Survival and internalization of Salmonella and Escherichia coli O157:H7 sprayed onto different cabbage cultivars during cultivation in growth chambers.

BACKGROUND: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant's antimicrobial content. RESULTS: Internalized Salmonella was detected in cabbage within 24 h with prevalence ranging from 62% (16 of 26) for the 'Super Red 80' cultivar to 92% (24 of 26) for the 'Red Dynasty' cultivar. Surface survival of pathogens on small cabbage plants over nine days was significantly affected by cultivar with both pathogens surviving the most on the 'Farao' cultivar and Salmonella and O157 surviving the least on the 'Super Red 80' and 'Capture' cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen's fate. An inverse relationship existed between antimicrobial levels and the pathogen's surface survival on cabbage heads (P < 0.05). CONCLUSIONS: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop. © 2019 Society of Chemical Industry.

Population genetic characterization of Cyclospora cayetanensis from discrete geographical regions.

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis.

Stray cats are more frequently infected with zoonotic protists than pet cats.

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.

Multilocus Sequence Typing Tool for Cyclospora cayetanensis.

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

Novel rat model for neurocysticercosis using Taenia solium.

Neurocysticercosis is caused by Taenia solium infecting the central nervous system and is the leading cause of acquired epilepsy and convulsive conditions worldwide. Research into the pathophysiology of the disease and appropriate treatment is hindered by lack of cost-effective and physiologically similar animal models. We generated a novel rat neurocysticercosis model using intracranial infection with activated T. solium oncospheres. Holtzman rats were infected in two separate groups: the first group was inoculated extraparenchymally and the second intraparenchymally, with different doses of activated oncospheres. The groups were evaluated at three different ages. Histologic examination of the tissue surrounding T. solium cysticerci was performed. Results indicate that generally infected rats developed cysticerci in the brain tissue after 4 months, and the cysticerci were observed in the parenchymal, ventricle, or submeningeal brain tissue. The route of infection did not have a statistically significant effect on the proportion of rats that developed cysticerci, and there was no dependence on infection dose. However, rat age was crucial to the success of the infection. Epilepsy was observed in 9% of rats with neurocysticercosis. In histologic examination, a layer of collagen tissue, inflammatory infiltrate cells, perivascular infiltrate, angiogenesis, spongy change, and mass effect were observed in the tissue surrounding the cysts. This study presents a suitable animal model for the study of human neurocysticercosis.

The food industry's current and future role in preventing microbial foodborne illness within the United States.

During the past century, the microbiological safety of the US food supply has improved; however, many foodborne illnesses and outbreaks occur annually. Hence, opportunities for the food industry to improve the safety of both domestic and imported food exist through the adoption of risk-based preventive measures. Challenging food safety issues that are on the horizon include demographic changes to a population whose immune system is more susceptible to foodborne and opportunistic pathogens, climate changes that will shift where food is produced, and consumers' preferences for raw and minimally processed foods. Increased environmental and product testing and anonymous data sharing by the food industry with the public health community would aid in identifying system weaknesses and enabling more targeted corrective and preventive actions. Clinicians will continue to play a major role in reducing foodborne illnesses by diagnosing and reporting cases and in helping to educate the consumer about food safety practices.

Contamination of knives and graters by bacterial foodborne pathogens during slicing and grating of produce.

Poor hygiene and improper food preparation practices in consumers' homes have previously been demonstrated as contributing to foodborne diseases. To address potential cross-contamination by kitchen utensils in the home, a series of studies was conducted to determine the extent to which the use of a knife or grater on fresh produce would lead to the utensil's contamination with Escherichia coli O157:H7 or Salmonella enterica. When shredding inoculated carrots (ca. 5.3 log CFU/carrot), all graters became contaminated and the number of E. coli O157:H7 present on the utensil was significantly greater than Salmonella (p < 0.05). Contamination of knives after slicing inoculated produce (4.9-5.4 log CFU/produce item) could only be detected by enrichment culture. After slicing tomatoes, honeydew melons, strawberries, cucumbers, and cantaloupes, the average prevalence of knife contamination by the two pathogens was 43%, 17%, 15%, 7%, and 3%, respectively. No significant increase in the incidence or level of contamination occurred on the utensils when residues were present (p > 0.05); however, subsequent contamination of 7 produce items processed with the contaminated utensils did occur. These results highlight the necessity of proper sanitization of these utensils when used in preparation of raw produce.

