Investigation of On-Farm Transmission Routes for Contamination of Dairy Cows with Top 7 Escherichia coli O-Serogroups.

Shiga toxin-producing Escherichia coli (STEC) are foodborne bacterial pathogens, with cattle a significant reservoir for human infection. This study evaluated environmental reservoirs, intermediate hosts and key pathways that could drive the presence of Top 7 STEC (O157:H7, O26, O45, O103, O111, O121 and O145) on pasture-based dairy herds, using molecular and culture-based methods. A total of 235 composite environmental samples (including soil, bedding, pasture, stock drinking water, bird droppings and flies and faecal samples of dairy animals) were collected from two dairy farms, with four sampling events on each farm. Molecular detection revealed O26, O45, O103 and O121 as the most common O-serogroups, with the greatest occurrence in dairy animal faeces (> 91%), environments freshly contaminated with faeces (> 73%) and birds and flies (> 71%). STEC (79 isolates) were a minor population within the target O-serogroups in all sample types but were widespread in the farm environment in the summer samplings. Phylogenetic analysis of whole genome sequence data targeting single nucleotide polymorphisms revealed the presence of several clonal strains on a farm; a single STEC clonal strain could be found in several sample types concurrently, indicating the existence of more than one possible route for transmission to dairy animals and a high rate of transmission of STEC between dairy animals and wildlife. Overall, the findings improved the understanding of the ecology of the Top 7 STEC in open farm environments, which is required to develop on-farm intervention strategies controlling these zoonoses.

Prevalence of Shiga toxin-producing Escherichia coli in pasture-based dairy herds.

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E. coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E. coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n = 36) and composite calf faeces (n = 12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.

Health, physiology, and behavior of dairy calves reared on 4 different substrates.

The objective of this study was to compare the health, physiology, and behavior of group-housed calves reared on wood shavings with those reared on alternative surfaces. At 1 wk of age, 80 calves were moved into 1 of 20 experimental pens (n = 4 calves/pen) where they remained until 6 wk of age. Pens had floors covered with pea gravel (PG), rubber chip (RC), sand (SA), or wood shavings (WS; n = 5 pens/substrate). Body weight, cleanliness, health, and skin surface and vaginal temperature were recorded at 1, 3, and 6 wk of age. Escherichia coli numbers were assessed on the skin surface of the shoulder and in the feces of calves at 3 and 6 wk of age. Blood samples were taken at 1, 3, and 6 wk of age to measure hematological values and cortisol, IgG, and lactate concentrations. Behaviors (lying, running, and self-grooming) were recorded in the home pen at 1, 3, and 6 wk of age using video recorders and accelerometer data loggers. At 6 wk of age, calves were tested individually in an arena test and behavior was recorded continuously for 20 min. Body weight did not differ among calves reared on PG, RC, SA, or WS, regardless of age. All calves were clean and no calves displayed any signs of lameness, leg lesions, or injuries at wk 1, 3, or 6, regardless of substrate. The number of E. coli recovered from a surface area of 100 cm2 on the shoulder of each calf was affected by rearing substrate, with more E. coli recovered from calves reared on WS than PG, RC, or SA at 3 and 6 wk of age. Fecal E. coli counts were not affected by rearing substrate at 3 or 6 wk of age. Over the entire study period, calves reared on PG and SA had lower skin temperatures than calves reared on RC or WS, but skin temperature was similar between calves reared on PG and SA. However, vaginal temperature did not differ among calves reared on different substrates at 1, 3, or 6 wk of age. Hematology values and cortisol, IgG, and lactate concentrations of calves were similar among rearing substrates over the 6-wk study period. In the home pen, rearing substrate did not influence time spent lying; however, calves reared on WS performed more lying bouts than calves reared on PG or SA. In addition, rearing substrate did not influence the time calves spent running; however, calves reared on WS spent more time self-grooming than calves reared on PG, RC, and SA. During a 20-min arena test, running, bucks, jumps, and kicks performed by calves was not affected by rearing substrate. In conclusion, the physiology and behavior of calves reared on PG, RC, and SA was similar to WS, which is considered the preferred rearing substrate to use when rearing calves. Therefore, PG, RC, and SA may be acceptable substrate options when rearing group-housed dairy calves.

Diversity and relatedness of Shiga toxin-producing Escherichia coli and Campylobacter jejuni between farms in a dairy catchment.

