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Salmonella DT104 and Dairy Farms:
Lessons from an Emerging Pathogen

1999 Dairy Farm Food Safety and Quality Assurance Symposium
Burlington, Vermont

John M. Gay, DVM PhD DACVPM

WSU Contributors: Clive Gay, Director; Tom Besser, Margaret Davis, Dale Hancock, Lori Pritchett, Dan Rice

Contents:

Note:

The following is an update on Salmonella DT104 intended for general audiences. This webpage is not intended to provide herd-specific advice and may not reflect the current state of the veterinary literature.


Introduction - What is Salmonellosis and Salmonella?

Salmonellosis is the name for the acute lower gastrointestinal tract disease caused by infection with the bacteria Salmonella. Compared to clinical disease, infection of humans and animals is usually several fold more common even in the midst of outbreaks. The infection is usually contracted by ingestion of Salmonella-contaminated foodstuffs but can be acquired by inhalation or by exposure of mucous membranes, such as by splashing of contaminated urine or the use of a contaminated rectal thermometer. The minimum oral dose required to infect healthy adults is typically 105 organisms but foodstuffs with buffers or high fat content or the consumption of oral antibiotics can reduce this threshold over a thousand fold. Salmonellosis is commonly manifested clinically in both man and animals by sudden onset, usually 12 to 72 hours after ingestion of the organism, of one or more signs of gastrointestinal infection including high fever, abdominal pain, diarrhea and sometimes vomiting. A clinical case usually lasts for several days but the clinical signs and disease course is variable, depending on host factors, bacterial strain and dose. Infected people and animals often shed the infectious agent intermittently in their feces for many days, a small portion becoming chronic carriers and shedding sporadically for months if not years. Some of these carriers may become latent, meaning they stop shedding for long periods only to resume after stresses such as calving. When severe, the diarrhea may contain mucous, blood and shreds of intestinal lining. Recovery from this form is often prolonged and may never be complete. Dehydration due to the diarrhea and vomiting may become severe, particularly in the very young. Although usually restricted to the intestinal tract, invasive forms of the infection can reach the bloodstream and can affect other body organs and cavities with severe consequences, sometimes without the presence of gastrointestinal signs. Cases experiencing invasive disease can shed the organism in all body secretions. The disease is most severe and the risk of death is the highest in the very young, the elderly, the debilitated and the immunocompromised. In this sense, salmonellosis is an opportunistic or secondary disease in adults, often following a primary problem. Severity of a case depends in large part upon the size of the infectious dose. As noted above most infections are sub-clinical rather than clinical, meaning that because the clinical signs aren't present in the infected person or animal special laboratory tests are required to detect it. Like other bacterial food-borne infections, clinical cases are more common during the summer than other seasons.

Salmonella, a member of the family Enterobacteriaceae that includes the ubiquitous Escherichia coli, is a gram negative, rod-shaped bacteria. It is capable of surviving for months if not years in many wet and dry environments typical of dairy farms as long as it isn’t exposed to direct sunlight or high temperatures (>145o F). The bacterial genus Salmonella consists of some 2,200 serovars that infect a wide variety of animals worldwide and multiply in their intestinal tracts. Until recently, these serovars were named individually, such as S. typhimurium or S. typhi. Due to recent molecular biology findings about the genetic relatedness within this genus, the common salmonella serotypes of concern to the livestock industries (e.g. S. anatum, dublin, montevideo, typhimurium) are now classified into a single species, Salmonella enterica, and are further subclassified by their traditional serovar name. Thus what in the research literature was S. typhimurium is now Salmonella enterica serovar Typhimurium or S. Typhimurium. Some serovars have a predilection for a particular animal host, such as S. typhi in humans, and seldom infect other species. Other serovars rarely infect humans or animals, appearing infrequently in submissions from clinical cases. The host-adapted serovars typically require lower doses to establish infection than do the non-host adapted serovars and have a greater propensity to establish chronic carrier states.

