Molecular characterization of Campylobacter spp. isolates obtained from commercial broilers and native chickens in Southern Thailand using whole genome sequencing.

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, blaOXA, 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 blaOXA 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.

Molecular identification and characterisation of Mannheimia haemolytica.

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.

LONEPINELLA SP. ISOLATED FROM WOUND INFECTIONS OF KOALAS.

We describe two cases of wound infections of koalas (Phascolarctos cinereus), one wild and one captive, in which Lonepinella-like organisms were involved. The wild adult koala was captured with bite wound injuries, as part of a koala population management program in Queensland, Australia. In both cases, there was evidence of physical trauma causing the initial wound. The captive koala suffered injury from the cage wire, and the wild koala had injuries suggestive of intermale fighting. Gram-negative bacteria isolated from both cases proved to be challenging to identify using routine diagnostic tests. The wound in the captive koala yielded a pure culture of an organism shown by whole genome sequence (WGS) analysis to be a member of the genus Lonepinella, but not a member of the only formally described species, L. koalarum. The wound of the wild koala yielded a mixed culture of Citrobacter koseri, Enterobacter cloacae and an organism shown by WGS analysis to be Lonepinella, but again not Lonepinella koalarum. Both cases were difficult to treat; the captive koala eventually had to have the phalanges amputated, and the wild koala required removal of the affected claw. The two Lonepinella isolates from these cases have a close relationship to an isolate from a human wound caused by a koala bite and may represent a novel species within the genus Lonepinella. Wound infections in koalas linked to Lonepinella have not been reported previously. Wildlife veterinarians need to be aware of the potential presence of Lonepinella-like organisms when dealing with wound infections in koalas, and the inability of commercial kits and systems to correctly identify the isolates.

Phase variation in the glycosyltransferase genes of Pasteurella multocida associated with outbreaks of fowl cholera on free-range layer farms.

Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene (htpE) (ST7 and ST274) or galactosyltransferase gene (gatG) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG. Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.

MOLECULAR IDENTIFICATION OF MEMBERS OF THE FAMILY PASTEURELLACEAE FROM THE ORAL CAVITY OF KOALAS (PHASCOLARCTOS CINEREUS) AND THEIR RELATIONSHIP WITH ISOLATES FROM KOALA BITE WOUNDS IN HUMANS.

A total of 22 Pasteurellaceae isolates obtained from the oral cavity of koalas (Phascolarctos cinereus) at different wildlife centers in Australia were investigated using amplification and sequencing of two housekeeping genes, rpoA and recN. The available sequences from the Lonepinella koalarum type strain (ACM3666T) and the recent isolates of Lonepinella-like bacteria obtained from human infected wounds associated with koala bites were also included. Phylogenetic analysis was performed on the concatenated rpoA-recN genes and genome relatedness was calculated based on the recN sequences. The oral cavity isolates, the koala bite wound isolates, and L. koalarum ACM3666T resulted in four clusters (Clusters 1-4). Clusters 1-3 were clearly not members of the genus Lonepinella. Cluster 1 was closely related to the genus Fredericksenia, and Clusters 2 and 3 appeared to be novel genera. Cluster 4 consisted of three subclusters: Cluster 4a with one koala bite wound isolate and L. koalarum ACM3666T, Cluster 4b with three oral cavity isolates and two Lonepinella-like wound isolates, and Cluster 4c with three nearly identical oral cavity isolates that may represent a different species within the genus Lonepinella. The rich Pasteurellaceae population, including potential novel taxa in the oral cavity of koalas supports an important role of these highly adapted microorganisms in the physiology of koalas. Moreover, the pathogenic potential of Lonepinella-like species is an important consideration when investigating infected koala bites in humans.

Molecular and serological characterization of Riemerella isolates associated with poultry in Australia.

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.

Glaesserella australis sp. nov., isolated from the lungs of pigs.

