria have functional implications in metabolic pathways associated with blood feeding. These results support the use of metaproteomics for the characterization of alphaproteobacteria associated with the D. gallinae microbiota that could provide relevant information for the understanding of mite-host interactions and the development of potential control interventions.

Research highlights

• Metaproteomics is a valid approach for microbiome characterization in ectoparasites.

• Alphaproteobacteria putative bacterial symbionts were identified in D. gallinae.

• Mite development and feeding were related to variations in bacterial community.

• Potentially pathogenic bacteria were identified in mite microbiota.

     

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

     

Dermanysuss gallinae attacks humans. Mind the gap!

ABSTRACT

Dermanyssus gallinae is a haematophagous ectoparasite primarily known as a pest of domestic and wild birds. It occasionally feeds on a range of mammals, and, more importantly, is of growing concern in human medicine. This review highlights mite attacks on people working with poultry, and updates the increasing incidence of dermanyssosis in urban environments in Europe. Although several cases of dermanyssosis have been documented, there are a number of reasons why diagnosis of D. gallinae infestations in humans is likely to be underestimated. Firstly, medical specialists are not well aware of D. gallinae infestations in humans. There is also a lack of collaboration with specialists from other disciplines. The problem is compounded by misdiagnoses and by the lack of diagnostic tools. We review the literature on human dermanyssosis cases in Europe, and also provide information on the epidemiology, clinical, histo-pathological and immunological aspects of dermanyssosis. We stress the need for improved recognition of this challenging infestation in humans, and provide straightforward recommendations for health practitioners, starting with collection of the correct anamnestic information and including appropriate management methods for case recognition and resolution. Finally, we indicate the most urgent areas to be addressed by future research.

RESEARCH HIGHLIGHTS

• Dermanyssus gallinae is of growing concern in human medicine.

• Most physicians are not well aware of dermanyssosis in humans.

• Bio-epidemiological and clinical aspects of this ectoparasitosis are highlighted.

• Practical key actions for diagnosis and correct management of infestation in humans are provided.

     

How can Dermanyssus gallinae (De Geer 1778) (Acari: Anactinotrichida: Dermanyssidae) walk upwards on slippery surfaces?

ABSTRACT

Scanning electron microscopy observations of the distal leg region of the poultry red mite Dermanyssus gallinae (De Geer 1778) identified the presence of a compound ambulacrum, the part of the leg that contacts the substratum when the mite walks. The ambulacrum is comprised of a praetarsus (the ambulacrum stalk), a pulvillus and two claws. The pulvillus is a weakly sclerotized structure that can be partly expanded or retracted in the praetarsus. When expanded, the pulvillus shows a cushion-like shape which can, as a result of its soft surface, function as a sucker, thus allowing D. gallinae to adhere to a smooth surface. When traversing an irregular surface, or clinging to a soft surface, the mite retracts the pulvillus and uses only its strongly sclerotized movable claws. These observed morphological adaptations explain the ability of D. gallinae to climb upwards on a slippery surface, resist an air flux, walk on smooth and rigid feathers of its avian hosts, and cling to the bird’s or human's soft skin. This knowledge is important to better understand the attachment mechanism of this species to its host and to the substratum on which it moves, and also to provide insight into the circumstances under which it is able to move.

RESEARCH HIGHLIGHTS

• The ambulacrum is the distal part of the leg touching the substratum

• D. gallinae’s ambulacrum consists of a praetarsus, a pulvillus and two claws

• The weakly sclerotized pulvillus can be part expanded/retracted in the praetarsus

• The expanded pulvillus functions as a sucker to adhere to smooth surfaces

• The claws are used to walk on an irregular surface or cling to a soft surface

     

Cross-species transmission of poultry pathogens in backyard farms: ducks as carriers of chicken viruses

ABSTRACT

In backyard farms of Lao People’s Democratic Republic, mixed-species rearing of poultry is a breeding-ground for cross-species transmission. Here, the epidemiology of viruses circulating among backyard poultry in Vientiane Province was assessed to guide future control strategies. Oral/tracheal and cloacal swabs, collected from 605 poultry (308 ducks, 297 chickens) between 2011 and 2015, were screened by PCR for Newcastle disease virus (NDV), coronavirus (CoV) and chicken anaemia virus (CAV). Chicken sera were screened for anti-NDV antibodies by ELISA. Statistical and phylogenetic analyses revealed transmission patterns and relationships.

