Questionnaire study suggests grave consequences of infectious laryngotracheitis, infectious coryza and mycoplasmosis in small chicken flocks.

BACKGROUND: A growing number of people in western countries keep small chicken flocks. In Sweden, respiratory disease is a common necropsy finding in chickens from such flocks. A respiratory real-time polymerase chain reaction (PCR) panel was applied to detect infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum (A. paragallinarum) and Mycoplasma gallisepticum (M. gallisepticum) in chickens from small flocks which underwent necropsy in 2017-2019 and had respiratory lesions. Owners (N = 100) of PCR-positive flocks were invited to reply to a web-based questionnaire about husbandry, outbreak characteristics and management. RESULTS: Response rate was 61.0%. The flocks were from 18 out of Sweden's 21 counties indicating that respiratory infections in small chicken flocks are geographically widespread in Sweden. Among participating flocks, 77.0% were coinfected by 2-3 pathogens; 91.8% tested positive for A. paragallinarum, 57.4% for M. gallisepticum and 50.8% for ILTV. Larger flock size and mixed-species flock structure were associated with PCR detection of M. gallisepticum (P = 0.00 and P = 0.02, respectively). Up to 50% mortality was reported by 63.9% of respondents. Euthanasia of some chickens was carried out in 86.9% of the flocks as a result of the outbreaks. Full clinical recovery was reported by 39.3% of owners suggesting chronic infection is a major challenge in infected flocks. Live birds had been introduced in many flocks prior to outbreaks, which suggested these as an important source of infection. Following the outbreaks, 36.1% replaced their flocks with new birds and 9.8% ceased keeping chickens. CONCLUSIONS: This study highlights the severity of respiratory outbreaks in small non-commercial chicken flocks and points to the need for more research and veterinary assistance to prevent and manage respiratory infections in small chicken flocks.

Ascaridia galli - An old problem that requires new solutions.

Reports of Ascaridia galli in laying hens in Europe have increased since the ban on conventional battery cages in 2012. As this parasite is transmitted directly via the faecal-oral route by parasite eggs containing a larva, it is reasonable to assume that the escalating problem is related to the increased exposure now occurring in modern welfare-friendly cage-free housing systems. On many farms, A. galli reappears in subsequent flocks, even though the birds have no access to the outdoors, biosecurity is high and empty houses are cleaned and disinfected during downtime. Since the egg production cycle lasts only ≈80 weeks and recombinant antigen production for helminth vaccines has not yet been solved, the development of a vaccine seems to be an unrealistic option. Therefore, disrupting the life cycle of the parasite by other means, including the strategic use of dewormers, appears to be the key to controlling infection. Of concern is that only one class of anthelmintics is licenced for poultry in Europe and that are usually administered indiscriminately through the birds' drinking water and often too late when the parasite is already established. If current calendar-based parasite control strategies are not changed, there is a risk that resistance to anthelmintics may develop, as has already been demonstrated with nematodes in livestock. We insist that treatments can be more effective and the risk of developing drug resistance can be mitigated if we invest in a better understanding of A. galli responses to more prudent and judicious use of anthelmintics. This review identifies knowledge gaps and highlights aspects of sustainable parasite control that require further research to support commercial egg producers.

Core genome multilocus sequence typing (cgMLST) confirms systemic spread of avian pathogenic Escherichia coli (APEC) in broilers with cellulitis.

