Salmonella has also been implicated in cases of osteomyelitis in children with co-existant sickle cell anemia. The top 4 Salmonella isolates that cause gastrointestinal illness are Salmonella typhimurium, Salmonella enteritidis, Salmonella Heidelberg and Salmonella newport. Other prominent members of the salmonella species that are implicated in gastrointestinal illness are Salmonella javiana, Salmonella poona and Salmonella montevedio. Raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, and chocolate from the bad bug book.
People of all age groups are susceptible to these bacteria; however immunocompromised, elderly and young children are at a higher risk. Incubation period The Incubation period for food borne salmonellosis is hours. Salmonella is transmitted to humans via the feco-oral route. An infected individual sheds the bacteria in his feces, and the bacterium is viable for months in the environment in water, soil, and manure. Salmonellosis can be diagnosed by isolating and culturing the bacteria from the stool or blood of the infected person.
Most people often recover from a bout of salmonellosis without a course of antibiotic treatment. Treatment for such individuals is only supportive, with intravenous or oral fluids, adequate nutrition and rest. Often times when the illness does get complicated a course of broad spectrum antibiotics might be necessary.
While all mice displayed similar colonization Fig 2B , there were some differences, which may be due to the VC inoculation, the prolonged streptomycin treatment in the drinking water, the prolonged fasting period or any combination of these [ 30 , 31 ]. Bacterial loads in tissues varied by 1—3 logs, with the exception of the feces and lungs of mice infected by OG which varied from below the limit of detection to 3.
Strikingly, two mice infected by OG contained particularly high bacterial numbers in the lungs suggesting possible unintended tracheal delivery of bacteria [ 21 ]. Decreased systemic spread of L.
Similarly, decreased systemic spread was observed in mice infected by VC of water contaminated with Salmonella compared to OG [ 32 ]. Altogether, these three studies indicate that infection by VC is a viable alternative to OG. For doses of 10 5 and 10 6 , the bacteria were rinsed once by centrifugation before dilution since we observed in preliminary experiments that otherwise mice were hesitant to consume chow inoculated with high doses data not shown.
By 3 days p. A Bacterial loads in tissues 3 days p. B Bacterial loads in tissues 3 days p. As expected, the bacterial loads in general were lower in the B6N mice Fig 3B , nonetheless, they followed the same trends seen in B6 mice, with the highest number of bacteria in the feces followed by the cecum, ileum, spleen, liver and blood similar to what has been shown previously [ 34 ].
Mice with an intact microbiota are also much less susceptible to oral infection with Salmonella. Therefore, we also infected B6 and B6N mice that were not pretreated with streptomycin hereafter referred to as strep- by VC. For these experiments mice were infected with 10 8 bacteria, the standard dose used in oral infections of mice with Salmonella see e. At 3 days p. In systemic tissues, no bacteria were detected in B6N mice and bacterial numbers were variable in B6 mice, with bacteria detectable in four out of five mice.
Bacterial numbers in tissues 3 days p. Filled symbols represent individual B6 mice and open symbols represent individual B6N mice. While it is difficult to determine the dose of nontyphoidal Salmonella required to cause gastroenteritis in humans, the dose is generally considered to be approximately 10 5 to 10 6 CFUs, although lower doses in susceptible individuals have been reported [ 36 , 37 ]. At the time of euthanasia, all of the B6 were infected, while in B6N mice bacterial loads were sometimes below the limit of detection Fig 5C and 5D.
In contrast, when mice were pretreated with streptomycin and inoculated with 10 4 CFU, both B6 and B6N mice developed clinical disease although this was slower in B6N day 11—13 compared to day 5—6 Fig 5A and 5B.
All of these mice had systemic disease with detectable CFUs in the spleen and liver as well as in the intestine Fig 5E and 5F. These data, together with the results of the early colonization studies Figs 3 and 4 , show that inoculation by VC does not alter the course of infection compared to inoculation by OG [ 6 , 35 , 38 ]. Open symbols in Fig 5D indicate the B6N mouse that was euthanized 15 days p. Mice are routinely fasted before oral inoculation with Salmonella , although the fasting times vary from 2—16 h [ 6 , 14 , 16 , 39 , 40 ].
