A cross-sectional investigation of Leptospira at the wildlife-livestock interface in New Zealand.

There has been a recent upsurge in human cases of leptospirosis in New Zealand, with wildlife a suspected emerging source, but up-to-date knowledge on this topic is lacking. We conducted a cross-sectional study in two farm environments to estimate Leptospira seroprevalence in wildlife and sympatric livestock, PCR/culture prevalence in wildlife, and compare seroprevalence and prevalence between species, sex, and age groups. Traps targeting house mice (Mus musculus), black rats (Rattus rattus), hedgehogs (Erinaceus europaeus) and brushtail possums (Trichosurus vulpecula) were set for 10 trap-nights in March-April 2017 on a dairy (A) and a beef and sheep (B) farm. Trapped wild animals and an age-stratified random sample of domestic animals, namely cattle, sheep and working dogs were blood sampled. Sera were tested by microagglutination test for five serogroups and titres compared using a Proportional Similarity Index (PSI). Wildlife kidneys were sampled for culture and qPCR targeting the lipL32 gene. True prevalence in mice was assessed using occupancy modelling by collating different laboratory results. Infection profiles varied by species, age group and farm. At the MAT cut-point of ≥ 48, up to 78% of wildlife species, and 16-99% of domestic animals were seropositive. Five of nine hedgehogs, 23/105 mice and 1/14 black rats reacted to L. borgpetersenii sv Ballum. The sera of 4/18 possums and 4/9 hedgehogs reacted to L. borgpetersenii sv Hardjobovis whilst 1/18 possums and 1/9 hedgehogs reacted to Tarassovi. In ruminants, seroprevalence for Hardjobovis and Pomona ranged 0-90% and 0-71% depending on the species and age group. Titres against Ballum, Tarassovi and Copenhageni were also observed in 4-20%, 0-25% and 0-21% of domestic species, respectively. The PSI indicated rodents and livestock had the most dissimilar serological responses. Three of nine hedgehogs, 31/105 mice and 2/14 rats were carrying leptospires (PCR and/or culture positive). True prevalence estimated by occupancy modelling in mice was 38% [95% Credible Interval 26, 51%] on Farm A and 22% [11, 40%] on Farm B. In the same environment, exposure to serovars found in wildlife species was commonly detected in livestock. Transmission pathways between and within species should be assessed to help in the development of efficient mitigation strategies against Leptospira.

"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.

Leptospirosis in Aotearoa New Zealand: Protocol for a Nationwide Case-Control Study.

BACKGROUND: In Aotearoa New Zealand, 90% of patients with notified leptospirosis (a zoonotic bacterial disease) have been men working in agricultural industries. However, since 2008, the epidemiology of notified cases has been gradually changing, that is, more women are affected; there are more cases associated with occupations traditionally not considered high risk in New Zealand; infecting serovars have changed; and many patients experience symptoms long after infection. We hypothesized that there is a shift in leptospirosis transmission patterns with substantial burden on affected patients and their families. OBJECTIVE: In this paper, we aimed to describe the protocols used to conduct a nationwide case-control study to update leptospirosis risk factors and follow-up studies to assess the burden and sources of leptospirosis in New Zealand. METHODS: This study used a mixed methods approach, comprising a case-control study and 4 substudies that involved cases only. Cases were recruited nationwide, and controls were frequency matched by sex and rurality. All participants were administered a case-control questionnaire (study 1), with cases being interviewed again at least 6 months after the initial survey (study 2). A subset of cases from two high-risk populations, that is, farmers and abattoir workers, were further engaged in a semistructured interview (study 3). Some cases with regular animal exposure had their in-contact animals (livestock for blood and urine and wildlife for kidney) and environment (soil, mud, and water) sampled (study 4). Patients from selected health clinics suspected of leptospirosis also had blood and urine samples collected (study 5). In studies 4 and 5, blood samples were tested using the microscopic agglutination test to test for antibody titers against Leptospira serovars Hardjo type bovis, Ballum, Tarassovi, Pomona, and Copenhageni. Blood, urine, and environmental samples were also tested for pathogenic Leptospira DNA using polymerase chain reaction. RESULTS: Participants were recruited between July 22, 2019, and January 31, 2022, and data collection for the study has concluded. In total, 95 cases (July 25, 2019, to April 13, 2022) and 300 controls (October 19, 2019, to January 26, 2022) were interviewed for the case-control study; 91 cases participated in the follow-up interviews (July 9, 2020, to October 25, 2022); 13 cases participated in the semistructured interviews (January 26, 2021, to January 19, 2022); and 4 cases had their in-contact animals and environments sampled (October 28, 2020, and July 29, 2021). Data analysis for study 3 has concluded and 2 manuscripts have been drafted for review. Results of the other studies are being analyzed and the specific results of each study will be published as individual manuscripts.. CONCLUSIONS: The methods used in this study may provide a basis for future epidemiological studies of infectious diseases. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/47900.