Broiler cellulitis has emerged as an important cause of economic losses for farmers and slaughter plants from carcass condemnation at processing. Avian pathogenic Escherichia coli (APEC) has been identified as the main causative agent. The aim was to characterize E. coli isolated from cellulitis and organs in broilers at slaughter by whole genome sequencing analysis to study if systemic spread could be confirmed. Isolates were collected post-mortem from 101 carcasses condemned due to dermatitis/cellulitis from five commercial farms and six flocks. Forty-six isolates were characterised to determine serotypes, sequence types and virulence-associated genes. Analysis by cgMLST was performed to study the genetic similarity between isolates from the same broiler, among birds from the same flock and between flocks. Escherichia coli was isolated from 90% of birds from subcutaneous samples. In 20 broilers, E. coli was isolated from organs in pure culture or mixed with sparse growth of other bacteria. In eight of these, there were post-mortem findings suggestive of systemic bacterial spread. The majority of the isolates from the same bird and flock belonged to the same serotype and sequence type and were genetically indistinguishable, but differed when compared between flocks. Common APEC virulence genes, i.e. chuA, fyuA, hlyF, iroN, irp2, iss, ompT, sitA, TerC, TraT, were present in > 87% of the isolates. We conclude that evidence of systemic spread of E. coli from cellulitis was present in some birds at time of slaughter but cannot be reliably detected at meat inspection.

Highly Pathogenic Avian Influenza (HPAI H5Nx, Clade 2.3.4.4.b) in Poultry and Wild Birds in Sweden: Synopsis of the 2020-2021 Season.

Highly pathogenic avian influenza (HPAI, Gs/Gd lineage) was introduced to Europe in 2005 and has since caused numerous outbreaks in birds. The 2020-2021 season was the hitherto most devastating when considering bird numbers and duration in Europe. Surveillance data, virologic results and epidemiologic investigations from the 2020-2021 outbreaks in Sweden were analysed. Subtypes H5N8 and H5N5 were detected on 24 farms with poultry or other captive birds. In wild birds, subtypes H5N8, H5N5, H5N1, H5N4, H5Nx were detected in 130 out of 811 sampled birds. There was a spatiotemporal association between cases in wild birds and poultry. Based on phylogeny and epidemiology, most of the introductions of HPAI to commercial poultry were likely a result of indirect contact with wild birds. A definite route of introduction to poultry could not be established although some biosecurity breaches were observed. No spread between farms was identified but airborne spread between flocks on the same farm was suspected. Our findings exemplify the challenges posed by the continuously changing influenza viruses that seem to adapt to a broader species spectrum. This points to the importance of wild bird surveillance, compliance to biosecurity, and identification of risk factors for introduction on poultry farms.

Anticoccidial Vaccination Is Associated with Improved Intestinal Health in Organic Chickens.

Eimeria spp. and Clostridium perfringens (CP) are pathogens associated with coccidiosis and necrotic enteritis (NE) in broiler chickens. In this study we evaluated the effect of anticoccidial vaccination on intestinal health in clinically healthy organic Ross 308 chickens. On each of two farms, one unvaccinated flock (A1 and B1) was compared to one vaccinated flock (A2 and B2) until ten weeks of age (WOA). Faecal oocysts were counted weekly, and species were identified by PCR (ITS-1 gene). Lesion scoring, CP quantification and PCR targeting the CP NetB toxin gene were performed at three, four, and six WOA and chickens were weighed. Necropsies were performed on randomly selected chickens to identify coccidiosis/NE. Oocyst shedding peaked at three WOA in all flocks. Later oocyst shedding (E. tenella/E. maxima) in unvaccinated flocks at 5-7 WOA coincided with coccidiosis/NE. Although results differed somewhat between farms, vaccination was associated with lower intestinal lesion scores, reduced caecal CP counts, lower proportions of netB-positive CP, lower body weight at three-four WOA, and similar or slightly increased body weight at six WOA. In conclusion, the intestinal health of organic broilers can benefit from anticoccidial vaccination when oocyst exposure levels are high.

Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate.

The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus "chicken embryo lethal orphan" (CELO). RESEARCH HIGHLIGHTSAGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing.Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1).Multiple point mutations, site insertions and deletions identified compared to CELO.Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

Occurrence and spread of a reassortant very virulent genotype of infectious bursal disease virus with altered VP2 amino acid profile and pathogenicity in some European countries.

