Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes - adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

Risk-based surveillance for avian influenza control along poultry market chains in South China: The value of social network analysis.

Over the past two decades, the poultry sector in China went through a phase of tremendous growth as well as rapid intensification and concentration. Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 was first detected in 1996 in Guangdong province, South China and started spreading throughout Asia in early 2004. Since then, control of the disease in China has relied heavily on wide-scale preventive vaccination combined with movement control, quarantine and stamping out. This strategy has been successful in drastically reducing the number of outbreaks during the past 5 years. However, HPAIV H5N1 is still circulating and is regularly isolated in traditional live bird markets (LBMs) where viral infection can persist, which represent a public health hazard for people visiting them. The use of social network analysis in combination with epidemiological surveillance in South China has identified areas where the success of current strategies for HPAI control in the poultry production sector may benefit from better knowledge of poultry trading patterns and the LBM network configuration as well as their capacity for maintaining HPAIV H5N1 infection. We produced a set of LBM network maps and estimated the associated risk of HPAIV H5N1 within LBMs and along poultry market chains, providing new insights into how live poultry trade and infection are intertwined. More specifically, our study provides evidence that several biosecurity factors such as daily cage cleaning, daily cage disinfection or manure processing contribute to a reduction in HPAIV H5N1 presence in LBMs. Of significant importance is that the results of our study also show the association between social network indicators and the presence of HPAIV H5N1 in specific network configurations such as the one represented by the counties of origin of the birds traded in LBMs. This new information could be used to develop more targeted and effective control interventions.

Impacts on nontarget avian species from aerial meat baiting for feral pigs

Bait containing sodium fluoroacetate (1080) is widely used for the routine control of feral pigs in Australia. In Queensland, meat baits are popular in western and northern pastoral areas where they are readily accepted by feral pigs and can be distributed aerially. Field studies have indicated some levels of interference and consumption of baits by nontarget species and, based on toxicity data and the 1080 content of baits, many nontarget species (particularly birds and varanids) are potentially at risk through primary poisoning. While occasional deaths of species have been recorded, it remains unclear whether the level of mortality is sufficient to threaten the viability or ecological function of species. A series of field trials at Culgoa National Park in south-western Queensland was conducted to determine the effect of broadscale aerial baiting (1.7 baits per km2) on the density of nontarget avian species that may consume baits. Counts of susceptible bird species were conducted prior to and following aerial baiting, and on three nearby unbaited properties, in May and November 2011, and May 2012. A sample of baits was monitored with remote cameras in the November 2011 and May 2012 trials. Over the three baiting campaigns, there was no evidence of a population-level decline among the seven avian nontarget species that were monitored. Thirty per cent and 15% of baits monitored by remote cameras in the November 2011 and May 2012 trials were sampled by birds, varanids or other reptiles. These results support the continued use of 1080 meat baits for feral pig management in western Queensland and similar environs.

Coronavirus Infection and Diversity in Bats in the Australasian Region

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

Avian influenza in Australia: a summary of 5 years of wild bird surveillance

BackgroundAvian influenza viruses (AIVs) are found worldwide in numerous bird species, causing significant disease in gallinaceous poultry and occasionally other species. Surveillance of wild bird reservoirs provides an opportunity to add to the understanding of the epidemiology of AIVs. MethodsThis study examined key findings from the National Avian Influenza Wild Bird Surveillance Program over a 5-year period (July 2007-June 2012), the main source of information on AIVs circulating in Australia. ResultsThe overall proportion of birds that tested positive for influenza A via PCR was 1.90.1%, with evidence of widespread exposure of Australian wild birds to most low pathogenic avian influenza (LPAI) subtypes (H1-13, H16). LPAI H5 subtypes were found to be dominant and widespread during this 5-year period. ConclusionGiven Australia's isolation, both geographically and ecologically, it is important for Australia not to assume that the epidemiology of AIV from other geographic regions applies here. Despite all previous highly pathogenic avian influenza outbreaks in Australian poultry being attributed to H7 subtypes, widespread detection of H5 subtypes in wild birds may represent an ongoing risk to the Australian poultry industry.

