Comparative examination of a rapid immunocytochemical test for the detection of highly pathogenic avian influenza virus in domestic birds in field outbreaks.

The quantitative real-time reverse polymerase chain reaction (RRT-PCR) is the preferred test method for the diagnosis of avian influenza (AI), but can be performed only in specialized laboratories. Different antigen detection methods for the diagnosis of AI were previously reported to be specific and sensitive in field outbreaks. These tests can be performed in basic countryside labs. Brain smears of domestic birds (n = 105) collected during AI field outbreaks were examined with immunocytochemistry (IC). The results were statistically analysed by comparing IC to brain histology (BH), and immunohistochemistry (IHC), to gross pathological examination (GP) (n = 105), and RRT-PCR (n = 91). AI was diagnosed with RRT-PCR in 66 cases. IC and IHC were positive in 59/66 (90%) and 60/66 (91%) cases, respectively. Lesions suspicious for AI were detected with GP and HP in 66/66 (100%) and 61/66 (92%) cases, respectively. An almost perfect agreement was found between RRT-PCR, IC, IHC, and HP. Substantial agreement was found between IC and GP, between IHC and GP, between HP and GP, and between RRT-PCR and GP. The chromogen-based IC test presented in this study produces durable staining, which can be evaluated using a simple brightfield microscope. The test is rapid (can be completed in 2 h), sensitive (90%), specific (100%), and cost-effective, which makes the method suitable for routine diagnostic tests in AI epidemics.RESEARCH HIGHLIGHTSAvian influenza virus (AIV) antigen detection was examined in field outbreaks.Bird brain smears were tested using immunocytochemistry (IC).IC results strongly correlated with real-time RT-PCR results.The IC method was rapid, specific, sensitive, and cost-effective in AIV field outbreaks.

Development and evaluation of temperature-sensitive Mycoplasma anserisalpingitidis clones as vaccine candidates.

Mycoplasma anserisalpingitidis is economically the most important pathogenic Mycoplasma species of waterfowl in Europe and Asia. The lack of commercially available vaccines against M. anserisalpingitidis had prompted this study with the aim to produce temperature-sensitive (ts+) clones as candidates for an attenuated live vaccine. The production of ts+ clones was performed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis of Hungarian M. anserisalpingitidis field isolates. The clones were administered via eye-drop and intracloacally to 33-day-old geese. Colonization ability was examined by PCR and isolation from the trachea and cloaca, while the serological response of the birds was tested by ELISA. Pathological and histopathological examinations were performed in the eighth week after inoculation. Whole-genome sequence (WGS) analysis of the selected clone and its parent strain was also performed. NTG-treatment provided three ts+ mutants (MA177/1/11, MA177/1/12, MA271). MA271 was detected at the highest rate from cloacal (86.25%) and tracheal (30%) samples, while MA177/1/12 and MA271 elicited remarkable serological responses with 90% of the birds showing seroconversion. Re-isolates of MA271 remained ts+ throughout the experiment. Based on these properties, clone MA271 was found to be the most promising vaccine candidate. WGS analysis revealed 59 mutations in the genome of MA271 when compared to its parent strain, affecting both polypeptides involved in different cellular processes and proteins previously linked to bacterial fitness and virulence. Although further studies are needed to prove that MA271 is in all aspects a suitable vaccine strain, it is expected that this ts+ clone will contribute to the control of M. anserisalpingitidis infection.RESEARCH HIGHLIGHTS Three M. anserisalpingitidis ts+ vaccine candidates were produced by NTG-mutagenesis.Clone MA271 was able to colonize geese and induce a serological response.MA271 re-isolates remained ts+ during the 8-week-long experiment.WGS analysis revealed 59 mutations in the genome of MA271.

Candidate 'Avian orthoreovirus B': An emerging waterfowl pathogen in Europe and Asia?

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.

First isolation of atypical enteropathogenic Escherichia coli from geese (Anser anser domestica) and first description of atypical EPEC from turkeys and pigeons in Hungary.