Reassortant strains of Infectious Bursal Disease Virus (IBDV) were detected in commercial broiler flocks in the Netherlands, Belgium, Denmark, Czech Republic and Germany and in layers and organic broilers in Sweden in the period of 2017-19. Genetic analysis, based on hypervariable region of VP2 gene showed grouping together with very virulent IBDV strains (vvIBDV, Genogroup 3), but these recent viruses formed a separate cluster, which was most closely related to Latvian IBDV strains from 2010-13. VP1 gene of these isolates was most closely related to D78 attenuated IBDV strain. The recently described reassortant IBDV strain (Bpop/03/PL) from Poland with similar genomic constellation (segment A from vvIBDV, segment B from attenuated strain) retained its pathogenicity (80 % mortality in SPF chickens). Infection with the North-West European reassortant IBDVs described in this study showed subclinical feature in the field (without complicating agents) and when tested under standardized pathogenicity test in SPF layer chickens (no mortality or clinical signs, but marked bursa atrophy was observed). Although these recent North-West European reassortant strains had all amino acid residues in their VP2 gene which are considered as markers of vvIBDV strains, they exhibited typical amino acid changes compared to vvIBDV reference strains that should contribute to the determination of pathogenicity. Diagnostic investigations indicated that co-infection with fowl adenovirus or chicken infectious anaemia virus exaggerated the outcome of the IBDV infection (10-20 % mortality). Widespread presence of this reassortant IBDV group in clinically healthy flocks draws attention to the importance of active surveillance.

Diagnostics, epidemiological observations and genomic subtyping in an outbreak of pullorum disease in non-commercial chickens.

Salmonella Gallinarum biovar Pullorum (S. Pullorum) is a poultry pathogen associated with significant economic losses and animal suffering. Strict eradication programmes have eliminated S. Pullorum from the commercial poultry sector in most regions, but occasional outbreaks still occur in the non-commercial population. In 2012, pullorum disease was diagnosed in a non-commercial flock in Sweden. Epidemiological, post-mortem and bacteriological investigations identified three more infected flocks. To study the epidemiological relationships between the flocks and evaluate different subtyping methods for S. Pullorum, 13 isolates from the four infected flocks were investigated by pulsed-field gel electrophoresis (PFGE) and multi-locus variable number tandem repeat analysis (MLVA). Four isolates collected from two non-commercial flocks in 2001 were also included. Six representative isolates from 2012 were further analysed by high-throughput sequencing. To differentiate between biovars Gallinarum and Pullorum, ten regions of difference (RODs) were investigated by in silico PCR. Three different PFGE-types were observed from the four epidemiologically linked farms in 2012 and MLVA further discriminated the isolates. SNP typing based on high-throughput sequencing clustered the four farms from the 2012 outbreak in two pairs. The PFGE, MLVA and high-throughput sequencing results suggested at least two different sources of infection or a common genetically mixed source in 2012. High-throughput sequencing is useful both for subtyping of S. Pullorum and may also be used for differentiating between the two biovars Pullorum and Gallinarum.

Post mortem findings and their relation to AA amyloidosis in free-ranging Herring gulls (Larus argentatus).

Since the late 1990s, high mortality and declining populations have been reported among sea birds including Herring gulls (Larus argentatus) from the Baltic Sea area in Northern Europe. Repeated BoNT type C/D botulism outbreaks have occurred, but it remains unclear whether this is the sole and primary cause of mortality. Thiamine deficiency has also been suggested as a causal or contributing factor. With this study, we aimed to investigate gross and microscopic pathology in Herring gulls from affected breeding sites in Sweden in search of contributing diseases. Herring gulls from Iceland served as controls. Necropsies and histopathology were performed on 75 birds, of which 12 showed signs of disease at the time of necropsy. Parasites of various classes and tissues were commonly observed independent of host age, e.g. oesophageal capillariosis and nematode infection in the proventriculus and gizzard with severe inflammation, air sac larid pentastomes and bursal trematodiasis in pre-fledglings. Gross and microscopic findings are described. Notably, amyloidosis was diagnosed in 93 and 33% of the adult birds from Sweden and Iceland, respectively (p<0.001), with more pronounced deposits in Swedish birds (p<0.001). Gastrointestinal deposits were observed in the walls of arteries or arterioles, and occasionally in villi near the mucosal surface. Amyloid was identified within the intestinal lumen in one severely affected gull suggesting the possibility of oral seeding and the existence of a primed state as previously described in some mammals and chickens. This could speculatively explain the high occurrence and previously reported rapid onset of amyloidosis upon inflammation or captivity in Herring gulls. Amyloid-induced malabsorbtion is also a possibility. The Herring gull SAA/AA protein sequence was shown to be highly conserved but differed at the N-terminus from other avian species.

