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A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from -proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.purine salvage ͉ lateral gene transfer ͉ Cryptosporidium parvum ͉ Toxoplasma gondii T he phylum Apicomplexa includes a large number of obligate intracellular parasites, among which are the human pathogens Plasmodium (malaria), Toxoplasma (AIDS-related encephalitis), Cryptosporidium, and Cyclospora (severe enteritis) as well as many parasites of substantial veterinary importance (Eimeria, Theileria, Sarcocystis, and Babesia). Toxoplasma gondii has emerged as a versatile genetic model organism to study the basic biology of this group of parasites (1). T. gondii is safe and easy to culture in vitro, and excellent animal models are available; it is also amendable to molecular genetic experimentation. The high transfection efficiencies, which can be achieved in this organism, have spurred the development of a powerful set of reverse genetic tools (2-4).Apicomplexa, as with most protists, diverged relatively early in the eukaryotic lineage and have many biological features not shared by major eukaryotic model systems (e.g., intracellular parasitism or the possession of secondary plastids). Several large scale sequencing efforts have increased the number of known apicomplexan genes drastically (5). Assigning biological functions to many of these genes, however, remains a major challenge. A forward genetic approach could bridge this gap and permit functional analysis of genes unique to Apicomplexa. The parasites maintain a haploid genome over most of their life cycle, which should greatly facilitate the generation of loss of function mutants. Indeed chemical as well as insertional mutagenesis has been applied successfully in several screens targeting nonessential genes involved in nucleotide biosynthesis (6-11). Parasites harboring conditional temperature-sens...

The hide and carcass hygiene of cull cattle at slaughter in four geographically distant regions of the United States was examined from July 2005 to April 2006 by measuring the aerobic plate counts (APC) and the prevalences and loads of Salmonella and Escherichia coli O157:H7. The geometric mean log 10 APC CFU/100 cm 2 levels on hides and preevisceration and postintervention carcasses ranged from 6.17 to 8.19, 4.24 to 6.47, and 1.46 to 1.96, respectively, and were highest in the summer (P < 0.0001). The average prevalences of Salmonella on hides and preevisceration and postintervention carcasses were 89.6% (95% confidence interval [CI], 85.1 to 94.0), 50.2% (95% CI, 40.9 to 59.5), and 0.8% (95% CI, 0.18 to 1.42), respectively. The prevalences of E. coli O157:H7 were 46.9% (95% CI, 37.3 to 56.6) and 16.7% (95% CI, 9.8 to 23.6) on hides and preevisceration carcasses, respectively. Examination of the concomitant incidence of Salmonella and E. coli O157:H7 showed that, on average, 33.3% (95% CI, 15.9 to 69.8) of cattle hide and 4.1% (95% CI, 0.98 to 17.3) of preevisceration carcass samples were contaminated with both pathogens. The pathogen prevalence on hides and carcasses was not significantly affected by the season; however, significant differences were observed between plants with respect to the incoming pathogen load and the ability to mitigate hide-to-carcass transfer. In spite of these differences, postintervention carcass contamination was significantly reduced (P < 0.001), likely as a result of the use of one or more of the processing interventions employed at each of the four processing plants examined.

In 1999 the foodborne pathogens Salmonella, Listeria, Campylobacter, and Escherichia coli (both O157 and non-O157) were estimated to cause more than 6 million illnesses and approximately 9000 deaths each year. However, the most recent Centers for Disease Control and Prevention report on the sources and incidence of foodborne disease, released in 2004, has shown a dramatic decrease in E. coli O157:H7 infections. Since raw beef products are the most frequently foodborne sources of these pathogens, the results of this report demonstrate that the microbiological quality of raw beef has improved greatly. During the intervening years, post-harvest interventions have continually improved, with new attention to hide decontamination and innovative treatments of carcasses. In addition, a system to hold and test beef trim or ground beef for E. coli O157:H7 before its release into commerce has provided an even greater level of safety. In this paper, we review the latest information on the prevalence of E. coli O157:H7 and other pathogens on beef, the evidence identifying the hide as the primary source of pathogens on beef carcasses, the efficacy of various hide and carcass interventions, and other developments that have led or have the potential to lead to even greater improvements in the microbial quality of beef.

