Molecular-based surveillance of campylobacteriosis in New Zealand--from source attribution to genomic epidemiology.

Molecular-based surveillance of campylobacteriosis in New Zealand contributed to the implementation of interventions that led to a 50% reduction in notified and hospitalised cases of the country's most important zoonosis. From a pre-intervention high of 384 per 100,000 population in 2006, incidence dropped by 50% in 2008; a reduction that has been sustained since. This article illustrates many aspects of the successful use of molecular-based surveillance, including the distinction between control-focused and strategy-focused surveillance and advances in source attribution. We discuss how microbial genetic data can enhance the understanding of epidemiological explanatory and response variables and thereby enrich the epidemiological analysis. Sequence data can be fitted to evolutionary and epidemiological models to gain new insights into pathogen evolution, the nature of associations between strains of pathogens and host species, and aspects of between-host transmission. With the advent of newer sequencing technologies and the availability of rapid, high-coverage genome sequence data, such techniques may be extended and refined within the emerging discipline of genomic epidemiology. The aim of this article is to summarise the experience gained in New Zealand with molecular-based surveillance of campylobacteriosis and to discuss how this experience could be used to further advance the use of molecular tools in surveillance.

Multiple-locus variable-number tandem-repeat analysis for discriminating within Salmonella enterica serovar Typhimurium definitive types and investigation of outbreaks.

The discriminatory power of multiple-locus variable-number tandem-repeat analysis (MLVA) needs to be evaluated for all Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) phage types so that the power of this methodology is understood and results can be interpreted correctly during outbreak investigations. We evaluated the ability of MLVA to characterize four definitive phage types (DT) problematic in New Zealand. MLVA discriminated between DT104 isolates although there was very limited variation in the MLVA profiles for isolates with an RDNC phage type (reacts but does not conform to a recognized Typhimurium phage pattern) first observed in New Zealand's Enteric Reference Laboratory in May 2006. Most DT101 isolates had indistinguishable MLVA profiles or profiles that differed at one or two loci. This was also observed in DT160 isolates. MLVA may not identify all common-source outbreaks although it provided valuable data when applied to case isolates from two S. Typhimurium outbreaks.

Characterization of Escherichia coli O157:H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis.

Recently, multiple-locus variable-number tandem-repeat analysis (MLVA) has been proposed as an alternative to pulsed-field gel electrophoresis (PFGE) for characterization of Escherichia coli O157:H7. In this study we characterized 118 E. coli O157:H7 isolates from cases of gastrointestinal disease in New Zealand using XbaI PFGE profiles and a MLVA scheme that assessed variability in eight polymorphic loci. The 118 isolates characterized included all 80 E. coli O157:H7 referred to New Zealand's Enteric Reference Laboratory in 2006 and 29 phage-type 2 isolates from 2005. When applied to these isolates the discriminatory power of PFGE and MLVA was not significantly different. However, MLVA data may be more epidemiologically relevant as isolates from family clusters of disease had identical MLVA profiles, even when the XbaI PFGE profiles differed slightly. Furthermore, most isolates with indistinguishable XbaI PFGE profiles that did not appear to be epidemiologically related had distinct MLVA profiles.

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium.

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.

Molecular and spatial epidemiology of human campylobacteriosis: source association and genotype-related risk factors.

The epidemiology of human campylobacteriosis is complex but in recent years understanding of this disease has advanced considerably. Despite being a major public health concern in many countries, the presence of multiple hosts, genotypes and transmission pathways has made it difficult to identify and quantify the determinants of human infection and disease. This has delayed the development of successful intervention programmes for this disease in many countries including New Zealand, a country with a comparatively high, yet until recently poorly understood, rate of notified disease. This study investigated the epidemiology of Campylobacter jejuni at the genotype-level over a 3-year period between 2005 and 2008 using multilocus sequence typing. By combining epidemiological surveillance and population genetics, a dominant, internationally rare strain of C. jejuni (ST474) was identified, and most human cases (65.7%) were found to be caused by only seven different genotypes. Source association of genotypes was used to identify risk factors at the genotype-level through multivariable logistic regression and a spatial model. Poultry-associated cases were more likely to be found in urban areas compared to rural areas. In particular young children in rural areas had a higher risk of infection with ruminant strains than their urban counterparts. These findings provide important information for the implementation of pathway-specific control strategies.

Novel clonal complexes with an unknown animal reservoir dominate Campylobacter jejuni isolates from river water in New Zealand.

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis.

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum.

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.

Quantification of historical livestock importation into New Zealand 1860-1979.

AIMS: To quantify the numbers of live cattle, sheep and poultry imported into New Zealand and, where possible, their country of origin from 1860 to 1979. METHODS: Information on the origin and number of live animal importations into New Zealand was collected for cattle, sheep and poultry for the period 1868-1979 from the annual reports compiled by the New Zealand Registrar General's Office, Government Statistician's Office, Census and Statistics Office, Census and Statistics Department, Customs Department and Department of Statistics. Census data from 1851 to 1871 were also used to estimate the livestock population during this period. The number of animals imported and the mean population for each species in a decade were determined, and the major countries of origin were identified. RESULTS: A large number of cattle (53,384) and sheep (604,525) were imported in the 1860s, and then there was a marked reduction in importations. Live poultry were imported in relatively small numbers (20,701) from 1880 to 1939, then 1,564,330 live poultry were imported between 1960 and 1979. Australia was the predominant country of origin for sheep between 1868 and 1959 (51,347/60,918; 84.3%) and of cattle between 1868 and 1979 (10,080/15,157; 66.5%). Only 6,712 (11.0%) sheep and 3,909 (25.8%) cattle were imported from the United Kingdom over the same periods, and even fewer from other countries. CONCLUSIONS: The collated data and historical reports show that from 1860 to 1979 Australia has been the main source of livestock introduced into New Zealand. The pattern of importation showed that large numbers of cattle and sheep were initially imported in the 1860s, probably in response to rapid agricultural expansion. Thereafter importations continued at much reduced numbers. In contrast, relatively small numbers of poultry were introduced until the 1960s when large numbers were imported as part of the development of a modern high-production industry. The overall pattern for both cattle and sheep was of a bottleneck event, as initially a relatively limited number of animals arrived from outside populations, followed by population expansion with ongoing but limited immigration (admixture). Investigation into the genetic population structure of New Zealand's cattle and sheep, as well as their host-associated microorganisms, could reflect the impact of these early historical events.