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers (anthelmintics). Benzimidazoles (BZ) are currently the only anthelmintic registered in the EU for use in controlling A. galli and there is an obvious risk of overuse of one drug class, selecting for resistance. Thus we developed a reference transcriptome of A. galli to investigate the response in gene expression before and after exposure to the BZ drug flubendazole (FLBZ). Transcriptional variations between treated and untreated A. galli showed that transcripts annotated as mitochondrial glutamate dehydrogenase and cytochrome P450 were significantly down-regulated in treated worms, whereas transcripts homologous to heat shock proteins (HSP), catalase, phosphofructokinase, and a multidrug resistance P-glycoprotein (PGP1) were significantly up-regulated in treated worms. Investigation of candidate transcripts responsible for anthelmintic resistance in livestock nematodes led to identification of several tubulins, including six new isoforms of beta-tubulin, and several ligand-gated ionotropic receptors and ABC-transporters. We discovered several transcripts associated with drug binding and processing genes, but further characterisation using a larger set of worms exposed to BZs in functional assays is required to determine how these are involved in drug binding and metabolism.

Occurrence of Salmonella spp.: a comparison between indoor and outdoor housing of broilers and laying hens.

BACKGROUND: Outdoor production of poultry is rapidly increasing, which could be associated with increased risks for exposure to different environmental sources of Salmonella. We report a comparison on the occurrence of Salmonella during 2007-2015 in broilers and laying hens in outdoor and indoor production subjected to the same requirements for the prevention and control of Salmonella as applied in Sweden. RESULTS: Our results give no indication that, during the period studied, the exposure to Salmonella in outdoor poultry production was higher than in the indoor production. The annual incidence of Salmonella infected flocks in outdoor production remained at a very low and at a similar level as for indoor production. For laying hens the annual proportion of birds in test positive flocks ranged from 0 to 1.3% for indoor production from 0 to 2.0% for outdoor production. For broilers the proportion of Salmonella infected flocks (2013-2015) was 0.16% for indoor, and 0% in outdoor production. The difference was not statistically significant and was further reduced when flocks infected due to vertical transmission or from a hatchery source were excluded. It should, however, be considered that the number of outdoor flocks included in this evaluation is very small and continuous evaluation is needed. CONCLUSIONS: New animal production systems, including those driven by consumer and welfare demands, may be associated with a higher risk for the exposure of potential pathogens to food animals and possibly also subsequent outbreaks of food borne infections. In this study no increase in the risk for exposure of flocks to Salmonella in outdoor poultry production was found. The situation may well change and the possibility of Salmonella contamination in outdoor poultry production requires continuous attention.

Intestinal spirochaetes (genus Brachyspira) colonise wild birds in the southern Atlantic region and Antarctica.

