- Email: [email protected]
Salmonella
MICROBIAL FOOD BORNE PATHOGENS
0749-0720/98 $8.00
+ .00
SALMONELLA Henry E. Ekperigin, DVM, MPVM, PhD, and Kakambi V. Nagaraja, DVM, MVSc, PhD
Salmonella is a genus of bacteria belonging to the family Enterobacteriaceae. Bacteria constituting the genus are gram-negative, nonsporeforming, usually motile bacilli. They are facultative anaerobes and contain three different types of antigens. The somatic (0) antigen is associated with the cell wall and composed of lipopolysaccharides. The flagellar (H) antigen is associated with the microbe's peritrichous flagella and is proteinaceous. The third type of antigen, a capsular (Vi) antigen, is found only in some Salmonella. 3, 4, 13, 14, 18, 19, 21, 27, 29 The agglutinating properties of the antigens are used to differentiate among the more than 2200 serologically distinct types of Salmonella. 18, 29 These serotypes are not biochemically defined subdivisions of the genus and are not regarded as distinct species even though their conventional names suggest otherwise (e.g., S. typhimurium, S. dublin, S. choleraesuis, S. enteritidis, S. newport, and so forth. The serotypes are classified into one of 50 groups based on the composition of their somatic (0) antigens. The groups are named after the letters of the alphabet, A-Z, and the numbers, 51-65. Thus Salmonella serotypes are classified into serogroup A if they possess the 2 antigen as the dominant antigen, and into serogroup B if they possess the 4 and 12 antigens. 4,18 A serotype can be further subdivided by using biotype, phage type, and plasmid content to identify phenotypic variations within the serotype. A biotype is the biochemical variation between two microbes of the same serotype, whereas the phage type reflects differences between two organisms with the same serotype but different susceptibilities to a lytic
From the Feed Safety Team, Division of Animal Feeds, Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland (HEE); and the Department of Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota (KVN)
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 14· NUMBER 1 • MARCH 1998
17
18
EKPERIGIN & NAGARAJA
bacteriophage. Examples include S. gallinarum and S. pullorum as biotypes of S. gallinarum-pullorum, and the 27 or more phage types of S. enteritidis. Plasmid analysis is useful in identifying strain differences.lO, 26, 29 The nutritional requirements of Salmonella are simple. For optimum growth, the microorganism requires a temperature of 3SOC to 37°C, a pH of 6.5 to 7.5, and a water activity of 0.930 or higher. However, some serotypes have been shown to grow in temperatures or pH as low as SoC or 4.5, or as hig~ 47°C or 9.0. Others have also been shown to survive freezing and long-term frozen storage. Salmonella can survive in dry foods but does not compete well with food spoilage microbes, Escherichieae, lactic acid bacteria, or other microbes that occur naturally in foods. 3 SALMONELLOSIS
Salmonella can gain entry into an animal (the host) through contaminated feeds or fomites. 3,28 The usual portal of entry is the host's oral cavity; however, pores in the shells of freshly laid eggs can provide another important portal of entry in poultry. After entry, the invading Salmonella is either overwhelmed by the host's defenses and destroyed or expelled, or succeeds in overcoming those defenses and establishing itself inside the host. The initial site of establishment of Salmonella within the host is usually the gastrointestinal tract, especially the ileum, where the microbe attaches itself to mucosal cells. 22 A host containing an established Salmonella colony is said to be infected. All food animals, except aquatic species in their usual habitats, are susceptible to natural infection with Salmonella. An animal infected with Salmonella mayor may not develop salmonellosis, the disease. Salmonellosis may occur in the host as a localized disease affecting the gastrointestinal tract and resulting in diarrhea alone or diarrhea together with fever, anorexia, depression, and shock. The localized form of salmonellosis is also referred to in literature as the subchronic, chronic, or enteric form. Salmonellosis may also occur as a generalized disease affecting several body systems. Depending on the body systems affected, symptoms of the generalized disease may include pneumonia, cough, central nervous system signs, abortion, etc. The generalized form of salmonellosis is also referred to in literature as the acute, systemic, or septicemic form. If infection does not progress into salmonellosis, Salmonella organisms remain in the gastrointestinal tract as part of the host's commensal flora and may be shed in feces. In cases in which infection develops into salmonellosis, Salmonella initiate the disease process by migrating to the lamina propria through the microvilli of mucosal cells and the tight junctions between those cells. 22 The mucosal cells are damaged in the process, and fibronecrotic plaques are formed. Damage to the mucosal cells distrupts the integrity of the mucosal lining of the gastrointestinal tract and causes materials that would ordinarily have been confined to or excluded from the gut lumen, to leak out or in. 23 Endotoxins and
SALMONELLA
19
other toxic materials seep out of the lumen into the body cavity while plasma proteins and other materials present in exudates from the inflamed lamina propria leak into the lumen. 23 A putrid diarrhea ensues accompanied by fever, anorexia, depression, and shock. Secretion of enterotoxins by Salmonella can also cause diarrhea to occur independent of the mucosal damage. Inside the lamina propria, Salmonella stimulate an inflammatory response and are subsequently engulfed by macrophages and neutrophils. The engulfed Salmonella are transported in the blood to the mesenteric lymph nodes and other organ systems where they can survive and multiply as facultative intracellular parasites and eventually cause embolic lesions. Examples of problems that can result from those lesions include respiratory difficulties, meningitis, abortion, and sudden death. The incubation period for the disease is about 1 to 4 days. The duration of the disease can be as long as 2 weeks. Animals that recover from the disease usually become carriers of Salmonella. These carrier animals appear to be in good health; however, they continually shed Salmonella into the environment and become sources of infection for other animals. 3, 9,18,27 EPIDEMIOLOGY Determinants of the Disease
Several factors determine whether or not an animal infected with Salmonella will develop salmonellosis. The factors include those attributable to Salmonella, to the host, and to the environment. Factors attributable to Salmonella include the microbe's serotype, virulence, and the number of viable cells present in the inoculum. Most of the 2200 or more serotypes of Salmonella have no predilection for any particular type of host and are capable of infecting all host species. However, a few serotypes are host-adapted and, under normal circumstances, will cause disease only in the host to which they are adapted. Examples include S. gallinarum-pullorum, whose biotypes will infect and cause fowl typhoid and pullorum disease in chickens and turkeys but will not infect cattle, and S. choleraesuis, which has a preference for pigs. With regards to serotype virulence, S. enteritidis phage type 4 is virulent to chickens and will infect and cause disease in the birds while most of the other phage types of the same serotype are less virulent and will infect but not cause disease in the birds. Although the number of viable Salmonella cells needed to initiate infection in animals has been shown to be as few as one colony forming unit (CFU) per 15 grams of feed,14 many more cells are usually needed to allow infection to progress into salmonellosis. This has been demonstrated in experiments in which the number of S. typhimurium that was enough to cause infection in pigs was found not to be enough to cause lesions in the same pigs until the microbes were allowed to increase in number to about 107 cfu of the organism per gram of intestinal content. 18, 27 In short, not only must Salmonella be present for infection to occur but also the number of viable cells present must be enough to
20
EKPERIGIN & NAGARAJA
initiate the infection and subsequent disease. The number of viable cells required varies with the virulence of the serotype involved. Host factors that determine whether or not infection of an animal will occur and subsequently develop into salmonellosis include species, breed, age, and health and immune status. Cattle are very susceptible to S. dublin, S. newport, and S. typhimurium but much less so to S. gallinarumpullorum. Chickens, on the other hand, are very susceptible to the two biotypes of S. gallinarum-pullorum but much less so to S. choleraesuis. Pigs are much more susceptible to S. choleraesius than other food animal species. With regard to age, younger animals are generally more susceptible to salmonellosis than older animals; however, there are instances in which the adult animal is equally or more susceptible to salmonellosis than the young. As an example, adult chickens are more susceptible to salmonellosis caused by the biotype S. gallinarum than young chicks are. With regard to health and immune status, malnourished, debilitated, or immunodeficient animals are more likely to succumb to infection with Salmonella than their properly nourished, healthy, or immunocompetent counterparts.3, 12, 13, 14, 27, 29 Environmental factors can determine whether or not infection of an animal by Salmonella will occur and develop into salmonellosis. The factors include the season of the year, and animal husbandry and management practices that affect the animal's immediate environment and well-being. Those practices include housing design and maintenance, feeds and feeding schedule, and vaccination programs. Salmonellosis has been shown to occur more frequently during the summer and fall. Also, animals are more likely than not to succumb to Salmonella infection and disease if they are undernourished, inadequately vaccinated against other diseases, easily accessible to delivery personnel and other visitors that can track infection between farms, and raised in unhygienic, rodent or fly-infested facilities with poor air quality and temperature. Frequency of Salmonellosis
There is currently no reliable information about the incidence of salmonellosis in food animals on a national basis in the United States. 3, 19,27 Survey data from various parts of the world indicate that New Zealand has an infection rate of 13% to 15% in dairy cows, calves, sheep, and beef cattle. For healthy pigs sent to slaughter, the infection rates were 25% in the Netherlands, 10% in New Zealand, 60/0 in the United Kingdom, and 10% to 13% in the United States. However, these data were collected at slaughter houses and could be overestimates of incidence because the stress of transportation and food withdrawal experienced by animals going to slaughter has been shown to increase the number of shedders of Salmonella among those animals. On the other hand, the data could be gross underestimates of incidence because the information was obtained using healthy pigs. The U.S. National Veterinary Services Laboratories (NVSL) issues
SALMONELLA
21
quarterly and annual reports that summarize the data on distribution and frequency of Salmonella serotypes. The reports are based on data obtained from the results of analyses conducted at NVSL to determine the serotypes of Salmonella cultures sent to the laboratory. As presented, the reports cannot be used to determine the incidence of Salmonella infections or salmonellosis; however, they are useful as a general indication of the frequency of occurrence of various Salmonella serotypes in various food animals species during a specified time period. During the period October 1, 1995 through September 30, 1996, for example, S. typhimurium was the predominant serotype found among clinical isolates from cattle and constituted 58.5% of the 1461 cultures submitted to the laboratory for serotyping. Similarly, S. choleraesuis (35% of 848 cultures), S. arizonae (55% of 49 cultures), and S. senftenberg (17% of 345 cultures) were the predominant serotypes among clinical isolates of Salmonella from swine, sheep, and turkeys, respectively. In chicken, S. heidelberg was the predominant serotype and constituted 21 % of 160 cultures submitted.