Twenty-nine isolates of an unknown haemophilic organism were isolated from the lungs of pigs from 14 farms in Australia. Phylogenetic analyses based on the 16S rRNA gene, recN and rpoA showed a monophyletic group that was most closely related to Glaesserella parasuis and [Actinobacillus] indolicus. Whole genome sequence analysis indicated that the Glaesserella parasuis and this group, using the type strain HS4635T for comparison, showed a similarity of 30.9 % DNA-DNA renaturation. The isolates were Gram-stain-negative, NAD-dependent, CAMP-negative and were oxidase-positive, catalase-negative and produced indole but not urease. The isolates could be separated from all currently recognized haemophilic and non-haemophilic members of the family Pastuerellaceae. Key phenotypic properties were the production of indole, the lack of urease activity, production of ß-galactosidase but not α-fucosidase, acid formation from (-)-d-arabinose, (+)-d-galactose, maltose and trehalose and a failure to produce acid from (-)-d-mannitol. Taken together, these data indicate that the isolates belong to a novel species for which the name Glaesserella australis sp. nov. is proposed. The type strain is HS4635T (=CCUG 71931T and LMG 30645T).

Phase variation in latB associated with a fatal Pasteurella multocida outbreak in captive squirrel gliders.

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.

Using genomics to understand inter- and intra- outbreak diversity of Pasteurella multocida isolates associated with fowl cholera in meat chickens.

Fowl cholera, caused by Pasteurella multocida, continues to be a challenge in meat-chicken-breeder operations and has emerged as a problem for free-range meat chickens. Here, using whole-genome sequencing (WGS) and phylogenomic analysis, we investigate isolate relatedness during outbreaks of fowl cholera on a free-range meat chicken farm over a 5-year period. Our genomic analysis revealed that while all outbreak isolates were sequence type (ST) 20, they could be separated into two distinct clades (clade 1 and clade 2) consistent with difference in their lipopolysaccharide (LPS) type. The isolates from the earlier outbreaks (clade 1) were carrying LPS type L3 while those from the more recent outbreaks (clade 2) were LPS type L1. Additionally, WGS data indicated high inter- and intra-chicken genetic diversity during a single outbreak. Furthermore, we demonstrate that while a killed autogenous vaccine carrying LPS type L3 had been successful in protecting against challenge from L3 isolates it might have driven the emergence of the closely related clade 2, against which the vaccine was ineffective. The genomic results also revealed a 14 bp deletion in the galactosyltransferase gene gatG in LPS type L3 isolates, which would result in producing a semi-truncated LPS in those isolates. In conclusion, our study clearly demonstrates the advantages of genomic analysis over the conventional PCR-based approaches in providing clear insights in terms of linkage of isolate within and between outbreaks. More importantly, it provides more detailed information than the multiplex PCR on the possible structure of outer LPS, which is very important in the case of strain selection for killed autogenous vaccines.

Novel insights into pasteurellosis in captive pinnipeds.

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.

Identification of Lonepinella sp. in Koala Bite Wound Infections, Queensland, Australia.

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.

Molecular epidemiology of an outbreak of clinical mastitis in sheep caused by Mannheimia haemolytica.

The aetiology and epidemiology of outbreaks of clinical mastitis in sheep under extensive pastoral conditions are incompletely understood. The objective of this study was to conduct a detailed investigation of a clinical mastitis outbreak that affected more than 10% of 230 at-risk ewes on a sheep and grain producing property in south east Australia during drought conditions in 2009. Milk samples were collected aseptically from all affected ewes and plated on sheep blood agar for bacterial identification. M. haemolytica was isolated from 80% of the samples that yielded cultivable microorganisms and thus was the main microorganism responsible for the outbreak. Analysis of the restriction endonuclease cleavage patterns of the isolates using pulsed field gel electrophoresis revealed some evidence of clonality, suggesting the possibility of horizontal transmission, but there was also considerable diversity between the clusters of closely related isolates. Multilocus sequence typing of the M. haemolytica isolates revealed most of the isolates belonged to ST1 with no association between the PFGE and MLST fingerprints of the isolates. Resistance to neomycin, streptomycin and sulphafurazole was detected in some of the isolates, but they were all susceptible to penicillin, ampicillin, ceftiofur, amoxycillin/clavulanic acid, ciprofloxacin, tetracycline, erythromycin and trimethoprim. This is the first published record of a comparison of the strains of M. haemolytica involved in a clinical mastitis outbreak in sheep and demonstrates the importance of this pathogen in sheep production systems, particularly during adverse climatic conditions and increased stocking rate.