Coriza infecciosa: revisión de métodos de diagnóstico y vacunas

La coriza infecciosa es una enfermedad respiratoria aguda de las gallinas domesti- cas causada por la bacteria Haemophilus parugallinarum. Excepcionalmente pueden enfermarse tambien los faisanes y gallinas de Guinea. El H. paragallinarum infecta al ave por via respiratoria y luego de un cor- to periodo de incubation, que varia entre 1 a 3 dias, produce una enfermedad que se manifiesta por inflamacion catarral de los senos paranasales. Este cuadro puede estar asociado a inflamacion de los barbillones, conjuntivitis o queratitis. Los casos de neu- nionia y aerosaculitis son menos frecuentes pero tambien suelen ocurrir en las infeccio- nes puras por estos hemofilos. En las gallinas en produccion causa alta morbilidad, baja o nula mortalidad y una importante perdida en la produccion de huevos, la que generalmente oscila entre 10% y 40%. En pollos parrilleros puede cau- sar un cuadro descrito como «cabeza hin- chada» y ocasionalmente tambien producir septicemia y muerte (48). Esta bacteria ge- neralmente se asocia con otros agentes bacterianos, viricos o parasitarios y cuan- do esto ocurre se agrava el curso de la en- fermedad. Entre los agentes bacterianos mas comunes deben mencionarse los mycoplasinas y las pasteurelas. Cuando H . paragallinarum se asocia con otros agentes esta enfermedad se denomina .«coriza infec- ciosa complicada» (48). En esta recopilacion se aportaran deta- lles sobre la clasificacion, identificacion y serotipificacion del agente causal. Tambien se resumira la informacion disponible sobre nuevos metodos de diagnostico y programas de vacunacion para prevenir esta enferme-dad. A lo largo de esta revision se hara re-ferencia a los hemofilos aviarios que, para el proposito de este trabajo, seran definidos como organisnios gram negativos aislados de aves y que necesariamente requieren factores de crecimiento in vitro. Los dos factores que pueden ser requeridos por los hemofilos para su crecimiento in vitro son hemina (factor X) y/o nicotin-adenin-dinucleirtido (NAD o factor V).

Presence of Nicotinamide Adenine Dinucleotide–Independent Haemophilus paragallinarum in Mexico

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent—growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3 wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NADindependent H. paragallinarum of serovar B has been reported. Abbreviations: NAD ¼ nicotinamide adenine dinucleotide; NAM ¼ nicotinamide; PCR ¼ polymerase chain reaction; TM ¼ complete growth medium without chicken serum or nicotinamide adenine dinucleotide; TM/SN ¼ complete growth medium that contains both chicken serum and nicotinamide adenine dinucleotide

Virulence of the Nine Serovar Reference Strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.

Cross-protection study of the nine serovars of Haemophilus paragallinarum in the Kume haemagglutinin scheme

The cross-protection and haemagglutination-inhibition antibodies present in chickens vaccinated with one of the nine currently recognized Kume haemagglutinin serovars of Haemophilus paragallinarum were investigated. The results confirmed the widely accepted dogma that serogroups A, B, and C represent three distinct immunovars. Within Kume serogroup A, there was generally good cross-protection among all four serovars. However, within Kume serogroup C, there was evidence of a reduced level of cross protection between some of the four serovars. The haemagglutination-inhibition antibody levels generally showed the same trend as with the cross-protection results. This study suggests that some apparent field failures of infectious coryza vaccines may be due to a lack of cross-protection between the vaccine strains and the field strains. Our results will help guide the selection of strains for inclusion in infectious coryza vaccines.

Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov.