S. Typhimurium is more cosmopolitan, commonly infecting a wide range of animal species worldwide. Of the 19,483 salmonella isolates obtained from agricultural animals in the United States that were submitted to the USDA for typing during a recent year, 23% were S. Typhimurium, the most common serovar. During that year, S. Typhimurium was the most frequently identified serovar submitted to the USDA from clinical cases in cattle, horses and pigs and was the fourth and sixth most common serovar isolated from turkeys and chickens respectively. Of the 82 different serovars submitted from clinical cases in cattle that year, it comprised fully 60% of the 2,050 submissions and the next most common, S. Dublin, comprised only 4%. A similar serovar pattern exists in humans. Of the 9,787 human foodborne infections detected in the seven states covered by the CDC FoodNet surveillance system in 1998, 29% were due to Salmonella serovars. Salmonella was the second most frequent species, the first being Campylobacter, comprising 41% of the isolates. Of these Salmonella isolates that were serotyped 30% were S. Typhimurium, the most common serovar found. Of note is that the largest known foodborne Salmonella outbreak in the U.S. was due to S. Typhimurium and that the food involved was pasteurized milk contaminated by an in-plant failure post-pasteurization.

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What is S. enterica var Typhimurium DT104 (S. typhimurium DT104)?

The epidemic strain of Salmonella DT104 is a specific subspecies within serorvar Salmonella Typhimurium and is a specific strain within DT104 whose full name is Salmonella enterica serovar Typhimurium variant DT104 R-ACSSuT (S. Typhimurium DT104). In the early 1990's this new strain of DT104 emerged as a pathogen for humans and farmed animal species in the U.S. and elsewhere. This epidemic strain is identified by the following characteristics. It is phage type DT (distinguished type) 104. The phage type is determined by identifying which bacteriophages, viruses that infect bacteria, infect and lyse the bacteria and which do not. The DT104 phage type has been known for many years but did not have the wide range of antibiotic resistance that the epidemic strain now has. The initial epidemic strain of DT104 had a broad antibiotic resistance pattern characterized by resistance to the antibiotics ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (R-type ACSSuT, sometimes indicated as "mr" for "multiply resistant"). Some descendants of the initial epidemic strain are acquiring resistance to other antibiotics while others are loosing resistance to some members of the initial antibiotic set. The R-type ACSSuT is almost unique among strains of serogroup B salmonella and thus is often used by diagnostic laboratories as an initial method for identifying the epidemic strain among group B isolates. The resistance to chloramphenicol includes florfenicol (Nuflor), which is also often used as a preliminary marker. The epidemic strain has a plasmid profile characterized by the presence of a single 60 megadalton plasmid. This plasmid profile has changed in those descendants that acquired additional antibiotic resistance genes through acquisition of plasmids.

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Where did Salmonella DT104 come from?

The epidemic strain of DT104 was first isolated in England in 1984 from a human specimen. In 1990 it began rising rapidly as a proportion of salmonella isolated from U.K. human cases, becoming second only to S. Enteriditis. The U.K. epidemic in humans appeared to peak in 1996 and continues to decline. Of note is that during this period the total number of human Typhimurium isolates submitted to public health laboratories in England did not increase significantly. The numbers of isolates from U.K. animals over time has followed a similar pattern. This suggests that this epidemic strain displaced other Typhimurium strains in humans rather than expanding the niches previously occupied by Typhimuriums or by occupying new ones and that this strain is being displaced in turn by other Typhimuriums as the epidemic subsides.

In the U.S., the CDC reported that human isolates of the R-type ACSSuT increased from 9% of S. Typhimurium isolates in the U.S. in 1990 to 32% in 1996, the year when the first human outbreak in a group was recognized. In a bank of S. Typhimurium isolates collected from Northwest animals, we found that R-type ACSSuT was absent in cattle isolates obtained prior to 1986, comprised 13% of the isolates prior to 1991, increased to 64% in 1994 and now appears to be declining. Of the S. Typhimurium isolated from animal infections and further classified by the USDA in 1998, approximately 16% were the epidemic strain of DT104. We have obtained S. Typhimurium with this R-type from a broad range of species in both farm and non-farm environments in the Northwest, having isolated it from the cow, horse, goat, emu, cat, dog, deer, elk, mouse, coyote, ground squirrel, raccoon, chipmunk and birds (pigeon, starling, pine siskin).

