Risk Assessment of Escherichia coli O157 illness from consumption of hamburgers in the United States made from Australian manufacturing beef.

We analyze the risk of contracting illness due to the consumption in the United States of hamburgers contaminated with enterohemorrhagic Escherichia coli (EHEC) of serogroup O157 produced from manufacturing beef imported from Australia. We have used a novel approach for estimating risk by using the prevalence and concentration estimates of E. coli O157 in lots of beef that were withdrawn from the export chain following detection of the pathogen. For the purpose of the present assessment an assumption was that no product is removed from the supply chain following testing. This, together with a number of additional conservative assumptions, leads to an overestimation of E. coli O157-associated illness attributable to the consumption of ground beef patties manufactured only from Australian beef. We predict 49.6 illnesses (95%: 0.0-148.6) from the 2.46 billion hamburgers made from 155,000 t of Australian manufacturing beef exported to the United States in 2012. All these illness were due to undercooking in the home and less than one illness is predicted from consumption of hamburgers cooked to a temperature of 68 °C in quick-service restaurants.

Microbiological Quality of Australian Beef, Sheep and Pork Carcases, Cuts and Offals.

A one-year survey was undertaken of the microbiological quality of carcases and the derived primal cuts, manufacturing meat and offals at twelve Australian export establishments (six beef, three sheep/lamb and three pork). A total of 27,157 microbiological results for aerobic plate count (APC) and generic Escherichia coli were gathered, 15,155 from beef, 8405 from sheep and 3597 from pig establishments. The mean log10 APCs on beef, sheep and pig carcases were 0.84, 1.60 and 1.30 log10 cfu/cm2, respectively. For primals, the mean log10 APC was higher for beef but was similar for sheep and pork primals, with 'outside' cuts having higher counts. For manufacturing meat, the concentration was 2-3 log10 cfu/g, irrespective of species. The prevalence (%) of generic E. coli from beef, sheep and pork was 2.3, 28.4 and 5.4 on carcases; 7.0, 20.6 and 3.2 on primals; and 5.8, 33.6 and 6.1 on manufacturing meat, respectively. The mean log10 APCs of beef, sheep and pork offal were 3.23, 3.18 and 3.37 log10 cfu/g, with tripes and tongues having APCs 1-2 log10 units higher than organ offals. The results reflect improvements in total bacterial loadings compared with previous national baseline surveys.

2009 pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States.

In the spring of 2009, a novel influenza A (H1N1) virus emerged in North America and spread worldwide to cause the first influenza pandemic since 1968. During the first 4 months, over 500 deaths in the United States had been associated with confirmed 2009 pandemic influenza A (H1N1) [2009 H1N1] virus infection. Pathological evaluation of respiratory specimens from initial influenza-associated deaths suggested marked differences in viral tropism and tissue damage compared with seasonal influenza and prompted further investigation. Available autopsy tissue samples were obtained from 100 US deaths with laboratory-confirmed 2009 H1N1 virus infection. Demographic and clinical data of these case-patients were collected, and the tissues were evaluated by multiple laboratory methods, including histopathological evaluation, special stains, molecular and immunohistochemical assays, viral culture, and electron microscopy. The most prominent histopathological feature observed was diffuse alveolar damage in the lung in all case-patients examined. Alveolar lining cells, including type I and type II pneumocytes, were the primary infected cells. Bacterial co-infections were identified in >25% of the case-patients. Viral pneumonia and immunolocalization of viral antigen in association with diffuse alveolar damage are prominent features of infection with 2009 pandemic influenza A (H1N1) virus. Underlying medical conditions and bacterial co-infections contributed to the fatal outcome of this infection. More studies are needed to understand the multifactorial pathogenesis of this infection.

Rickettsia 364D: a newly recognized cause of eschar-associated illness in California.

