Molecular characterization of antimicrobial resistance in Brachyspira species isolated from UK chickens: Identification of novel variants of pleuromutilin and beta-lactam resistance genes.

Brachyspira species are Gram negative, anaerobic bacteria that colonise the gut of many animals, including poultry. In poultry, Brachyspira species can be commensal (B. innocens, B. murdochii, 'B. pulli') or pathogenic (B. pilosicoli, B. intermedia, B. alvinipulli or rarely B. hyodysenteriae), the latter causing avian intestinal spirochaetosis (AIS). Antimicrobial therapy options for treatment is limited, frequently involving administration of the pleuromutilin, tiamulin, in water. In this study 38 Brachyspira isolates from chickens in the UK, representing both commensal and pathogenic species, were whole genome sequenced to identify antimicrobial resistance (AMR) mechanisms and the minimum inhibitory concentration (MIC) to a number of antimicrobials was also determined. We identified several new variants of blaOXA in B. pilosicoli and B. pulli isolates, and variations in tva which led to two new tva variants in B.murdochii and B.pulli. A number of isolates also harboured mutations known to encode AMR in the 16S and 23S rRNA genes. The percentage of isolates that were genotypically multi-drug resistance (MDR) was 16%, with the most common resistance profile being: tetracycline, pleuromutilin and beta-lactam, which were found in three 'B. pulli' and one B. pilosicoli. There was good correlation with the genotype and the corresponding antibiotic MIC phenotypes: pleuromutilins (tiamulin and valnemulin), macrolides (tylosin and tylvalosin), lincomycin and doxycycline. The occurrence of resistance determinants identified in this study in pathogenic Brachyspira, especially those which were MDR, is likely to impact treatment of AIS and clearance of infections on farm.

Survey of respiratory disease associated with Mycoplasma gallisepticum in British gamebirds (2016-2019): In vitro antibiotic sensitivity, pathology and detection of other pathogens.

BACKGROUND: The causes of respiratory disease in British gamebirds were investigated during 2016-2019 following concerns about poorer responses to antibiotic treatment. Emphasis was placed on Mycoplasma gallisepticum, but other possible bacterial and viral causes were included, along with gross and histopathological examination. METHODS: Clinical respiratory disease outbreaks were investigated. RESULTS: Mycoplasma gallisepticum was detected by PCR in 65 of 69 outbreaks in pheasants and partridges and isolated from 56 of these. Partial mgc2 gene sequences from 28 M. gallisepticum isolates were compared, and 26 proved identical, suggesting the prevalence of a dominant sequence type. Minimum inhibitory concentration values for tiamulin, tylosin, tylvalosin, doxycycline and tetracycline were significantly higher than the reference strain but could not be correlated with treatment failures. Other bacterial species were isolated from sinuses but were not consistently correlated with disease. RT-PCRs detected coronaviruses in 18% of 49 outbreaks and avian metapneumovirus in 8%. Histopathological lesions were typical of M. gallisepticum sinusitis and significantly associated with M. gallisepticum PCR outbreak positivity. CONCLUSION: Mycoplasma gallisepticum remains an important cause of respiratory disease in gamebirds. Synergism with other pathogens may have played a role in some outbreaks. Specific reasons for variable responses to antibacterial treatment were not identified.

Looking out for avian influenza in smallholding poultry flocks.

This focus article has been prepared by David Welchman, Rowena Hansen, Lévon Stephan and Anna Brzozowska of the APHA. It is an update of a focus article first published in December 2017 (VR, 9 December 2017, vol 181, pp 618-620).

Backyard poultry cases in UK small animal practices: Demographics, health conditions and pharmaceutical prescriptions.

BACKGROUND: Backyard poultry ownership is of keen interest in the United Kingdom. However, despite this, little is known about veterinary care engagement and outcomes of visits in this group of species. METHODS: This study described and characterised veterinary practice-visiting backyard poultry, utilising electronic health record data supplied by veterinary practices voluntarily participating in the Small Animal Veterinary Surveillance Network between 1st April 2014 and 31st March 2019. RESULTS: In total, 4424 recorded poultry consultations originating from 197 veterinary practices (352 sites) were summarised. Chicken consultation (n = 3740) peak incidence was in early summer (April-June), relative to all recorded species. More chickens resided in rural (incident rate ratio = 2.5, confidence interval [CI] 2.3-2.6, p <0.001) or less deprived areas. Non-specific clinical signs were commonly recorded (17.6% of chicken consultations, CI 15.9-19.2), as were those indicative of advanced disease. This latter finding was reflected in prescribed management strategies, with euthanasia comprising 29.8% (CI 27.0-32.6) of consultations. Antimicrobials were commonly prescribed (33.0% of consultations, CI 29.8-36.2), 43.8% of which included antimicrobials considered 'highest priority critically important' by the World Health Organisation. CONCLUSION: Our findings indicate a need to tailor antimicrobial prescription guidance to the backyard poultry setting. In addition, late presentation of disease, vague clinical descriptions in clinical narratives and high euthanasia rates show that disease identification, management and knowledge of poultry health and welfare among owners and veterinary surgeons can be improved.