Distribution of Salmonellosis
Salmonellosis occurs worldwide. However, the pattern of distribution might differ for individual Salmonella serotypes. As an example, S. typhimurium affects all animal species and has a worldwide distribution. On the other hand, the patterns of distribution for host-adapted serotypes like S. choleraeusuis and S. dublin are patchy and generally match the patterns of distribution of the hosts to which they are adapted. Figure 1 shows the general cycle of infection for Salmonella using poultry as an example and portraying the inter-relationships between the three main determinants of salmonellosis: Salmonella, the host, and the environment.
CLINICAL SIGNS, SYMPTOMS, AND DIAGNOSIS
In all animals, the signs and symptoms of Salmonellosis usually reflect the location of the disease. If the disease is localized to the gastrointestinal tract, the signs and symptoms usually include a profuse, watery, and fetid diarrhea. In cases in which endotoxins and other toxic materials leak out of the host's gastrointestinal tract into the body, the diarrhea may be accompanied by fever, anorexia, depression, and shock. If bacteremia occurs and the disease spreads throughout the body of the host, the signs and symptoms will depend on the organs affected and the severity of embolic lesions formed in those organs. The signs and symptoms may include dyspnea and other respiratory problems, abortion, occasional diarrhea, and sudden death.
22
EKPERIGIN & NAGARAJA
---~--. ) ~
Feed
VVater
(
)l~ Feed Water
~
~ W j
·Meat, bone CONTAMINATED and feather..... FEED
~~Co
me~
Figure 1. Salmonella cycle of infection for poultry
Cattle
The most common Salmonella serotypes infecting cattle include S. dublin, S. typhimurium, S. newport, and S. montevideo. S. dublin is adapted to cattle, and the disease it causes in them can become endemic in a herd or farm because cattle recovering from salmonellosis caused by the serotype become true carriers and, for a long time, continually shed this microbe into the environment through feces and milk. Neonates (usually 1 to 2 months of age) and adult cattle are both susceptible to infection with Salmonella. 16 The infection can develop into salmonellosis after an incubation period of 1 to 4 days. Clinical findings include a watery to fibrino-mucohemorrhagic diarrhea with a fetid odor, anorexia, marked depression, fever, and shock. In cases with bacteremia, there may also be dyspnea, incoordination, nystagmus, polyarthritis, decreased milk production, and abortion. Affected animals become recumbent and die within 24 hours (adults) or 3 to 7 days (calves). Animals that recover become true (S. dublin) or passive (S. typhimurium and other non-cattle-adopted serotypes) carriers. Unlike the persistent infection that occurs in a true carrier, infection in a passive carrier lasts only for a relatively short period of time and is cleared from the animal after 3 to 16 weeks of shedding. A single carrier can shed as much as one million cfu of S. dublin per gram of feces, or 100 to 100,000 cfu per milliliter of raw milk. Is
SALMONELLA
23
Necropsy findings include emaciated carcasses with serous atrophy of fat, fibrino-necrotic plaques that adhere to a thickened and hemorrhagic mucosa (especially in the ileum), fibrinous sheets especially in the area of Peyer's patches, enlarged and darkened mesenteric lymph nodes, and watery bowel contents that may contain fibrin or blood. There also may be splenomegaly and an edematous lung with hemorrhagic or congested foci. The gall bladder wall may be thickened and hemorrhagic, and the bile is often inspissated into a firm coagulum. A tentative diagnosis of bovine salmonellosis can be made from the history of the herd (including disclosure of recent stress factors like calving, transportation, vaccination, addition of new calves, commingling of calves from many different sources, water and food deprivation), and from clinical and necropsy findings. A definitive diagnosis depends on the isolation and identification of the causative Salmonella. The disease should be differentiated from similar enteric diseases of calves including those caused by E. coli, rotaviruses, coronaviruses, bracken fern and other poisonings, Cryptosporidium sp, and Eimeria sp. The pneumonic form of calf salmonellosis should also be differentiated from pneumonic pasteurellosis. In adult cattle, salmonellosis should also be differentiated from bovine viral diarrhea, winter dysentery, and feedind uced indigestion. Poultry
Over 200 Salmonella serotypes have been isolated from chickens and turkeys in the United States. Most of these were isolated from the intestines and internal organs of birds suffering from acute, fatal salmonellosis. One serotype, S. gallinarum-pullorum (which, as mentioned earlier, consists of two biotypes that have traditionally been identified as S. gallinarum and S. pullorum), is adapted to poultry and is nonmotile. In addition to the fecal-oral route, transmission of Salmonella in poultry can also occur through the egg. Egg transmission is achieved in two ways. In one, Salmonella in the blood of bacteremic hens are transported to the reproductive system, where they are incorporated into developing ova. In the other method of egg transmission, Salmonellacontaminated fomites are sucked through the pores of freshly laid eggs as those eggs cool down to ambient temperature. In either case, Salmonella becomes part of the egg or, if the egg is fertile, part of the developing embryo. The incubation period varies with the causative serotype but is usually about 4 to 5 days. The diseases of poultry caused by the two biotypes of S. gallinarum-pullorum are called pullorum disease and fowl typhoid. That caused by S. arizonae is called avian arizonosis, whereas those caused by the other serotypes are collectively referred to as fowl paratyphoid. The clinical signs and symptoms are basically the same for all and are compatible with those of the localized or generalized forms of salmonellosis described earlier. The birds show somnolence, weakness,
24
EKPERIGIN & NAGARAJA
anorexia, drooped wings, and ruffled feathers. They huddle together and their vents are pasted with chalk-white excreta that is sometimes stained greenish brown. Other signs that can occur include labored breathing or gasping, retarded growth, blindness, and lameness due to swelling of joints and adjacent synovial sheaths. Mortality is variable, and there are variable effects on egg production, fertility, and hatchability. Pullorum disease and fowl paratyphoid generally affect younger birds, whereas fowl typhoid is encountered more frequently in adult chickens and turkeys. Since the early 1970s, all major commercial breeding flocks have been free of pullorum disease. There have also not been any reported outbreaks of fowl typhoid in the United States since 1980. 19 Flock history and clinical signs and symptoms are of limited value in arriving at a diagnosis of the diseases because of their similarity to the history, and signs and symptoms of several other diseases, including diseases caused by coliforms, staphylococcus, and Mycoplasma synoviae. Definitive diagnosis requires isolation and identification of the causative Salmonella serotype. Swine
S. choleraesuis and S. typhimurium are the Salmonella serotypes most commonly associated with the generalized form of salmonellosis in swine, whereas S. typhimurium is more commonly associated with the localized form. S. choleraesuis is the serotype most frequently isolated from pigs; it is swine-adapted. Porcine salmonellosis occurs most often in intensively reared, weaned pigs less than 5 months old, only occasionally in market-size swine and adult breeding stock, and rarely in conventionally reared suckling pigs. The incubation period is 24 to 48 hours. In the localized form, a watery, yellow, fetid diarrhea lasts 3 to 7 days and then may recur one or more times. Blood mayor may not appear in the watery feces. There is also a moderate fever, anorexia, and dehydration. The pigs assume a "tucked abdomen" look and emit a complaining grunt and squeal. Mortality is low and occurs only after several days of watery feces. In the generalized form, there is dyspnea, anorexia, fever of 105 to 107°F (40.5-41.6°C), reluctance to move, shallow moist cough, and huddling. Adult pregnant pigs may abort. There may also be central nervous system signs including tremor, weakness, paralysis, and convulsion. Diarrhea does not occur until the third or fourth day after onset of the disease and is watery yellow. Death occurs in 2 to 4 days. Dead pigs have purple extremities and abdomens. Mortality is high and can reach 100%, especially in outbreaks with the central nervous system signs. Morbidity is variable but usually less than 10%. Pigs that recover from both forms of the disease become carriers and will intermittently shed Salmonella for five months afterwards or longer. Necropsy findings include cyanosis of the ears, feet, tail, and ventral abdominal skin. There is splenomegaly, hepatomegaly with miliary white foci of necrosis, and congestion to infarction of the gastric fundic
SALMONELLA
25
