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Pasteurella multocida isolates from dairy cattle on a farm in Spain were associated with pneumonia of calves (six isolates) and mastitis of heifers (five isolates). The objective was to determine if the P. multocida isolates retrieved from both disease scenarios were the same strain or whether more than one strain was present. The isolates were identified by a species-specific polymerase chain (PCR) assay, serotyped by the Heddleston scheme and then typed by a number of molecular genotyping assays including multi-locus sequence typing (MLST). The 11 isolates were confirmed as P. multocida but failed to react with any of the 16 Heddleston antisera. The PCR targeting the genes associated with the lipopolysaccharide outer core biosynthesis locus assigned all the isolates to L3–the type that contains Heddleston serovars 3 and 4. The MLST analysis showed all isolates belonging to ST 79 within the clonal complex of ST13. Only one of the isolates showed a slight different profile by the repetitive extragenic palindromic PCR. The conclusion was that the same strain was associated with pneumonia in calves and mastitis in heifers. [ABSTRACT FROM AUTHOR]

Simple Summary: At the farm-level, all 40 farms sampled were positive for avian pathogenic E. coli (APEC), and the mean within-farm prevalence was 63%. Higher APEC within-farm bird-level prevalence was significantly associated with the usage of well water as a source of drinking water, failure to disinfect the waterline after each flock, farm visitors not showering before entering the shed, distances greater than 20 m between the car park and the poultry shed, and the presence of wild birds within 50 m of the shed. Chlorinating the drinking water combined with automatic water filtration reduced within-farm bird-level APEC prevalence. Based on the results concluded from the multivariable model, improving biosecurity and water treatments might reduce APEC prevalence, decrease the risk of colibacillosis, reduce the use of antimicrobials, improve food safety, and positively influence poultry and public health. Avian pathogenic E. coli (APEC) can cause avian colibacillosis, an economically important disease that contributes to bird mortality and the high costs associated with prevention and treatment. Little is known about APEC in the context of Australian conditions. The current study aimed to estimate the prevalence of APEC and determine the risk factors associated with cloacal carriage of APEC among commercial healthy meat chickens in Queensland. Cloacal swabs were collected at slaughter from 400 healthy meat chickens (ten per farm) originating from 40 farms. A total of 2200 E. coli isolates were selected from cultured swabs and screened for the presence of five APEC-associated virulence genes (VGs). Farm-level data were collected using a questionnaire. Binominal general linear models were used to identify farm-level risk factors associated with bird-level APEC prevalence. Thirty-four per cent of the cultured E. coli isolates (n = 751) were classified as APEC, with all farms testing positive for APEC, and the overall bird-level prevalence of APEC was 63.0%. Higher APEC within-farm bird-level prevalence was positively associated with the usage of well water as a source of drinking water (OR = 6.2, 95% CI: 2.3, 16.5, p < 0.001); not having shower facilities available for farm visitors (OR = 3.6, 95% CI: 1.8, 7.1, p < 0.001); distances greater than 20 m between the car park and the poultry shed (OR = 2.2, 95% CI: 1.4, 3.4, p = 0.001); not applying water line disinfection after each flock cycle (OR = 2.2, 95% CI: 1.4, 3.5, p = 0.001); the presence of wild birds within 50 m of the poultry shed (OR = 2.3, 95% CI: 1.4, 3.7, p = 0.001). Chlorine combined with automatic drinking water filtration reduced within-farm bird-level APEC prevalence (OR = 0.07, 95% CI: 0.02, 0.34, p = 0.001). This study identified a number of important factors associated with APEC and showed that improving biosecurity and water treatments might reduce the prevalence of APEC. The notable high APEC prevalence on all farms requires further epidemiological investigations. [ABSTRACT FROM AUTHOR]