BACKGROUND: Four spotted fever group rickettsiae (SFGR) are known to infect humans in the United States. A member of the SFGR designated 364D and detected in Dermacentor occidentalis ticks has not previously been identified as a human pathogen. METHODS: An 80-year-old man from a rural northern California community presented with an eschar on his forearm. A skin punch biopsy of the lesion was evaluated by immunohistochemistry and molecular analysis. Serum specimens obtained from the patient and 3 other area residents with similar illnesses were tested by immunofluorescence and Western immunoblot for antibodies to SFGR. Ticks were collected near the patient's residence and tested for SFGR. RESULTS: Abundant intracellular rickettsiae and fragmented rickettsial antigens were observed in the mononuclear inflammatory infiltrates of the biopsy. Nucleotide sequences of DNA fragments amplified from the biopsy were identical to those of 364D. Convalescent sera from all four patients exhibited high immunoglobulin G titers to Rickettsia rickettsii, Rickettsia rhipicephali, and 364D antigens. Three adult D. occidentalis were positive for 364D, R. rhipicephali, and an unidentified Rickettsia species. CONCLUSIONS: This is the first confirmation of human disease associated with the SFGR 364D, which was likely transmitted by D. occidentalis. Although the patients described here presented with a single cutaneous eschar as the principal manifestation, the full spectrum of illness associated with 364D has yet to be determined. Possible infection with 364D or other SFGR should be confirmed through molecular techniques in patients who present with "spotless" Rocky Mountain spotted fever or have serum antibodies to R. rickettsii with group-specific assays.

Isolation of Rickettsia parkeri and identification of a novel spotted fever group Rickettsia sp. from Gulf Coast ticks (Amblyomma maculatum) in the United States.

Until recently, Amblyomma maculatum (the Gulf Coast tick) had garnered little attention compared to other species of human-biting ticks in the United States. A. maculatum is now recognized as the principal vector of Rickettsia parkeri, a pathogenic spotted fever group rickettsia (SFGR) that causes an eschar-associated illness in humans that resembles Rocky Mountain spotted fever. A novel SFGR, distinct from other recognized Rickettsia spp., has also been detected recently in A. maculatum specimens collected in several regions of the southeastern United States. In this study, 198 questing adult Gulf Coast ticks were collected at 4 locations in Florida and Mississippi; 28% of these ticks were infected with R. parkeri, and 2% of these were infected with a novel SFGR. Seventeen isolates of R. parkeri from individual specimens of A. maculatum were cultivated in Vero E6 cells; however, all attempts to isolate the novel SFGR were unsuccessful. Partial genetic characterization of the novel SFGR revealed identity with several recently described, incompletely characterized, and noncultivated SFGR, including "Candidatus Rickettsia andeanae" and Rickettsia sp. Argentina detected in several species of Neotropical ticks from Argentina and Peru. These findings suggest that each of these "novel" rickettsiae represent the same species. This study considerably expanded the number of low-passage, A. maculatum-derived isolates of R. parkeri and characterized a second, sympatric Rickettsia sp. found in Gulf Coast ticks.

New approaches to detection and identification of Rickettsia africae and Ehrlichia ruminantium in Amblyomma variegatum (Acari: Ixodidae) ticks from the Caribbean.

Imported from Africa in the 1700s and despite frequent modern eradication efforts, Amblyomma variegatum (F.) spread through the Caribbean by cattle transport, small ruminants, and migrating birds. A. variegatum is a vector for Rickettsia africae, the causative agent of African tick bite fever, and Ehrlichia ruminantium, the causative agent of heartwater. We examined 95 A. variegatum and six Rhipicephalus (Boophilus) microplus (Canestrini) collected from cattle at an abattoir in Antigua. Engorged tick extracts adsorbed on Nobotu filter paper strips and new nested polymerase chain reaction (PCR) assays for E. ruminantium and Dermatophilus congolensis were used to evaluate these ticks for the presence of these pathogenic bacteria. Amblyomma ticks (62.4%) contained R. africae DNA by PCR/restriction fragment length polymorphism analysis and DNA sequencing of the OmpA and 17-kDa antigen genes. Twenty Amblyomma and two Rh. microplus contained E. ruminantium DNA. No E. chaffeensis, Anaplasma phagocytophilum, Coxiella burnetii, or D. congolensis DNA was detected in these ticks. The continued presence of Am. variegatum in the Caribbean poses a significant risk of infection in cattle with E. ruminantium and in humans by R. africae. Eradication efforts are essential to prevent the further spread of Am. variegatum.