The upper respiratory tract is a natural reservoir of haemolytic Mannheimia species associated with ovine mastitis.

Lamb suckling has been suggested to be an important way of infecting a ewe's udder with different bacteria, including Mannheimia haemolytica. To test the potential role of lambs in transferring Mannheimia species to the ewe's udder, the restriction endonuclease cleavage patterns of isolates obtained from nasopharyngeal swabs were compared with those obtained from cases of mastitis. Sterile cotton swabs were used to collect nasopharyngeal samples from 50 ewes and 36 lambs from three flocks. M. haemolytica and Mannheimia glucosida as well as haemolytic Mannheimia ruminalis-like organisms were detected in the upper respiratory tract of lambs and ewes. Comparison of the restriction endonuclease cleavage patterns of the isolates suggested that the M. haemolytica isolates obtained from different milk samples from ewes with mastitis were more clonal than those obtained from the nasal swabs. However, some nasal isolates within both Mannheimia species had restriction endonuclease cleavage patterns identical to those obtained from milk samples from ewes with mastitis, indicating that lambs may have a role in transferring these organisms to the udder. More clonality was observed between the M. glucosida isolates than between M. haemolytica isolates.

Human Wound Infection with Mannheimia glucosida following Lamb Bite.

Mannheimia spp. are veterinary pathogens that can cause mastitis and pneumonia in domestic cattle and sheep. While Mannheimia glucosida can be found as normal flora in oral and respiratory mucosa in sheep, there have been no reported cases of human infection with this organism.

Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep.

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles.

Molecular epidemiology of Mannheimia haemolytica and Mannheimia glucosida associated with ovine mastitis.

While Mannheimia haemolytica and Mannheimia glucosida have been recognized as causes of intramammary infection in sheep, there has been no investigation of the epidemiology of the strains involved. Pulsed field gel electrophoresis was used to study the molecular epidemiology of isolates of these 2 species associated with ovine mastitis. Ten distinct strains were recognized among 12 M. haemolytica isolates, and 7 distinct strains among 13 M. glucosida isolates. The results demonstrate a high diversity of isolates with the ability to cause ovine mastitis. However, the presence of some identical isolates may suggest the possibility of horizontal transmission of these species in some flocks, possibly through lamb sucking, and/or differences in the capacity of some isolates to cause mastitis in sheep.

The role of Mannheimia species in ovine mastitis.

Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. It usually causes severe clinical mastitis, followed by toxaemia and gangrenous necrosis of the udder. However there are limited data available on the epidemiology and pathogenesis of mastitis associated with Mannheimia species. These organisms can be more significant as a cause of mastitis than Staphylococcus aureus in some flocks. Some data suggest the possibility of horizontal transmission of Mannheimia species between ewes via lamb sucking. There is no vaccine available for prevention, and the sudden onset of mastitis and its peracute nature renders most treatments unsuccessful. This review examines the significance of the species within this genus in sheep mastitis.

Mannheimia species associated with ovine mastitis.

Mannheimia glucosida, M. haemolytica, and M. ruminalis were isolated from cases of acute mastitis in ewes. M. glucosida was found to be a common cause of clinical mastitis in sheep. Selected phenotypic tests in addition to genotyping were needed to definitively identify Mannheimia species causing ovine mastitis.