Chicken is a major reservoir and source of human infections from zoonotic Campylobacter. In southern Thailand, chicken is the most popular meat consumed; however, little is known about the antimicrobial resistance (AMR) of Campylobacter spp. in chicken production systems. We aimed to investigate the prevalence and characteristics of AMR in Campylobacter isolates from commercial broiler and native chicken farms in southern Thailand. A total of 286 Campylobacter isolates (adjusted prevalence: 60.3%, SE ± 0.01) were recovered, of which 268 were identified as Campylobacter jejuni and 18 as Campylobacter coli. The adjusted prevalence of resistance was highest for nalidixic acid (98.6%), followed by ciprofloxacin (97.2%), enrofloxacin (96.4%), tetracycline (74.6%), tylosin (38.3%), and erythromycin (10.6%). Commercial broilers had resistance levels 7–11% higher to quinolones and 35–47% lower to macrolides and tetracycline (all P ≤ 0.025) compared to native chickens. Genotypic resistance analyses revealed high prevalence rates (>86%) for gyrA mutant, efflux pump CmeABC, and point mutation A2075G in the 23S rRNA gene. High homogeneity was observed among isolates within study farms. Commercial broilers exhibited higher homogeneity in AMR patterns than native chickens. The best agreement was observed between tetracycline resistance and tet(O) genes (Kappa: k = 0.811, P < 0.001), while the fair agreement was found for quinolone resistance and gyrA mutant genes (k = 0.260–0.271, P < 0.001). No association was detected between macrolide resistance and point mutations in the 23S rRNA gene. These findings fill knowledge gaps on AMR in Campylobacter in Thailand, emphasizing the need for targeted AMR mitigation strategies in both commercial broilers and native chickens. High Campylobacter prevalence in chickens; C. jejuni more prevalent than C. coli. Susceptibility to macrolides but resistance to quinolones/tetracyclines in isolates. Homogeneous resistance patterns within farms; higher in broilers than in native birds. Partial association between phenotypic and genotypic resistance among isolates. [ABSTRACT FROM AUTHOR]

The authors reflect on the studies comparing and contrasting virulent and non-virulent strains and isolates of Haemophilus parasuis. They mention the degrees of virulence in virulent H. parasuis as virulent vs. non-virulent does not reflect the complexity of infections in the field. They suggest that the use of colostrum deprived piglets and virulence within 'virulent' H. parasuis can be used for future progress.

Simple Summary: Skin lesions in cattle associated with feeding by Haematobia fly species are a significant health issue in northern Australian cattle. These lesions have most commonly been attributed to the effects of Stephanofilaria nematodes vectored by the Haematobia spp. flies. However, the exact etiology of these lesions and reasons for the variation between the cattle in the severity of lesions was unclear. This study investigated the cutaneous responses to fly and nematode antigens in lesion-resistant and susceptible cattle, which indicated that differences in the hypersensitivity response to buffalo fly (Haematobia irritans exigua) antigens is a key factor underlying the variation amongst the cattle in susceptibility-to-lesion development. There was no association found between the intensity of fly infestation and lesion susceptibility. The findings from this study suggest that further characterization of the skin response could lead to the identification of the biomarkers for selecting cattle with an increased resistance to buffalo fly lesion development. This study investigated the role of cattle immune responses in the pathogenesis of buffalo fly (Haematobia irritans exigua) (BF) lesions. Brangus steers phenotyped for lesion development were divided into three groups: high lesion susceptibility (HL), low lesion susceptibility (LL) and no lesions (NL), based on lesion severity scores. Each steer was injected intradermally with different concentrations of BF, Onchocerca gibsoni (Og), and Musca domestica (Md) antigens. At 1 h post-injection, wheal areas at BF injection sites were found to be significantly larger in HL than NL cattle, but there were no significant differences (p < 0.05) found between either the HL or NL cattle and LL cattle. At 24, 48, and 72 h post-injection, the skinfold thickness response to both BF and Md antigens was significantly greater in the HL group than the NL group. However, skin thickness was significantly greater for the BF antigens than the Md antigens (p < 0.05). There were no significant differences found between the LL and NL animals in response to the BF antigens at any time, and no significant differences were determined between any of the lesion groups in response to the Og antigens. Histological examination of skin sections taken from the BF antigen injection sites in HL cattle at 72 h post-injection revealed necrosis of the epidermis and superficial dermis, along with severe eosinophilic inflammation. This study suggests that differences in the hypersensitivity to BF antigens underlie differences amongst the cattle in their susceptibility to the development of BF lesions, and breeding for immune-related biomarkers may assist in selecting more BF lesion-resistant cattle. [ABSTRACT FROM AUTHOR]

