C. Turni

Validation of a real-time PCR for Haemophilus parasuis

Aims:  To validate a real‐time PCR test for the diagnosis of Glässer’s disease, a major pig disease caused by Haemophilus parasuis.

Antimicrobial resistance in bacteria associated with porcine respiratory disease in Australia

The porcine respiratory disease complex greatly affects the health and production of pigs. While antimicrobial agents are used to treat the respiratory infections caused by bacterial pathogens, there is no current information on antimicrobial resistance in Australian pig respiratory bacterial isolates. The aim of this study was to determine the antimicrobial resistance profiles, by determining the minimum inhibitory concentration of nine antimicrobial agents for 71 Actinobacillus pleuropneumoniae, 51 Pasteurella multocida and 18 Bordetella bronchiseptica cultured from Australian pigs. The majority of A. pleuropneumoniae isolates were resistant to erythromycin (89%) and tetracycline (75%). Resistance to ampicillin (8.5%), penicillin (8.5%) and tilmicosin (25%) was also identified. The P. multocida isolates exhibited resistance to co-trimoxazole (2%), florfenicol (2%), ampicillin (4%), penicillin (4%), erythromycin (14%) and tetracycline (28%). While all the B. bronchiseptica isolates showed resistance to beta-lactams (ampicillin, ceftiofur and penicillin), some were resistant to erythromycin (94%), florfenicol (6%), tilmicosin (22%) and tetracycline (39%). The incidence of multiple drug resistance (MDR) varied across the species - in B. bronchiseptica, 27.8% of resistant isolates showed MDR, while 9.1% of the resistant isolates in A. pleuropneumoniae, and 4.8% in P. multocida showed MDR. This study illustrated that Australian pig strains of bacterial respiratory pathogens exhibited low levels of resistance to antimicrobial agents commonly used in the pig industry.

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

ABSTRACT Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the “gold standard.” The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Serovar profiling of Haemophilus parasuis on Australian farms by sampling live pigs

OBJECTIVE Investigate the diversity of serovars of Haemophilus parasuis (Hps) present in Australian pig herds. DESIGN Nasal swabs were used to obtain multiple isolates of Hps, which were grouped first by genotyping and then by serotyping representative isolates. PROCEDURE Swabs were taken from the nasal cavity of just-weaned healthy pigs from multiparous sows on 12 farms and from post-weaned pigs of multiparous sows on 1 farm. On 5 of the 13 farms, nasal swabs were also obtained from pigs showing clinical signs suggestive of Glässers disease. On a further 7 farms, nasal swabs were obtained only from pigs with clinical signs suggestive of Glässers disease. RESULTS A total of 556 Hps isolates were genotyped, and 150 isolates were serotyped. Hps was detected on 19 of the 20 farms, including 2 farms with a long history of freedom from Glässers disease. Isolates of Hps belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glässers disease outbreaks. Sampling 213 sick pigs yielded 115 isolates of Hps, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these 213 sick pigs were of a serovar known to be non-pathogenic. CONCLUSION Healthy pigs contain a range of Hps serovars, even on farms free of Glässers disease. Nasal swabbing of both healthy and sick pigs seems a useful method of serovar profiling of farms.

Parasites of the bridled nailtail wallaby (Onychogalea fraenata) (Marsupialia : Macropodidae)

The bridled nailtail wallaby (Onychogalea fraenata), an endangered macropod, has been reintroduced into the wild after a captive-breeding program. As part of a management program to assess the risks to its survival O. fraenata were trapped and examined for ecto- and endoparasites. From February to September 1996, 55 wallabies from Taunton National Park, central Queensland, some trapped more than once, were visually examined for ectoparasites. The blood of 39 O. fraenata was tested for antibodies against Toxoplasma gondii and Echinococcus granulosus and a total of 82 faecal samples were examined microscopically. In addition, in a second study a complete carcase, three complete gastro-intestinal tracts, and a single stomach, obtained from various sources, including Idalia National Park, were examined for helminth parasites. The most prevalent ectoparasites were the ticksAmbylomma triguttatum and Haemaphysalis bancrofti. Other ectoparasites included four species of trombiculid mites and a louse, Heterodoxus sp. A single instance of the nippoboscid fly, Ortholfersia minuta, was found. From the serological surveys, antibodies against Toxoplasma and Echinococcus were detected in 15% and 21% respectively. No trematode or cestode eggs or protozoal cysts were found in faeces. Nematode eggs had a prevalence of 92% with a mean egg density of 500 eggs per gram. Strongyloides sp. (larvae) was the most prevalent nematode in faeces. In the postmortem study, seven nematode species (Cloacina polyxo, Hypodontus macropi, Labiostrongylus onychogale, Macropostrongyloides baylisi, Macropoxyuris sp., Rugopharynx australis and Zoniolaimus buccalis) and four cestode species (Progamotaenia bancrofti, P. zschokkei, P. abietiformis and larval E. granulosus) were found. Six of the nematode species are new host records. The presence of infection with the introduced parasites T. gondii and E. granulosus, both recognised as serious pathogens, is of management significance. Since the definitive hosts of these parasites are cats and canids respectively, control of cat, dog and dingo populations within the Park will lessen the incidence of infection with these parasites.

Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non‐A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species‐specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar‐specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations.

