Characterisation of a Rhizobium loti nodulation mutant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University

Please note: Published papers - consult the print copy held in the LibraryThe aim of the project was to characterise the Rhizobiurn loti Nod- Tn5 mutant strain, PN233. The Tn5 insertion had been previously localised to a 7.1 kb Eco RI chromosomal fragment. This fragment was sub-cloned and a Bam HI/Sal I endonuclease restriction map for the region was determined. Hind III digests were utilised to identify the approximate location of the Tn5 233 insertion and those of four other Tn5 insertions (4016, 4019, 4047 and 4053) in the 7.1 kb region. The 233 mutation was found to map to a 1.45 kb Sal I fragment and that of an overlapping 2.8 kb Barn HI fragment. The 7.1 kb Eco RI fragment and a larger 22.7 kb fragment that encompassed this region, had been cloned into pLAFRl. The construct carrying the 22.7 kb fragment (pPN305) was crossed into four R.l. bv. trifolii strains, each mutant in one of the four common nod genes, A,B,C, and D. The construct was able to complement the nodC mutation indicating the presence of a nodC gene somewhere on the 22.7 kb region. The mutations 4047 and 4053 had been found to map to either side of the 233 Tn5 insertion. Both insertions affected nodule formation and were thus included in further plant complementation tests. These experiments involved crossing both the pPN305 and a construct bearing the smaller 7.1 kb Eco RI fragment (pPN25) into the R. loti and R.l. bv. trifolii Tn5 mutants. What was unusual about the results was that, while the 7.1 kb fragment was able to complement the mutations, the larger 22.7 kb fragment which encompasses that region could complement PN4047 and PN4053 but was unable to complement the PN233 mutant. The 2.8 kb Barn HI and 1.45 kb Sal I fragments, to which the 233 insertion was mapped, and that of an adjacent 1.2 kb Sal I fragment, were sub-cloned and then Bal 31 digested in both orientations to create a series of overlapping fragments. These fragments were then sequenced. The data revealed that the 233 Tn5 had inserted into the R. loti node gene. It was determined that the 4047 Tn5 was also located in this gene, slightly upstream of 233, while 4053 had inserted into the 5'-region of nodI which is downstream of nodC. Nod.A was identified upstream of nodC indicating an arrangement of common nod genes different from the conventional nod.ABeIJ found in other rhizobia. The promoter for these nod genes, the nod box, was located upstream of the nodA gene. A particularly puzzling aspect of the results is that, while PN4047 is complemented by both pPN305 and pPN25, PN233, which has an insertion in the same gene, could only be complemented by the smaller fragment carried by the pPN25 construct. To explain this result, it is proposed that PN233 is producing a mutant NodC protein and that this, in combination with doubled copies of a gene or genes present elsewhere on the 22.7 kb fragment, is responsible for interfering with complementation in this mutant. Alternatively, it may be that the imbalance of doubled copies of downstream, co-transcribed genes in the presence of one copy of a functional node gene causes complementation failure

Whole-Genome Comparison of Two Campylobacter jejuni Isolates of the Same Sequence Type Reveals Multiple Loci of Different Ancestral Lineage

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Seroprevalence of Leptospira in Racehorses and Broodmares in New Zealand

A cross-sectional survey was conducted to determine the seroprevalence of Leptospira in a cohort of horses and to evaluate potential risk factors for Leptospira seropositivity in horses in New Zealand. The convenience sample included 499 Thoroughbred racing and breeding horses from 25 commercial properties in North Island, New Zealand. A questionnaire was used to collect demographic data on horses and property-level information on grazing and management practices, pest (rodent) management, access to natural waterways, other livestock on the property, and possible contact with wildlife. The microscopic agglutination test was used to test sera for serovars Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi. Logistic regression was used to investigate the risk factors for Leptospira seropositivity to at least one serovar and for each serovar individually. A total of 124 (25%, 95% confidence interval (CI) 21–29%) horses had positive titres to any one of the five serovars. The seroprevalence of Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi was 5% (95% CI 3–7%), 9% (95% CI 7–12%), 6% (95% CI 4–8%), 6% (95% CI 4–8%), and 6% (95% CI 4–8%), respectively. Broodmares, compared to racehorses and alternately grazing horses with sheep, increased the odds of exposure to any one serovar, whilst grazing the same time as sheep and alternately grazing horses with cattle increased the odds of exposure to Ballum and Hardjo (bovis), respectively. Historical exposure to Leptospira in racing and breeding horses was identified, and risk factors were consistent with pasture-based exposure

“We don't really do doctors.” messages from people diagnosed with occupational leptospirosis for medical professionals on infection, hospitalisation, and long-term effects