Cattle enterprises are one of the major livestock production systems globally and are forecasted to have stable growth in the next decade. To facilitate sustainable live weight production, optimal reproductive performance is essential. Microbial colonisation in the reproductive tract has been demonstrated as one of the factors contributing to bovine reproductive performance. Studies also implied that reproductive metagenomes are different at each stage of the estrous cycle. This study applied Oxford Nanopore Technologies' adaptive long-read sequencing to profile the bovine reproductive microbiome collected from tropical cattle in northern Queensland, Australia. The microbiome samples were collected from cattle of different sexes, reproductive status and locations to provide a comprehensive view of the bovine reproductive microbiome in northern Australian cattle. Ascomycota, Firmicutes and Proteobacteria were abundant phyla identified in the bovine reproductive metagenomes of Australian cattle regardless of sexes, reproductive status and location. The species level taxonomical investigation suggested that gastrointestinal metagenome and the surrounding environment were potentially the origins of the bovine reproductive metagenome. Functional profiles further affirmed this implication, revealing that the reproductive metagenomes of the prepubertal and postpartum animals were dominated by microorganisms that catabolise dietary polysaccharides as an energy substrate while that of the pregnant animals had the function of harvesting energy from aromatic compounds. Bovine reproductive metagenome investigations can be employed to trace the origins of abnormal metagenomes, which is beneficial for disease prevention and control. Additionally, our results demonstrated different reproductive metagenome diversities between cattle from two different locations. The variation in diversity within one location can serve as the indicator of abnormal reproductive metagenome, but between locations inferences cannot be made. We suggest establishing localised metagenomic indices that can be used to infer abnormal reproductive metagenomes which contribute to abortion or sub-fertility. [ABSTRACT FROM AUTHOR]

Chickens are the primary reservoirs of Campylobacter spp., mainly C. jejuni and C. coli , that cause human bacterial gastrointestinal infections. However, genomic characteristics and antimicrobial resistance of Campylobacter spp. in low- to middle-income countries need more comprehensive exploration. This study aimed to characterize 21 C. jejuni and 5 C. coli isolates from commercial broilers and native chickens using whole genome sequencing and compare them to 28 reference Campylobacter sequences. Among the 26 isolates, 13 sequence types (ST) were identified in C. jejuni and 5 ST in C. coli. The prominent ST was ST 2274 (5 isolates, 19.2%), followed by ST 51, 460, 2409, and 6455 (2 isolates in each ST, 7.7%), while all remaining ST (464, 536, 595, 2083, 6736, 6964, 8096, 10437, 828, 872, 900, 8237, and 13540) had 1 isolate per ST (3.8%). Six types of antimicrobial resistance genes (ant(6)-Ia, aph(3')-III , bla OXA , cat, erm (B), and tet (O)) and one point mutations in the gyrA gene (Threonine-86-Isoleucine) and another in the rpsL gene (Lysine-43-Arginine) were detected. The bla OXA resistance gene was present in all isolates, the gyrA mutations was in 95.2% of C. jejuni and 80.0% of C. coli , and the tet (O) resistance gene in 76.2% of C. jejuni and 80.0% of C. coli. Additionally, 203 virulence-associated genes linked to 16 virulence factors were identified. In terms of phenotypic resistance, the C. jejuni isolates were all resistant to ciprofloxacin, enrofloxacin, and nalidixic acid, with lower levels of resistance to tetracycline (76.2%), tylosin (52.3%), erythromycin (23.8%), azithromycin (22.2%), and gentamicin (11.1%). Most C. coli isolates were resistant to all tested antimicrobials, while 1 C. coli was pan-susceptible except for tylosin. Single-nucleotide polymorphisms concordance varied widely, with differences of up to 13,375 single-nucleotide polymorphisms compared to the reference Campylobacter isolates, highlighting genetic divergence among comparative genomes. This study contributes to a deeper understanding of the molecular epidemiology of Campylobacter spp. in Thai chicken production systems. [ABSTRACT FROM AUTHOR]

Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium , which was identified at low abundances and categorized under Order "Unclassified Clostridiales " when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations. [ABSTRACT FROM AUTHOR]

A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars. [ABSTRACT FROM AUTHOR]