BACKGROUND: Escherichia coli is a bacterial species widely distributed among mammals and avian species, and also a member of the normal intestinal microbiota. However, some E. coli strains of different pathotypes can cause disease in both humans and animals. Atypical enteropathogenic E. coli (aEPEC) can infect both animals and humans or influence the severity of other ongoing infections. RESULTS: In the present study, a total of 332 samples were collected from ducks, geese, turkeys, chickens, and pigeons from the Hungarian Veterinary Diagnostic Directorate, two slaughterhouses, two pigeon keepers and one backyard chicken farm. E. coli was isolated and verified from 319 samples. The isolates were screened by PCR for diarrheagenic E. coli pathotypes. Altogether seven atypical enteropathogenic E. coli (aEPEC) strains were identified: two from four-week-old dead turkeys, two from force-fed geese, and three from pigeons. No further pathotypes were identified in the collection. The atypical EPEC strains were classified phylogenetically to B1, B2, and F, and four out of the seven aEPEC isolates proved to be multidrug resistant. Serotypes of aEPEC strains were uniform collected from same farms and showed diversity between their origins with O76, O145, O109 serogroups. CONCLUSIONS: This is the first report in the literature about aEPEC in goose (Anser anser domestica). Furthermore, this is the first isolation of aEPEC from turkeys and pigeons in Hungary. The uneven distribution of aEPEC in different age groups of poultry suggests that aEPEC disappears with growing up, but stress (e.g.: force-feeding) and concurrent diseases might promote its reappearance in the intestine.

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary.

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A-E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.

Comparative Analysis of Pasteurella multocida Isolates from Acute and Chronic Fowl Cholera Cases in Hungary During the Period 2005 Through 2010.

Fowl cholera (FC) is a highly contagious and economically important disease of poultry worldwide. This study was performed on 218 Pasteurella multocida isolates collected from separated breeding farms or backyards with acute and chronic FC cases in multiple localities across Hungary during the period 2005-2010. All isolates were characterized by a broad range of biochemical, serological, and molecular methods, as well as their antibiotic susceptibility to aminoglycosides (A), macrolides (M), penicillins (P), quinolones (Q), cephalosporins, sulphonamides (S), and tetracyclines (T) was determined. Fifty-two percent of all isolates belonged to a well-defined type that was highly virulent, caused acute FC, and had the same character: fermented L-arabinose, possessed capsule type A, identified as Heddleston serotype 1, and possessed allele type A of the ptfA fimbrial gene. This type was widely distributed among poultry in Hungary, especially in waterfowl flocks. Isolates collected from the chronic FC cases were more diverse: none of them fermented L-arabinose; they possessed capsule type A (76%), F (9%), or was non-typeable (15%) with different Heddleston serotypes, mainly 1, 3, 4, and 5, or 7 and 16; in addition, possessed allele type B of ptfA fimbrial gene. Only 26 isolates presented characters similar to any of the chronic FC cases but caused severe disease. The antibiotic susceptibility assay presented that 80% of all isolates were resistant to 1-5 of the studied antimicrobial agents. During the survey, after two years, there was a dramatic decline both in the number of the multi-drug resistance phenotypes and the prevalence of the highly virulent type of the isolates. In the next four years, multiresistant isolates were almost completely removed, whereas the number of isolates resistant to 1 or 2 drugs was constant. Reduced frequency of antibiotic multiresistant, mostly L-arabinose-fermenting isolates, has been observed since 2007. This reduction may be a consequence of the elimination of multiple waterfowl flocks in Hungary during avian influenza outbreaks, which possibly created a break in the "transmission chain" of pathogenic P. multocida isolates.

Neuroinvasive influenza virus A(H5N8) in fattening ducks, Hungary, 2015.

Highly pathogenic avian influenza (HPAI) A virus H5N8 was detected in far east Asian countries during 2014 and emerged in late 2014 in European countries. Hungary reported a HPAI A(H5N8) outbreak during late winter of 2015 at a Pekin duck fattening facility. Epidemiologic monitoring was extended to holdings in neighboring areas and nearby habitats used by wild birds but failed to identify the source of infection. In addition to respiratory symptoms, the affected birds showed lethargy and neuronal signs, including torticollis. Consistent with this finding, influenza A virus antigen was detected in large quantity in the brain. Molecular analysis of the identified strain showed very close genetic relationship (and >99% nucleotide sequence identity) with co-circulating HPAI A(H5N8) strains. A number of unique or rarely detected amino acid changes was detected in the HA (T220I, R512G), the M2 (I39M), the NA (T211I), the NS1 (P85T), and the PB2 (I261V) proteins of the Hungarian strain. Further studies are needed to demonstrate whether any of these mutations can be linked to neuroinvasiveness and neurovirulence in ducks.