Ehrlichia ewingii in an immunocompetent adult.

The third Ehrlichia infection described in humans in the United States is reviewed. This rare zoonosis was first published in 1999. This case report that expands the clinical paradigm is described. A 57-year-old immunocompetent adult, unlike previously published reports, did not cross react with Ehrlichia chaffeensis.

Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever.

BACKGROUND: Rickettsia parkeri rickettsiosis, a recently identified spotted fever transmitted by the Gulf Coast tick (Amblyomma maculatum), was first described in 2004. We summarize the clinical and epidemiological features of 12 patients in the United States with confirmed or probable disease attributable to R. parkeri and comment on distinctions between R. parkeri rickettsiosis and other United States rickettsioses. METHODS: Clinical specimens from patients in the United States who reside within the range of A. maculatum for whom an eschar or vesicular rash was described were evaluated by > or =1 laboratory assays at the Centers for Disease Control and Prevention (Atlanta, GA) to identify probable or confirmed infection with R. parkeri. RESULTS: During 1998-2007, clinical samples from 12 patients with illnesses epidemiologically and clinically compatible with R. parkeri rickettsiosis were submitted for diagnostic evaluation. Using indirect immunofluorescence antibody assays, immunohistochemistry, polymerase chain reaction assays, and cell culture isolation, we identified 6 confirmed and 6 probable cases of infection with R. parkeri. The aggregate clinical characteristics of these patients revealed a disease similar to but less severe than classically described Rocky Mountain spotted fever. CONCLUSIONS: Closer attention to the distinct clinical features of the various spotted fever syndromes that exist in the United States and other countries of the Western hemisphere, coupled with more frequent use of specific confirmatory assays, may unveil several unique diseases that have been identified collectively as Rocky Mountain spotted fever during the past century. Accurate assessments of these distinct infections will ultimately provide a more valid description of the currently recognized distribution, incidence, and case-fatality rate of Rocky Mountain spotted fever.

Rickettsia parkeri in Argentina.

Clinical reports of an eschar-associated rickettsiosis in the Paraná River Delta of Argentina prompted an evaluation of Amblyomma triste ticks in this region. When evaluated by PCR, 17 (7.6%) of 223 questing adult A. triste ticks, collected from 2 sites in the lower Paraná River Delta, contained DNA of Rickettsia parkeri.

Microbial profiles of carcasses and minced meat from kangaroos processed in South Australia.

The microbiological profiles of kangaroo carcasses and minced meat at game meat processing plants in South Australia were determined in surveys undertaken in 2002 and 2004. In 2002 mean values for log(10) total viable counts (TVC) on carcasses at individual plants ranged from 0.9 to 3.9 log(10) cfu/cm(2), with the mean for all plants being 2.3 log(10) cfu/cm(2). In 2004 the between plant range was narrower, by about 1 log unit, and the mean value for carcasses at all plants was 1.2 log(10) cfu/cm(2). Minced kangaroo meat, was sampled in 2002 only. The overall mean log(10) TVC was 3.9 log(10) cfu/g, with mean counts at individual plants ranging from 3.1 to 4.6 log(10) cfu/g. The overall prevalence of E. coli was 70%, with mean numbers of 2.1 log(10) cfu/g on positive samples. Salmonella was not detected in any of 60 samples from carcasses in 2002. However, in 2004 Salmonella was detected in 4/385 samples (1.04%, 95% CI: 0.28%-2.64%). In minced kangaroo meat, Salmonella was detected in 9/50 (18%, 95% CI: 9%-31%) samples. The abdominal cavity, sampled in 2004, was found to be highly contaminated, with E. coli isolated from 46% of samples and the mean number for positive samples being 2.7 log(10) cfu/cm(2); Salmonella was isolated from 14/120 (12%; 95% CI: 6.52%-18.80%) of abdominal cavities. The practice of collecting carcasses together and pushing grouped carcasses into the chiller likely leads to cross contamination of carcasses from the abdominal cavities of others. To align results of sampling by swabbing for domestic purposes with excision sampling, required for export purposes, both methods were used to sample opposite sides of each of the 50 carcasses sampled in 2004. The results obtained with the two methods of sampling were similar.