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica -specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease. • A species-specific PCR targeting the rpoB gene sequence was developed to identify M. haemolytica exclusively, yielding negative results for other Mannheimia species. • Whole genome sequencing and bioinformatic analysis were successfully used to serotype confirmed M. haemolytica isolates that could not be assigned to serovars 1, 2 or 6 using an existing multiplex PCR. • Whole genome sequencing and bioinformatic analysis were successfully used to identify those Mannheimia isolates that were not M. haemolytica , while matrix-assisted laser desorption-ionization time of flight analysis lacked the discriminative capacity to identify the isolates to the species level. [ABSTRACT FROM AUTHOR]

Highlights • Two different disease manifestations in 2 fur seals associated with P. multocida. • Whole genome sequencing revealed the presence of within-host diversity of P. multocida , a novel finding for this important pathogen. • The isolates from several internal organs of the two seals belonged to the same genetic lineage. • Results suggest that transiting birds could have been responsible for introducing the outbreak strain. Abstract Pasteurella multocida is a heterogeneous bacterium, which has the capacity to cause disease in a wide range of host species and is also recognized as an important zoonotic pathogen. Two sequential deaths in captive fur seals occurred at Sea World, Australia during December 2017. A fibrinosuppurative bronchopneumonia in a Subantarctic fur seal (Arctocephalus tropicalis) resulted in death within 24 h of nonspecific signs of illness, whereas a septic peritonitis in a New Zealand fur seal (Arctocephalus forsteri) resulted in death within 12 h of clinical presentation. The cases happened within three days in two different pool locations, although both had previously been housed in the same area. A total of six Pasteurella multocida isolates were obtained from several internal organs at necropsy in both cases and were subjected to whole genome sequencing and phylogenomic analysis. In-silico typing of the isolates revealed that all belonged to Multi-Locus Sequence Type 7 and carried lipopolysaccharide outer core biosynthesis loci Type 3. Phylogenomic analysis of the isolates confirmed that the isolates were near identical at the core genome level, suggesting acquisition from a common source. The results also revealed the presence of within host and across animal diversity of P. multocida isolates for the first time even in a clearly connected outbreak. [ABSTRACT FROM AUTHOR]

We report 3 cases of koala bite wound infection with Lonepinella koalarum-like bacteria requiring antimicrobial and surgical management. The pathogens could not be identified by standard tests. Phylogenetic analysis of 16S rRNA and housekeeping genes identified the genus. Clinicians should isolate bacteria and determine antimicrobial susceptibilities when managing these infections. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. [ABSTRACT FROM AUTHOR]

Macrolides are often used to treat and control bacterial pathogens causing respiratory disease in pigs. This study analyzed the whole genome sequences of one clinical isolate of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, and Bordetella bronchiseptica, all isolated from Australian pigs to identify the mechanism underlying the elevated minimum inhibitory concentrations (MICs) for erythromycin, tilmicosin, or tulathromycin. The H. parasuis assembled genome had a nucleotide transition at position 2059 (A to G) in the six copies of the 23S rRNA gene. This mutation has previously been associated with macrolide resistance but this is the first reported mechanism associated with elevated macrolide MICs in H. parasuis. There was no known macrolide resistance mechanism identified in the other three bacterial genomes. However, strA and sul2, aminoglycoside and sulfonamide resistance genes, respectively, were detected in one contiguous sequence (contig 1) of A. pleuropneumoniae assembled genome. This contig was identical to plasmids previously identified in Pasteurellaceae. This study has provided one possible explanation of elevated MICs to macrolides in H. parasuis. Further studies are necessary to clarify the mechanism causing the unexplained macrolide resistance in other Australian pig respiratory pathogens including the role of efflux systems, which were detected in all analyzed genomes. [ABSTRACT FROM AUTHOR]