mucosa. The mesenteric lymph nodes are moist and swollen, and the lungs are firm and diffusely congested. The lungs also often have interlobular edema and hemorrhage. The history of the herd and the clinical and necropsy findings are helpful but are usually not enough to make a definitive diagnosis of salmonellosis because of the similarity between these findings and those attributable to other diseases. Those other diseases, which need to be ruled out, include hog cholera, swine dysentery, and diseases caused by erysipelas, streptococci, and Actinobacillus pleuropneumonia. A definitive diagnosis is made after the isolation and identification of the causative Salmonella. Sheep and Goats
The major Salmonella serotype associated with salmonellosis in sheep is S. typhimurium. Other serotypes that have been found in sheep include S. arizonae, S. abortus ovis, and S. dublin. S. abortus ovis is sheepadapted. It is endemic in the United Kingdom and other parts of Europe, but has not been reported in the United States. Ovine salmonellosis occurs most frequently in feeder lambs; however, ewes and lamb flocks are also susceptible. The clinical findings in feeder lambs include watery scours that start within 24 hours of placement in the feed lot, dullness, depression, drooped ears, anorexia, coughing, and dehydration. The diarrhea is watery and greenish-yellow. There is also a 2° to 4° rise in temperature and increased heartbeat and respiration. Symptoms may persist for 1 to 2 weeks, and pregnant young ewes may abort. Morbidity ranges from 5% to 50 %, and mortality is usually not over 5% to 10%. Necropsy findings include emaciated, dehydrated cadavers, congested and hemorrhagic mucosal linings in stomach and intestines, and enlarged and congested lymph nodes. Pneumonia may be present. Diagnosis requires differentiation from diseases with similar signs and symptoms. Examples include coccidiosis in which scouring and other signs begin 2 to 4 weeks after placement in the feedlot; enterotoxemia in which the lambs are on full feed and not emaciated; and lamb dysentery that affects unweaned lambs. Definitive diagnosis of salmonellosis is based on history of that herd, clinical signs and symptoms, and necropsy findings followed by isolation and identification of causative Salmonella. Reports of naturally occurring cases of salmonellosis in goats are scanty. Among reported cases, S. dublin is the usual pathogen. There have also been reports specifying S. typhimurium as a cause. The signs and lesions observed were similar to those in cattle. FOOD SAFETY CONCERNS
As mentioned earlier, food animals whose infection with Salmonella does not progress into salmonellosis, and those that recover from the disease, become carriers of Salmonella. Although these animals appear
26
EKPERIGIN & NAGARAJA
healthy, they constitute a very large reservoir of Salmonella and continually shed the microbe into their environment. The frequency of shedding is influenced by several factors, including stress. It is a common practice to withhold feed for 6 hours or more from animals that are ready to be processed for food, and to transport them from the ranches, farms or feedlots on which they were reared to slaughter houses that are usually located fairly far away. The stress of transportation and feed deprivation can cause the carriers among these animals to shed Salmonella and infect other animals in the herd or flock being transported. This spread of infection among live animals awaiting slaughter can cause a significant increase in the likelihood that carcasses emerging from the slaughter house will be contaminated with Salmonella. The rate of contamination of such carcasses by Salmonella has been shown to range from 0% to 90%. The use of meats from such contaminated carcasses has also been shown to be one of the major factors contributing to the occurrence of outbreaks of food borne human salmonellosis in the United States. 2,24 About 50 such outbreaks are reported annually, in addition to the very large number of sporadic cases that occur independently of the recognized outbreaks. Human salmonellosis is one of the most commonly reported bacterial diseases in the United States. 25 It can be fatal. 24 Humans can become infected by most, possibly all serotypes of Salmonella. In the United States, however, the most common serotypes among the 40,000 isolates reported from humans each year are S. typhimurium, S. enteritidis, S. heidelberg, S. hadar and S. newport. Poultry is the food animal reservoir for S. heidelberg and S. infantis, and egg-laying hens the reservoir for S. enteritidis. For S. newport and S. dublin, the reservoir is cattle. S. typhimurium has no preference for any particular animal species and is equally infectious to all. Just like in other animals, Salmonella infection in humans does not always result in salmonellosis. When it does, the result is identical to that in other animals: a disease that is either localized (gastroenteritis) or generalized (septicemia). The very young, the very old and the immunosuppressed are most susceptible. 25 Apart from concerns about humans contracting Salmonella infections through foods of animal origin, there is the additional concern that a sizable proportion of those infections could be caused by antibioticresistant Salmonella. There have been reports of isolation of gentamicinresistant S. arizonae from turkeys5,8 and of multidrug-resistant S. newport from cattle. 24 The ingestion of contaminated meats from such animals could result in human infections with gentamicin-resistant Salmonella and become a major problem if the infections progressed into salmonellosis that required treatment with gentamicin. Even in those cases in which the infection did not progress into salmonellosis, treatment of the infected individual with gentamicin to control another ailment could suppress the normal intestinal flora, disrupt their protective effect, and give a competitive advantage to the gentamicin-resistant Salmonella and facilitate its ability to cause salmonellosis. More recently, a multidrug-
SALMONELLA
27
resistant S. typhimurium, known as Definitive Type 104 (S. typhimurium DT104), has been isolated in the United Kingdom and the United States from humans, poultry, sheep, pigs, cats, wild birds, rodents, foxes, and badgers, and has been shown to be transmitted from cattle and sheep to humans. The isolates in the United Kingdom are highly resistant to many valuable antimicrobial agents and frequently demonstrate a pattern of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracyclines and more recently, trimethoprim and fluoroquinolones. THERAPY, PREVENTION, AND CONTROL
Therapy and control involve taking action against a microbe that has already gained, or stands a very good chance of gaining, access to an animal, whereas prevention involves taking steps to keep the microbe away from the animal. From the food animal practitioner's point of view, anyone of these actions is considered successful if it proves effective against the Salmonella serotype causing the disease. From a food safety standpoint, however, any such action would be considered successful only if it were effective against all Salmonella serotypes. In therapy, action is taken after the microbe has infected and caused disease in the animal. Therapy involves the use of drugs to counteract the effects of the disease, halt its progress and, if possible, reverse its course. For salmonellosis, the therapeutic compounds used include antimicrobial drugs, fluids and electrolytes, and nonsteroidal anti-inflammatory drugs. Therapy has been shown to be effective in specific cases especially if started early and the antimicrobial drugs selected for use are administered properly and are those to which the causative Salmonella is sensitive. 1, 6, 18 Because Salmonella is intracellular during clinical disease, the effectiveness of therapy is also enhanced if the selected antimicrobial drugs achieve good intracellular levels and have large volume distribution. 18, 27 Combinations of trimethoprim and sulfonamides satisfy these requirements and are relatively inexpensive (especially when they are given orally). Cephalosporins such as ceftiofur and amoxicillin are also considered to be good choices for therapy against Salmonella. Other antimicrobial drugs to which Salmonella are generally susceptible include gentamicin, amikacin, and apramycin. Although nitrofurazones are effective, they are often toxic. The down side of therapy is that carrier animals and drug-resistant Salmonella often result. In control, contact between the microbe and the host animal is expected. However, action is taken to ensure that the microbe either does not succeed in establishing itself in the animal or is immediately neutralized if it does. Tools utilized include vaccines and competitive exclusion products. The use of vaccines is effective under certain circumstances, especially when the vaccines are designed to combat hostadapted serotypes, contain the Salmonella serotypes of interest, and are used to inoculate dams with the intention of providing immunity to the neonate. Most of the current commercial vaccines are killed bacterins.