Effect of incubation temperature on aerobic plate counts of beef and sheep carcasses.

Australian regulations for microbiological testing of carcasses specify a number of incubation temperatures and media for meat processed at both domestic and export establishments. Accordingly, the effect of incubation temperature and media on aerobic plate counts of samples from beef and sheep carcasses was investigated. For both species, aerobic plate counts on Petrifilm incubated at 35 degrees C were significantly lower than those counts on Petrifilm and pour plates incubated at 25 and 30 degrees C, reflecting the inability of many psychrotrophs to grow at 35 degrees C. When samples were taken from carcasses that had been stored in abattoir chillers for periods between 16 h and 5 days, difference between counts at 35 degrees C versus those incubated at 25 and 30 degrees C became greater as the period of refrigerated storage increased. For export beef carcasses, the effect of this difference is minimal, since the vast majority of counts incubated at 35 degrees C are done on carcasses that have been chilled for less than 24 h and will not have a large proportion of psychrotrophs.

A national survey of the microbiological quality of retail raw meats in Australia.

A national survey of the microbiology of meat (ground beef and diced lamb) at the retail level in Australia was undertaken. For ground beef samples (n = 360), the mean aerobic plate count (APC) was 5.79 log CFU/g, and Escherichia coli was detected in 17.8% of samples; the mean population for these positive samples was 1.49 log CFU/g. Enterobacteriaceae were detected in 96.9% of samples (mean for positive samples, 3.01 log CFU/g), and coagulase-positive staphylococci were detected in 28.1% of samples (mean for positive samples, 2.18 log CFU/g). For diced lamb samples (n = 360), the mean APC was 5.71 log CFU/g, and E. coli was detected in 16.7% of samples (mean for positive samples, 1.67 log CFU/g). Enterobacteriaceae were detected in 91.1% of samples (mean for positive samples, 2.85 log CFU/g), and coagulase-positive staphylococci were detected in 22.5% of samples (mean for positive samples, 2.34 log CFU/g). Salmonella was recovered from 4 (1.1%) of the 360 ground beef samples (isolates were Salmonella Typhimurium phage types), and E. coli O157 was recovered from 1 (0.3%) of 357 samples; Campylobacter and Clostridium perfringens were not recovered from any of the 91 and 94 samples tested, respectively. Salmonella was recovered from 2 (0.6%) of the 360 diced lamb samples (serovars were Salmonella Infantis and Salmonella Typhimurium), Campylobacter was recovered from 1 (1.1%) of 95 samples, and C. perfringens was recovered from 1 (1.1%) of 92 samples.

An alternative process for cleaning knives used on meat slaughter floors.