What region of the world the epidemic strain of DT104 actually originated from or when it actually originated is unclear and will likely remain so. Few countries have a Salmonella surveillance system as thorough as the United Kingdom's, which likely accounts for the strain's apparent emergence there. Studies have shown that DT104 is worldwide. Further, the fact that all DT104 isolates from around the world are virtually identical suggests that it evolved fairly recently and spread world-wide fairly rapidly. This rapid spread suggests that a species migrating world-wide, such as humans or some migratory bird species, were responsible. Other animal species, such as cattle, may serve as local reservoirs and local multipliers of the infection once they become infected. Studies have shown that cross species infection likely occurs, including from humans to cattle.

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Why is Salmonella DT104 of such public health concern?

Compared with the other Typhimuriums, this variant created great concern among the public health community for the following reasons. First, the genes encoding this broad antibacterial resistance are integrated into the bacterial chromosome. This is unique among Salmonella as most acquire antibiotic resistance genes by acquiring plasmids, which is extra-chromosomal DNA. To some experts, this suggests that this broad resistance will likely be retained by descendants of the epidemic strain even in the absence of the selective pressure of antibiotic use. With plasmid-mediated resistance, the conventional paradigm is that descendants of the resistant bacteria will discard the plasmid carrying a resistance gene against an antibiotic in the absence of exposure to that antibiotic. This loss is believed to occur because replication of the plasmid places that strain at a competitive disadvantage compared to non-plasmid carrying bacteria. Thus, the conventional wisdom is that if the antibiotic resistance is phage mediated then in the absence of antibiotic usage the antibiotic resistance of the strain will decline. With the resistance genes integrated into the chromosome, this mechanism of loss won't occur. Further, these genes are part of a "cassette" or integron, a mechanism that enables easy exchange of genes between quite different, unrelated bacterial species. This means of bacterial genetic exchange was recognized only recently so the implications are not yet fully known. It is conceivable that this "cassette" could appear in other completely unrelated bacterial pathogens that are currently susceptible to these commonly used antibiotics.

Of great concern to the public health community is that S. Typhimurium DT104 strains have emerged in Britain that have acquired plasmid-mediated resistant to fluroquinolone antibiotics. This family of antibiotics are often the only effective drugs for treating invasive salmonellosis in humans. This has very serious implications for antibiotic use in food animals, particularly of the fluroquinolones such as Baytril and Saraflox and in part accounts for the close monitoring of and severe regulatory sanctions against off-label usage of this family of antibiotics in the livestock industries. On the other hand, it should be noted that no studies have found that antibiotic use was a risk factor for obtaining or maintaining DT104 in a livestock herd. Rather some other characteristic of DT104 that affected the host-bacteria relationship is more likely to account for the epidemic.

Initial British reports suggested that this strain had a significantly higher human morbidity and case mortality than other Typhimurium strains, although this has yet to be confirmed by subsequent publications. Evidence from two large human outbreaks in the U.S. associated with DT104-contaminated cheese produced from incubated raw milk suggests that this strain is more similar to other Typhimurium strains with respect to human morbidity and case mortality than initially believed. On the other hand, studies from Britain, Scotland and the U.S. have shown that human cases are significantly associated with direct cattle contact. In-contact children appear to be at significantly greater risk of clinical infection than in-contact adults. This propensity to cause clinical disease in healthy children compared to healthy adults is not dissimilar from other Typhimurium strains but the association with direct cattle contact is. Also of concern is that we and others have isolated the agent from clinically normal pet dogs and cats around infected herds and have detected it in the associated household environment, presenting another potential route of significant exposure for young children.

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What are the consequences of Salmonella DT104 entering a dairy herd?