Leptospirosis is largely an occupational disease for people working with livestock in Aotearoa New Zealand. Introduction of livestock vaccination and use of personal protective equipment has been associated with a reduction in the incidence. However, the incidence of occupational leptospirosis remains high, with significant burdens for affected families and healthcare system. For this article, a subset of thirteen participants from a nationwide leptospirosis case-control study (2019–2021) who were diagnosed with leptospirosis and worked with livestock at the time of illness were invited and agreed to a semi-structured interview. Interviewees reflected on their experiences as messages for medical professionals. The analysis of transcripts reveals widely shared experiences with infection, hospitalisation, and treatment, as well as long-term effects and recovery. Conclusions for medical professionals include that ill workers continue to have their diagnosis of leptospirosis delayed. This delay may contribute to more than half the people ill with leptospirosis hospitalised. Further, medical professionals' communication and relationship with ill people strongly colours the latter's experience, for good or for bad. Moreover, most interviewees experienced a recovery process that took several months of feeling tired, which undermined professional performance and emotional wellbeing

Sero-Prevalence and Risk Factors for Leptospirosis in Abattoir Workers in New Zealand

Leptospirosis is an important occupational disease in New Zealand. The objectives of this study were to determine risk factors for sero-prevalence of leptospiral antibodies in abattoir workers. Sera were collected from 567 abattoir workers and tested by microscopic agglutination for Leptospira interrogans sv. Pomona and Leptospira borgpetersenii sv. Hardjobovis. Association between prevalence and risk factors were determined by species specific multivariable analysis. Eleven percent of workers had antibodies against Hardjobovis or/and Pomona. Workers from the four sheep abattoirs had an average sero-prevalence of 10%–31%, from the two deer abattoirs 17%–19% and the two beef abattoirs 5%. The strongest risk factor for sero-positivity in sheep and deer abattoirs was work position. In sheep abattoirs, prevalence was highest at stunning and hide removal, followed by removal of the bladder and kidneys. Wearing personal protective equipment such as gloves and facemasks did not appear to protect against infection. Home slaughtering, farming or hunting were not significantly associated with sero-prevalence. There is substantial risk of exposure to leptospires in sheep and deer abattoirs in New Zealand and a persisting, but lower risk, in beef abattoirs. Interventions, such as animal vaccination, appear necessary to control leptospirosis as an occupational disease in New Zealand

Molecular Epidemiology of Campylobacter jejuni in a Geographically Isolated Country with a Uniquely Structured Poultry Industry▿

In New Zealand the number of campylobacteriosis notifications increased markedly between 2000 and 2007. Notably, this country's poultry supply is different than that of many developed countries as the fresh and frozen poultry available at retail are exclusively of domestic origin. To examine the possible link between human cases and poultry, a sentinel surveillance site was established to study the molecular epidemiology of Campylobacter jejuni over a 3-year period from 2005 to 2008 using multilocus sequence typing. Studies showed that 60.1 to 81.4% of retail poultry carcasses from the major suppliers were contaminated with C. jejuni. Differences were detected in the probability and level of contamination and the relative frequency of genotypes for individual poultry suppliers and humans. Some carcasses were contaminated with isolates belonging to more than one sequence type (ST), and there was evidence of both ubiquitous and supplier-associated strains, an epidemiological pattern not recognized yet in other countries. The common poultry STs were also common in human clinical cases, providing evidence that poultry is a major contributor to human infection. Both internationally rare genotypes, such as ST-3069 and ST-474, and common genotypes, such as ST-45 and ST-48, were identified in this study. The dominant human sequence type in New Zealand, ST-474, was found almost exclusively in isolates from one poultry supplier, which provided evidence that C. jejuni has a distinctive molecular epidemiology in this country. These results may be due in part to New Zealand's geographical isolation and its uniquely structured poultry industry

Assigning the source of human campylobacteriosis in New Zealand : a comparative genetic and epidemiological approach

Integrated surveillance of infectious multi-source diseases using a combination of epidemiology, ecology, genetics and evolution can provide a valuable risk-based approach for the control of important human pathogens. This includes a better understanding of transmission routes and the impact of human activities on the emergence of zoonoses. Until recently New Zealand had extraordinarily high and increasing rates of notified human campylobacteriosis, and our limited understanding of the source of these infections was hindering efforts to control this disease. Genetic and epidemiological modeling of a 3-year dataset comprising multilocus sequence typed isolates from human clinical cases, coupled with concurrent data on food and environmental sources, enabled us to estimate the relative importance of different sources of human disease. Our studies provided evidence that poultry was the leading cause of human campylobacteriosis in New Zealand, causing an estimated 58–76% of cases with widely varying contributions by individual poultry suppliers. These findings influenced national policy and, after the implementation of poultry industry-specific interventions, a dramatic decline in human notified cases was observed in 2008. The comparative-modeling and molecular sentinel surveillance approach proposed in this study provides new opportunities for the management of zoonotic diseases