INTRODUCTION: The genus Brachyspira contains well-known enteric pathogens of veterinary significance, suggested agents of colonic disease in humans, and one potentially zoonotic agent. There are recent studies showing that Brachyspira are more widespread in the wildlife community than previously thought. There are no records of this genus in wildlife from the southern Atlantic region and Antarctica. Our aim was therefore, to determine whether intestinal spirochaetes of genus Brachyspira colonise marine and coastal birds in this region. METHOD: Faecal samples were collected from marine and coastal birds in the southern Atlantic region, including sub-Antarctic islands and Antarctica, in 2002, 2009, and 2012, with the aim to isolate and characterise zoonotic agents. In total, 205 samples from 11 bird species were selectively cultured for intestinal spirochaetes of genus Brachyspira. To identify isolates to species level, they were subjected to phenotyping, species-specific polymerase chain reactions, sequencing of partial 16S rRNA, NADH oxidase (nox), and tlyA genes, and phylogenetic analysis. Antimicrobial susceptibility tests were performed. RESULTS: Fourteen unique strains were obtained from 10 birds of three species: four snowy sheathbills (Chionis albus), three kelp geese (Chloephaga hybrida subsp. malvinarum), and three brown skua (Stercorarius antarcticus subsp. lonnbergi) sampled on the Falkland Islands, Tierra del Fuego in Argentina, South Georgia, South Shetland Islands, and the Antarctic Peninsula. Five Brachyspira strains were closely related to potentially enteropathogenic Brachyspira sp. of chickens: B. intermedia (n=2, from snowy sheathbills), and B. alvinipulli (n=3, from a kelp goose and two snowy sheathbills). Three strains from kelp geese were most similar to the presumed non-pathogenic species 'B. pulli' and B. murdochii, whereas the remaining six strains could not be attributed to currently known species. No isolates related to human strains were found. None of the tested strains showed decreased susceptibility to tiamulin, valnemulin, doxycycline, tylvalosin, lincomycin, or tylosin. CONCLUSIONS: This is the first report of intestinal spirochaetes from this region. Despite limitations of current diagnostic methods, our results, together with earlier studies, show that Brachyspira spp., including potentially pathogenic strains, occur globally among free-living avian hosts, and that this genus encompasses a higher degree of biodiversity than previously recognised.

Brachyspira suanatina sp. nov., an enteropathogenic intestinal spirochaete isolated from pigs and mallards: genomic and phenotypic characteristics.

BACKGROUND: The genus Brachyspira currently encompasses seven valid species that colonize the intestines of mammals and birds. In a previous study a group of strongly haemolytic isolates from pigs and mallards was provisionally described as a new species within genus Brachyspira, "B. suanatina", and enteropathogenic properties were demonstrated in a porcine challenge model. METHODS: In the current study characterization of B. suanatina was performed on the basis of cell morphology, growth characteristics, enzyme profiles, DNA-DNA hybridization (DDH) and whole genome comparisons. The draft genome sequence of B. suanatina strain AN4859/03 was determined and compared with the available genomes of all valid species of Brachyspira. RESULTS: According to morphological traits, growth characteristics and enzymatic profiles, B. suanatina was similar to the type strain of B. hyodysenteriae, but using the recommended threshold value of 70% similarity by DDH it did not belong to any of the recognized Brachyspira species (range 16-64% similarity). This was further supported by average nucleotide identity values. Phylogenetic analysis performed using housekeeping genes and core genomes of all valid Brachyspira sp. and "B. hampsonii" revealed that B. suanatina and B. intermedia formed a clade distinct from B. hyodysenteriae. By comparing the genomes of the three closely related species B. intermedia, B. hyodysenteriae and B. suanatina similar profiles of general genomic features and distribution of genes in different functional categories were obtained. However, the genome size of B. hyodysenteriae was smallest among the species, suggesting the possibility of reductive evolution in the divergence of this species. A bacteriophage region and a putative plasmid sequence were also found in the genome of B. suanatina strain AN4859/03. CONCLUSIONS: The results of our study suggest that despite being similar to B. hyodysenteriae phenotypically, B. suanatina should be regarded as a separate species based on its genetic characteristics. Based on characteristics presented in this report we propose that strains AN4859/03, AN1681:1/04, AN2384/04 and Dk12570-2 from pigs in Sweden and Denmark, and strains AN3949:2/02 and AN1418:2/01 isolated from mallards in Sweden, represent a unique species within genus Brachyspira. For this new species we propose the name B. suanatina for which the type strain is AN4859/03T (=ATCC® BAA-2592™=DSM 100974T).

Investigations into an Outbreak of Botulism Caused by Clostridium botulinum Type C/D in Laying Hens.