DT104 is not a superbug compared to other Typhimurium strains in dairy herds. After S. Typhimurium DT104 enters a dairy herd, usually via an infected animal, the disease course is highly variable. In some herds a significant proportion of the cows, including mid-lactation cows, and an even larger proportion of the calves are affected. In other herds only a few postpartum cases or only cases in calves are observed even though the environment is heavily contaminated. Our preliminary data suggests that the median duration of herd infection is about 12 months after the index clinical case is recognized and that infection is still present in about one-third of the herds 24 months after this index case. Based on preliminary data, in dairy herds experiencing DT104 outbreaks the morbidity in calves or cows is about one-half that of dairy herds experiencing non-DT104 Typhimurium outbreaks while the case fatality rate due to DT104 appears to be about twice as high. An S. Typhimurium DT104 enteric infection persisting for four months and udder infection persisting for eight months, including the dry period, have been documented. In our studies we have observed a clinically normal carrier cow shedding over a million DT104 per gram of feces for over six months. At necropsy, all intestinal lumens including her rumen were DT104 positive, suggesting that her saliva was also likely intermittently positive, potentially contaminating items in her environment such as water troughs. The frequency of these clinically normal, long term chronic carriers has not been established but is likely low. The reasons for such a wide variation of subclinical and clinical syndromes between herds have not been delineated, but the existence of such variability suggests that factors in a farm's environment and management modulate the herd's experience with this agent to a large degree.

In British herds, active surveillance and investigation of farm DT104 outbreaks by government personnel has been occurring since the epidemic was first recognized in the early 1990’s. In one outbreak, 15 isolations were made from 22 normal in-contact calves, indicating that subclinical bovine infection by DT104 is likely common. We have observed a similar occurrence in an outbreak affecting a group of 20 in-contact cows, most of which shed on at least one occasion over six months but none of which ever exhibited clinical signs even after calving. In another U.K. outbreak, index bovine cases shed DT104 for four months, showing the persistence of infection in individual cattle. The introduction of a large number of feral cats to control a rat infestation was associated with one livestock DT104 outbreak. The agent was isolated from the cat feces, suggesting that cats were an established part of the salmonella cycle on that premises. DT104 was isolated from a cat with intermittent bloody diarrhea, the cat never being not associated with cattle, as well as from pigeons, and from rabbits, one of which was associated with a secondary human case. Cats have been implicated as a common source of infection for humans. Another human outbreak of approximately 20 cases was traced to milk from a producer whose bulk tank milk was DT104 positive. In other cattle outbreaks, the agent was isolated from a healthy dog, healthy ducks, and from pond water accessible to cattle. Two cattle DT104 outbreaks were associated with exposure to human sewage, suggesting a waterborne route of transfer from humans to cattle. A large number of starlings were noted in one outbreak, leading investigators to suggest their role in heavily contaminating the environment and to point out the potential risk they present to surrounding farms. Even though only two clinical cases had been noted in the cattle, DT104 was found in the bird feces from various places on the premises, within the silage and throughout the buildings. In our studies, we have isolated DT104 from normal members of a wide range of species associated with outbreak farms.

In a large case-control study of S. Typhimurium DT104 in British cattle herds, statistically significant associations were found between clinical case occurrence and seven risk factors. These were being a cattle dealer (OR = 14.25) as well as a farmer, introducing newly purchased animals (OR = 2.51), being in the calving period for seasonally calving herds (OR = 2.48), birds accessing stored feeds (OR = 1.67), cattle being housed (OR = 1.51), lack of isolation facilities for sick animals (OR = 1.51) and cats accessing stored feeds (OR = 1.35). This suggests that enhancing livestock feed and water safety by preventing fecal contamination by any species is an important component of controlling and of preventing this infection on a farm. A preliminary analysis of our case-control data suggests that the strongest risk factors are purchasing animals and using a common area for both calving cows and housing sick cows. We have not detected any association between prior antibiotic usage and outbreak occurrence.

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Evidence from dairy herd outbreaks of other Salmonellas