Genetic recombination and Cryptosporidium hominis virulent subtype IbA10G2.

Little is known about the emergence and spread of virulent subtypes of Cryptosporidium hominis, the predominant species responsible for human cryptosporidiosis. We conducted sequence analyses of 32 genetic loci of 53 C. hominis specimens isolated from a longitudinally followed cohort of children living in a small community. We identified by linkage disequilibrium and recombination analyses only limited genetic recombination, which occurred exclusively within the 60-kDa glycoprotein gene subtype IbA10G2, a predominant subtype for outbreaks in industrialized nations and a virulent subtype in the study community. Intensive transmission of virulent subtype IbA10G2 in the study area might have resulted in genetic recombination with other subtypes. Moreover, we identified selection for IbA10G2 at a 129-kb region around the 60-kDa glycoprotein gene in chromosome 6. These findings improve our understanding of the origin and evolution of C. hominis subtypes and the spread of virulent subtypes.

Survey and genetic characterization of wastewater in Tunisia for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp.

The microbial diversity of wastewater used for irrigation and fertilization was assessed using specific polymerase chain reaction (PCR) assays to detect and genotype several pathogenic protists including Cryptosporidium spp., Giardia duodenalis, Cyclospora spp., Eimeria spp. and Enterocytozoon bieneusi. A total of 220 wastewater samples (110 raw, 110 treated) and 12 sludge samples were collected from 2005 to 2008 from 18 treatment plants located throughout Tunisia. Except for Cyclospora, which was detected only once, E. bieneusi (61%), G. duodenalis (28%), Cryptosporidium spp. (27%) and Eimeria spp. (45%) were frequently observed in wastewater and sludge. Sequencing of PCR products showed that C. hominis, C. andersoni, G. duodenalis sub-assemblage A-II and E. bieneusi genotypes D and IV were the most prevalent. An analysis of the distribution of 209 internal transcribed spacer sequences of E. bieneusi originating from wastewater at the 18 treatment plants showed a similar genetic diversity, regardless of the geographical location. The identification of these parasite species and genotypes and of host-specific Eimeria species indicates that the microbial quality of wastewater was impacted by humans, livestock and rodents. Given the public health risks that some of these parasites represent, guidelines on wastewater usage are needed to minimize human exposure to these pathogens.

Cryptosporidium tyzzeri and Cryptosporidium muris originated from wild West-European house mice (Mus musculus domesticus) and East-European house mice (Mus musculus musculus) are non-infectious for pigs.

Three and 8 week old pigs were inoculated with Cryptosporidium muris HZ206 (Mus musculus musculus isolate), Cryptosporidium tyzerri CR2090 (M. m. musculus isolate) or C. tyzzeri CR4293 (isolate from a hybrid between Mus musculus domesticus and M. m. musculus) at a dose of 1 × 10(7) oocysts per animal. Inoculated pigs showed no detectable infection and no clinical symptoms of cryptosporidiosis during 30 days post infection (DPI), and no macroscopic changes were detected in the digestive tract following necropsy. Developmental stages were not detected in gastrointestinal tract tissue by histology or PCR throughout the duration of the experiment. The infectivity of isolates was verified on SCID mice, in which oocysts shedding started from 4 to 8 DPI. Based on our findings, it can be concluded that pigs are not susceptible to C. muris or C. tyzzeri infection.

Update on Cyclospora cayetanensis, a food-borne and waterborne parasite.

The coccidian parasite Cyclospora cayetanensis is recognized as an emerging pathogen that causes protracted diarrhea in humans. The first cases of Cyclospora infection were reported in the late 1970s and were observed among expatriates and travelers in regions where infections are endemic. Since then, Cyclospora has been considered a cause of traveler's diarrhea. Epidemiological investigations were reported and examined in areas of endemicity even before the true identity of Cyclospora was elucidated. Cyclospora was fully characterized in the early 1990s, but it was not until the 1995 Cyclospora outbreak in the United States and Canada that it caught the attention of the public and physicians. The biology, clinical presentation, epidemiology, diagnosis, treatment, and control of cyclosporiasis are reviewed, with a focus on diagnostic assays currently being used for clinical and environmental samples. Challenges and limitations in working with Cyclospora are also discussed.

Molecular analysis of household transmission of Giardia lamblia in a region of high endemicity in Peru.