Traditionally on slaughter floors operator knives are cleaned by rinsing in hand wash water at 20-40 degrees C followed by brief immersion in baths termed "sterilisers" which contain water no cooler than 82 degrees C. Under Australian legislation, both domestic and export, it is possible for a meat processing establishment to apply to the Controlling Authority for permission to implement an alternative procedure providing that it is at least the equivalent of that legislated. No firm evidence appears to exist for the 82 degrees C requirement and the possibility of replacing this element of the knife cleaning procedure with an alternative procedure using 60 degrees C water and a longer immersion time was investigated at an abattoir slaughtering cattle and sheep. Knives were tested at a range of work stations located along beef and mutton slaughter floors for Aerobic Plate Counts (APCs) and E. coli. For knives used on the beef chain the mean log APC/cm(2) was 2.18 by the current knife cleaning process and 1.78 by the alternate procedure (P<0.001). Using the current system E. coli was isolated from cleaned knives on 20/230 (8.7%) occasions compared with 21/230 (9.1%) occasions using the alternative system. The mean log E. coli of positive knives was 0.43/cm(2) and 0.61/cm(2) from the current and alternative systems, respectively. On the mutton chain the mean log APC/cm(2) was 1.95 using the current knife cleaning process and 1.69 by the alternative procedure (P=0.014). Using the current system E. coli was isolated from cleaned knives on 24/130 (18.5%) occasions compared with 29/130 (22.3%) occasions using the alternative system. The mean log E. coli of positive knives was 0.90/cm(2) and 0.76/cm(2) from the current and alternative systems, respectively. It is concluded that using two knives alternatively, rinsing them in hand wash water, then immersing them between uses in 60 degrees C water provides a microbiological outcome equivalent to rinsing them and momentary dipping in 82 degrees C water.

Effect of acidified sodium chlorite treatment on chicken carcases processed in South Australia.

A trial on the effectiveness of acidified sodium chlorite (ASC) on Salmonella and Campylobacter was undertaken on chicken carcases after they exited the screw chiller of a commercial premises in Adelaide, Australia. On untreated carcases mean log10 total viable count (25 degrees C) was 2.78/cm2 compared with 1.23/cm2 on treated carcases. Prevalence of E. coli, Salmonella and Campylobacter was 100%, 90% and 100% respectively, on untreated carcases and 13%, 10% and 23% respectively, on treated carcases. The distributions of E. coli, Salmonella and Campylobacter (mean log10 of positive samples) from untreated carcases were 1.55, -1.80 and 1.59/cm2 respectively, and -0.64, -1.85 and -2.21/cm2 respectively, on treated carcases. On untreated carcases S. Sofia and S. Infantis were isolated from 73% and 37% of carcases, respectively; only S. Sofia was isolated from treated carcases. The significant reductions in both prevalence and concentration demonstrated in the present trial indicate that ASC is a risk management option immediately available to the poultry industry.

Diagnosis of invasive group a streptococcal infections by using immunohistochemical and molecular assays.

Invasive group A streptococcus (GAS) infections cause 1,100 to 1,300 deaths annually in the United States. Diagnosis is made when Streptococcus pyogenes is isolated from pus or body fluids; however, cultures are not always obtained, and antibiotic treatment can preclude bacterial growth. An immunohistochemical assay for GAS was applied to formalin-fixed tissue samples from 122 patients with suspect GAS infection. Immunohistochemical staining of well-defined cocci and small, granular antigen fragments was observed in 27 cases. S pyogenes was isolated in 18 cases, whereas in 8 cases, immunohistochemical staining was confirmed by amplification of the sepB gene of S pyogenes from paraffin-embedded samples in a heminested polymerase chain reaction (PCR) assay. A primary focus of infection (respiratory, mucocutaneous, or gynecologic) was present in 22 patients, whereas 5 had no identifiable primary focus of infection. Eighteen patients had systemic infection. Immunohistochemical analysis and PCR can be used for diagnosis of GAS infections in formalin-fixed, paraffin-embedded samples.

Isolation of Rickettsia akari from eschars of patients with rickettsialpox.

Rickettsialpox is a cosmopolitan, mite-borne, spotted fever rickettsiosis caused by Rickettsia akari. The disease is characterized by a primary eschar, fever, and a papulovesicular rash. Rickettsialpox was first identified in New York City in 1946 and the preponderance of recognized cases in the United States continues to originate from this large metropolitan center. The most recently isolated U.S. strain of R. akari was obtained more than a half century ago. We describe the culture and initial characterization of five contemporaneous isolates of R. akari obtained from eschar biopsy specimens from New York City patients with rickettsialpox. This work emphasizes the importance and utility of culture-and molecular-based methods for the diagnosis of rickettsialpox and other eschar-associated illnesses.

