Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx(1), and stx(2) genes and isolates harboring the eaeA, stx(1), and hly(933) genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.     

Sorbitol-fermenting, β-glucuronidase-positive, Shiga toxin-negative Escherichia coli O157:H7 in free-ranging red deer in South-Central Spain

We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24?h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.     

Laboratory investigation of an E. coli O157:H7 outbreak associated with swimming in Battle Ground Lake,Vancouver, Washington

Escherichia coli O157:H7 has been associated with a number of waterborne outbreaks, but it has never been recovered from an implicated environment. This paper reports on an August 1999 outbreak of E. coli O157:H7 associated with swimming in Battle Ground Lake in Clark Country, Washington. E. coli O157:H7 was isolated from duck feces, as well as from two water samples. The authors used pulsed-field gel electrophoresis to compare these isolates with patient isolates for genetic homology. All the isolates yielded the same restriction fragment patterns. In addition, using polymerase chain reaction, the authors found patient isolates and environmental isolates to have the same virulence factors (Stx, eaeA, and hly).     

Further evidence of constrained radiation in the evolution of pathogenic Escherichia coli O157:H7

Escherichia coli O157:H7 is a human pathogen that has emerged from its less pathogenic progenitor, E. coli O55:H7, to form the EHEC 1 clade. In its emergence, E. coli O157:H7 formed three distinct clusters, each of which exists today. Sequencing and SNP analysis of Cluster 1 of this clade demonstrated constrained radiation from the cluster founder. Here we investigated the diversity of Cluster 2 strains by sequencing signature SNPs in six strains collected throughout Washington State. Our results suggest that successful Cluster 2 strains have radiated on only two branches from their founder; one of these two branches leads to Cluster 3. Constrained radiation appears to be a common theme among this pathogenic clade.     

Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.     

Geographic Divergence of Bovine and Human Shiga Toxin–Producing Escherichia coli O157:H7 Genotypes,New Zealand

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a zoonotic pathogen of public health concern worldwide. To compare the local and large-scale geographic distributions of genotypes of STEC O157:H7 isolates obtained from various bovine and human sources during 2008–2011, we used pulsed-field gel electrophoresis and Shiga toxin–encoding bacteriophage insertion (SBI) typing. Using multivariate methods, we compared isolates from the North and South Islands of New Zealand with isolates from Australia and the United States. The STEC O157:H7 population structure differed substantially between the 2 islands and showed evidence of finer scale spatial structuring, which is consistent with highly localized transmission rather than disseminated foodborne outbreaks. The distribution of SBI types differed markedly among isolates from New Zealand, Australia, and the United States. Our findings also provide evidence for the historic introduction into New Zealand of a subset of globally circulating STEC O157:H7 strains that have continued to evolve and be transmitted locally between cattle and humans.     

Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates

Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances.     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

2007年浙江省衢州地区动物中产志贺毒素大肠埃希菌O157:H7感染状况

目的:了解2007年浙江省衢州地区产志贺毒素大肠埃希菌O157:H7在动物中的分布情况及其耐药性、PFGE分型及毒力基因携带状况。方法:按全国O157:H7监测方案于5~10月份肠道传染病高发季节,在衢州地区采集各种动物粪便/肛拭,用免疫磁珠富集后进行O157:H7分离培养、鉴定,可疑菌株以PCR法检测O、H抗原及志贺样毒素(SLT1和SLT2)、粘附抹平因子(eaeA)及溶血素(hly)4种毒力基因。用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法进行同源性分析,同时选择14种抗生素进行药敏试验,分析分离所得菌株的耐药状况。结果:共监测动物粪便标本300份,分离得产志贺毒素大肠埃希菌O157:H7菌株16株,分离率为5.33%。16株O157:H7菌株,毒力基因Hly、eaeA、SLT2均阳性,SLT1均阴性。脉冲场凝胶电泳分型显示,16株O157:H7菌株可分2个PFGE基因型,型间差异较小。耐药性分析显示这些菌株对红霉素、利福平的耐药率最高,达100.0%,对其他受试抗生素均敏感。结论:该地区动物中产志贺毒素大肠埃希菌O157:H7带菌率较高,所分离菌株主要携带SLT2基因,因此推测该地区存在发生产志贺毒素大肠埃希菌O157:H7感染暴发或流行的潜在危险,需增加对动物源性O157:H7的监测力度。     

