Physiological,biochemical and histopathological changes associated with heatstroke in the galah (Eolophus roseicapilla) and rock dove (Columba livia)

The pathophysiology of heat illnesses in birds has not been well characterized. In this study, we describe the changes in heart rate, respiratory rate, blood biochemistry and histopathological findings in galahs and rock doves after heat exposure under standardized conditions designed to induce heatstroke. Birds in the heat-exposed group were exposed to environmental heat stress and compared to control birds. Both groups of birds were under general anaesthesia throughout the experiment and serial blood collections were performed for biochemical analyses, while organs were collected at the end of the experiment for histopathology. No electromyography traces consistent with the onset of heat cramps were observed in any of the birds. Biochemical changes suggestive of skeletal muscle and hepatocellular injury, including hyperkalaemia and increased serum muscle and hepatic enzyme activities, were often observed in heat-exposed galahs and rock doves at the onset of heatstroke. Microscopic analyses did not reveal any significant cardiac changes, although some lungs had signs of acute congestion. Some heat-exposed rock doves had microscopic changes indicative of necrosis in the pectoral muscle. There were significant hepatic changes in some heat-exposed galahs, but not in rock doves. This suggests that there may be species differences amongst birds in the organs most affected by heatstroke. The observed species differences in the physiological, biochemical and histopathological changes indicate that bird species should be studied separately for clinical syndromes such as heatstroke.

RESEARCH HIGHLIGHTS

• Biochemical changes suggestive of skeletal muscle and hepatocellular injury in heat-exposed galahs and rock doves at the onset of heatstroke

• No electromyography traces consistent with the onset of heat cramps were observed

• Some heat-exposed rock doves had indications of necrosis in the pectoral muscle

• There were significant hepatic changes in some heat-exposed galahs

     

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).

     

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.

     

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

     

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

     

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.

     

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

     

Biological control as an alternative to synthetic fungicides for the management of grey and blue mould diseases of table grapes: a review

Abstract

Grey and blue mould diseases are among the most important diseases of grapes worldwide. They are causing extensive decay in postharvest grapes. Chemical fungicides remain the primary treatment for managing these diseases. However, consumer’s interest in organic produce, restrictions on chemical use by some countries, and concerns about human and environmental safety have driven research to identify safe and effective alternatives. Among several alternative approaches, the use of biological control agents (BCAs) is getting more acceptance and has been comprehensively studied. This review summarizes the use of BCAs as a postharvest treatment to control the postharvest grey and blue mould of grapes. The review also emphasizes the economic importance of these two major postharvest diseases of grapes. In addition, other non-chemical postharvest treatments, the advantage of an integrated approach and finally problems, challenges, and future trends of the BCAs are described. Several yeasts that have a promising result to control grey and blue mould disease of grapes are thoroughly reviewed. The current market share of BCAs and their future directions on commercialization are also suggested in this review.

• Biocontrols can be a potential control method for postharvest diseases of fruits and vegetables in place of chemical fungicides.

• Antagonistic yeasts have high ability to suppress fungal growth.

• Integrated approach utilizes a combination approaches, which often results in a synergistic effect to control the diseases.

• USA and Europe are currently the largest markets for biocontrol products.

     

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.

     

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.

     

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

     

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK

ABSTRACT

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT–PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT–PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT–PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.

RESEARCH HIGHLIGHTS

• TVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.

• TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.

• CPNV was detected in proventriculi with both TVP and LP-lesions.

• Viruses other than CPNV may be responsible for some TVP-affected cases.

     

Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5?dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China.

RESEARCH HIGHLIGHTS

• A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China.

• The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains.

• The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection.

• These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

     

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.

     

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.

     

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.

     

MinION sequencing to genotype US strains of infectious laryngotracheitis virus

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing.

RESEARCH HIGHLIGHTS

• A multi-allelic assay identified nine ILTV genotypes circulating in the US

• Single-allele genotyping is congruent with whole genome phylogenetic partitioning

• US ILTV strains can be grouped into five genotypes using the single-allele assay

• The single-allele assay can be done using MinION sequencing of barcoded amplicons

     

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.

     

Antigenicity,pathogenicity and immunosuppressive effect caused by a South American isolate of infectious bursal disease virus belonging to the “distinct” genetic lineage

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious immunosuppressive disease affecting young chickens. The recently described “distinct IBDV” (dIBDV) genetic lineage encompasses a group of worldwide distributed strains that share conserved genetic characteristics in both genome segments making them unique within IBDV strains. Phenotypic characterization of these strains is scarce and limited to Asiatic and European strains collected more than 15 years ago. The present study aimed to assess the complete and comprehensive phenotypic characterization of a recently collected South American dIBDV strain (1/chicken/URY/1302/16). Genetic analyses of both partial genome segments confirmed that this strain belongs to the dIBDV genetic lineage and that it is not a reassortant. Antigenic analysis with monoclonal antibodies indicated that this strain has a particular antigenic profile, similar to that obtained in a dIBDV strain from Europe (80/GA), which differs from those previously found in the traditional classic, variant and very virulent strains. Chickens infected with the South American dIBDV strain showed subclinical infections but had a marked bursal atrophy. Further analysis using Newcastle disease virus-immunized chickens, previously infected with the South American and European dIBDV strains, demonstrated their severe immunosuppressive effect. These results indicate that dIBDV strains currently circulating in South America can severely impair the immune system of chickens, consequently affecting the local poultry industry. Our study provides new insights into the characteristics and variability of this global genetic lineage and is valuable to determine whether specific control measures are required for the dIBDV lineage.

Research Highlights

• A South American strain of the dIBDV lineage was phenotypically characterized.

• The strain produced subclinical infections with a marked bursal atrophy.

• Infected chickens were severely immunosuppressed.

• The dIBDV strains are antigenically divergent from other IBDV lineages.