Closely related strains co-circulated in chickens and ducks. While CoV RNA was detected in oral/tracheal swabs of 9.3% of the chickens and 2.4% of the ducks, rates were higher in faecal swabs of both species (27.3% and 48.2%). RNA of infectious bronchitis virus (IBV) and duck CoV was found in faecal swabs of chickens (19.7% and 7.1%) and ducks (4.1% and 44.1%). Moreover, DNA of the generally chicken-specific CAV was detected in oral/tracheal swabs of chickens (18.1%) and, sporadically, of ducks (2.4%). Despite serological evidence of NDV circulation or vaccination (86.9%), NDV RNA was not detected. We found a high prevalence and indication for cross-species transmission of different CoV strains in backyard poultry. Interestingly, ducks served as biological, or at least mechanical, carriers of viral strains closely related not only to IBV, but also to CAV. Bird containment and poultry species separation could be first steps to avoid cross-species transmission and emergence of novel strains with broad host range and enhanced pathogenicity.

RESEARCH HIGHLIGHTS

• High rates of avian viruses were detected by PCR in backyard poultry from Lao PDR.

• Diverse coronavirus and chicken anemia virus strains co-circulated.

• Phylogenetic analyses suggested virus transmission between chickens and ducks.

• Serological evidence of Newcastle disease was found, but viral RNA was not detected.

     

Parasitological and molecular survey of scattered parasitism by trichomonads in some avian species in Iran

ABSTRACT

Outbreaks of avian trichomonosis are being reported worldwide; meanwhile, the genetic and virulence variations are under investigation. In this study, the occurrence and genetic variability of oral or faecal trichomonads among various avian species were investigated. Samples obtained from either the oropharyngeal cavity, crop/oesophagus, droppings/cloaca, or conjunctival swabs of avian species were inspected for flagellates. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples was performed to investigate the genetic diversity of the isolates. Investigation of 737 birds revealed an infection rate of 15.7% in the upper gastrointestinal tract, 7.3% in the faecal samples, and 0.7% involvement of the conjunctiva. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples, identified genotypes A and B of Trichomonas gallinae and genogroups A-C and E of Tetratrichomonas gallinarum. A novel ITS genotype of intestinal trichomonads was also detected in hooded crow (Corvus cornix) and common mynah (Acridotheres tristis). In the present study, in addition to Columbiformes and Falconiformes, trichomonads were detected in Passeriformes and Galliformes with the involvement of organs other than the gastrointestinal tract. Genotype A T. gallinae was detected in domestic pigeons (Columba livia domestica), a laughing dove (Spilopelia senegalensis), a common kestrel (Falco tinnunculus), a budgerigar (Melopsittacus undulates), and a canary (Serinus canaria). Distinct genotype B was detected in a common mynah and a budgerigar. Genogroups A-C of T. gallinarum were also demonstrated in Galliformes and Anseriformes. Furthermore, two novel trichomonad ITS genotypes were detected in hooded crows and a common mynah warranting detailed multi-locus molecular analysis.

RESEARCH HIGHLIGHTS

• ITS diversity of trichomonads was shown in various avian species.

• Diversity of the parasites’ target organ and clinical manifestations was demonstrated.

• Two novel ITS genotype trichomonads from common mynah and hooded crow were identified.

     

Genotyping of Riemerella anatipestifer by ERIC-PCR and correlation with serotypes

Riemerella anatipestifer (RA) is a widely distributed bacterial pathogen of birds responsible for remarkable losses to poultry production, especially among waterfowl. We characterized the genomic diversity of 166 field isolates of RA, collected from geese and ducks, using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR). The field strains and five reference strains showed 17 distinct patterns consisting of five to 12 bands ranging from approximately 150–1800bp. The majority of the strains belonged to two closely related ERIC-PCR types (A and B), while the other types contained only a few isolates each. There was no association between ERIC-PCR type and host species, place, or year of isolation; however the ERIC-PCR pattern was correlated with serotype for most isolates. The majority of serotype 1 strains (101/107) belonged to ERIC-PCR type A while the remaining six strains represented five different ERIC-PCR types (D, G, L, M, and O). Serotypes 1,7 and 7 corresponded to ERIC-PCR types B and C, respectively. Serotypes 2, 4, and 10 could be subdivided by ERIC-PCR revealing two to four patterns within each serotype. These results indicate that ERIC-PCR may be a suitable technique for the molecular identification of RA serotypes, and the detection of subtypes within certain serotypes may aid further epidemiological investigations.