Surveillance of Escherichia coli O157 isolation and confirmation, United States, 1988

In 1989, to examine patterns of testing for Escherichia coli O157:H7 in state public health laboratories (SPHLs), CDC conducted a survey to determine the availability and type of Escherichia coli O157:H7 testing in SPHLs during 1988 and the number of isolates confirmed at SPHLs if such testing was available. The results were compared with information on isolates submitted for confirmatory testing at CDC in 1988. Thirty-nine (78%) of the 51 SPHLs were testing for E. coli O157:H7 in 1988; 26 confirmed at least one E. coli O157 isolate in that year. CDC confirmed isolates from three additional states. A total of 489 E. coli O157:H7 or E. coli O157:NM isolates were identified, with the largest numbers being reported from Washington (156), Oregon (64), Minnesota (63), and Massachusetts (36). These results show that E. coli O157 has been detected in most areas of the United States. Infections are apparently concentrated in northern states; however, improved surveillance data are needed to determine regional incidence and trends.     

徐州市2000年肠出血性大肠埃希菌O157：H7感染性腹泻的调查

目的 了解徐州市丰县和铜山县肠出血性大肠埃希菌（EHEC）O157：H7引起的出血性结肠炎（HC）在腹泻病患者中所占的比例，以及临床症状和肾功能变化的情况。方法 使用EHECO157胶体金快速检测试剂筛选粪便标本，阳性者使用免疫磁珠方法分离病原菌。对经过细菌学证实的EHECO157：H7引起的HC患者，进行临床症状的观察和生化检验指标分析。结果 2000年5月份，丰县由EHECO157：H7引起的HC占腹泻病患者0.98%，6月份铜山县的HC患者占腹泻病患者5.89%。在出现腹泻病症状的同时，18.5%患者的肾功能已经出现异常，表现为尿蛋白，血清肌苷或血尿素氮等指标的升高，在27例HC患者中，有14和13例分别分离到不产生志贺毒素和产生志贺毒素的EHECO157：H7菌株，根据分离菌株是否产生志贺毒素将患者分为两组进行比较，尿蛋白阳性患者的比例分别为4/13和1/14，血小板减少患者的比例分别为6/13和3/14，统计学分析有显著意义。提示分离到产生志贺毒素的EHECO157：H7菌株的患者，发生肾功能异常的可能性较大，结论 此次调查证实了EHECO157：H7感染所引起的HC在整个腹泻病患者中所占的比例随季节的变化而不同，感染产生志贺毒素EHECO157：H7菌株的患者，发生肾功能异常的可能性要比不产生志贺毒素的大，还证实了在该菌感染的初期，患者的肾功能就已经出现了异常。     

我国部分产生志贺毒素肠出血性大肠埃希菌O157：H7脉冲电场凝胶电泳分型

目的 探讨我国部分地区产生志贺毒素的肠出血性大肠埃希菌（EHEC）O157：H7菌株的分布，流行以及分型情况。方法 用脉冲电场凝胶电泳（PFGE）方法进行分型，辅以药物敏感性试验对51株产生志贺毒素的EHECO157：H7菌株进行研究。结果 根据细菌染色体DNA的XbaI酶切图谱，可将从江苏省徐州市等地分离的51株产生志贺毒素的EHECO157：H7菌株分成8个PFGE型别（PFGE1-PFGE8），宁夏菌株为PFGE1型和2型，徐州菌株有6个PFGE型，1986-1988年的分离菌株属于PFGE7型，1999、2000年病人分离株的优势型别分别是PFGE5型与3型，1999年爆发性流行期间从患者分离的5株菌属于PFGE5型，从家畜家禽的粪便，食品，蔬菜等标本分离的19株菌，可以分为PFGE3-6等4个型别，其中，PFGE5型占优势（10株菌）。结论 爆发性流行的发生与携带病原菌的家畜家禽粪便污染的食品，蔬菜等有关，从我国部分地区分离的EHECO157：H7菌株的PFGE型别有地区性差异。     

