Chapter 24 ENTERIC INFECTIONS & SALMONELLOSIS

 

24.1Summary

One of the important groups of bacteria found in the intestinal tract of man and animal is the enterobacteriaceae or as they are most commonly called the enteric bacteria. In this group we find some microbes that may lead to significant losses. Some of the more important are Salmonella sp., Escherichia coli, Klebseilla. Less significant genuses but may be present alone or in mixed infection are the Proteus, Airobacter and Shigella. Not all Salmonella or E. coli are pathogenic. Some non-enteropathogenic species that normally inhabit the intestinal cause infection only on rare occasions when the body is stressed.

Enterobacteriaceae are gram-negative rods that may or may not be motile with peritrichous flagella. They usually grow on all types of artificial media and all species form acid and gas from glucose. All of the species except these belonging to the genus Erwinia produce nitrate from nitrite.

Note:Although Difco Handbook may be available to aid you in the identification of various organisms we have attempted to include some descriptive reactions that may be helpful to you.

The antigenic composition is a mosic that results in the serological interrelationship among several general as well as among families. In the classification that has been established the term paracolon has been used to designate many of the organisms as has been pointed out by Edward and Eming (two outstanding enteric microbiologists) "We showed not mistake" this "designation of convenience" to mean that such a group actually occur.

The identification of species in the various genera may be made by utilizing biochemical tests and serological typing. This is very important in Salmonella where more than 5 serologic groups are recognized and more than 300 antigenic types have been characterized.

Another infectious bacteria that will be discussed in this chapter, the genus Vibrio of the family Spirillaceae which similarly infect animals has been changed to Campylobacter. The spirillaceae are a group of morphological related organism characterized by being simple gram-negative curved or spiral rods which are sometimes found in chains of actually growing cultures. They process one to three polar flagella which accounts for their motility. Most of the Campylobacter are aerobic or facultatively anaerobic but some genera contain anaerobic species. A good representative of this family are saprophytes that are found in water, however some notable members are pathogens for man and other animals. 

24.2 Other Campylobacters: 

Campylobacter metschnikovii was reported as a cause of a cholera like disease in chickens. The original report was probably in error as C. metschnikovii seems to be, at this time, a nonpathogenic inhabitant of the intestinal tract of normal birds. The symptomology has been attributed to concurrent infection with a Salmonella.

 Campylobacter jejuni causes winter dysentery in stabled cattle. Campylobacter suis causes a form of dysentery in swine. It is H2S positive and catalase positive. It may be the same as the European C. coli.

 Campylobacter hepatitis causes avian vibrionic hepatitis, a serious contagious disease of chickens. The gross lesions are irregularly shaped grayish white lesions on the surface of the liver. In acute cases, the liver is swollen, congested and studded with necrotic areas. In chronic cases, varying degrees of ascities and cardiac hypertrophy may be present. Histological changes show generalized congestion, localized hemorrhages and many areas of necrosis. The incubation period varies from 2 to more than 10 days depending on the age of the bird. Prevention must be based on good management and hygiene. Treatment is usually followed by limited success.

24.4 Enteric Infections

Enteric agents with the primary habitat in the intestinal tract and members of the family Enterobacteriaceae.

These bacteria, with a few exceptions, cause a similar set of symptoms. Infection is primarily by ingestion. The incubation period is variable as is the disease state which follows. The symptoms of an enteric infection are diarrhea with possible septicemia. A common sequellae of septicemia is localization in joints and thrombophlebitis.

A proportion of the pathogenicity of Enterobactericeae is due to endotoxins. These are complex lipopolysaccharides of the bacterial cell wall. Endotoxins are released into the surrounding media during culture only when the bacteria autolyze or are disrupted by mechanical means. Endotoxins are characterized as follows:

  24.5 Endotoxins:

• 1. They are produced primarily by Gram negative bacteria.
• 2. They are complex macromolecules which contain phospholipid and polysaccharide portions.
• 3. They are an integral part of the bacterial cell wall and are released only when the integrity of the cell is disturbed.
• 4. They are relatively heat stable.
• 5. They are less potent toxins than exotoxins and much less specific in their activities.
• 6. The toxic part is the phospholipid portion and the specific antigenic part is the polysaccharide portion.
• 7. They do not form toxoids. They are apparently not O (somatic) antigens as was first believed.

The numerous physiological effects include irreversible shock accompanied by severe diarrhea when organisms are injected in sufficient amounts. In lesser quantities they cause fever and transient leukopenia, followed by leukocytosis, hyperglycemia, hemorrhagic necrosis of tumors, abortion, altered resistance to many if not most bacterial infections, a wide variety of circulatory disorders and vascular hyper-reactivity to adrenergic drugs.