This case report describes a recent botulism outbreak in commercial laying hens with a history of increased mortality and flaccid paralysis. Routine diagnostic gross examination and microscopy from seven hens were inconclusive, but botulinum neurotoxin (BoNT) in peripheral blood was neutralized with both type C and type D antitoxins in the mouse bioassay. During a farm visit, 10 additional hens from a 34-wk-old flock on the farm were selected for clinical examination and further sampling. Nine hens were observed in sternal recumbency, with flaccid paralysis of the neck, drooping wings and tail, inability to escape, and bilateral ptosis, and one hen showed nonspecific clinical signs. Samples from cecum and liver were collected, and the gene coding for BoNT was detected by PCR in all 10 cecal samples and in four of the liver samples. Clostridium botulinum mosaic type C/D was isolated from 5 out of 10 hens from either cecum or liver, and the isolates were subjected to pulsed-field gel electrophoresis subtyping. All five isolates produced the same banding pattern, which was identical or showed >90% similarity to isolates from three different outbreaks on broiler farms in Sweden and Denmark during the 2007-10 period. However, they were clearly distinguishable from the predominantly reported pulsotype associated with avian botulism outbreaks in Europe. The authors are unaware of any previous report of C. botulinum mosaic type C/D isolates from laying hens.

Environmental tolerance of free-living stages of the poultry roundworm Ascaridia galli.

The poultry roundworm Ascaridia galli is re-emerging in laying hens in many European countries due to the increase in non-caged housing. A series of in vitro experiments was carried out to study the in ovo larval development (embryonation) under different environmental conditions. Between 83% and 96% of the eggs developed to L3 within 7-21 days of incubation in water between 20 and 30°C. Twenty-six percent completed development at 33°C and 4% at 35°C after 31 days. At 15°C parasite egg development was low with 8% L3 after 56 days. In another trial larval development occurred, when parasite eggs were exposed to freeze-thaw cycle (30' to 12h) followed by incubation for 2 weeks at 25°C. Alkaline and acidic conditions in the range of pH 2.5-12.5 had no adverse effect on development. Oxygen and relative humidity above 70% were necessary for development to occur. Thus, some A. galli eggs may complete development at conditions prevailing in poultry barns in temperate climate zones throughout the year. Although exposure to a 1% or 2% dilution of the broad-spectrum disinfectant chlorocresol for 4h or longer was ovicidal, further work is required to improve the method of application in the field.

Erysipelothrix rhusiopathiae contamination in the poultry house environment during erysipelas outbreaks in organic laying hen flocks.

This study investigated organic laying hen farms for the presence of Erysipelothrix rhusiopathiae in the house environment and from potential carriers (i.e. insects and mice) during ongoing erysipelas outbreaks, and compared the obtained isolates with those from laying hens. The samples were investigated by selective culture followed by species-specific polymerase chain reaction on cultures. E. rhusiopathiae was isolated from the spleen, jejunal contents, manure, dust and swabs from water nipples. Three more samples from the house environment tested positive by polymerase chain reaction compared with selective culture alone. Selected isolates were investigated by pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). One farm was represented by isolates from laying hens only, and one of these isolates differed in one PFGE band from the others. Different banding patterns were observed for isolates from laying hens and manure on one farm. On the remaining two farms, the isolates from the house environment and laying hens were identical but differed between farms. Outbreaks reoccurred in the next flock on two of the farms, and different PFGE types were isolated from consecutive flocks. Our results suggest an external source of infection, which would explain the previously reported increased risk of outbreaks in free-range flocks. Contaminated manure and dust may represent sources of transmission. For the isolates, MALDI-TOF MS and biochemical typing results were in agreement but, since the type strain of Erysipelothrix tonsillarum was typed as E. rhusiopathiae using MALDI-TOF MS, further studies into this method are needed.

Population genetic structure of Ascaridia galli re-emerging in non-caged laying hens.