Results from investigations of other Salmonella strain and serotype outbreaks may be useful for those dealing with DT104 outbreaks. In numerous instances investigators have reported that animals other than cattle are associated with the salmonella cycle on farms. These include an association between various bird species contaminating feeds and the transmission of salmonella to cattle. The avian species are among the many animal species that have been identified as susceptible to infection and could be involved in the spread and survival of S. Typhimurium DT104. The mobility of avian species makes them of particular concern if they are carriers and shedders of S. Typhimurium DT104 for extended periods. As birds follow their seasonal migration patterns and mating instincts, they could potentially disperse S. Typhimurium DT104 over wide geographic regions. Of particular suspicion are those species that are commonly found on or near farms and are known to feed on dead animal tissue (e.g. dead carcasses, placentas or mucous shreds), animal feeds or on material in livestock droppings (e.g., undigested feed, invertebrates living off of or in the dung). The Corvidae (crows, ravens, magpies), known carrion feeders and prevalent in the farm environment, could readily ingest such animal tissues and become infected. Starlings, blackbirds and pigeons are common pests on many farms and feed either directly from feed bunks or search for food in livestock droppings. Feeding on the later is likely a means of ingesting large numbers of fecal microorganisms. Infected starlings, blackbirds or pigeons could readily contaminate cattle feed as well as the environment. California investigators found that 4% of such birds were infected with Salmonella on some dairy farms. Infected droppings of wild birds have been found in feedmill environments and the presence of salmonella-contaminated feeds of swine farms has been shown to be associated with the lack of bird-proofing. Infected gulls have been implicated as the source of bovine salmonella outbreaks in a number of reports. This suggests that bird-proofing of livestock feed and water sources and of feeding areas will enhance herd resistance against Salmonella.

Rodents have been implicated in outbreaks in a dairy herd, in a beef herd and in poultry flocks. A single rodent fecal pellet from infected mice typically contains up to 104 salmonella. Large rodent populations can be present before their signs (e.g., rodent droppings and runways) are obvious. In our ongoing studies of affected herds, we have found several in which all rodent fecal collections were DT104 positive. Live-trapped raccoons have been reported to harbor S. Typhimurium. In one outbreak, we found that the feces of raccoons living in the bale stacks were DT104 positive. Insects may also be vectors. Flies may function as a biologic vector as well as a mechanical vector, with S. Typhimurium replicating in flies under the right conditions. In our ongoing studies of affected herds, we have found several in which all fly collections (primarily the house fly) were DT104 positive.

That feedstuffs are often contaminated with salmonella and that outbreaks in livestock can be caused by this contamination is well established. In fact, given the frequency of feedstuff contamination, the unanswered question is why outbreaks due to this contamination aren't recognized more frequently. Although the ruminant protein feeding ban eliminated one common source of foodborne exposure, recent evidence suggests that both animal and vegetable fat sources may be involved in salmonella infection and in outbreaks of clinical disease. The epidemic DT104 strain has been reported both in off-farm and on-farm feed samples. In the later study, S. Typhimurium DT104 replicated in 9 of 10 mixed rations from dairy farms when inoculated in the laboratory.

Other factors in the farm environment may also perpetuate the salmonella cycle. In a review of salmonellae in the environment, Murray (1991) states "the predominate feature of Salmonella spread is human influence on the natural environment, including animal management practices, waste management and effluent control, all which contribute significantly to the spread of salmonellae." As one expert put it, the epidemiology of salmonella is the epidemiology of fecal pollution. The use of recycled flush water may have maintained it on one dairy farm. For other strains of salmonella, the agent has been shown to persist in cattle or the farm environment for many months or even years, sometimes persisting after the clinical syndrome has ceased. In one study, a strain of S. Typhimurium (not DT104) persisted in a herd for 3.5 years.

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Conclusion

Given that the DT104 epidemic is declining as evidenced by the declining number of DT104 isolates being submitted to human and animal diagnostic laboratories, can dairy producers and the dairy industry reduce their vigilence? No! Most important is to recognize that new infectious agents like DT104 will continue to emerge and that the best protection is to enhance and to maintain herd biosecurity. This enhancement should be both against introducing new infectious agents, where most people focus, and, more importantly, against survival and transmission of infectious agents within the herd. Because we can’t see the bacteria around us, we overlook them when developing and executing our management practices and technology. Johnathan Mann wrote that the history of our time will be marked by recurrent eruptions of newly discovered diseases, epidemics of diseases migrating to new areas, diseases which become important due to new technologies, and diseases that occur due to changes in habitats. The fundamental reason for this continuing emergence is that bacteria and other microbes are very adaptable and they are moved rapidly worldwide by man and other species. Under optimal conditions, many bacteria produce a new generation every 30 minutes or approximately 4,380 generations per year, each generation providing a new opportunity to evade our defenses or to take advantage of weaknesses in our management practices or technology.

