Sakchai Himathongkham

Ultraviolet Inactivation of Feline Calicivirus, Human EntericViruses and Coliphages¶

Norwalk and Norwalk‐like viruses (NLV) are major causes of food‐ and water‐related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque‐forming units/mL) of UV‐inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight‐line curves represent exponential inactivation, so UV inactivation can be said to show “one‐hit kinetics.” The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and ϕX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures.

Exponential inactivation was observed for Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure with decimal reduction times ranging from half a day at 37 C to 1-2 wk at 4 C. There was no material difference in inactivation rates between S. typhimurium and E. coli O157:H7. Inactivation was slower in slurries made by mixing two parts of water with one part of manure; decimal reduction times (time required for 90% destruction) ranged from 1-2 days at 37 C to 6-22 wk at 4 C. Escherichia coli O157:H7 consistently exhibited slightly slower inactivation than S. typhimurium. Log decimal reduction time for both strains was a linear function of storage temperature for manure and slurries. Chemical analysis indicated that accumulation of free ammonia in poultry manure was an important factor in inactivation of the pathogens. This finding was experimentally confirmed for S. typhimurium by adding ammonia directly to peptone water or to bovine manure, which was naturally low in ammonia, and adjusting pH to achieve predetermined levels of free ammonia.

Efficacy of disinfection of shell eggs externally contaminated with Salmonella enteritidis: Implications for egg testing

Experimental contamination of the surface of shell eggs by dipping in a culture of Salmonella enteritidis resulted in the presence of Salmonella enteritidis in/on the shells as well as shell membranes but not in the egg content. Disinfection with Lugols solution, chlorhexidine, ethanol, quarternary ammonium solutions or flaming after dipping in ethanol failed to achieve complete decontamination of the shell and membranes with resulting false positives when eggs were broken for culturing of the content. Dipping eggs for three seconds in boiling water resulted in complete destruction of Salmonella enteritidis in shells and membranes but sometimes caused the eggs to crack. A method of aseptically opening eggs without risk of contaminating the content from the shell or membrane was developed. Salmonella enteritidis deposited in/on the shell and membranes did not multiply during storage of the eggs at 20 degrees C for four weeks, the counts seemed to decrease. No Salmonella enteritidis was detected in the contents of any contaminated eggs.

Heat Destruction of Salmonella in Poultry Feed: Effect of Time, Temperature, and Moisture

Salmonella enteriditis in poultry feed declines with increasing time of exposure to heat. The interactions of temperature, time, and moisture and their effect on the thermal death of S. enteriditis were established in a factorial randomized experiment. Two other serotypes were tested, and though there was some variation, the thermal death rate followed the same basic pattern. A number of samples of poultry feed were collected and dried. After drying, the water was added back to give specific percentages of moisture contents. The feed was then inoculated with salmonella and heated at specific temperatures, with samples being removed at certain time intervals. These samples were then cultured, and the surviving salmonella were counted. A linear relationship was obtained when the logarithm of survivors was plotted against the logarithm of exposure time. These results permitted the construction of a graph depicting that the rate of reduction in numbers of S. enteriditis when plotted against increasing temperatures is linear. This linear relationship is apparent for other salmonella serotypes such as S. typhimurium and S. haardt. Our results show that the thermal death rate of salmonella in poultry feed can now be predicted at varying time, temperature, and moisture contents.

Reduction of Escherichia coli O157:H7 and Salmonella Typhimurium in Artificially Contaminated Alfalfa Seeds and Mung Beans by Fumigation with Ammonia

Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium. Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins. Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting. Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide. Samples were taken after intervals up to 22 h at 20 degrees C. Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans. Greater kills are apparently associated with lower initial bacterial loads. Germination of these seeds was unaffected by the treatment. It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.

Destruction of Salmonella enteritidis in poultry feed by combination of heat and propionic acid.

A factorial laboratory experiment was conducted to assess the effects of heating times of 0, 20, 40, and 80 sec at 160 F and propionic acid concentrations of 0, 0.1%, and 0.2% on reduction of Salmonella enteritidis in poultry feed with approximately 15% moisture. The results showed that after 80 sec heating time an approximately 10,000-fold reduction in living salmonella was obtained in the samples with 0.2% propionic acid. Survival in the 0.2% acid group was 2 log10 lower than in the 0.1% and control groups. This difference was statistically significant. Multivariate analysis with repeated measures showed there was no interaction between heating time and propionic acid concentration (P = 0.4113). There were overall significant effects for both acid concentration (P < 0.00001) and heating time (P < 0.0001).

Drag Swab Efficiency Factors when Sampling Chicken Manure

This study examines drag swabbing distance, media for moistening the drag swabs, and site selection when sampling a laying facility by drag swabbing manure piles. Manure piles at a laying facility in Californias San Joaquin Valley were sampled with drag swabs over various distances. Samples were cultured for Salmonella spp. with standard laboratory methods, and most probable number calculations. Salmonella spp. counts were expected to be highly variable because of reported clustering. Therefore, total bacteria and Escherichia coli, which were assumed to have a more uniform distribution on the surface of the manure, were additionally used as proxies for Salmonella. Media for moistening the swabs were compared by seeding postswabbing samples with Salmonella typhimurium, and culturing at different delay times. Total bacterial counts were compared between samples that were obtained from either wet or dry surfaces. Numbers of Salmonella spp. and total bacteria peaked within 120 feet of swabbing distance. Higher total bacteria counts were obtained by swabbing wet areas rather than dry areas, but the distance that could be swabbed effectively was shorter in wet areas. Moistening media selected for the swab resulted in statistically different culture counts, but did not show any important difference in maintaining Salmonella viability over a 48-hr period when the samples were kept at refrigerated temperatures. Once swabs became fully loaded with fecal material, bacterial numbers failed to increase with further use. Overuse of a swab may result in failure to detect Salmonella enteritidis on chicken manure if the distribution of this organism is clustered.