In their paper describing VC inoculation with L. They also showed that 0—4 h of fasting was not sufficient to get bacterial colonization of the intestine but when food was withheld overnight 16 h the colons of all mice were colonized [ 24 ]. Based on their findings we used a 20 h fast initiated between pm for the initial experiments, although mice were fasted in a clean cage with bedding instead of an elevated wire floor. While fasting overnight is a standard procedure, the nocturnal eating pattern of mice can lead to weight loss and stress [ 41 ].
Therefore, to determine whether a reduced fasting period could be used, without significantly affecting the time taken to consume inoculated chow, mice were fasted for 14 h 6pm— 8am or 4 h 8am— 12pm before inoculation 10 4 CFUs. Mice fasted for 14 h consumed chow within 2 min, while those fasted for 4 h took up to 12 min Fig 6A.
Mice eat more in dark phase vs light phase so the short consumption time after 14 h may, at least in part, be due to fasting taking place overnight rather than during the day. To minimize the time taken to eat chow after 4 h fasting we slightly modified the feeding procedure. Mice were moved into individual clean cages, given a fragment of chow and then left undisturbed until consuming the whole chow fragment. A Time required for mice to completely consume fragments of chow with the indicated inoculum and fasting time.
B Consumption time left panel and bacterial loads right panel in tissues 3 days p. To determine whether mice would eat a higher inoculum after a short period of fasting, B6 mice were fasted for 4 h, and given chow inoculated with 10 8 CFUs.
Despite the higher inoculum, these mice consumed the fragments of chow in a time frame similar to mice fed 10 4 CFUs Fig 6B , left panel. Comparison of the bacterial loads in tissues from mice fasted for 4 h Fig 6B , right panel with mice fasted for 20 h Fig 4 revealed less systemic dissemination at 3 days p. Since these experiments were not performed side by side the differences in organ loads may be due to experimental variation but overall these data indicate that shorter fasting times result in delayed, or less efficient, dissemination of Salmonella.
In the method described for VC inoculation of mice with L. In contrast, the mice in our study showed no reluctance to eat inoculated food, possibly because we used their regular chow rather than food they were unfamiliar with buttered bread. In summary, oral infection of mice by VC mimics the natural route of infection for Salmonella and results in reproducible colonization of tissues. This method is straightforward to carry out and may avoid the stress and potential adverse side effects of OG.
Further refinements to the method are possible, such as; adjusting fasting times; the concentration of streptomycin in drinking water; and the time allowed for mice to access water containing streptomycin. This approach should also work for other intestinal pathogens. Protocol number E. Animals were euthanized either before the development of clinical disease or at the defined humane endpoint development of clinical disease: ruffled fur, hunched posture, lethargy.
Salmonella Typhimurium strain SL was used for all experiments. For inoculation of 10 5 CFU or higher, a wash step was included prior to dilution. Prepared pieces were kept separated in a petri dish during transport to the animal facility. One fragment of inoculated chow was retained for estimation of the dose by plating.
The B6 mice used in this study were either from a colony of mice originally purchased from The Jackson Laboratory Bar Harbor, ME and maintained at the Rocky Mountain Laboratories or purchased from The Jackson Laboratory and used immediately after arrival.
Except where specified, mice had unlimited access to food and water. Mice were then moved to a clean cage to limit coprophagy and access to cached food , containing normal drinking water but no chow. After a period of 18—22 h typically 20 h individual mice were put in a clean empty cage without bedding material and a fragment of inoculated chow placed on the floor of the cage next to a side. Typically, mice ate the fragment of chow immediately or within a couple of min.
For the 4 h fasting mice were left undisturbed until the chow was eaten. Immediately after the inoculated chow was consumed, mice were returned to their cage with unlimited access to food and water. Mice were fasted for 4 h prior to all gavages. For infections without streptomycin treatment, mice were only fasted prior to feeding.
Mice were euthanized by isoflurane inhalation followed by exsanguination. Tubes were weighed before and after organ collection. We thank the members of the Steele-Mortimer laboratory, Karin Peterson and Clayton Winkler for critical review of the manuscript, and Ryan Kissinger and Anita Mora for assistance with figures.
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