This paper describes a phenotypic and genotypic investigation of the taxonomy of [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium, a major subcluster within the avian 16S rRNA cluster 18 of the family Pasteurellaceae. An extended phenotypic characterization was performed of the type strain of [Haemophilus] paragallinarum, which is NAD-dependent, and eight NAD-independent strains of [Haemophilus] paragallinarum. Complete 16S rRNA gene sequences were obtained for one NAD-independent and four NAD-dependent [Haemophilus] paragallinarum strains. These five sequences along with existing 16S rRNA gene sequences for 11 other taxa within avian 16S rRNA cluster 18 as well as seven other taxa from the Pasteurellaceae were subjected to phylogenetic analysis. The analysis demonstrated that [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium formed a monophyletic group with a minimum of 96·8% sequence similarity. This group can also be separated by phenotypic testing from all other recognized and named taxa within the Pasteurellaceae. As both genotypic and phenotypic testing support the separate and distinct nature of this subcluster, the transfer is proposed of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium to a new genus Avibacterium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. The type strains are NCTC 1118T (Avibacterium gallinarum), NCTC 11296T (Avibacterium paragallinarum), NCTC 11297T (Avibacterium avium) and NCTC 3438T (Avibacterium volantium). Key characteristics that separate these four species are catalase activity (absent only in Avibacterium paragallinarum) and production of acid from galactose (negative only in Avibacterium paragallinarum), maltose (negative only in Avibacterium avium) and mannitol (negative in Avibacterium gallinarum and Avibacterium avium).

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera

A 5′ Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5′ Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5′ Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Distributions of lesser mealworm (Coleoptera: Tenebrionidae) in litter of a compacted earth floor broiler house in subtropical Queensland, Australia

Distributions of lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), in litter of a compacted earth floor broiler house in southeastern Queensland, Australia, were studied over two flocks. Larvae were the predominant stage recorded. Significantly low densities occurred in open locations and under drinker cups where chickens had complete access, whereas high densities were found under feed pans and along house edges where chicken access was restricted. For each flock, lesser mealworm numbers increased at all locations over the first 14 d, especially under feed pans and along house edges, peaking at 26 d and then declining over the final 28 d. A life stage profile per flock was devised that consisted of the following: beetles emerge from the earth floor at the beginning of each flock, and females lay eggs, producing larvae that peak in numbers at 3 wk; after a further 3 to 4 wk, larvae leave litter to pupate in the earth floor, and beetles then emerge by the end of the flock time. Removing old litter from the brooder section at the end of a flock did not greatly reduce mealworm numbers over the subsequent flock, but it seemed to prevent numbers increasing, while an increase in numbers in the grow-out section was recorded after reusing litter. Areas under feed pans and along house edges accounted for 5% of the total house area, but approximately half the estimated total number of lesser mealworms in the broiler house occurred in these locations. The results of this study will be used to determine optimal deployment of site-specific treatments for lesser mealworm control.

The vaccination-challenge trial: the gold standard test to evaluate the protective efficacy of infectious coryza vaccines

Infectious coryza is an upper respiratory tract disease of chickens with the major impact occurring in multi-age flocks. We investigated the relationship between the level of antibodies, as detected by a haemagglutination-inhibition (HI) assay, in infectious coryza-vaccinated chickens and the protection against challenge in those chickens. In one experiment, chickens given a single dose of either of two infectious coryza vaccines lacked a detectable HI response to vaccination but showed significant levels of protection 11 weeks after vaccination. In contrast, in chickens given two doses of an infectious coryza vaccine and challenged 3 weeks after the second vaccine dose, there was a strong serological response with 36/40 birds having a HI titre of 1/20 or greater. In this trial there was an apparent relationship between titre and subsequent protection, with none of the 32 chickens with a titre of 1/40 or 1/80 showing any clinical signs and only one of the same group yielding the challenge organism on culture. In contrast, three of the four vaccinated chickens with a HI titre less than 1/5 developed the typical clinical signs of coryza and yielded the challenge organism on culture. Overall, our results suggest that HI titres cannot be regarded as a definitive predictor of vaccine efficacy. We suggest that the vaccination-challenge trial is the gold standard for the evaluation of the immune response to infectious coryza vaccines.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.