BACKGROUND: Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. METHODS: Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. RESULTS: Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. CONCLUSIONS: In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

Survival of Salmonella and Shiga Toxin-Producing Escherichia coli and Changes in Indigenous Microbiota during Fermentation of Home-Brewed Kombucha.

ABSTRACT: Survival and growth of Salmonella and Shiga toxin-producing Escherichia coli (STEC) were investigated in kombucha prepared from four brands of commercially available kombucha kits intended for use by home brewers. Changes in populations of the indigenous microbiota responsible for fermentation of kombucha were also determined. An initial population of Salmonella (6.77 log CFU/mL) decreased to below the detection limit (0.30 log CFU/mL) within 10 days in kombucha prepared from two of the test brands. Populations of 1.85 and 1.20 log CFU/mL were detected in two brands fermented for 14 days. An initial STEC population of 7.02 log CFU/mL decreased to <0.30 log CFU/mL in two brands within 14 days; 0.77 and 0.87 log CFU/mL were detected in kombucha prepared from the other two brands. Salmonella and STEC increased within 1 day in three brands of base tea used to prepare kombucha and were stable throughout 14 days of incubation. Both pathogens steadily declined in base tea prepared from one brand of kombucha kit. Inactivation of the pathogens occurred as the pH of the kombucha decreased, but a clear correlation between rates of inactivation among different brands of kits and decrease in pH was not evident. Growth and peak populations of mesophilic aerobic microorganisms, yeasts, lactic acid bacteria, and acetic acid bacteria varied depending on the kombucha kit brand. No strong evidence was found of a correlation between the behavior of Salmonella or STEC and that of any of these groups of indigenous microbiota. Results of this study show that survival of Salmonella and STEC in kombucha and base tea used to prepare kombucha is dependent on inherent differences in commercially available kombucha kits intended for use in home settings. Strict application of hygienic practices is essential for preventing contamination with Salmonella or STEC and reducing the risk of illness associated the consumption of kombucha.

Hyperendemic H. pylori and tapeworm infections in a U.S.-Mexico border population.

OBJECTIVE: A higher incidence of infectious disease has been documented in U.S. regions bordering Mexico compared with non-border areas. We assessed the prevalence of important gastrointestinal infections in Ciudad Juarez, Mexico, and El Paso, Texas, the largest binational community along the U.S.-Mexico border. METHODS: Fecal specimens from a sample of the asymptomatic population representing all ages were tested for Helicobacter pylori (H. pylori), Cryptosporidium spp., Giardia spp., and other intestinal parasitic pathogens using flotation, immunoassays, and/or polymerase chain reaction. We also measured indicators of microbiological contamination of drinking water, hands of food preparers, and kitchen surfaces. RESULTS: Overall, of the 386 participants, H. pylori was present in 38.2%, Taenia spp. in 3.3%, Giardia spp. in 2.7%, Cryptosporidium spp. in 1.9%, Entamoeba dispar in 1.3%, and Ascaris lumbricoides and Necator americanus in 0.3% of the study subjects; Cyclospora spp. and Entamoeba histolytica were not found. H. pylori infection was associated with handwashing (prevalence ratio [PR] = 1.3, 95% confidence interval [CI] 1.0, 1.8). Taenia spp. was found more often on the U.S. side (PR=8.6, 95% CI 2.3, 30.8). We did not find an association between these infections and the occurrence of total coliforms or fecal coliforms on kitchen surfaces. In addition, Escherichia coli was not found in any drinking water sample. CONCLUSION: The study results indicated that H. pylori and Taenia spp. infections may be highly prevalent along the U.S.-Mexico border. Additional research is necessary to adequately characterize the prevalence, as well as determine whether interventions that reduce these infections are warranted.

Frequency of diarrhoea as a predictor of elevated blood pressure in children.