The aim of this study was to examine the population structure, transmission and spatial relationship between genotypes of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, on 20 dairy farms in a defined catchment. Pooled faecal samples (n = 72) obtained from 288 calves were analysed by real-time polymerase chain reaction (rtPCR) for E. coli serotypes O26, O103, O111, O145 and O157. The number of samples positive for E. coli O26 (30/72) was high compared to E. coli O103 (7/72), O145 (3/72), O157 (2/72) and O111 (0/72). Eighteen E. coli O26 and 53 C. jejuni isolates were recovered from samples by bacterial culture. E. coli O26 and C. jejuni isolates were genotyped using pulsed-field gel electrophoresis and multilocus sequence typing, respectively. All E. coli O26 isolates could be divided into four clusters and the results indicated that E. coli O26 isolates recovered from calves on the same farm were more similar than isolates recovered from different farms in the catchment. There were 11 different sequence types of C. jejuni isolated from the cattle and 22 from water. An analysis of the population structure of C. jejuni isolated from cattle provided evidence of clustering of genotypes within farms, and among groups of farms separated by road boundaries.

Prevalence, concentration and genotypes of Campylobacter jejuni in faeces from dairy herds managed in farm systems with or without housing.

AIM: To determine the faecal excretion of Campylobacter jejuni by dairy cows that used housing in combination with outdoor grazing. METHODS AND RESULTS: Campylobacter jejuni prevalence and concentration were measured in a total of 990 cow faecal samples collected from seven herd home farms (HH), seven stand-off pad farms (SOP) and seven pasture farms (P) over a 2-year period. On all the farms, cows had access to pasture but were restricted to narrow grazing strips in winter. The overall Camp. jejuni prevalence was 55, 49 and 54% on HH, SOP and P farms, respectively. The Camp. jejuni concentration ranged from 0 to 6·7 log10 g(-1) faeces and was not statistically different among the farm systems. However, Camp. jejuni prevalence (P = 0·014) and concentration (P = 0·0001) were significantly greater in winter and early spring after intensive use of HH, SOP and strip-grazing. Typing of 30 Camp. jejuni isolates revealed a dominance of ruminant types (MLST CC-61, CC-21, CC-42 and CC-48), which are associated with human disease. CONCLUSION: No overall difference was observed among systems, but seasonal management practices that force cows close together increased the prevalence and concentration of Camp. jejuni in faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are important when identifying farm practices that reduce Camp. jejuni excretion and the associated risk to human health.

Effect of using a stand-off pad on Campylobacter jejuni strain diversity in a herd of dairy cows.

AIM: To determine the effect of stand-off pad (SOP) use on the prevalence and strain diversity of Campylobacter jejuni in a small herd of dairy cows. METHODS AND RESULTS: Faecal samples were collected from 21 cows on four sampling occasions (events), one in each season, over 1 year. The cows usually grazed on pasture but during winter they spent 18 h a day on a SOP. Campylobacter prevalence ranged from 48-52% on pasture but was 62% on the SOP. The diversity of 386 C. jejuni isolates was determined using Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (ERIC/PCR). There were 11 ERIC types identified for the herd over the course of the study. Of those 11, four to seven (per event) were present when the cows were grazing pasture but only two during SOP use. CONCLUSIONS: The use of the SOP was associated with an increase in prevalence and a reduction in diversity of C. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction in ERIC types on the SOP indicated an increase in transfer of only some strains of C. jejuni among the cows. One of these strains persisted throughout the study. The zoonotic potential of this strain warrants further investigation.

Campylobacter jejuni inactivation in New Zealand soils.

AIM: The study was undertaken to determine the inactivation rate of Campylobacter jejuni in New Zealand soils. METHODS AND RESULTS: Farm dairy effluent (FDE) inoculated at c. 10(5) ml(-1) with C. jejuni was applied to intact soil cores at a rate of 2 l m(-2). Four soils were used: Hamilton (granular); Taupo (pumice); Horotiu and Waihou (allophanic). After FDE application cores were incubated at 10 degrees C for up to 32 days. For all four soils all the FDE remained within the cores and at least 99% of C. jejuni were retained in the top 5 cm. Campylobacter jejuni had declined to the limit of detection (two C. jejuni 100 g(-1)) by 25 days in Hamilton and Taupo soils and by 32 days in Waihou soil. In contrast, in Horotiu soil the decline was only three orders of magnitude after 32 days. Simulated heavy rainfall was applied 4 and 11 days after FDE application and only about 1% of the applied C. jejuni were recovered in leachates. CONCLUSIONS: This study demonstrated that at least 99% of applied C. jejuni were retained in the top 5 cm of four soils where they survived for at least 25 days at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil retention of C. jejuni is efficient at FDE application rates that prevent drainage losses. The low infectious dose of C. jejuni and its ability to survive up to 25 days have implications for stock management on dairy farms.