Longitudinal Testing of Leptospira Antibodies in Horses Located near a Leptospirosis Outbreak in Alpacas.

The objectives of this study were to determine if horses located near an outbreak of leptospirosis in alpacas had Leptospira titres indicative of a previous or current infection and, if so, to determine the magnitude in change of titres over time. Further, the objective was to determine if horses with high titre results were shedding Leptospira in their urine. Blood samples were collected from twelve horses located on or next to the farm with the outbreak in alpacas, on day zero and at four subsequent time points (two, four, six and nine weeks). The microscopic agglutination test was used to test sera for five serovars endemic in New Zealand: Ballum, Copenhageni, Hardjo, Pomona and Tarassovi. A reciprocal MAT titre cut-off of ≥1:100 was used to determine positive horses. Seven out of twelve horses (58%) were positive to at least one serovar during one of the time points. Two horses recorded titres of ≥1600, one for both Pomona and Copenhageni and the other for Hardjo, and these two horses were both PCR positive for Leptospira in their urine samples. For five out of seven horses, the titres either remained the same or changed by one dilution across the sampling time points. The study confirmed endemic exposure to five endemic Leptospira serovars in New Zealand in a group of horses located near a confirmed leptospirosis outbreak in alpacas.

Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding.

Leptospirosis is a zoonotic disease of global importance. The breadth of Leptospira diversity associated with both human and animal disease poses major logistical challenges to the use of classical diagnostic techniques, and increasingly molecular diagnostic tools are used for their detection. In New Zealand, this has resulted in an increase in positive cases reported nationally that have not been attributed to the infecting serovar or genomospecies. In this study, we used data from all pathogenic Leptospira genomes to identify a partial region of the glmU gene as a suitable locus for the discrimination of the infecting species and serovars of New Zealand-endemic Leptospira. This method can be used in culture and culture-independent scenarios making it flexible for diagnostics in humans, animals, and environmental samples. We explored the use of this locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION. Sequences obtained by this method allowed specific identification of Leptospira species in mixed and enriched environmental cultures, however read error inherent in the MinION sequencing system reduced the accuracy of strain/variant identification. Using this approach to characterise Leptospira in enriched environmental cultures, we detected the likely presence of Leptospira genomospecies that have not been reported in New Zealand to date. This included a strain of L. borgpetersenii that has recently been identified in dairy cattle and sequences similar to those of L. mayottensis. L. tipperaryensis, L. dzianensis and L. alstonii.

Transmission Dynamics of Shiga Toxin-Producing Escherichia coli in New Zealand Cattle from Farm to Slaughter.

Cattle are asymptomatic carriers of Shiga toxin-producing Escherichiacoli (STEC) strains that can cause serious illness or death in humans. In New Zealand, contact with cattle feces and living near cattle populations are known risk factors for human STEC infection. Contamination of fresh meat with STEC strains also leads to the potential for rejection of consignments by importing countries. We used a combination of PCR/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and whole-genome sequencing (WGS) to evaluate the presence and transmission of STEC on farms and in processing plants to better understand the potential pathways for human exposure and thus mitigate risk. Animal and environmental samples (n = 2,580) were collected from six farms and three meat processing plants in New Zealand during multiple sampling sessions in spring of 2015 and 2016. PCR/MALDI-TOF analysis revealed that 6.2% were positive for "Top 7" STEC. Top 7 STEC strains were identified in all sample sources (n = 17) tested. A marked increase in Top 7 STEC prevalence was observed between calf hides on farm (6.3% prevalence) and calf hides at processing plants (25.1% prevalence). Whole-genome sequencing was performed on Top 7 STEC bacterial isolates (n = 40). Analysis of STEC O26 (n = 25 isolates) revealed relatively low genetic diversity on individual farms, consistent with the presence of a resident strain disseminated within the farm environment. Public health efforts should focus on minimizing human contact with fecal material on farms and during handling, transport, and slaughter of calves. Meat processing plants should focus on minimizing cross-contamination between the hides of calves in a cohort during transport, lairage, and slaughter.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) strains, which can cause serious illness or death in humans. Contact with cattle feces and living near cattle are known risk factors for human STEC infection. This study evaluated STEC carriage in young calves and the farm environment with an in-depth evaluation of six farms and three meat processing plants over 2 years. An advanced molecular detection method and whole-genome sequencing were used to provide a detailed evaluation of the transmission of STEC both within and between farms. The study revealed widespread STEC contamination within the farm environment, but no evidence of recent spread between farms. Contamination of young dairy calf hides increased following transport and holding at meat processing plants. The elimination of STEC in farm environments may be very difficult given the multiple transmission routes; interventions should be targeted at decreasing fecal contamination of calf hides during transport, lairage, and processing.