BACKGROUND: The poultry roundworm Ascaridia galli has reappeared in hens kept for egg production in Sweden after having been almost absent a decade ago. Today this is a frequent intestinal nematode parasite in non-caged laying hens. The aim of this study was to investigate the genetic diversity (Fst) in A. galli collected from different poultry production sites in southern Sweden, to identify possible common routes of colonization. METHODS: Adult parasites (n = 153) from 10 farms, including both broiler breeder parents and laying hens, were investigated by amplified restriction fragment length polymorphism analysis (AFLP). Worms from a Danish laying hen farm were also included for comparison. Most of the farms were represented by worms from a single host, but on two farms multiple samples from different hosts were assessed in order to study flock variation. RESULTS: A total of 97 fragments (loci) were amplified among which 81% were variable alleles. The average genetic diversity was 0.13 (range = 0.09-0.38), which is comparable to other AFLP studies on nematodes of human and veterinary importance. Within-farm variation showed that worms harboured by a single hen in a flock covered most of the A. galli genetic variation within the same flock (Fst = 0.01 and 0.03 for two farms). Between-farm analysis showed a moderate population genetic structure (Fst = 0.13), along with a low mutational rate but high gene flow between different farms, and absence of strong genetic selection. Network analysis showed repeated genetic patterns among the farms, with most worms on each farm clustering together as supported by high re-allocation rates. CONCLUSIONS: The investigated A. galli populations were not strongly differentiated, indicating that they have undergone a genetic bottlenecking and subsequent drift. This supports the view that the investigated farms have been recently colonized, and that new flocks are reinfected upon arrival with a stationary infection.

Characterization of encephalitis in wild birds naturally infected by highly pathogenic avian influenza H5N1.

During the outbreak of highly pathogenic avian influenza (HPAI) H5N1 in Sweden in 2006, disease and mortality were observed in a number of wild bird species. Encephalitis was one of the most consistent and severe findings in birds submitted for postmortem examination. However, the distribution and severity of the inflammation varied among individuals. This study characterized the encephalitis and the phenotype of the cellular infiltrate in brains of 40 birds of various species naturally infected with HPAI H5N1. Brain sections stained with hematoxylin and eosin and immunostained for influenza A viral antigen were evaluated in parallel to brain sections immunostained with antibodies against T lymphocytes (CD3+), B lymphocytes (CD79a+), macrophages (Lectin RCA-1+), and astrocytes expressing glial fibrillary acidic protein. The virus showed marked neurotropism, and the neuropathology included multifocal to diffuse areas of gliosis and inflammation in the gray matter, neuronal degeneration, neuronophagia, vacuolation of the neuropil, focal necrosis, perivascular cuffing, and meningitis. Broad ranges in severity, neuroanatomical distribution, and type of cellular infiltrate were observed among the different bird species. Since neurotropism is a key feature of HPAI H5N1 infection in birds and other species and because the clinical presentation can vary, the characterization of the inflammation in the brain is important in understanding the pathogenesis of the disease and also has important diagnostic implications for sample selection.

Emergence of novel strains of avian infectious bronchitis virus in Sweden.

Infectious bronchitis virus (IBV) causes avian infectious bronchitis, an important disease that produces severe economic losses in the poultry industry worldwide. Recent IBV infections in Sweden have been associated with poor growth in broilers, drop in egg production and thin egg shells in layers. The complete spike gene of selected isolates from IBV cases was amplified and sequenced using conventional RT-PCR. Nucleotide and amino acid sequence comparisons have shown that the recent isolates bear 98.97% genetic similarity with strains of the QX-like genotype. The phylogenetic analysis revealed that strains predominant in the nineties, which were of the Massachusetts type, have been replaced by D388/QX-like strains, however the evolutionary link could not be established. The homology between the two genotypes was 79 and 81%. Remarkably, a strong positive selection pressure was determined, mostly involving the S1 subunit of the S gene. This strong selective pressure resulted in recombination events, insertions and deletions in the S gene. Two new isolates generated from recombination were found with nucleotide sequence diverging 1.7-2.4% from the D388/QX-like branch, indicating the emergence of a new lineage. The study demonstrates a constant evolution of IBV that might be in relation to increased poultry farming, trade and vaccine pressure. The findings underscore the importance of continuous monitoring to control spread of infections, as well as to timely adjust diagnostic methods, molecular epidemiological studies, development and use of vaccines that are adapted to the changing disease scenario.