Phosphorylation of Ca2+/calmodulin-dependent protein kinase type ii and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet.

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c-fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Arranging child care.

More than half of the children in families supported by welfare are under age six, and another third are in grade school. The mothers of these children cannot leave welfare for employment unless they can find and pay for child care. Yet, as this article points out, the child care needs of these families are not easily met: Many require care for infants and toddlers, care at odd hours, and care in poor neighborhoods-all of which are scarce. Evidence reviewed by the authors indicates that problems with child care affordability, availability, and quality impede mothers from participating in the labor force and in job training programs. Recent public finding for child care subsidies has helped families leaving welfare to afford the child care they need, although the demand for financial assistance outstrips available funding. This article urges that policymakers work to facilitate access to subsidies, increase the supply of care that can meet the needs of poor working families, and guard against exposure to poor-quality care that can jeopardize both children's well-being and parents' employment.

Quality control of bacterial enumeration.

Standard bacterial suspensions can be used to assess test method performance, via control charts, and inhibition of recovery when analyzing water samples. Variability in standard suspensions prepared from different strains and species and the use of frozen environmental samples for quality control for spore and bacteriophage analyses are also discussed.

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms.

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.

Regulation of tryptophan biosynthesis in Caulobacter crescentus.

We present an analysis of the expression of the trpE gene and the trpFBA operon in the dimorphic bacterium Caulobacter crescentus. The catalytic activity of component I of anthranilate synthase, the product of the trpE gene, was efficiently inhibited by tryptophan, the end product of the pathway, which suggests that tryptophan biosynthesis is likely controlled at the pathway level in C. crescentus. However, trpFBA mRNA levels and trpE enzyme levels did not vary significantly in wild-type C. crescentus in response to the presence of tryptophan in the growth medium or to growth in minimal versus rich medium. This lack of regulation of the trpE, trpF, trpB, and trpA genes is consistent with the idea that oligotrophic bacteria, such as C. crescentus, do not utilize regulatory mechanisms that greatly alter the biosynthetic capabilities in exponentially growing cells. In contrast, mRNA levels from the 5'-untranslated region and the upstream gene (usg) coding region increased dramatically in C. crescentus trpD or hisB auxotrophs starved for tryptophan or histidine, respectively. Surprisingly, concomitant increases in mRNA levels were not detected from the trpF, trpB, or trpA coding regions downstream in the operon. Thus, severe starvation of C. crescentus for amino acids appears to elicit a strong, general transcriptional response that is not observed in bacteria growing exponentially in medium lacking amino acids.

Structure of the Caulobacter crescentus trpFBA operon.

The DNA sequences of the Caulobacter crescentus trpF, trpB, and trpA genes were determined, along with 500 base pairs (bp) of 5'-flanking sequence and 320 bp of 3'-flanking sequence. An open reading frame, designated usg, occurs upstream of trpF and encodes a polypeptide of 89 amino acids which seems to be expressed in a coupled transcription-translation system. Interestingly, the usg polypeptide is not homologous to any known tryptophan biosynthetic enzyme. S1 nuclease mapping of in vivo transcripts indicated that usg, trpF, trpB, and trpA are arranged into a single operon, with the transcription initiation site located 30 bp upstream from the start of usg. Sequences centered at -30 and -6 bp upstream from the transcription initiation site are somewhat homologous to the Escherichia coli promoter consensus sequence and are homologous to sequences found upstream of genes from several organisms which are evolutionarily related to C. crescentus. Furthermore, the trpFBA operon promoter sequence lacks homology to promoter sequences identified for certain developmentally regulated C. crescentus genes. The structures of the C. crescentus usg, trpF, trpB, and trpA genes were further analyzed in terms of codon usage, G+C content, and genetic signals and were related to genetic signals previously identified in C. crescentus and other bacteria. Taken together, these results are relevant to the analysis of gene expression in C. crescentus and the study of trp gene structure and regulation.

Interpreting changes in the economic status of the elderly, 1949-1979.

"This paper analyzes changes in the economic well-being of the elderly using data from the [U.S.] Decennial Censuses of 1950 through 1980. We find that the economic status of each elderly cohort is higher on average than that of the preceding cohort. Certain events associated with age--retirement for both men and women and widowhood for women--are associated with declining incomes. Controlling for sex, labor force participation, and marital status, however, the economic well-being of elderly cohorts generally increases with age."