RESEARCH HIGHLIGHTS

• ERIC-PCR analysis of field R. anatipestifer strains revealed 17 distinct patterns

• Most strains belonged to two closely related ERIC-PCR types

• Serotype 1 was the most prevalent serotype representing 64.5% of the strains

• ERIC-PCR may be suitable for molecular identification of R. anatipestifer serotypes

     

Experimental model of oral transmissible AA amyloidosis in quails

ABSTRACT

In poultry and zoo birds, mass outbreaks of amyloid A (AA) amyloidosis are often reported, and horizontal transmission is considered as one of the causes. However, oral transmission of avian AA amyloidosis in nature has been unclear. In order to clarify the horizontal transmission of avian AA amyloidosis, basic research using an appropriate oral transmission model is necessary. In this study, we developed an oral transmission model of AA amyloidosis using quails, and assessed the oral transmission efficiency of AA amyloidosis in quails and mice. Young quails, adult quails, and young mice received inflammatory stimulation with lipopolysaccharide; simultaneously, homogeneous amyloid fibrils were orally or intravenously administered. By histological examination, induction of amyloidosis by oral or intravenous administration of amyloid was confirmed in all species. Furthermore, both quail and murine AA amyloidosis were orally transmitted in a dose-dependent manner. These results support the possibility of horizontal transmission of avian AA amyloidosis in nature. This model will be able to contribute to the elucidation of spontaneous horizontal transmission of avian AA amyloidosis in the future.

RESEARCH HIGHLIGHTS

• Quail AA amyloidosis was orally transmitted in a dose-dependent manner.

• Oral transmission was less efficient than intravenous transmission.

• In-cage horizontal transmission did not occur during 4-week cohabitation.

• Amyloid deposition in tissues of quail was grossly visible.

     

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).

     

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

RESEARCH HIGHLIGHTS

• Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.

• Virus shedding was significantly (P?<?0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.

• Serum antibody titres were not a reliable indicator of protection.

• An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

     

Synergistic effect of a combination of nicarbazin and monensin against coccidiosis in the chicken caused by Eimeria spp.

ABSTRACT

A clinical study was made into the abilities of nicarbazin and monensin and a nicarbazin?+?monensin combination to control Eimeria acervulina, E. maxima, and E. tenella in chickens. When included in the feed, at concentrations of 40?ppm nicarbazin or 40?ppm monensin, these products showed partial efficacy evaluated by daily weight gain (DWG) but no activity judged by daily feed intake (DFI) or feed conversion ratio (FCR). By contrast, the combination of 40?ppm nicarbazin?+?40?ppm monensin provided complete control of infection judged by greater DWG and DFI, and lower FCR. Monensin at a concentration of 40?ppm was ineffective in preventing lesions caused by all three species. Nicarbazin at a concentration of 40?ppm was unable to suppress lesions of E. acervulina and E. maxima but was able to suppress lesions caused by E. tenella. Nicarbazin 40?ppm?+?monensin 40?ppm suppressed lesions of all three species.

RESEARCH HIGHLIGHTS

• Nicarbazin or monensin at 40 ppm gave only partial control of Eimeria spp.

• A combination of 40 ppm nicarbazin + 40 ppm monensin controlled DWG, DFI and FCR.

• Nicarbazin or monensin at 40 ppm did not suppress all Eimeria spp. lesions.

• Nicarbazin 40 ppm + monensin 40 ppm suppressed lesions of all three species.

     

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.

     

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

     

Individual and stable autoantibody repertoires in healthy individuals

In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year’s time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0–16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals.

• Highlights

• A unique longitudinal profiling of autoantibody repertoires in healthy individuals

• Autoantibody profiles are highly individual and stable over time

• All individuals display IgG binding to human protein fragments

• The specificity of disease associated autoantigens needs to be thoroughly characterized

• The identification of a small set of highly reactive autoantigens

• Importance of stringent antigen and sample specific cut-offs for defining reactivity

     

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.

     

Pigeon circoviruses from feral pigeons in Australia demonstrate extensive recombination and genetic admixture with other circoviruses

ABSTRACT

Like other avian circovirus species, Pigeon circovirus (PiCV) is known to be genetically diverse with a relatively small circular single-stranded DNA genome of 2?kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Recent paleoviral evidence hints towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and dispersal of their hosts. Limited availability of PiCV genome sequence data in Australia has hindered phylogeographic studies in this species, so we screened clinically normal rock doves (Columba livia) in regional New South Wales, and demonstrated a high prevalence (76%) of PiCV infection by PCR. We also recovered 12 complete novel PiCV genomes and phylogenetic analyses revealed that PiCV circulating in Australian feral pigeons formed two strongly supported monophyletic clades. One clade resided with PiCV genomes from Poland, Australia, United Kingdom, Belgium, China, and Japan, and another basal clade was more closely related to PiCV genomes from Poland. A novel more distantly-related PiCV rep gene formed a solitary clade with weak posterior support. So we further analysed all selected partial rep gene sequences to demonstrate a likely naturally occurring spillover infection from a passerine circovirus candidate. The findings suggest that there is a high degree of genetic variation within PiCV in Columbiformes with potential greater admixture between avian circoviruses within Australia than previously known.

RESEARCH HIGHLIGHTS

• Confirmed high prevalence rate of PiCV circulating in Australian wild pigeons.

• Highlighted extensive recombination events within Australian PiCV.

• Demonstrated a likely naturally occurring spillover infection from a passerine circovirus candidate.

     

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.

     

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.