Use of routine beef carcase Escherichia coli monitoring data to investigate the relationship between hygiene status of incoming stock and processing efficacy.

In Australian export-registered abattoirs microbiological monitoring is carried out within the E. coli and Salmonella Monitoring (ESAM) program. During the calendar year 2003, the ESAM database indicated a national prevalence of Escherichia coli of around 3.0% for steers/heifers and 7.1% for cows/bulls. An investigation was carried out to attempt to elucidate why some establishments had E. coli prevalence markedly higher or markedly lower than the national average. The investigation was based on a questionnaire completed by fifteen export establishments which provided data on livestock, processing, operator training and management. The responses were verified by site visits and then evaluated for their relationship with ESAM data on E. coli in two stages. In stage 1, E. coli prevalence for each abattoir was plotted against each variable recorded by the questionnaire; no single variable was a reasonable predictor for prevalence of E. coli on carcases. In stage 2, variables influencing contamination were grouped under two categories: contamination on incoming livestock (Problem variables) together with the ability of the plant's process to deal with such contamination (Process variables). The analysis prompted two main conclusions. Firstly, plants with a large incoming problem with livestock (long haul, high tag score and proportion of cows/bulls slaughtered) plus "poor" processes had higher than average E. coli prevalence. Secondly, plants with hot water decontamination systems had low E. coli prevalence even when there was a substantial incoming problem with livestock, such as a relatively high proportion of cows/bulls.

A national survey of the microbiological quality of beef carcasses and frozen boneless beef in Australia.

The third national baseline microbiological survey of Australian beef carcasses and frozen boneless beef was conducted in 2004. Carcasses (n=1155) sampled at 27 slaughter establishments had a mean aerobic plate count (at 25 degrees C) of 1.3 log CFU/cm2. Escherichia coli was isolated from 8.0% of the cacasses, with a mean count of -0.8 log CFU/cm2 for positive samples. On samples from 24 boning (fabrication) plants (n=1082), the mean aerobic plate count for frozen boneless beef was 1.3 log CFU/g, and the mean count for the 1.8% of samples with detectable E. coli was 1.5 log CFU/g. E. coli O157: H7 was isolated from 1 of 1,143 carcasses and from 0 of 1082 boneless samples. Salmonella was isolated from 0 of 1155 carcasses and from 1 of 1082 samples of boneless product. No Campylobacter spp. were isolated from carcasses or boneless beef. Coagulase-positive staphylococci were isolated from 28.7% of beef carcasses and 20.3% of boneless beef samples, and positive samples had a mean count of 0.3 log CFU/cm2 and 0.8 log CFU/g, respectively.

Phylogenetic analysis of a novel molecular isolate of spotted fever group Rickettsiae from northern Peru: Candidatus Rickettsia andeanae.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

Using national microbiological data to set meaningful performance criteria for slaughter and dressing of animals at Australian export abattoirs.

Slaughter establishments in Australia that export meat to the USA are required by the controlling authority, the Australian Quarantine and Inspection Service (AQIS), to test carcases under the Escherichia coli and Salmonella monitoring (ESAM) program and to use statistical process control techniques to ensure meat is produced hygienically. However, analysing the ESAM database for E. coli using standard statistical techniques proved difficult because of inter-plant variability and because the vast majority of results were below the limits of detection. As well, it is likely that, in slaughter and dressing, higher than normal microbiological counts can often be random events, for which there is neither logical explanation nor obvious management reaction. One approach to statistical process control is to set performance criteria so that a high proportion of establishments are likely to pass, while prompting individual plants to improve the process if they cannot meet the criteria. A spreadsheet-based tool was developed in Visual Basic in order to interrogate the ESAM database and to identify those plants with microbiological performance significantly different from the norm. The present paper describes how performance criteria for cattle, sheep, pigs and goats and for sub-categories within a species (e.g. sheep/lambs, cows/bulls) were established.