28
EKPERIGIN & NAGARAJA
However, an attenuated live vaccine (Strain 51) is used in the United Kingdom and is reported to be safe and effective against S. typhimurium and S. dublin when it is used to vaccinate 2 to 4-week-old calves. 1, 14, 18 Until vaccines are developed that are effective against all Salmonella serotypes, vaccines will continue to be of limited value in the effort to control Salmonella. Similarly, the usefulness of competitive exclusion products in effectively inhibiting the ability of Salmonella to establish itself in the animal will depend on the development of products that contain well-defined microflora. The products should also not be used concurrently with systemic or oral antibiotics. Prevention involves keeping Salmonella away from the animal, and its effectiveness depends on a knowledge of the epidemiology of the microbe or the diseases it causes. For example, since the 1980s chickens and turkeys in the United States have been rendered free of pullorum disease and fowl typhoid by establishing breeding flocks that are free of the diseases and by hatching and rearing their progeny under circumstances that preclude direct or indirect contact with infected chickens and turkeys. This approach is based on the knowledge that the host range of S. gallinarum-pullorum is basically limited to chickens and turkeys and that a major mode of its transmission is via the egg. 17, 20 It is possible to apply the same principles to other Salmonella because the sources of Salmonella infection are known to be the animal and its feed and environment. Basically, the strategy would involve producing Salmonella-free animals, raising the animals in an environment from which Salmonella is excluded, and feeding them Salmonella-free feed. As described previously, it is possible to produce chickens and turkeys that are free of Salmonella. It is also possible to use appropriate housing construction to keep sources of Salmonella contamination (rodents, wild birds, and insects) away from the immediate environment of animals. Finally, there have been reports of pelleting techniques for producing Salmonella-free feeds. 6, 11 Recently, the United States Food and Drug Administration approved the use of a 37% solution of formaldehyde (2.5 kg, or 5.4 Ibs per ton) for preventing recontamination of such feeds. References 1. Blood DC, Radostits OM, Henderson JA: Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, ed 6. Philadelphia, Bailliere Tindall 1985, pp 576-589 2. Bryan FL: Factors that contribute to outbreaks of foodborne disease. J Food Prot 41:816-827, 1978 3. Bryan FL, Fanelli MJ, Riemann H: Salmonella infections. In Riemann H, Bryan FL (eds): Food-borne infections and intoxications, ed 2. New York, Academic Press, 1979, pp 73-130 4. Doyle MP, Cliver DO: Salmonella. In Cliver DO (ed): Foodborne Diseases. San Diego, Academic Press, Inc., 1990, pp 185-204 . 5. Ekperigin HE, Jang S, McCapes RH: Effective control of a gentamicin-resistant Salmonella arizonae infection in turkey poults. Avian Dis 27:822-829, 1983 6. Ekperigin HE, McCapes RH, Redus R, et al: Research note: Microcidal effects of a new pelleting process. Poultry Science 69:1595-1598, 1990 7. Hibbs CM, Kennedy GA: Salmonellosis, Current Veterinary Therapy, Food Animal Practice. Philadelphia, WB Saunders Co., 1981, pp 703-706
SALMONELLA
29
8. Hirsh DC, Ikheda JS, Martin LD, et al: R plasmid-mediated gentamicin resistance in Salmonella isolated from turkeys and their environment. Avian Dis 27:766-722, 1983 9. Jennings WE: Food-borne illness. In Libby IA (ed): Meat Hygiene, 4th ed. Philadelphia, Lea and Febiger, pp 277-286, 1975 10. LeMinor L: Genus III. Salmonella. In Krieg NR, Holt JE (eds): Bergey's Manual of Systematic Bacteriology, vol 1. Baltimore, Williams and Wilkins, 1984, pp 427-458 11. McCapes RH, Ekperigin HE, Cameron WJ, et al: Effect of a new pelleting process on the level of contamination of poultry mash by Escherichia coli and Salmonella. Avian Dis 33:103-111, 1989 12. Merchant lA, Barner RD: Infectious Diseases of Domestic Animals, ed 3. Ames, Iowa, Iowa State University Press, 1964, pp 90-101 13. Nagaraja KV, Pomeroy BS, Williams JE: Paratyphoid infections. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 99-130 14. Nagaraja KV, Pomeroy BS, Williams JE: Arizonosis. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, p 130-137 15. National Academy of Sciences: Meat and Poultry Inspection: The scientific basis of the nationOs program. Washington, D.C., National Academy Press, 1985, pp 21-42, 68-79 16. Naylor JM: Diarrhea in neona~al ruminants. In Smith BP (ed): Large animal internal medicine. St. Louis, The c.v. Mosby Company, 1990, pp 348-367 17. Pomeroy BS, Nagaraja KV: Fowl typhoid. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 87-99 18. Smith BP: Salmonellosis. In Smith BP (ed): Large animal internal medicine. St. Louis, The C.V. Mosby Company, 1990, pp 818-822 19. Snoeyenbos GH: Pullorium disease. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 73-86 20. Snoeyenbos GH: Salmonella infection at the farm level. In Proceedings of the international symposium on Salmonella and prospects for control, Guelph, 1977, pp 841-47 21. Snoeyenbos GH: Salmonellosis: Introduction. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, p 72 22. Takeuchi A: Electron microscope studies of experimental Salmonella infection. I: Penetration into the intestinal epithelium by Salmonella typhimurium. Am J PathoI50:109136, 1967 23. Takeuchi A, Sprinz H: Electron microscope studies of experimental Salmonella infection in the preconditioned guinea pig, II. Response of the intestinal mucosa to the invasion by Salmonella typhimurium. Am J Pathol 51:137-161, 1967 24. Tauxe RV: Salmonella, a postmodern pathogen. J Food Prot 54:563-568, 1991 25. Tauxe RV, Cohen ML: Epidemiology of diarrhea diseases in developed countries. In Blaser MJ, Smith PD, Ravdin JI, et al (eds): Infections of the Gastrointestinal Tract. New York, 1995, pp 37-51 26. Threfall EJ, Hall MLM, Rowe B: Lactose-fermenting Salmonellae in Britain, FEMS. Microbiol Lett 17:127-130, 1983 27. Wilcock BP, Schwartz KJ: Salmonellosis. In Allen DL, Straw BE, Mengeling WL, et al (eds): Diseases of Swine, ed 7. Ames, Iowa, Iowa State University Press, 1992, pp 570-583 28. Zecha BC, McCapes RH, Dungan WM, et al: The Dillon Beach project-a five-year epidemiological study of naturally occurring Salmonella infection in turkeys and their environment. Avian Dis 21:141-159, 1977 29. Ziprin RL: Salmonella. In Hui YH, Gorham JR, Murrell KD, et al (eds): Foodborne disease handbook-diseases caused by bacteria. New York, Marcel Dekker, Inc., 1994, pp 253-318
Address reprint requests to Henry E. Ekperigin, DVM, MPVM, PhD Feed Safety Team, HFV-222 Division of Animal Feeds Center for Veterinary Medicine Food and Drug Administration 7500 Standish Place Rockville, Maryland 20855
0749-0720/98 $8.00
+ .00
SALMONELLA Henry E. Ekperigin, DVM, MPVM, PhD, and Kakambi V. Nagaraja, DVM, MVSc, PhD
Salmonella is a genus of bacteria belonging to the family Enterobacteriaceae. Bacteria constituting the genus are gram-negative, nonsporeforming, usually motile bacilli. They are facultative anaerobes and contain three different types of antigens. The somatic (0) antigen is associated with the cell wall and composed of lipopolysaccharides. The flagellar (H) antigen is associated with the microbe's peritrichous flagella and is proteinaceous. The third type of antigen, a capsular (Vi) antigen, is found only in some Salmonella. 3, 4, 13, 14, 18, 19, 21, 27, 29 The agglutinating properties of the antigens are used to differentiate among the more than 2200 serologically distinct types of Salmonella. 18, 29 These serotypes are not biochemically defined subdivisions of the genus and are not regarded as distinct species even though their conventional names suggest otherwise (e.g., S. typhimurium, S. dublin, S. choleraesuis, S. enteritidis, S. newport, and so forth. The serotypes are classified into one of 50 groups based on the composition of their somatic (0) antigens. The groups are named after the letters of the alphabet, A-Z, and the numbers, 51-65. Thus Salmonella serotypes are classified into serogroup A if they possess the 2 antigen as the dominant antigen, and into serogroup B if they possess the 4 and 12 antigens. 4,18 A serotype can be further subdivided by using biotype, phage type, and plasmid content to identify phenotypic variations within the serotype. A biotype is the biochemical variation between two microbes of the same serotype, whereas the phage type reflects differences between two organisms with the same serotype but different susceptibilities to a lytic
From the Feed Safety Team, Division of Animal Feeds, Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland (HEE); and the Department of Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota (KVN)
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 14· NUMBER 1 • MARCH 1998
17
18
EKPERIGIN & NAGARAJA
bacteriophage. Examples include S. gallinarum and S. pullorum as biotypes of S. gallinarum-pullorum, and the 27 or more phage types of S. enteritidis. Plasmid analysis is useful in identifying strain differences.lO, 26, 29 The nutritional requirements of Salmonella are simple. For optimum growth, the microorganism requires a temperature of 3SOC to 37°C, a pH of 6.5 to 7.5, and a water activity of 0.930 or higher. However, some serotypes have been shown to grow in temperatures or pH as low as SoC or 4.5, or as hig~ 47°C or 9.0. Others have also been shown to survive freezing and long-term frozen storage. Salmonella can survive in dry foods but does not compete well with food spoilage microbes, Escherichieae, lactic acid bacteria, or other microbes that occur naturally in foods. 3 SALMONELLOSIS