BACKGROUND: Diarrhoeal illness is a major public health problem for children worldwide, particularly among developing countries, and is a proxy condition for severe dehydration. It has been hypothesized that severe dehydration in the first 6 months of life could be associated with increased blood pressure later in life. This study aimed to explore whether frequency of diarrhoea is associated with elevated blood pressure in children in a setting with a high incidence of diarrhoeal disease. METHODS: The present study is a cross-sectional study of blood pressure among children from a longitudinal child diarrhoeal disease cohort in Lima, Peru. From 2001 to 2006, daily diarrhoeal surveillance was made. Children were revisited in 2006 and blood pressure was measured. Diarrhoeal exposures were evaluated in terms of total number of diarrhoea days, number of episodes of diarrhoea, persistent diarrhoeal episodes and by the quartiles of daily incidence and episode incidence of diarrhoea. RESULTS: The overall incidence of diarrhoeal episodes at age under 1 year was 4.35 (95% confidence interval: 3.79-4.98) and under 5 years was 2.80 (95% confidence interval: 2.69-2.92). No association was observed between the total number of diarrhoeal days, diarrhoeal episodes or diarrhoeal incidence rates with childhood blood pressure. There was weak evidence that hospital admission due to severe dehydration in the first year of life showed a gradient towards an increase in both, systolic and diastolic blood pressure. CONCLUSION: In the first study to date to examine the association in a setting with a high incidence of diarrhoeal disease, diarrhoeal frequency did not show an association with increased blood pressure. Our observations of elevated levels of blood pressure among those admitted into hospitals in the first year of life are in line with the original hypothesis of dehydration in early infancy and high blood pressure. However, the effect of episodes of severe dehydration on later blood pressure remains uncertain.

Mitochondrial genome sequence variation as a useful marker for assessing genetic heterogeneity among Cyclospora cayetanensis isolates and source-tracking.

BACKGROUND: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks. METHODS: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products. RESULTS: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking. CONCLUSIONS: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.

Cryptosporidium species and subtypes and clinical manifestations in children, Peru.

To determine whether clinical manifestations are associated with genotypes or subtypes of Cryptosporidium spp., we studied a 4-year longitudinal birth cohort of 533 children in Peru. A total of 156 infection episodes were found in 109 children. Data from first infections showed that C. hominis was associated with diarrhea, nausea, vomiting, general malaise, and increased oocyst shedding intensity and duration. In contrast, C. parvum, C. meleagridis, C. canis, and C. felis were associated with diarrhea only. C. hominis subtype families were identified (Ia, Ib, Id, and Ie); all were associated with diarrhea. Ib was also associated with nausea, vomiting, and general malaise. All C. parvum specimens belonged to subtype family IIc. Analysis of risk factors did not show associations with specific Cryptosporidium spp. genotypes or subtypes. These findings strongly suggest that Cryptosporidium spp. and subtypes are linked to different clinical manifestations in children.

Use of PCR to improve diagnostic yield in an outbreak of cyclosporiasis in Lima, Peru.

Protozoal diseases are increasingly recognized as the cause of diarrhoeal outbreaks in both developed and developing countries. Cyclospora cayetanensis has been responsible for several epidemics in the last decade. In March 2005, an outbreak of diarrhoea was identified in recruits at the Ancon Naval Base in Lima, Peru. A case-control study was carried out. The overall diarrhoea attack rate was 53% (45/85). Complete data from 52 recruits were available for the analysis; 37 met the criteria for case and 15 for control. The epidemic curve indicated a point source transmission, with cases occurring over 9 days with a peak on the fifth day. Cyclospora cayetanensis was found in 7/37(18.9%) cases and 1/15 (6.7%) controls via standard microscopic techniques. PCR for C. cayetanensis detected 20/35 (57.1%) cases and 3/15 (20%) controls, demonstrating the improved diagnostic yield of this technique. This is the second report to characterize an outbreak of diarrhoea due to C. cayetanensis in Peru among a local population. The epidemiology and clinical course were similar to other reported outbreaks in developed regions. PCR greatly increased the number of C. cayetanensis cases detected during this outbreak, allowing the correct identification of its aetiology.

Efficacy of gaseous chlorine dioxide as a sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on produce.

The efficacy of gaseous chlorine dioxide to reduce parasite and bacterial burden in produce was studied. Basil and lettuce leaves were inoculated with Cryptosporidium parvum and Cyclospora cayetanensis oocysts, Encephalitozoon intestinalis spores, and a cocktail of two isolates of nalidixic acid-resistant Escherichia coli O157:H7. The inoculated samples were then treated for 20 min with gaseous chlorine dioxide at 4.1 mg/liter. Cryptosporidium had a 2.6 and 3.31 most-probable-number log reduction in basil and lettuce, respectively. Reduction of Encephalitozoon in basil and lettuce was 3.58 and 4.58 CFU/g respectively. E. coli loads were significantly reduced (2.45 to 3.97 log), whereas Cyclospora sporulation was not affected by this treatment.