Pasteurella multocida is a major animal pathogen that causes a range of diseases including fowl cholera. P. multocida infections result in considerable losses to layer and breeder flocks in poultry industries worldwide. Both killed whole-cell and live-attenuated vaccines are available; these vaccines vary in their protective efficacy, particularly against heterologous strains. Moreover, until recently there was no knowledge of P. multocida LPS genetics and structure to determine precisely how LPS structure affects the protective capacity of these vaccines. In this study we show that defined lipopolysaccharide (LPS) mutants presented as killed whole-cell vaccines elicited solid protective immunity only against P. multocida challenge strains expressing highly similar or identical LPS structures. This finding indicates that vaccination of commercial flocks with P. multocida killed cell formulations will not protect against strains producing an LPS structure different to that produced by strains included in the vaccine formulation. Conversely, protective immunity conferred by vaccination with live P. multocida strains was found to be largely independent of LPS structure. Birds vaccinated with a range of live mutants belonging to the L1 and L3 LPS genotypes, each expressing a specific truncated LPS structure, were protected against challenge with the parent strain. Moreover, birds vaccinated with any of the five LPS mutants belonging to the L1 LPS genotype were also protected against challenge with an unrelated strain and two of the five groups vaccinated with live LPS mutants belonging to the L3 genotype were protected against challenge with an unrelated strain. In summary, vaccination with live P. multocida aroA mutants producing full-length L1 or L3 LPS or vaccination with live strains producing shortened L1 LPS elicited strong protective immunity against both homologous and heterologous challenge. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance in bacterial porcine respiratory pathogens has been shown to exist in many countries. However, little is known about the variability in antimicrobial susceptibility within a population of a single bacterial respiratory pathogen on a pig farm. This study examined the antimicrobial susceptibility of Actinobacillus pleuropneumoniae using multiple isolates within a pig and across the pigs in three different slaughter batches. Initially, the isolates from the three batches were identified, serotyped, and subsample genotyped. All the 367 isolates were identified as A. pleuropneumoniae serovar 1, and only a single genetic profile was detected in the 74 examined isolates. The susceptibility of the 367 isolates of A. pleuropneumoniae to ampicillin, tetracycline and tilmicosin was determined by a disc diffusion technique. For tilmicosin, the three batches were found to consist of a mix of susceptible and resistant isolates. The zone diameters of the three antimicrobials varied considerably among isolates in the second sampling. In addition, the second sampling provided statistically significant evidence of bimodal populations in terms of zone diameters for both tilmicosin and ampicillin. The results support the hypothesis that the antimicrobial susceptibility of one population of a porcine respiratory pathogen can vary within a batch of pigs on a farm. [ABSTRACT FROM AUTHOR]

Histophilus somni is a prevalent commensal organism of the upper respiratory tract of cattle and a major causative agent of bovine respiratory disease (BRD) and other syndromes including myocarditis and infectious thromboembolic meningoencephalitis. This study investigated the antimicrobial susceptibility and phylogenetic relationships of H. somni isolates obtained from lung, heart, and other tissues at post-mortem as well as nasal mucosa swabs from cases of BRD in Australian feedlots (2004–2019). Broth microdilution Minimal Inhibitory Concentration (MIC) assays were determined for 19 antimicrobials using three different media (CLSI approved Veterinary Fastidious Medium [VFM], Mueller-Hinton fastidious broth medium supplemented with yeast extract [MHF-Y] and Columbia Broth [CB] supplemented with 5% lysed horse blood). For all antimicrobials, MICs obtained using CB medium were identical or within 1 dilution step of the MICs obtained for VFM and MHF-Y media. Therefore, CB may be a suitable medium for H. somni antimicrobial susceptibility testing similar to MHF-Y medium. None of the 70 Australian H. somni isolates exhibited resistance to antimicrobials with CLSI breakpoints including those commonly used in the treatment of BRD in Australia (first-line tetracyclines [chlortetracycline and oxytetracycline], second-line macrolides [tulathromycin], and third-line extended-spectrum cephalosporin [ceftiofur]). Whole-genome sequence analysis of 65 H. somni isolates for genomic single nucleotide polymorphism differences identified four phylogenetic clusters, each containing isolates from different Australian states, feedlots and tissue sources that clustered together. These findings demonstrate limited genetic diversity and the absence of significant antimicrobial resistance among Australian isolates of H. somni isolated from feedlot cattle. • Histophilus somni a primary causative agent of bovine respiratory disease (BRD). • H. somni isolates samples highly collected from lung tissue compared with heart, other tissue and nasal swab. • Australian isolates of H. somni during (2004-2019) are pan-susceptible to the antimicrobial agents that are currently used to treat infections. • H. somni seems to be relatively homogeneous and showed no geographic clustering, in comparison with international isolates. [ABSTRACT FROM AUTHOR]