Salmonella can gain entry into an animal (the host) through contaminated feeds or fomites. 3,28 The usual portal of entry is the host's oral cavity; however, pores in the shells of freshly laid eggs can provide another important portal of entry in poultry. After entry, the invading Salmonella is either overwhelmed by the host's defenses and destroyed or expelled, or succeeds in overcoming those defenses and establishing itself inside the host. The initial site of establishment of Salmonella within the host is usually the gastrointestinal tract, especially the ileum, where the microbe attaches itself to mucosal cells. 22 A host containing an established Salmonella colony is said to be infected. All food animals, except aquatic species in their usual habitats, are susceptible to natural infection with Salmonella. An animal infected with Salmonella mayor may not develop salmonellosis, the disease. Salmonellosis may occur in the host as a localized disease affecting the gastrointestinal tract and resulting in diarrhea alone or diarrhea together with fever, anorexia, depression, and shock. The localized form of salmonellosis is also referred to in literature as the subchronic, chronic, or enteric form. Salmonellosis may also occur as a generalized disease affecting several body systems. Depending on the body systems affected, symptoms of the generalized disease may include pneumonia, cough, central nervous system signs, abortion, etc. The generalized form of salmonellosis is also referred to in literature as the acute, systemic, or septicemic form. If infection does not progress into salmonellosis, Salmonella organisms remain in the gastrointestinal tract as part of the host's commensal flora and may be shed in feces. In cases in which infection develops into salmonellosis, Salmonella initiate the disease process by migrating to the lamina propria through the microvilli of mucosal cells and the tight junctions between those cells. 22 The mucosal cells are damaged in the process, and fibronecrotic plaques are formed. Damage to the mucosal cells distrupts the integrity of the mucosal lining of the gastrointestinal tract and causes materials that would ordinarily have been confined to or excluded from the gut lumen, to leak out or in. 23 Endotoxins and
SALMONELLA
19
other toxic materials seep out of the lumen into the body cavity while plasma proteins and other materials present in exudates from the inflamed lamina propria leak into the lumen. 23 A putrid diarrhea ensues accompanied by fever, anorexia, depression, and shock. Secretion of enterotoxins by Salmonella can also cause diarrhea to occur independent of the mucosal damage. Inside the lamina propria, Salmonella stimulate an inflammatory response and are subsequently engulfed by macrophages and neutrophils. The engulfed Salmonella are transported in the blood to the mesenteric lymph nodes and other organ systems where they can survive and multiply as facultative intracellular parasites and eventually cause embolic lesions. Examples of problems that can result from those lesions include respiratory difficulties, meningitis, abortion, and sudden death. The incubation period for the disease is about 1 to 4 days. The duration of the disease can be as long as 2 weeks. Animals that recover from the disease usually become carriers of Salmonella. These carrier animals appear to be in good health; however, they continually shed Salmonella into the environment and become sources of infection for other animals. 3, 9,18,27 EPIDEMIOLOGY Determinants of the Disease
Several factors determine whether or not an animal infected with Salmonella will develop salmonellosis. The factors include those attributable to Salmonella, to the host, and to the environment. Factors attributable to Salmonella include the microbe's serotype, virulence, and the number of viable cells present in the inoculum. Most of the 2200 or more serotypes of Salmonella have no predilection for any particular type of host and are capable of infecting all host species. However, a few serotypes are host-adapted and, under normal circumstances, will cause disease only in the host to which they are adapted. Examples include S. gallinarum-pullorum, whose biotypes will infect and cause fowl typhoid and pullorum disease in chickens and turkeys but will not infect cattle, and S. choleraesuis, which has a preference for pigs. With regards to serotype virulence, S. enteritidis phage type 4 is virulent to chickens and will infect and cause disease in the birds while most of the other phage types of the same serotype are less virulent and will infect but not cause disease in the birds. Although the number of viable Salmonella cells needed to initiate infection in animals has been shown to be as few as one colony forming unit (CFU) per 15 grams of feed,14 many more cells are usually needed to allow infection to progress into salmonellosis. This has been demonstrated in experiments in which the number of S. typhimurium that was enough to cause infection in pigs was found not to be enough to cause lesions in the same pigs until the microbes were allowed to increase in number to about 107 cfu of the organism per gram of intestinal content. 18, 27 In short, not only must Salmonella be present for infection to occur but also the number of viable cells present must be enough to
20
EKPERIGIN & NAGARAJA
initiate the infection and subsequent disease. The number of viable cells required varies with the virulence of the serotype involved. Host factors that determine whether or not infection of an animal will occur and subsequently develop into salmonellosis include species, breed, age, and health and immune status. Cattle are very susceptible to S. dublin, S. newport, and S. typhimurium but much less so to S. gallinarumpullorum. Chickens, on the other hand, are very susceptible to the two biotypes of S. gallinarum-pullorum but much less so to S. choleraesuis. Pigs are much more susceptible to S. choleraesius than other food animal species. With regard to age, younger animals are generally more susceptible to salmonellosis than older animals; however, there are instances in which the adult animal is equally or more susceptible to salmonellosis than the young. As an example, adult chickens are more susceptible to salmonellosis caused by the biotype S. gallinarum than young chicks are. With regard to health and immune status, malnourished, debilitated, or immunodeficient animals are more likely to succumb to infection with Salmonella than their properly nourished, healthy, or immunocompetent counterparts.3, 12, 13, 14, 27, 29 Environmental factors can determine whether or not infection of an animal by Salmonella will occur and develop into salmonellosis. The factors include the season of the year, and animal husbandry and management practices that affect the animal's immediate environment and well-being. Those practices include housing design and maintenance, feeds and feeding schedule, and vaccination programs. Salmonellosis has been shown to occur more frequently during the summer and fall. Also, animals are more likely than not to succumb to Salmonella infection and disease if they are undernourished, inadequately vaccinated against other diseases, easily accessible to delivery personnel and other visitors that can track infection between farms, and raised in unhygienic, rodent or fly-infested facilities with poor air quality and temperature. Frequency of Salmonellosis
There is currently no reliable information about the incidence of salmonellosis in food animals on a national basis in the United States. 3, 19,27 Survey data from various parts of the world indicate that New Zealand has an infection rate of 13% to 15% in dairy cows, calves, sheep, and beef cattle. For healthy pigs sent to slaughter, the infection rates were 25% in the Netherlands, 10% in New Zealand, 60/0 in the United Kingdom, and 10% to 13% in the United States. However, these data were collected at slaughter houses and could be overestimates of incidence because the stress of transportation and food withdrawal experienced by animals going to slaughter has been shown to increase the number of shedders of Salmonella among those animals. On the other hand, the data could be gross underestimates of incidence because the information was obtained using healthy pigs. The U.S. National Veterinary Services Laboratories (NVSL) issues
SALMONELLA
21
quarterly and annual reports that summarize the data on distribution and frequency of Salmonella serotypes. The reports are based on data obtained from the results of analyses conducted at NVSL to determine the serotypes of Salmonella cultures sent to the laboratory. As presented, the reports cannot be used to determine the incidence of Salmonella infections or salmonellosis; however, they are useful as a general indication of the frequency of occurrence of various Salmonella serotypes in various food animals species during a specified time period. During the period October 1, 1995 through September 30, 1996, for example, S. typhimurium was the predominant serotype found among clinical isolates from cattle and constituted 58.5% of the 1461 cultures submitted to the laboratory for serotyping. Similarly, S. choleraesuis (35% of 848 cultures), S. arizonae (55% of 49 cultures), and S. senftenberg (17% of 345 cultures) were the predominant serotypes among clinical isolates of Salmonella from swine, sheep, and turkeys, respectively. In chicken, S. heidelberg was the predominant serotype and constituted 21 % of 160 cultures submitted.