Retrospective analysis of necrotizing typhlitis cases associated with Brachyspira spp. in British rheas.

This paper describes a retrospective analysis of necrotizing typhlitis in common rheas (Rhea americana) diagnosed in the United Kingdom by the Animal & Plant Health Agency (APHA). From January 2008 to January 2020, seven cases of spirochaetal typhlitis associated with Brachyspira spp. were identified using the Veterinary Investigation Diagnosis Analysis database. Gross examination was combined with selective anaerobic culture, polymerase chain reaction, and histopathology to diagnose typhlitis associated with spirochaetal infection. Whole-genome sequencing was subsequently utilized on archived isolates from six of the seven submissions, overcoming issues with traditional testing methods and yielded gains in the identification of Brachyspira to species level. Brachyspira hyodysenteriae, an organism traditionally associated with typhlitis in rheas, was isolated in three sequenced submissions. One of these also demonstrated co-infection with Brachyspira intermedia. Brachyspira suanatina, Brachyspira hampsonii, and Brachyspira alvinipulli were identified by sequencing as single infections in the remaining three animals. This report demonstrates the ability of Brachyspira species other than B. hyodysenteriae to colonize the caeca of rheas presenting with typhlitis. Additionally, the B. alvinipulli isolate harboured a tva(A) gene, indicating higher potential pleuromutilin resistance, which has not previously been described in this Brachyspira species. This study discusses the epidemiology of examined cases and examines the potential role other species may play in these outbreaks.

Avian leukosis virus subgroup J-associated myelocytoma in a hobby chicken.

The avian leukosis viruses (ALVs) are a major group of retroviruses associated with neoplastic diseases in poultry. The ALV-J strain was identified as a cause of myelocytomas in broiler breeder and broiler chickens in the UK in the 1980s; however, following eradication of the virus,commercial broilers have remained free of infection since the early 2000s. A pet chicken was submitted to Animal and Plant Health Agency (APHA) in 2013 with a history of croaking respirations, abnormality of the left eye and apparent paralysis. Postmortem examination of the bird showed widespread tumour-like infiltration of many organs, including the pectoral muscles, internal organs, sternum and ribs. Histopathological examination of the affected tissues revealed myelocytoma formation typical of the lesions associated with ALV-J, and the virus was confirmed by PCR testing and sequencing. Virus was not detected in blood samples in the other five chickens remaining in the flock. The source of infection was not established. This was the first time ALV-J had been seen in the UK since its eradicationand the case highlights the importance of continued surveillance of backyard and hobby chickens to detect potential new and re-emerging disease threats, such as ALV-J, which may be of significance to the wider poultry population.

Looking out for avian influenza in backyard and small poultry flocks.

This article has been prepared by David Welchman, veterinary lead of the Avian Expert Group, and Anna Brzozowska, surveillance data scientist, of the APHA's Surveillance Intelligence Unit.

Detection of Theileria luwenshuni in sheep from Great Britain.

BACKGROUND: Theileria spp. are tick-borne protozoan parasites of the Phylum Apicomplexa, Order Piroplasmida that infect a wide range of wild and domestic animals. In Great Britain, Theileria spp. have been reported from livestock associated with transmission by the tick Haemaphysalis punctata. However, these reports have not been associated with disease. This study has investigated the cause of a disease outbreak accompanied by mortality in a flock of sheep grazing reclaimed marshland in north Kent. FINDINGS: A polymerase chain reaction-reverse line blot assay indicated the presence of Theileria spp. in blood samples from five animals. Subsequent testing with a pan-piroplasm PCR of a larger panel of blood samples detected a piroplasm amplicon in 19 of 21 sheep submitted from the affected flock. Automated sequencing confirmed that these amplicons shared 99-100% identity with T. luwenshuni. CONCLUSIONS: The clinical and PCR data suggest infection with T. luwenshuni was associated with disease and mortality in this flock.

Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection.

Peptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila.

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish(E20) flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish(E20) flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.

Insect immunity: from pattern recognition to symbiont-mediated host defense.

The Jacques Monod conference "Insect Immunity in Action: From Fundamental Mechanisms of Host Defense to Resistance Against Infections in Nature," organized by Ulrich Theopold (Stockholm University, Sweden) and Dominique Ferrandon (CNRS, France), was held in May 2009 in Aussois, France. Here, we review key topics and concepts that were presented and highlight emerging trends in the field of insect immunity.