Pasteurella multocida Heddleston Serovar 3 and 4 Strains Share a Common Lipopolysaccharide Biosynthesis Locus but Display both Inter- and Intrastrain Lipopolysaccharide Heterogeneity

Pasteurella multocida is a Gram-negative multispecies pathogen and the causative agent of fowl cholera, a serious disease of poultry which can present in both acute and chronic forms. The major outer membrane component lipopolysaccharide (LPS) is both an important virulence factor and a major immunogen. Our previous studies determined the LPS structures expressed by different P. multocida strains and revealed that a number of strains belonging to different serovars contain the same LPS biosynthesis locus but express different LPS structures due to mutations within glycosyltransferase genes. In this study, we report the full LPS structure of the serovar 4 type strain, P1662, and reveal that it shares the same LPS outer core biosynthesis locus, L3, with the serovar 3 strains P1059 and Pm70. Using directed mutagenesis, the role of each glycosyltransferase gene in LPS outer core assembly was determined. LPS structural analysis of 23 Australian field isolates that contain the L3 locus revealed that at least six different LPS outer core structures can be produced as a result of mutations within the LPS glycosyltransferase genes. Moreover, some field isolates produce multiple but related LPS glycoforms simultaneously, and three LPS outer core structures are remarkably similar to the globo series of vertebrate glycosphingolipids. Our in-depth analysis showing the genetics and full range of P. multocida lipopolysaccharide structures will facilitate the improvement of typing systems and the prediction of the protective efficacy of vaccines.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks

Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.

Epidemiology of Fowl Cholera in Free Range Broilers

SUMMARY Fowl cholera, caused by Pasteurella multocida, remains a major problem of poultry worldwide. In the current report, we describe an outbreak in free range organic broilers. In addition to culturing samples from dead broilers, we attempted to isolate P. multocida from feral cats trapped on the farm. The isolates were identified by PCR as P. multocida and then serotyped using the Heddleston scheme and genotyped using both a multilocus sequence typing (MLST) method and an enterobacterial repetitive intergenic consensus (ERIC)-PCR method. A total of 123 isolates of P. multocida were recovered from 12 broilers. All 123 isolates were examined by ERIC-PCR, and only one pattern was identified. A subset of seven broiler isolates were examined by MLST and all were typed as sequence type (ST) 20. A total of 28 isolates of P. multocida were recovered from 17 cats, and five ERIC-PCR genotypes were identified, with one genotype (E-1, shared by 19 isolates) being the same as the ERIC-PCR pattern associated with the broilers. One representative cat strain for each ERIC-PCR pattern was subjected to MLST. The cat isolate with the same ERIC-PCR genotype as the broiler isolates was confirmed as having the same MLST result, ST 20. The other five cat ERIC-PCR patterns were allocated to four STs: E-2 and E-5 to ST 265, E-3 to ST 30, E-4 to ST 20, and E-6 to ST 264. Both genotyping methods confirmed that isolates of P. multocida were common between the feral cats and the chickens. It was not clear whether the strain was transmitted from the cats to the chicken or whether the cats obtained the strain preying on chicken. The study has shown that cats can harbor P. multocida strains with the same genotype found in chickens affected with fowl cholera. RESUMEN Epidemiología del cólera aviar en pollos orgánicos—Estudio de un caso. El cólera aviar, causada por Pasteurella multocida, sigue siendo un problema importante en la avicultura a nivel mundial. En el presente reporte, se describe un brote en pollos de engorde de tipo orgánico. Además de cultivar muestras de pollos muertos, se intentó aislar P. multocida de gatos salvajes atrapados en la granja. Los aislamientos fueron identificados por PCR como P. multocida y luego fueron serotipificados utilizando el esquema de Heddleston y genotipificados utilizando tanto el método de tipificación por secuencia multilocus (MLST) y mediante el método del consenso intergénico repetitivo de enterobacerias (ERIC)-PCR. Se recuperaron un total de 123 aislamientos de P. multocida de 12 pollos de engorde. Todos los 123 aislamientos fueron examinados por ERIC-PCR, y se identificó un solo patrón. Un subconjunto de siete aislamientos de pollos de engorde fueron examinados por el método MLST y todos se tipificaron como secuencia tipo (ST) 20. Un total de 28 aislamientos de P. multocida se recuperaron a partir de 17 gatos, y se identificaron cinco genotipos por ERIC-PCR, con un genotipo (E-1, compartida por 19 aislamientos) siendo el mismo que el patrón de ERIC-PCR asociado con los pollos de engorde. Una cepa representativa de gato de cada patrón de ERIC-PCR fue sometida a MLST. El aislamiento de gato con el mismo genotipo ERIC-PCR del aislado de pollo de engorde se confirmó que mostraba el mismo resultado por MLST, ST 20. Los otros cinco patrones de ERIC-PCR de aislamientos de gatos fueron asignados a cuatro serotipos: E-2 y E-5 a ST 265, E-3 a ST 30, E-4 a ST 20, y E-6 a ST 264. Ambos métodos de genotipificación confirmaron que los aislamientos de P. multocida eran comunes entre los gatos silvestres y los pollos. No estuvo claro si las cepas se transmitían de los gatos al pollo, o si los gatos la obtenían mediante la predación de los pollos. El estudio ha demostrado que los gatos pueden albergar cepas de P. multocida con el mismo genotipo encontrado en los pollos afectados con el cólera aviar.