Characterization of Pasteurella multocida strains isolated from geese.

Phenotypic and genotypic diversity of forty-two Pasteurella multocida isolates from geese were characterized by analysis of their capsular type, Heddleston serotype, biotype, fimbrial gene allele type, comparative outer membrane protein (OMP) electrophoresis patterns, and were analyzed using PCR for the presence of virulence-associated genes (toxA, tbpA, pfhA, hgbA, hgbB, nanH, nanB, fimA, hsf-1, and pmHAS). A sequence comparison of the thdF and rpoB housekeeping genes of twenty representative P. multocida strains from three different OMP groups demonstrated that seventeen strains were closely related phylogenetically to previously published strains of P. multocida subsp. multocida and P. multocida subsp. gallicida, and only three strains from geese were grouped with previously published strains of P. multocida subsp. septica. This study is the first report regarding the characterization of phenotypic and genotypic features of different P. multocida field strains isolated from geese with the different clinical representation of pasteurellosis and provide our overview on the usefulness of these in vitro tests for primary characterization of P. multocida strains for the epidemiological studies of fowl cholera.

Experimental inoculation of day-old ducks with Brachyspira pilosicoli and B. alvinipulli.

Two groups of one-day-old Peking ducklings (Groups I and II, 12 birds/group) were inoculated orally with Brachyspira pilosicoli and two groups with B. alvinipulli (Groups III and IV, 12 birds/group). T-2 toxin was added to the feed of Groups II and IV in a dose of 1 mg/kg of feed. Groups V and VI served as uninfected control groups (ducks of Group VI received T-2 toxin). The body weight gain of the ducks was measured and clinical signs were monitored continuously. The birds were sacrificed and necropsied on days 7, 14, 21, and 28 post infection (PI). The liver, spleen, kidney, thymus, bursa of Fabricius, ileum, caecum and colon were examined histologically. Culturing of Brachyspira spp. and immunohistochemistry were performed from the sampled parts of the intestines as well. No gross pathological or histological lesions that could be associated with B. pilosicoli or B. alvinipulli were detectable in the intestinal mucous membrane including the colonised intestinal glands. Mortality did not occur during the experimental period. Decrease in body weight gain was significant in the T-2-toxin-treated groups, and it was slight (not significant) in the Brachyspira-infected groups. Crust on the beaks, necrosis, crusting and ulceration in the mucous membrane of the oral cavity and on the skin of the feet, atrophy of the thymus and bursa of Fabricius due to the effect of T-2 toxin, accompanied by lymphocyte depletion, were observed. These lesions were most prominent on days 14 and 21 PI but were seen on day 28 PI as well. Immunohistochemical detection and reisolation of B. pilosicoli and B. alvinipulli were successful on days 7, 14, 21 and 28 days from different segments of the intestine of certain birds, but no significant difference was observed in the colonisation rate between the T-2-toxin-treated and the untreated groups.

Typhlocolitis associated with spirochaetes in duck flocks.

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunopositive neuronal elements in the lateral septum: rostrocaudal distribution in the male rat.

The morphological features and distribution of cocaine- and amphetamine-regulated transcript peptide immunoreactivity (CART-IR) were studied in the lateral septum (LS) of male rats using light and electron microscopic immunocytochemistry and computer-aided densitometry. CART-IR was detected along the rostrocaudal axis of the LS in varicose axonal fibers only, although immunoreactive cell bodies and dendrites were not detected. Pericellular basket-like arrangements around immunonegative cell bodies were present. From among the targets of such pericellular baskets, glutamic acid decarboxylase (GAD)-immunopositive and NPY-immunoreactive somata were identified. Thin varicose axons were present in each section, whereas thick varicose axons were restricted to the sections of rostral position only. CART-IR was observed in varicose fibers forming a dense subependymal plexus, from which solitary varicose fibers entered the ependymal layer. The fine structure of varicosities was similar to that of other neuropeptide-containing fibers. Small varicosities established asymmetrical synaptic contacts mainly with dendrites and dendritic spines, and larger varicosities established symmetrical synapses with somata and dendritic shafts. CART-to-CART connections were not revealed. The density curve of the CART-IR along the rostrocaudal axis of LS was found to be paraboloid. CART is known as one of the most anorexigenic peptides. These results serve as basis for further physiological studies concerning the biological significance of lateral septal CART peptide in the regulation of food intake.