Buffalo flies (Haematobia irritans exigua) are ectoparasites of major animal health and production concern in north Australian beef herds. Skin lesions associated with buffalo fly infestation, cause hide damage and welfare issues and are manifested as dermatitis or ulcerated areas found most commonly near the medial canthus of the eye, along the lateral and ventral neck and on the abdomen. Buffalo flies can transmit a nematode, Stephanofilaria sp., which has been considered the main aetiological agent for buffalo fly lesions, but the role of nematodes in the development of the lesions has not been defined. To investigate the geographical distribution of Stephanofilaria , swabs were taken from the surface of lesions and buffalo flies were netted from the backs of beef cattle from 20 properties located in northern, central and southern Queensland. Swabs and buffalo flies were then tested for the presence of Stephanofilaria by qPCR. In northern and central Queensland, all properties except one, tested positive for the presence of Stephanofilaria in either buffalo flies or swabs, or in both. The infection rate varied amongst sites ranging from 0% to 100% in lesions and 0–34% in female buffalo flies. No nematodes were found in male buffalo flies. In contrast, none of the 66 lesion swabs or 1220 buffalo flies collected from southern Queensland tested positive for Stephanofilaria infection despite the frequent occurrence of lesions in the herds from which samples were collected. These findings suggest that infection with Stephanofilaria , although frequently detected, is not essential for the development of buffalo fly lesions and other factors may contribute to the initiation of lesions. This study also confirmed the potential for using surface swabs as a quicker and less invasive means of sampling lesions than dermal biopsies when testing for the presence of Stephanofilaria by qPCR, but further studies will be required to estimate the sensitivity of this technique. Understanding the pathogenesis of buffalo fly lesions will aid the development of optimal treatment and control strategies. • Haematobia flies vector Stephanofilaria nematodes found in cattle skin lesions. • Lesion swabbing and qPCR detected Stephanofilaria in lesions and flies. • In some areas Stephanofilaria was not found despite frequent occurrence of lesions. • Stephanofilaria is not always the cause of Haematobia -associated skin lesions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Pasteurella multocidawas 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) ofP. multocidain 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 ofP. multocidacan 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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Glaesserella australis , a newly described bacterial species, has been isolated from pig lungs that displayed lesions very similar to those caused by Actinobacillus pleuropneumoniae , prompting the need for a validated diagnostic tool. In this work, we have altered a multiplex PCR used for the identification of cultures of G. australis , A. pleuropneumoniae and Pasteurella multocida to be more sensitive and then evaluated the use of the altered diagnostic tool on cultures and directly on tissues. The altered multiplex PCR was validated using 47 related species, both type/reference strains and field isolates. The sensitivity was assessed by serial dilutions and used a mixture of target bacteria in different concentrations. Further, 166 lung samples from 54 farms from four Australian States were used to validate the ability of the multiplex PCR to detect bacteria in lung swabs. The multiplex PCR was specific for the three target species. The assay could detect a minimum of 40 colony forming units (CFU) of G. australis , 786 CFU of A. pleuropneumoniae and 238 CFU of P. multocida. The multiplex PCR yielded more positives than coventional bacteriological examination. From a total of 166 lung samples, 51.9%, 51.9% and 5.6% of farms were PCR positive for P. multocida , A. pleuropneumoniae and G. australis , respectively. The results suggested that the new multiplex PCR was specific, sensitive and out performed traditional culture. The prevalence of G. australis was not very high, but it was the dominant pathogen in infected pigs. • Improved multiplex PCR for G. australis performed better than traditional culture. • G. australis prevalence not very high, but dominant pathogen in infected pig herds. • Most prevalent on 54 farms were Pasteurella multocida L6 and Actinobacillus pleuropneumoniae serovar 15. [ABSTRACT FROM AUTHOR]