Distribution of Salmonellosis
Salmonellosis occurs worldwide. However, the pattern of distribution might differ for individual Salmonella serotypes. As an example, S. typhimurium affects all animal species and has a worldwide distribution. On the other hand, the patterns of distribution for host-adapted serotypes like S. choleraeusuis and S. dublin are patchy and generally match the patterns of distribution of the hosts to which they are adapted. Figure 1 shows the general cycle of infection for Salmonella using poultry as an example and portraying the inter-relationships between the three main determinants of salmonellosis: Salmonella, the host, and the environment.
CLINICAL SIGNS, SYMPTOMS, AND DIAGNOSIS
In all animals, the signs and symptoms of Salmonellosis usually reflect the location of the disease. If the disease is localized to the gastrointestinal tract, the signs and symptoms usually include a profuse, watery, and fetid diarrhea. In cases in which endotoxins and other toxic materials leak out of the host's gastrointestinal tract into the body, the diarrhea may be accompanied by fever, anorexia, depression, and shock. If bacteremia occurs and the disease spreads throughout the body of the host, the signs and symptoms will depend on the organs affected and the severity of embolic lesions formed in those organs. The signs and symptoms may include dyspnea and other respiratory problems, abortion, occasional diarrhea, and sudden death.
22
EKPERIGIN & NAGARAJA
---~--. ) ~
Feed
VVater
(
)l~ Feed Water
~
~ W j
·Meat, bone CONTAMINATED and feather..... FEED
~~Co
me~
Figure 1. Salmonella cycle of infection for poultry
Cattle
The most common Salmonella serotypes infecting cattle include S. dublin, S. typhimurium, S. newport, and S. montevideo. S. dublin is adapted to cattle, and the disease it causes in them can become endemic in a herd or farm because cattle recovering from salmonellosis caused by the serotype become true carriers and, for a long time, continually shed this microbe into the environment through feces and milk. Neonates (usually 1 to 2 months of age) and adult cattle are both susceptible to infection with Salmonella. 16 The infection can develop into salmonellosis after an incubation period of 1 to 4 days. Clinical findings include a watery to fibrino-mucohemorrhagic diarrhea with a fetid odor, anorexia, marked depression, fever, and shock. In cases with bacteremia, there may also be dyspnea, incoordination, nystagmus, polyarthritis, decreased milk production, and abortion. Affected animals become recumbent and die within 24 hours (adults) or 3 to 7 days (calves). Animals that recover become true (S. dublin) or passive (S. typhimurium and other non-cattle-adopted serotypes) carriers. Unlike the persistent infection that occurs in a true carrier, infection in a passive carrier lasts only for a relatively short period of time and is cleared from the animal after 3 to 16 weeks of shedding. A single carrier can shed as much as one million cfu of S. dublin per gram of feces, or 100 to 100,000 cfu per milliliter of raw milk. Is
SALMONELLA
23
Necropsy findings include emaciated carcasses with serous atrophy of fat, fibrino-necrotic plaques that adhere to a thickened and hemorrhagic mucosa (especially in the ileum), fibrinous sheets especially in the area of Peyer's patches, enlarged and darkened mesenteric lymph nodes, and watery bowel contents that may contain fibrin or blood. There also may be splenomegaly and an edematous lung with hemorrhagic or congested foci. The gall bladder wall may be thickened and hemorrhagic, and the bile is often inspissated into a firm coagulum. A tentative diagnosis of bovine salmonellosis can be made from the history of the herd (including disclosure of recent stress factors like calving, transportation, vaccination, addition of new calves, commingling of calves from many different sources, water and food deprivation), and from clinical and necropsy findings. A definitive diagnosis depends on the isolation and identification of the causative Salmonella. The disease should be differentiated from similar enteric diseases of calves including those caused by E. coli, rotaviruses, coronaviruses, bracken fern and other poisonings, Cryptosporidium sp, and Eimeria sp. The pneumonic form of calf salmonellosis should also be differentiated from pneumonic pasteurellosis. In adult cattle, salmonellosis should also be differentiated from bovine viral diarrhea, winter dysentery, and feedind uced indigestion. Poultry
Over 200 Salmonella serotypes have been isolated from chickens and turkeys in the United States. Most of these were isolated from the intestines and internal organs of birds suffering from acute, fatal salmonellosis. One serotype, S. gallinarum-pullorum (which, as mentioned earlier, consists of two biotypes that have traditionally been identified as S. gallinarum and S. pullorum), is adapted to poultry and is nonmotile. In addition to the fecal-oral route, transmission of Salmonella in poultry can also occur through the egg. Egg transmission is achieved in two ways. In one, Salmonella in the blood of bacteremic hens are transported to the reproductive system, where they are incorporated into developing ova. In the other method of egg transmission, Salmonellacontaminated fomites are sucked through the pores of freshly laid eggs as those eggs cool down to ambient temperature. In either case, Salmonella becomes part of the egg or, if the egg is fertile, part of the developing embryo. The incubation period varies with the causative serotype but is usually about 4 to 5 days. The diseases of poultry caused by the two biotypes of S. gallinarum-pullorum are called pullorum disease and fowl typhoid. That caused by S. arizonae is called avian arizonosis, whereas those caused by the other serotypes are collectively referred to as fowl paratyphoid. The clinical signs and symptoms are basically the same for all and are compatible with those of the localized or generalized forms of salmonellosis described earlier. The birds show somnolence, weakness,
24
EKPERIGIN & NAGARAJA
anorexia, drooped wings, and ruffled feathers. They huddle together and their vents are pasted with chalk-white excreta that is sometimes stained greenish brown. Other signs that can occur include labored breathing or gasping, retarded growth, blindness, and lameness due to swelling of joints and adjacent synovial sheaths. Mortality is variable, and there are variable effects on egg production, fertility, and hatchability. Pullorum disease and fowl paratyphoid generally affect younger birds, whereas fowl typhoid is encountered more frequently in adult chickens and turkeys. Since the early 1970s, all major commercial breeding flocks have been free of pullorum disease. There have also not been any reported outbreaks of fowl typhoid in the United States since 1980. 19 Flock history and clinical signs and symptoms are of limited value in arriving at a diagnosis of the diseases because of their similarity to the history, and signs and symptoms of several other diseases, including diseases caused by coliforms, staphylococcus, and Mycoplasma synoviae. Definitive diagnosis requires isolation and identification of the causative Salmonella serotype. Swine
S. choleraesuis and S. typhimurium are the Salmonella serotypes most commonly associated with the generalized form of salmonellosis in swine, whereas S. typhimurium is more commonly associated with the localized form. S. choleraesuis is the serotype most frequently isolated from pigs; it is swine-adapted. Porcine salmonellosis occurs most often in intensively reared, weaned pigs less than 5 months old, only occasionally in market-size swine and adult breeding stock, and rarely in conventionally reared suckling pigs. The incubation period is 24 to 48 hours. In the localized form, a watery, yellow, fetid diarrhea lasts 3 to 7 days and then may recur one or more times. Blood mayor may not appear in the watery feces. There is also a moderate fever, anorexia, and dehydration. The pigs assume a "tucked abdomen" look and emit a complaining grunt and squeal. Mortality is low and occurs only after several days of watery feces. In the generalized form, there is dyspnea, anorexia, fever of 105 to 107°F (40.5-41.6°C), reluctance to move, shallow moist cough, and huddling. Adult pregnant pigs may abort. There may also be central nervous system signs including tremor, weakness, paralysis, and convulsion. Diarrhea does not occur until the third or fourth day after onset of the disease and is watery yellow. Death occurs in 2 to 4 days. Dead pigs have purple extremities and abdomens. Mortality is high and can reach 100%, especially in outbreaks with the central nervous system signs. Morbidity is variable but usually less than 10%. Pigs that recover from both forms of the disease become carriers and will intermittently shed Salmonella for five months afterwards or longer. Necropsy findings include cyanosis of the ears, feet, tail, and ventral abdominal skin. There is splenomegaly, hepatomegaly with miliary white foci of necrosis, and congestion to infarction of the gastric fundic
SALMONELLA
25