Clade analysis of enterohemorrhagic Escherichia coli serotype O157:H7/H- strains and hierarchy of their phylogenetic relationships

Enterohemorrhagic Escherichia coli serotype O157:H7/H-(O157) strains isolated in Chiba prefecture, Japan, during 2002–2009 were studied by lineage, subgroup, cluster, and clade analysis. Lineage analysis of 470 O157 strains with no known epidemiological relationships using lineage specific polymorphism assay-6 showed that there were 242 lineage I strains, 160 lineage I/II strains, 67 lineage II strains, and 1 atypical strain. Clade analysis of these strains by single nucleotide polymorphism in eight loci showed that lineage I contained all the clade 1, clade 2, and clade 3 strains, and some of the clade 4/5 strains. In contrast, clade 7, clade 8, and the remaining clade 4/5 strains were divided between lineage I/II and II, and clade 6 was in lineage I/II, suggesting paraphyletic evolution of these lineages. Cluster and subgroup analysis of the stx phage insertion site showed that all lineage I strains were cluster 3 and all lineage I/II and II strains, with the exception of clade 9, were in cluster 1. Clade analysis also indicated that there were three phylogenetic groups of clade 4/5 strains: ancestral groups containing lineage I/IIand II strains and a descendant group containing lineages I. Analysis of stx2c gene distribution showed that stx2c was in ancestral clade 4/5 strains but not in descendant 4/5 strains, suggesting that the ancestral group may be clade 4 as reported by Manning et al. The results with the markers used in this study suggested that the hierarchy of O157 phylogenetic relationships was lineage as the upper level, followed by subgroup and then cluster, and clade as the lowest level. The need for refinement of clade definition and modification of the model of the O157 evolution have been discussed.     

Identification of intermediate in evolutionary model of enterohemorrhagic Escherichia coli O157

Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H(-) consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H(-) and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H(-) isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H(-). The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.     

Improved use of SNP information to detect the role of genes

A topical question in genetic association studies is the optimal use of the information provided by genotyped single-nucleotide polymorphisms (SNPs) in order to detect the role of a candidate gene in a multifactorial disease. We propose a strategy called "combination test" that tests the association between a quantitative trait and all possible phased combinations of various numbers of SNPs. We compare this strategy to two alternative strategies: the association test that considers each SNP separately, and a multilocus genotype-based test that considers the phased combination of all SNPs together. To compare these three tests, a quantitative trait was simulated under different models of correspondence between phenotype and genotype, including the extreme case when two SNPs interact with no marginal effects of each SNP. The genotypes were taken from a sample of 290 independent individuals genotyped for three genes with various number of SNPs (from 5-8 SNPs). The results show that the "combination test" is the only one able to detect the association when the two SNPs involved in disease susceptibility interact with no marginal effects. Interestingly, even in the case of a single etiological SNP, the "combination test" performed well. We apply the three tests to Genetic Analysis Workshop 12 (Almasy et al. [2001] Genet. Epidemiol. 21:332-338) simulated data, and show that although there was no interactions between the etiological SNPs, the "combination test" was preferable to the two other compared methods to detect the role of the candidate gene.     