Glaesserella parasuis consists of 15 serovars with some of them highly virulent and some of them avirulent. As killed vaccines do not provide crossprotection across serovars, serotyping is of importance. Serotyping, previously done by gel diffusion, is now done by multiplex PCR followed by electrophoresis. Accurately differentiating 15 serovars by electrophoresis is problematic. To overcome this problem, a Luminex microbead-based multiplex assay was used to differentiate the serovars. The assay consisted of a multiplex PCR assay followed by hybridisation to microbeads which were then analysed on a Luminex machine. The newly developed assay was compared to the multiplex serotyping PCR and the gel diffusion/indirect haemagglutination assay (GD/IHA). The microbead-based assay worked very well for the 15 reference strains but when used on the 74 Australian field strains displayed some problems. The main problems were with the eight out of nine serovar 4 field isolates and the five serovar 7 and three serovar 14 field isolates. While the microbead-based assay could differentiate between the serovar 5 and 12 reference strains, which the serovar multiplex PCR could not, all four field isolates identified by GD/IHA as serovar 12 were identified as serovar 5 by the microbead-based assay. Serovar 4 has been noted to have a high diversity especially among strains from different countries. Our work clearly shows that the diversity of strains at both the national and the international level has to be taken into account when developing diagnostic assays. • Development of a microbead-based assay for serotyping the 15 serovars of Glaesserella parasuis. • Works well for reference strains from Germany, Japan, Sweden, Switzerland and USA. • Genetic variation in Australian isolates resulted in cross-reactions and false positive and negative results. • Before the development of a gene based assay the genetic variability of a species needs to be fully explored. [ABSTRACT FROM AUTHOR]

• ST372 was the predominant ST according to both WGS and ST-specific PCR • The remaining B2 isolates were from diverse minor STs, including ST,73 ST12, ST80 and two novel STs • Dog ST73 isolates showed considerable overall phylogenetic diversity and split mostly into species-specific clusters • Some intermingling of closely related human and animal (dog > cat) strains SNP-based core genome phylogeny occurred • Most animal and human ST73 isolates segregated into distinct host-specific sub-clusters This study investigated the prevalence of extraintestinal pathogenic E. coli (ExPEC)-associated sequence types (STs) from phylogenetic group B2 among 449 fluoroquinolone-susceptible dog clinical isolates from Australia. Isolates underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 317 so-identified group B2 isolates, 77 underwent whole genome sequencing (WGS), whereas the remainder underwent PCR-based screening for ST complexes (STc) STc12, STc73, STc372, and ST131. The predominant ST was ST372 according to both WGS (31 % of 77) and ST-specific PCR (22 % of 240), followed by (per WGS) ST73 (17 %), ST12 (7 %), and ST80 (7 %). A WGS-based phylogenetic comparison of ST73 isolates from dogs, cats, and humans showed considerable overall phylogenetic diversity. Although most clusters were species-specific, some contained closely related human and animal (dog > cat) isolates. For dogs in Australia these findings both confirm ST372 as the predominant E. coli clonal lineage causing extraintestinal infections and clarify the importance of human-associated group B2 lineage ST73 as a cause of UTI, with some strains possibly being capable of bi-directional (i.e., dog-human and human-dog) transmission. [ABSTRACT FROM AUTHOR]

• ST73 was the predominant ST according to both WGS and ST-specific multiplex PCR. • The remaining B2 isolates were from diverse minor STs, including ST12, ST372, and three novel STs. • Cat ST73 isolates were highly diverse, and split into serotype-specific clusters in a SNP-based core genome phylogeny. • Most cat and human ST73 isolates segregated into distinct host-specific subclusters. Extraintestinal pathogenic Escherichia coli (ExPEC) can cause urinary tract and other types of infection in cats, but the relationship of cat ExPEC to human ExPEC remains equivocal. This study investigated the prevalence of ExPEC-associated sequence types (STs) from phylogenetic group B2 among fluoroquinolone-susceptible cat clinical isolates. For this, 323 fluoroquinolone-susceptible cat clinical E. coli isolates from Australia underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 274 group B2 isolates, 53 underwent whole genome sequencing (WGS), whereas 221 underwent PCR-based screening for (group B2) sequence type complexes (STc) STc12, STc73, ST131, and STc372. Group B2 was the dominant phylogenetic group (274/323, 85 %), whereas within group B2 ST73 dominated, according to both WGS (43 % of 53; followed by ST127, ST12, and ST372 [4/53, 8 % each]) and ST-specific PCR (20 % of 221). In WGS-based comparisons of cat and reference human ST73 isolates, cat isolates had a relatively conserved virulence gene profile but were phylogenetically diverse. Although in the phylogram most cat and human ST73 isolates occupied host species-specific clusters within serotype-specific clades (O2:H1, O6:H1, O25:H1, O50/O2:H1), cat and human isolates were intermingled within two serotype-specific clades: O120:H31 (3 cat and 2 human isolates) and O22:H1 (3 cat and 5 human isolates). These findings confirm the importance of human-associated group B2 lineages as a cause of urinary tract infections in cats. The close genetic relationship of some cat and human ST73 strains suggests bi-directional transmission may be possible. [ABSTRACT FROM AUTHOR]