Still 'dairy farm fever'? A Bayesian model for leptospirosis notification data in New Zealand.

Routinely collected public health surveillance data are often partially complete, yet remain a useful source by which to monitor incidence and track progress during disease intervention. In the 1970s, leptospirosis in New Zealand (NZ) was known as 'dairy farm fever' and the disease was frequently associated with serovars Hardjo and Pomona. To reduce infection, interventions such as vaccination of dairy cattle with these two serovars was implemented. These interventions have been associated with significant reduction in leptospirosis incidence, however, livestock-based occupations continue to predominate notifications. In recent years, diagnosis is increasingly made by nucleic acid detection which currently does not provide serovar information. Serovar information can assist in linking the recognized maintenance host, such as livestock and wildlife, to infecting serovars in human cases which can feed back into the design of intervention strategies. In this study, confirmed and probable leptospirosis notification data from 1 January 1999 to 31 December 2016 were used to build a model to impute the number of cases from different occupational groups based on serovar and month of occurrence. We imputed missing occupation and serovar data within a Bayesian framework assuming a Poisson process for the occurrence of notified cases. The dataset contained 1430 notified cases, of which 927 had a specific occupation (181 dairy farmers, 45 dry stock farmers, 454 meatworkers, 247 other) while the remaining 503 had non-specified occupations. Of the 1430 cases, 1036 had specified serovars (231 Ballum, 460 Hardjo, 249 Pomona, 96 Tarassovi) while the remaining 394 had an unknown serovar. Thus, 47% (674/1430) of observations had both a serovar and a specific occupation. The results show that although all occupations have some degree of under-reporting, dry stock farmers were most strongly affected and were inferred to contribute as many cases as dairy farmers to the burden of disease, despite dairy farmer being recorded much more frequently. Rather than discard records with some missingness, we have illustrated how mathematical modelling can be used to leverage information from these partially complete cases. Our finding provides important evidence for reassessing the current minimal use of animal vaccinations in dry stock. Improving the capture of specific farming type in case report forms is an important next step.

Diverse Epidemiology of Leptospira serovars Notified in New Zealand, 1999-2017.

Leptospirosis in New Zealand has been strongly associated with animal-contact occupations and with serovars Hardjo and Pomona. However, recent data suggest changes in these patterns, hence, serovar-specific epidemiology of leptospirosis from 1999 to 2017 was investigated. The 19-year average annual incidence is 2.01/100,000. Early (1999-2007) and late (2008-2017) study period comparisons showed a significant increase in notifications with serovar Ballum (IRR: 1.59, 95% CI: 1.22-2.09) in all cases and serovar Tarassovi (IRR: 1.75, 95% CI: 1.13-2.78) in Europeans and a decrease in notifications with serovars Hardjo and Pomona in all cases. Incidences of Ballum peaked in winter, Hardjo peaked in spring and Tarassovi peaked in summer. Incidence was highest in Maori (2.24/100,000) with dominant serovars being Hardjo and Pomona. Stratification by occupation showed meat workers had the highest incidence of Hardjo (57.29/100,000) and Pomona (45.32/100,000), farmers had the highest incidence of Ballum (11.09/100,000) and dairy farmers had the highest incidence of Tarassovi (12.59/100,000). Spatial analysis showed predominance of Hardjo and Pomona in Hawke's Bay, Ballum in West Coast and Northland and Tarassovi in Waikato, Taranaki and Northland. This study highlights the serovar-specific heterogeneity of human leptospirosis in New Zealand that should be considered when developing control and prevention strategies.

Combining MALDI-TOF and genomics in the study of methicillin resistant and multidrug resistant Staphylococcus pseudintermedius in New Zealand.