Genetic characterization of a diverse Escherichia coli O157:H7 population from a variety of farm environments

Many of the current studies on the genetic diversity of Escherichia coli O157:H7 have focused on pathogenic clinical, veterinary, or food isolates. These studies did not explore the diversity of the larger population in the farm environment. Research on selected farm isolates address this wider diversity but have typically been limited to a specific geographic locale or farm type, thus giving limited insight into the greater diversity across geographic regions and varied environments. The objective of this study was to evaluate a diverse population of E. coli O157:H7 collected from a variety of locations and farm environments. Eighty-eight isolates were collected from four farm types (swine, dairy, beef, and poultry) across the southeastern and western United States. Eighteen farms were sampled every 3 months over a period of 24 months. Isolates were analyzed by ribotyping and pulsed field gel electrophoresis (PFGE). Real-time PCR was used to determine the presence or absence of key pathogenic genes (stx1, stx2, and eae). The data indicate a significant amount of genetic diversity, however, ribotype analysis revealed meaningful clusters within the larger population. These groupings were consistent with PFGE analysis. Most of these isolates were clustered by location (i.e. from the same state or region) or farm type. Of the isolates in these clusters, most did not contain pathogenic genes. Of notable interest is a single group in which the majority of isolates, collected from four of the five states sampled, contained at least one stx gene and the eae gene suggesting the existence of a specific pathogenic cluster. These data suggest that, while there is notable diversity within the broader E. coli O157:H7 population, pathogenic isolates may be limited to a subset of strains within the population.     

Antimicrobial susceptibility and mechanism of resistance to antibiotics for Escherichia coli O157 and verotoxin-producing E. coli isolated in northern Kyushu and Yamaguchi area

The purpose of this study was to investigate the biochemical characteristics and antimicrobial susceptibility of Escherichia (E.) coli O157 and verotoxin-producing E. coli isolates from the Northern Kyushu Island and Yamaguchi area of Japan. A total of 54 isolates- 50 E. coli O157, 3 verotoxin-producing E. coli O26 and 1 verotoxin-producing E. coli O111 - were used in this study. Regarding H antigen, H7 type in E. coli O157 accounted for 98% (49/50), and residual 1 strain of E. coli O157 was untypable H type. Two of 3 E. coli O26 isolates were H11 type, residual 1 strain of E. coli O26 was untypable H type, and E. coli O111 isolate was non-motile strain. All 54 isolates were susceptible to cephems, fosfomycin, kanamycin, amikacin and co-trimoxazole. Tetracycline-resistant isolates existed in 13 of all 54 isolates, 5 of those 13 isolates had tetA, and the other 7 isolates had tetB. Eight amoxicillin-resistant isolates had TEM-1 beta-lactamase. Four of the 5 isolates that had tetA also had TEM-1 beta-lactamase. Nalidixic acid and 6 fluoroquinolone used had no insensitive or resistant isolates. Kanamycin-resistant isolates, fosfomycin- and nalidixic acid-insensitive isolates have been reported, so we must notice the antibiogram of such strains. It is important that the surveillance of antimicrobial susceptibility of enterohemorragic E. coli should be continued after this.     

Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers

The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle.     

An outbreak of Escherichia coli O157:H7 infections and haemolytic uraemic syndrome associated with consumption of unpasteurized apple cider

During October 1996, an outbreak of Escherichia coli O157:H7 infections among Connecticut residents occurred. An epidemiologic investigation included enhanced surveillance and a case-control study. Clinical isolates of Escherichia coli O157:H7 were typed by pulsed-field gel electrophoresis (PFGE). Implicated cider samples were analysed by culture and polymerase chain reaction (PCR). Consumption of implicated cider was associated with illness; (matched odds ratio = undefined, 95 % confidence interval = 3.5-infinity). Ultimately, a total of 14 outbreak-associated patients were identified. All isolates analysed by PFGE yielded the outbreak-associated subtype. Escherichia coli O157:H7 was not cultured from three cider samples; PCR analysis detected DNA fragments consistent with Escherichia coli O157:H7 in one. This outbreak was associated with drinking one brand of unpasteurized apple cider. PFGE subtyping supported the epidemiologic association. PCR analysis detected microbial contaminants in the absence of live organisms. Washing and brushing apples did not prevent cider contamination.     

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.