Pork is the main meat produced and consumed in the Philippines. The majority of pigs are raised by smallholders who experience a range of constraints to their pig production. This study presents the findings of the first part of an overarching project that used an Ecohealth approach and aimed to improve the production and competitiveness of the smallholder pig system in an area of the Philippines. A participatory approach was embraced, combining conventional and participatory epidemiology methods followed by a stakeholder discussion. The first aim was to identify management and health-related constraints to pig production among smallholder famers in San Simon, Pampanga, Philippines. The second aim was for the project team and stakeholders to jointly prioritise activities for the immediate future to address these constraints. Key management and health-related constraints identified included inadequate water supply to pigs, particularly lactating and gestating sows, and a range of feeding-related issues. Diarrhoea was recognised as the disease syndrome of highest priority and limited record keeping meant that farmers were unable to assess the productivity and profitability of their pig farming enterprises. Actions jointly prioritised by stakeholders and the project team were: the appointment of a project coordinator within each barangay; conduct two sets of seminars, the first covering water and nutrition and the second piglet management and diarrhoea, to be delivered by technical experts but with farmer "trusted sources" also sharing their experiences; development of easily understandable leaflets and posters covering key technical information; promotion of nipple drinkers attached to five-gallon water containers and creep boxes for piglets, and conduct of a record keeping workshop with a small group of innovative farmers to develop a useful and usable tool for record keeping. The use of multiple approaches to data-gathering enabled triangulation of study findings. Without any one of these components the understanding of the pig production system would have been less complete and it is possible that the proposed actions would not have been as well-tailored to the needs of the farmers. The participatory approach, in particular the stakeholder discussion, provided the opportunity to embrace the "deciding together" and "acting together" stances of participation rather than the lower "information giving" stance, thereby giving stakeholders greater ownership of the future activities of the overarching project and beyond. [ABSTRACT FROM AUTHOR]

• A septicaemic outbreak by P. multocida occurred in a resident group of squirrel gliders following importing two squirrel gliders from another zoo. • Whole genome sequencing and phylogenomic analysis confirmed that the outbreak strain was introduced through animal movement between the zoos. • A comparison of P. multocida strains from fatal and asymptomatic cases revealed structural differences in their latB , a structural LPS gene. • Isolates collected from fatal cases always had a fully intact latB gene, which is predicted to code for an acyltransferase. A septicaemic disease outbreak caused by Pasteurella multocida at a zoo in Western Australia (Zoo A) occurred in a resident group of squirrel gliders (Petaurus norfolcensis) following the introduction of two squirrel gliders imported from another zoo (Zoo B). P. multocida isolates obtained from the affected animals and asymptomatic, cohabiting marsupials at both zoos were typed via lipopolysaccharide outer core biosynthesis locus (LPS) typing, repetitive extragenic palindromic PCR (Rep-PCR) typing, and multilocus sequence typing (ST). Investigation of isolate relatedness via whole genome sequencing (WGS) and phylogenomic analysis found that the outbreak isolates shared the same genetic profile as those obtained from the imported gliders and the positive marsupials at Zoo B. Phylogenomic analysis demonstrated that these isolates belonged to the same clone (named complex one), confirming that the outbreak strain originated at Zoo B. As well, the carriage of multiple different strains of this pathogen in a range of marsupials in a zoo setting has been demonstrated. Importantly, the genomic investigation identified a missense mutation in the latB, a structural LPS gene, resulting in introduction of an immediate stop codon in the isolates carried by asymptomatic squirrel gliders in Zoo B. The identified diversity in the latB gene of LPS outer core biosynthesis loci of these isolates is consistent with a novel phase variable mechanism for virulence in P. multocida. Our study demonstrates the benefit of WGS and bioinformatics analysis in epidemiological investigations of pasteurellosis and its potential to reveal unexpected insights into bacterial virulence. [ABSTRACT FROM AUTHOR]

A correction to the article "Nucleic acid purification from plants, animals and microbes in under 30 seconds" published in a previous issue is presented.