Staphylococcus pseudintermedius is an opportunistic and emerging zoonotic pathogen that primarily colonises the skin of dogs. Many common variants are methicillin resistant (MRSP) or multidrug resistant (MDR), and drug resistance is increasingly reported across the globe. In New Zealand, MRSP isolation remains rare in clinics. To pre-emptively inform diagnostic and antimicrobial stewardship practices, we examine isolates of S. pseudintermedius, MRSP and MDR-MRSP from New Zealand dogs using a combination of methodologies. Genetic and genomic data combined with antimicrobial susceptibility screening identify common drug-resistance profiles and their genetic determinants. We demonstrate that sensitive and specific species-level identification of S. pseudintermedius can be achieved using Bruker MALDI-TOF MS and, further, that this technique can be used to identify some common subtype variants, providing a level of categorical precision that falls somewhere between single-locus and multi-locus sequence typing. Comparative genomics analysis of global S. pseudintermedius data shows that MRSP moves frequently across the globe, but that horizontal gene transfer events resulting in the acquisition of the SCCmec cassette (responsible for beta-lactam antibiotic resistance) are infrequent. This suggests that biosecurity and surveillance in addition to antibiotic stewardship should play important roles in mitigating the risk of MRSP, especially in countries such as New Zealand where MRSP is still rare.

BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli.

UNLABELLED: Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from the bfpA gene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP. IMPORTANCE: Bundle-forming pili contribute to the host colonization strategy of enteropathogenic Escherichia coli. The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development.

Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.

A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli.

Enteropathogenic Escherichia coli inhibits type I interferon- and RNase L-mediated host defense to disrupt intestinal epithelial cell barrier function.

Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-ß is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-ß induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-ß and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function.

In vitro evolution of an archetypal enteropathogenic Escherichia coli strain.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal(r)) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal(r) strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str(r)) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal(r) clone has a lower growth rate than the Str(r) isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal(r) clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.

The E. coli effector protein NleF is a caspase inhibitor.

Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) can cause severe and potentially life-threatening infections. Their pathogenicity is mediated by at least 40 effector proteins which they inject into their host cells by a type-III secretion system leading to the subversion of several cellular pathways. However, the molecular function of several effectors remains unknown, even though they contribute to virulence. Here we show that one of them, NleF, binds to caspase-4, -8, and -9 in yeast two-hybrid, LUMIER, and direct interaction assays. NleF inhibits the catalytic activity of the caspases in vitro and in cell lysate and prevents apoptosis in HeLa and Caco-2 cells. We have solved the crystal structure of the caspase-9/NleF complex which shows that NleF uses a novel mode of caspase inhibition, involving the insertion of the carboxy-terminus of NleF into the active site of the protease. In conformance with our structural model, mutagenized NleF with truncated or elongated carboxy-termini revealed a complete loss in caspase binding and apoptosis inhibition. Evasion of apoptosis helps pathogenic E. coli and other pathogens to take over the host cell by counteracting the cell's ability to self-destruct upon infection. Recently, two other effector proteins, namely NleD and NleH, were shown to interfere with apoptosis. Even though NleF is not the only effector protein capable of apoptosis inhibition, direct inhibition of caspases by bacterial effectors has not been reported to date. Also unique so far is its mode of inhibition that resembles the one obtained for synthetic peptide-type inhibitors and as such deviates substantially from previously reported caspase-9 inhibitors such as the BIR3 domain of XIAP.

Targeting the chromosome partitioning protein ParA in tuberculosis drug discovery.

OBJECTIVE: To identify inhibitors of the essential chromosome partitioning protein ParA that are active against Mycobacterium tuberculosis. METHODS: Antisense expression of the parA orthologue MSMEG_6939 was induced on the Mycobacterium smegmatis background. Screening of synthetic chemical libraries was performed to identify compounds with higher anti-mycobacterial activity in the presence of parA antisense. Differentially active compounds were validated for specific inhibition of purified ParA protein from M. tuberculosis (Rv3918c). ParA inhibitors were then characterized for their activity towards M. tuberculosis in vitro. RESULTS: Under a number of culture conditions, parA antisense expression in M. smegmatis resulted in reduced growth. This effect on growth provided a basis for the detection of compounds that increased susceptibility to expression of parA antisense. Two compounds identified from library screening, phenoxybenzamine and octoclothepin, also inhibited the in vitro ATPase activity of ParA from M. tuberculosis. Structural in silico analyses predict that phenoxybenzamine and octoclothepin undergo interactions compatible with the active site of ParA. Octoclothepin exhibited significant bacteriostatic activity towards M. tuberculosis. CONCLUSIONS: Our data support the use of whole-cell differential antisense screens for the discovery of inhibitors of specific anti-tubercular drug targets. Using this approach, we have identified an inhibitor of purified ParA and whole cells of M. tuberculosis.

Modifying culture conditions in chemical library screening identifies alternative inhibitors of mycobacteria.

In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.