mucosa. The mesenteric lymph nodes are moist and swollen, and the lungs are firm and diffusely congested. The lungs also often have interlobular edema and hemorrhage. The history of the herd and the clinical and necropsy findings are helpful but are usually not enough to make a definitive diagnosis of salmonellosis because of the similarity between these findings and those attributable to other diseases. Those other diseases, which need to be ruled out, include hog cholera, swine dysentery, and diseases caused by erysipelas, streptococci, and Actinobacillus pleuropneumonia. A definitive diagnosis is made after the isolation and identification of the causative Salmonella. Sheep and Goats
The major Salmonella serotype associated with salmonellosis in sheep is S. typhimurium. Other serotypes that have been found in sheep include S. arizonae, S. abortus ovis, and S. dublin. S. abortus ovis is sheepadapted. It is endemic in the United Kingdom and other parts of Europe, but has not been reported in the United States. Ovine salmonellosis occurs most frequently in feeder lambs; however, ewes and lamb flocks are also susceptible. The clinical findings in feeder lambs include watery scours that start within 24 hours of placement in the feed lot, dullness, depression, drooped ears, anorexia, coughing, and dehydration. The diarrhea is watery and greenish-yellow. There is also a 2° to 4° rise in temperature and increased heartbeat and respiration. Symptoms may persist for 1 to 2 weeks, and pregnant young ewes may abort. Morbidity ranges from 5% to 50 %, and mortality is usually not over 5% to 10%. Necropsy findings include emaciated, dehydrated cadavers, congested and hemorrhagic mucosal linings in stomach and intestines, and enlarged and congested lymph nodes. Pneumonia may be present. Diagnosis requires differentiation from diseases with similar signs and symptoms. Examples include coccidiosis in which scouring and other signs begin 2 to 4 weeks after placement in the feedlot; enterotoxemia in which the lambs are on full feed and not emaciated; and lamb dysentery that affects unweaned lambs. Definitive diagnosis of salmonellosis is based on history of that herd, clinical signs and symptoms, and necropsy findings followed by isolation and identification of causative Salmonella. Reports of naturally occurring cases of salmonellosis in goats are scanty. Among reported cases, S. dublin is the usual pathogen. There have also been reports specifying S. typhimurium as a cause. The signs and lesions observed were similar to those in cattle. FOOD SAFETY CONCERNS
As mentioned earlier, food animals whose infection with Salmonella does not progress into salmonellosis, and those that recover from the disease, become carriers of Salmonella. Although these animals appear
26
EKPERIGIN & NAGARAJA
healthy, they constitute a very large reservoir of Salmonella and continually shed the microbe into their environment. The frequency of shedding is influenced by several factors, including stress. It is a common practice to withhold feed for 6 hours or more from animals that are ready to be processed for food, and to transport them from the ranches, farms or feedlots on which they were reared to slaughter houses that are usually located fairly far away. The stress of transportation and feed deprivation can cause the carriers among these animals to shed Salmonella and infect other animals in the herd or flock being transported. This spread of infection among live animals awaiting slaughter can cause a significant increase in the likelihood that carcasses emerging from the slaughter house will be contaminated with Salmonella. The rate of contamination of such carcasses by Salmonella has been shown to range from 0% to 90%. The use of meats from such contaminated carcasses has also been shown to be one of the major factors contributing to the occurrence of outbreaks of food borne human salmonellosis in the United States. 2,24 About 50 such outbreaks are reported annually, in addition to the very large number of sporadic cases that occur independently of the recognized outbreaks. Human salmonellosis is one of the most commonly reported bacterial diseases in the United States. 25 It can be fatal. 24 Humans can become infected by most, possibly all serotypes of Salmonella. In the United States, however, the most common serotypes among the 40,000 isolates reported from humans each year are S. typhimurium, S. enteritidis, S. heidelberg, S. hadar and S. newport. Poultry is the food animal reservoir for S. heidelberg and S. infantis, and egg-laying hens the reservoir for S. enteritidis. For S. newport and S. dublin, the reservoir is cattle. S. typhimurium has no preference for any particular animal species and is equally infectious to all. Just like in other animals, Salmonella infection in humans does not always result in salmonellosis. When it does, the result is identical to that in other animals: a disease that is either localized (gastroenteritis) or generalized (septicemia). The very young, the very old and the immunosuppressed are most susceptible. 25 Apart from concerns about humans contracting Salmonella infections through foods of animal origin, there is the additional concern that a sizable proportion of those infections could be caused by antibioticresistant Salmonella. There have been reports of isolation of gentamicinresistant S. arizonae from turkeys5,8 and of multidrug-resistant S. newport from cattle. 24 The ingestion of contaminated meats from such animals could result in human infections with gentamicin-resistant Salmonella and become a major problem if the infections progressed into salmonellosis that required treatment with gentamicin. Even in those cases in which the infection did not progress into salmonellosis, treatment of the infected individual with gentamicin to control another ailment could suppress the normal intestinal flora, disrupt their protective effect, and give a competitive advantage to the gentamicin-resistant Salmonella and facilitate its ability to cause salmonellosis. More recently, a multidrug-
SALMONELLA
27
resistant S. typhimurium, known as Definitive Type 104 (S. typhimurium DT104), has been isolated in the United Kingdom and the United States from humans, poultry, sheep, pigs, cats, wild birds, rodents, foxes, and badgers, and has been shown to be transmitted from cattle and sheep to humans. The isolates in the United Kingdom are highly resistant to many valuable antimicrobial agents and frequently demonstrate a pattern of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracyclines and more recently, trimethoprim and fluoroquinolones. THERAPY, PREVENTION, AND CONTROL
Therapy and control involve taking action against a microbe that has already gained, or stands a very good chance of gaining, access to an animal, whereas prevention involves taking steps to keep the microbe away from the animal. From the food animal practitioner's point of view, anyone of these actions is considered successful if it proves effective against the Salmonella serotype causing the disease. From a food safety standpoint, however, any such action would be considered successful only if it were effective against all Salmonella serotypes. In therapy, action is taken after the microbe has infected and caused disease in the animal. Therapy involves the use of drugs to counteract the effects of the disease, halt its progress and, if possible, reverse its course. For salmonellosis, the therapeutic compounds used include antimicrobial drugs, fluids and electrolytes, and nonsteroidal anti-inflammatory drugs. Therapy has been shown to be effective in specific cases especially if started early and the antimicrobial drugs selected for use are administered properly and are those to which the causative Salmonella is sensitive. 1, 6, 18 Because Salmonella is intracellular during clinical disease, the effectiveness of therapy is also enhanced if the selected antimicrobial drugs achieve good intracellular levels and have large volume distribution. 18, 27 Combinations of trimethoprim and sulfonamides satisfy these requirements and are relatively inexpensive (especially when they are given orally). Cephalosporins such as ceftiofur and amoxicillin are also considered to be good choices for therapy against Salmonella. Other antimicrobial drugs to which Salmonella are generally susceptible include gentamicin, amikacin, and apramycin. Although nitrofurazones are effective, they are often toxic. The down side of therapy is that carrier animals and drug-resistant Salmonella often result. In control, contact between the microbe and the host animal is expected. However, action is taken to ensure that the microbe either does not succeed in establishing itself in the animal or is immediately neutralized if it does. Tools utilized include vaccines and competitive exclusion products. The use of vaccines is effective under certain circumstances, especially when the vaccines are designed to combat hostadapted serotypes, contain the Salmonella serotypes of interest, and are used to inoculate dams with the intention of providing immunity to the neonate. Most of the current commercial vaccines are killed bacterins.