Utilizing a combination of molecular and spatial tools to assess the effect of a public health intervention.

Until recently New Zealand had one of the highest rates of human campylobacteriosis reported by industrialized countries. Since the introduction of a range of control measures in the poultry production chain a reduction in human cases of around 50% has been observed nationwide. To inform risk managers a combination of spatial, temporal and molecular tools - including minimum spanning trees, risk surfaces, rarefaction analysis and dynamic source attribution modelling - was used in this study to formally evaluate the reduction in disease risk that occurred after the implementation of control measures in the poultry industry. Utilizing data from a sentinel surveillance site in the Manawatu region of New Zealand, our analyses demonstrated a reduction in disease risk attributable to a reduction in the number of poultry-associated campylobacteriosis cases. Before the implementation of interventions poultry-associated cases were more prevalent in urban than rural areas, whereas for ruminant-associated cases the reverse was evident. In addition to the overall reduction in prevalence, this study also showed a stronger intervention effect in urban areas where poultry sources were more dominant. Overall a combination of molecular and spatial tools has provided evidence that the interventions aimed at reducing Campylobacter contamination of poultry were successful in reducing poultry-associated disease and this will inform the development of future control strategies.

Wide geographical distribution of internationally rare Campylobacter clones within New Zealand.

During the southern hemisphere winter of 2006 New Zealand experienced a significant increase in the number of reported cases of Campylobacter infection. In total, 112 Campylobacter isolates from eight district health boards (DHBs) located across New Zealand were submitted for PFGE, MLST and Penner serotyping analysis. Distinct clusters of Campylobacter isolates were identified, several of which were composed of isolates from up to five different DHBs located on both the North and South islands of New Zealand. One sequence type, ST-474, was identified in 32 of the 112 isolates and may represent an endemic sequence type present in New Zealand. The spatial pattern of genotypes, combined with the generalized increase in notifications throughout the country is consistent with a common source epidemic, most likely from a source contaminated with the dominant sequence types ST-474 and ST-190 and may also represent widely distributed stable clones present in New Zealand.

Glucagon for hypoglycaemia in infants small for gestational age.

Twenty five infants who were small for gestational age received glucagon (0.5 mg/day by continuous infusion) in the treatment of hypoglycaemia. Twenty responded within three hours with a rise in blood glucose concentration to above 4 mmol/l. Five subjects subsequently required hydrocortisone to maintain glucose concentrations. Rebound hypoglycaemia occurred in nine infants after rapid discontinuation of glucagon or interruption of the intravenous infusions. Response was poor after maternal beta blockade.

Genomic and cDNA cloning of the human C1 inhibitor. Intron-exon junctions and comparison with other serpins.

Amino acid sequencing of trypsin fragments of C1 inhibitor gave regions of low codon degeneracy that were used for oligonucleotide probes. Human liver cDNA libraries gave clones containing most of the protein sequence, showing that the inhibitory domain belongs to the 'serpin' class of protein inhibitors. Fragments of these cDNA clones were used to probe human genomic cosmid libraries. The genomic sequence was found to be about 17 X 10(3) base pairs, with a coding sequence of approximately 1800 base pairs containing introns at amino acid positions--6, 162, 207, 275, 321, 395, and one in the 5' non-coding region. There is very little similarity of intron position amongst the serpin genes. All but one of the intron positions in the C1 inhibitor structural gene correspond to surface residues if C1 inhibitor is considered to have a structure similar to the cleaved form of alpha 1-antiproteinase. The serine and threonine residues in the N-terminal 100 amino acids of the sequence thought to carry complex carbohydrates are found in a single exon.

Coagulase gemne variants associated with distinct populations of Staphylococcus aureus.

An identifying characteristic of Staphylococcus aureus is the production of staphylocoagulase (coagulase). The aim of this study was to determine the clonal distribution of coagulase gene (coa) variants within populations of S. aureus defined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and protein A variation. The N-terminal region of the coa gene from 43 methicillin-susceptible (MSSA) and 252 methicillin-resistant (MRSA) S. aureus human isolates and 9 animal S. aureus isolates was amplified and digested with HinfI. Twelve types were identified amongst the MSSA isolates and the majority (93%) of MRSA isolates were assigned to 5 of the 12 types. MLST and PFGE analysis identified epidemic populations of MRSA and each epidemic population was characterized by a different coagulase type. Nine of the 12 MLST-defined clonal complex ancestral genotypes recently described each carried a different coagulase type suggesting that coagulase evolution and the evolution of the clonal complexes are intimately related.

The TATA-binding protein (TBP) from the human fungal pathogen Candida albicans can complement defects in human and yeast TBPs.

Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed the C. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal delta spt15 mutation in Saccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene.

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV-VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.

Structure and activity of C1r and C1s.

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.

The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%).