Commercially produced ground beef samples (n ‫؍‬ 4,136) were collected from seven regions of the United States over a 24-month period (July 2005 to June 2007) and analyzed for the presence of Salmonella enterica by using methods that concurrently provided total prevalence and enumerable levels. The overall prevalence of Salmonella strains was 4.2%. Enumeration showed that 94.2% were present at levels below 2 CFU/g. Regional monthly prevalences of Salmonella strains varied from 1.8% to 6.5% but were not statistically different (P > 0.05). All Salmonella isolates were serotyped and their antibiotic susceptibilities determined and analyzed by pulsed-field gel electrophoresis (PFGE). The most common serotypes identified were Salmonella enterica serotypes Montevideo, Anatum, Muenster, and Mbandaka, with these accounting for one-half of the isolates obtained. The prevalence of multidrug-resistant (MDR) Salmonella was determined to be 0.6%. The most common MDR serotypes were Salmonella enterica serotypes Dublin, Reading, and Typhimurium. MDR strains had resistance to between 2 and 10 antibiotics. There were no regional differences in prevalence of MDR Salmonella. PFGE analysis revealed that indistinguishable XbaI and AvrII restriction digest patterns (RDPs) could be observed in isolates of the same serotype found in different regions and months of sampling. The RDPs of 19 Salmonella strains were compared to RDPs in the PulseNet USA database. Thirteen were indistinguishable from existing patterns, and the number of records for each ranged from 1 to 478. These data show that Salmonella prevalence in commercial ground beef is low and suggest that attempts to identify sources contributing to Salmonella in ground beef by serotype, antibiogram, and PFGE cannot be made without additional documented evidence.

Hide has been established as the main source of carcass contamination during cattle processing; therefore, it is crucial to minimize the amount of Escherichia coli O157:H7 on cattle hides before slaughter. Several potential sources of E. coli O157: H7 are encountered during transportation and in the lairage environment at beef-processing facilities that could increase the prevalence and numbers of E. coli O157:H7 on the hides of cattle. On three separate occasions, samples were obtained from cattle at the feedlot and again after cattle were stunned and exsanguinated at the processing plant (286 total animals). The prevalence of E. coli O157:H7 on hides increased from 50.3 to 94.4% between the time cattle were loaded onto tractor-trailers at the feedlot and the time hides were removed in the processing plant. Before transport, nine animals had E. coli O157:H7 in high numbers (Ͼ0.4 CFU/cm 2 ) on their hides. When sampled at the slaughter facility, the number of animals with high hide numbers had increased to 70. Overall, only 29% of the E. coli O157:H7 isolates collected postharvest (221 of 764) matched pulsed-field gel electrophoresis types collected before transport. The results of this study indicate that transport to and lairage at processing plants can lead to increases in the prevalence and degree of E. coli O157:H7 contamination on hides and the number of E. coli O157:H7 pulsed-field gel electrophoresis types associated with the animals. More study is needed to confirm the mechanism by which additional E. coli O157:H7 strains contaminate cattle hides during transport and lairage and to design interventions to prevent this contamination.Recent work has revealed that the major source of beef carcass contamination is cattle hides (4,7,8,20). In a study done by , the carcass contamination rates during processing could not be accounted for by the fecal prevalence of Escherichia coli O157:H7, but could be accounted for by hide prevalence. Bosilevac et al. (7,8) and Nou et al. (20) extended these findings by demonstrating that antimicrobial interventions targeting cattle hides lead to drastic reductions in the rates of carcass contamination with E. coli O157:H7.Because hide is the main source of carcass contamination, it is crucial to minimize the amount of E. coli O157: H7 on cattle hides before slaughter. Cattle can shed E. coli O157:H7 in their feces at concentrations as high as 10 6 CFU/g (24). One such high shedder could contaminate the hides of many animals, especially in high-density situations like the transport truck or lairage environment. Few studies have addressed the effects of external contamination sources on E. coli O157:H7 hide prevalence during transport and lairage. Barham et al. (3) isolated E. coli O157:H7 from samples taken from truck trailers before cattle were loaded. It was not

Aim:  To develop and validate high throughput methods for the direct enumeration of viable and culturable Salmonella and Escherichia coli O157:H7 in ground beef, carcass, hide and faecal (GCHF) samples from cattle. Methods and Results:  The hydrophobic grid membrane filtration (HGMF) method and the spiral plate count method (SPCM) were evaluated as rapid tools for the estimation of pathogen load using GCHF samples spiked with known levels of Salmonella serotype Typhimurium. Validation studies showed that for a single determination of each sample type the low end of the detection limits were approx. 2·0 × 100 CFU g−1 for ground beef, 5·0 × 10−1 CFU (100 cm2)−1 for Salmonella and 8·0 × 10−1 CFU (100 cm2)−1 for E. coli O157:H7 on carcasses, 4·0 × 101 CFU (100 cm2)−1 for hide and 2·0 × 102 CFU g−1 for faecal samples. In addition, ground beef (n = 609), carcass (n = 1520) and hide (n = 3038) samples were collected from beef‐processing plants and faecal samples (n = 3190) were collected from feed‐lot cattle, and these samples were tested for the presence of Salmonella and E. coli O157:H7 by enrichment and enumeration methods. Conclusions:  The direct enumeration methods described here are amenable to high throughput sample processing and were found to be cost‐effective alternatives to other enumeration methods for the estimation of Salmonella and E. coli O157:H7, in samples collected during cattle production and beef processing. Significance and Impact of the Study:  Use of the methods described here would allow for more routine testing and quantification data collection, providing useful information about the effectiveness of beef processing intervention strategies.