28
EKPERIGIN & NAGARAJA
However, an attenuated live vaccine (Strain 51) is used in the United Kingdom and is reported to be safe and effective against S. typhimurium and S. dublin when it is used to vaccinate 2 to 4-week-old calves. 1, 14, 18 Until vaccines are developed that are effective against all Salmonella serotypes, vaccines will continue to be of limited value in the effort to control Salmonella. Similarly, the usefulness of competitive exclusion products in effectively inhibiting the ability of Salmonella to establish itself in the animal will depend on the development of products that contain well-defined microflora. The products should also not be used concurrently with systemic or oral antibiotics. Prevention involves keeping Salmonella away from the animal, and its effectiveness depends on a knowledge of the epidemiology of the microbe or the diseases it causes. For example, since the 1980s chickens and turkeys in the United States have been rendered free of pullorum disease and fowl typhoid by establishing breeding flocks that are free of the diseases and by hatching and rearing their progeny under circumstances that preclude direct or indirect contact with infected chickens and turkeys. This approach is based on the knowledge that the host range of S. gallinarum-pullorum is basically limited to chickens and turkeys and that a major mode of its transmission is via the egg. 17, 20 It is possible to apply the same principles to other Salmonella because the sources of Salmonella infection are known to be the animal and its feed and environment. Basically, the strategy would involve producing Salmonella-free animals, raising the animals in an environment from which Salmonella is excluded, and feeding them Salmonella-free feed. As described previously, it is possible to produce chickens and turkeys that are free of Salmonella. It is also possible to use appropriate housing construction to keep sources of Salmonella contamination (rodents, wild birds, and insects) away from the immediate environment of animals. Finally, there have been reports of pelleting techniques for producing Salmonella-free feeds. 6, 11 Recently, the United States Food and Drug Administration approved the use of a 37% solution of formaldehyde (2.5 kg, or 5.4 Ibs per ton) for preventing recontamination of such feeds. References 1. Blood DC, Radostits OM, Henderson JA: Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, ed 6. Philadelphia, Bailliere Tindall 1985, pp 576-589 2. Bryan FL: Factors that contribute to outbreaks of foodborne disease. J Food Prot 41:816-827, 1978 3. Bryan FL, Fanelli MJ, Riemann H: Salmonella infections. In Riemann H, Bryan FL (eds): Food-borne infections and intoxications, ed 2. New York, Academic Press, 1979, pp 73-130 4. Doyle MP, Cliver DO: Salmonella. In Cliver DO (ed): Foodborne Diseases. San Diego, Academic Press, Inc., 1990, pp 185-204 . 5. Ekperigin HE, Jang S, McCapes RH: Effective control of a gentamicin-resistant Salmonella arizonae infection in turkey poults. Avian Dis 27:822-829, 1983 6. Ekperigin HE, McCapes RH, Redus R, et al: Research note: Microcidal effects of a new pelleting process. Poultry Science 69:1595-1598, 1990 7. Hibbs CM, Kennedy GA: Salmonellosis, Current Veterinary Therapy, Food Animal Practice. Philadelphia, WB Saunders Co., 1981, pp 703-706
SALMONELLA
29
8. Hirsh DC, Ikheda JS, Martin LD, et al: R plasmid-mediated gentamicin resistance in Salmonella isolated from turkeys and their environment. Avian Dis 27:766-722, 1983 9. Jennings WE: Food-borne illness. In Libby IA (ed): Meat Hygiene, 4th ed. Philadelphia, Lea and Febiger, pp 277-286, 1975 10. LeMinor L: Genus III. Salmonella. In Krieg NR, Holt JE (eds): Bergey's Manual of Systematic Bacteriology, vol 1. Baltimore, Williams and Wilkins, 1984, pp 427-458 11. McCapes RH, Ekperigin HE, Cameron WJ, et al: Effect of a new pelleting process on the level of contamination of poultry mash by Escherichia coli and Salmonella. Avian Dis 33:103-111, 1989 12. Merchant lA, Barner RD: Infectious Diseases of Domestic Animals, ed 3. Ames, Iowa, Iowa State University Press, 1964, pp 90-101 13. Nagaraja KV, Pomeroy BS, Williams JE: Paratyphoid infections. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 99-130 14. Nagaraja KV, Pomeroy BS, Williams JE: Arizonosis. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, p 130-137 15. National Academy of Sciences: Meat and Poultry Inspection: The scientific basis of the nationOs program. Washington, D.C., National Academy Press, 1985, pp 21-42, 68-79 16. Naylor JM: Diarrhea in neona~al ruminants. In Smith BP (ed): Large animal internal medicine. St. Louis, The c.v. Mosby Company, 1990, pp 348-367 17. Pomeroy BS, Nagaraja KV: Fowl typhoid. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 87-99 18. Smith BP: Salmonellosis. In Smith BP (ed): Large animal internal medicine. St. Louis, The C.V. Mosby Company, 1990, pp 818-822 19. Snoeyenbos GH: Pullorium disease. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, pp 73-86 20. Snoeyenbos GH: Salmonella infection at the farm level. In Proceedings of the international symposium on Salmonella and prospects for control, Guelph, 1977, pp 841-47 21. Snoeyenbos GH: Salmonellosis: Introduction. In Diseases of Poultry, ed 9. Ames, Iowa, Iowa State University Press, 1991, p 72 22. Takeuchi A: Electron microscope studies of experimental Salmonella infection. I: Penetration into the intestinal epithelium by Salmonella typhimurium. Am J PathoI50:109136, 1967 23. Takeuchi A, Sprinz H: Electron microscope studies of experimental Salmonella infection in the preconditioned guinea pig, II. Response of the intestinal mucosa to the invasion by Salmonella typhimurium. Am J Pathol 51:137-161, 1967 24. Tauxe RV: Salmonella, a postmodern pathogen. J Food Prot 54:563-568, 1991 25. Tauxe RV, Cohen ML: Epidemiology of diarrhea diseases in developed countries. In Blaser MJ, Smith PD, Ravdin JI, et al (eds): Infections of the Gastrointestinal Tract. New York, 1995, pp 37-51 26. Threfall EJ, Hall MLM, Rowe B: Lactose-fermenting Salmonellae in Britain, FEMS. Microbiol Lett 17:127-130, 1983 27. Wilcock BP, Schwartz KJ: Salmonellosis. In Allen DL, Straw BE, Mengeling WL, et al (eds): Diseases of Swine, ed 7. Ames, Iowa, Iowa State University Press, 1992, pp 570-583 28. Zecha BC, McCapes RH, Dungan WM, et al: The Dillon Beach project-a five-year epidemiological study of naturally occurring Salmonella infection in turkeys and their environment. Avian Dis 21:141-159, 1977 29. Ziprin RL: Salmonella. In Hui YH, Gorham JR, Murrell KD, et al (eds): Foodborne disease handbook-diseases caused by bacteria. New York, Marcel Dekker, Inc., 1994, pp 253-318
Address reprint requests to Henry E. Ekperigin, DVM, MPVM, PhD Feed Safety Team, HFV-222 Division of Animal Feeds Center for Veterinary Medicine Food and Drug Administration 7500 Standish Place Rockville, Maryland 20855