Although antibiotics have a role, we should focus on management and housing changes that enhance biosecurity. Bacteria have the potential to either acquire or develop antibiotic resistance rapidly. Many of the antibiotics we currently use are similar to those used by microbes in their competition with each other over the eons. Some experts state that we have identified only 5% of the bacteria in the world and we understand the function of only 25% of the genome of those that we have identified. Others suggest that there isn’t an antibiotic that man can develop that hasn’t evolved somewhere sometime in the microbial community. If it has, the resistance mechanism also had to be developed. Because bacteria have several mechanisms to exchange genes between unrelated species, the pathogens we are targeting may acquire that resistance mechanism from an unrelated, unknown organism upon exposure to that new antibiotic.

With innovations in dairy technology, we inadvertently provide new niches for microbes or enhance their opportunities for survival and for transmission. For example, TMR’s (total mixed rations) provided a new opportunity for bacterial replication that didn’t exist when moist and dry feeds were fed separately. Recycling flush water provided a new, very efficient transmission route for infectious agents that spread by the fecal oral route and that survive well in water (e.g., salmonella, Mycobacteria paratuberculosis, cryptosporidia) to get back to the mouth of the susceptible cow. High animal densities in housing markedly enhance transmission opportunities, particularly for aerosol-borne viruses and bacteria in enclosed spaces that are poorly ventilated, compared to the environment in which the ancestors of the cow and its infectious agents co-evolved. On the other hand, alarm and panic is not justified. With respect to bacteria and other microbes our current situation is as it always been since the domestication of animals began. Now we just understand the enemy better and recognize the magnitude of the challenge they present. We only need to act accordingly.

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Selected Print References:

Akkina JE, AT Hogue, FJ Angulo, et al. (1999). Epidemiologic aspects, control and importance of multiple-resistant Salmonella Typhimurium DT104 in the United States. J Am Vet Med Assoc 214:790-798.

Evans S (1996). A case control study of multiple-resistant Salmonella typhimurium DT104 infection of cattle in Great Britain. Brit Cattle Vet Assoc 4: Part 3:259-266.

Glynn MK, C Bopp, W Dewitt, et al. (1998). Emergence of multidrug-resistant Salmonella enterica serotype typhimurium DT104 infections in the United States. N Engl J Med 338:1333-8.

House JK, BP Smith (1998). Current strategies for managing salmonella infections in cattle. Vet Med 93:756-764.

McDonough, PL (1995). Salmonellosis: Diagnostic approach to disease control and epidemiology in the bovine animal. Proc 27th Annual Convention AABP 27:61-68.

Poppe C, N Smart, R Khakhria, et al. (1998). Salmonella typhimurium DT104: a virulent and drug-resistant pathogen. Can Vet J 39:559-65.

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Selected On-line References:

Bovine Herd Salmonellosis, Including DT104: Eleven Characteristics to Keep in Mind (an earlier document intended more for veterinary practitioners than for a general audience and that contains more complete references)
http://www.vetmed.wsu.edu/courses-jmgay/FDIUHerdSalmonella.htm

Epidemiology Concepts for Disease in Animal Groups
http://www.vetmed.wsu.edu/courses-jmgay/EpiMod2.htm

Emerging Quinolone-Resistant Salmonella in the United States (CDC Emerging Infectious Diseases 3(3))
http://www.cdc.gov/ncidod/EID/vol3no3/hayes.htm

Salmonella typhimurium DT 104 (Institute of Food Science and Technology (UK))
http://www.easynet.co.uk/ifst/hottop20.htm

The Veterinarian's Role in Diagnosis, Treatment, and Prevention of Multidrug Resistant Salmonella typhimurium DT104 (document is a link off of this URL)
http://www.aphis.usda.gov/vs/ceah/cahm/cahm-act.htm

USDA FSIS Situation Assessment: Salmonella Typhimurium DT104 (review of information as of December, 1997)
http://www.fsis.usda.gov/ophs/stdt104.htm

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