The Schwartzman Phenomenon (Schwartzman Reaction) occurs when a small dose of endotoxin is injected subcutaneously resulting in a mild inflammation. When a second dose of endotoxin is injected intravenously, the original skin site injection site becomes hemorrhagic within a few hours. This reaction cannot be explained on the basis of immunity and is probably essentially toxic in nature.

The phenomenon of tolerance is quite nonspecific. It affects the response of an animal to any type of endotoxin. It occurs in individuals receiving repeated exposure to endotoxin. After a period of time, these individuals become unresponsive to the pyrogenic and other biochemical effects of endotoxin. This tolerance tends to fade away after a few weeks without exposure to endotoxin.

All these bacteria are rather small Gram negative rods. Most are motile, having peritrichous flagella. Smooth variants of all species form shiny convex colonies on agar. Rough stains are granular. Encapsulated variants give large mucoid colonies which may even run like drops of water or drip onto the lid of the petri dish. They all grow well on common agars and chemically defined media. They are facultative, catalase positive and oxidase negative.

There are many varied and ingenious selective and differential media for enteric bacteria. Selective media utilize inhibitory substances, bacteriostatic dyes and bile salts to inhibit less pathogenic bacteria (Escherichia and Enterobacter species) while allowing the more pathogenic Salmonella and Shigella to grow.

 24.6 Antigenic Structure

The enteric bacteria have four common kinds of antigens.

H Antigen:

The first antigen is the flagellar antigen, termed the H antigen. It may or may not be present and consists of flagellar protein. There are many distinct antigenic variants. The H antigen is preserved in formalin-killed bacterial cells and is present in intact flagellar bacteria. H antigen is destroyed by alcohol or heat.

 O Antigen:

The second important antigen is the somatic or surface antigen, termed the O antigen. It is polysaccharide in nature. It is not damaged by alcohol or boiling for hours, but is destroyed by acid hydrolysis. 

R Antigen:

The "core" of the surface antigens are the R antigens. These R antigens are found in mutants which do not possess O antigens. They may or may not possess H antigens. Apparently these R antigens are the core to which O side chains are added. There are apparently only two R antigens, I and II. These rough bacteria tend toward spontaneous agglutination in physiological saline solutions. 

Vi Antigen:

The Vi antigen is more uncommon. In some species, it may build up to form a visible capsule. It is a simple polysaccharide. When present, it forms a layer over the O antigen. This layer acts to prevent formation of aggregates with O antiserum. However, the Vi layer does not completely cover the O antigen, for although aggregates are not formed, O antibody is still absorbed. The H antigen when reacted with homologous antibody tends to form loose, fluffy aggregates. O antigen when reacted with homologous antigen in the presence of H antigen tends to form dense granular aggregates as does Vi antigen in the presence of homologous antiserum. The R antigen causes spontaneous agglutination in the absence of antibody. Under usual conditions H antigen interferes with agglutination caused by Vi or O antigen mechanically.

Enteric bacteria elaborate toxins other than endotoxins. Colicins--highly specific proteins which kill other enteric bacteria-- are the chief class. The colicins are in part responsible for the bacterial antagonism which stabilizes the enteric flora of the adult.

The genetic interrelations of these bacteria are quite complex. The high population number yields frequent spontaneous mutations. Many of these also harbor bacteriophages (viruses which attack bacteria) which can mutate at high rates. Genetic material may also be transmitted by transduction, lysogenation and conjugation.

With this rapid genetic change, the Enterobacteriaceae are best visualized as a related group of bacterial species which tend to have slightly blurred dividing lines. Species and genera are manmade distinctions. There are always a few individuals which blur the nice, cleancut distinctions. With the enteric bacteria, the distinctions are very fuzzy, and the either/or species are very numerous.

Infection with all these species is primarily by ingestion. These bacteria survive well in water, etc., outside the host's body. Clinical infection depends upon host resistance as well as strain virulence. Many are considered either nonpathogenic or at best opportunists; however, in very young or debilitated individuals some strains cause serious disease. On the other extreme of pathogenicity, the more pathogenic strains many cause only inapparent infections in healthy individuals. The carrier state is more common for those strains considered pathogens.

 24.7 Biochemical Activities

Family ENTEROBACTERICEAE--Gram negative, facultative, asporogenous, rod-shaped bacteria that grow well on artificial media. Some species are atrichous and nonmotile variants of motile species also may occur. Motile forms are peritrichously flagellated. Nitrates are reduced to nitrites, and glucose is utilized fermentatively with the formation of acid or acide and gas. The nitrophenol oxidase test is negative, and alginate is not liquefied. Pectinate is liquefied by members of only genus, Pectinobacterium.

 Tribe I: ESCHERICHIEAE

Nonmotile or motile bacteria. The methyl red reaction is positive, and the Voges-Proskauer test is negative. Urease phenylalanine deaminase and H2S are not produced. Sodium malonate or citrate are not utilized. Gelatin is not liquefied nor does growth occur on medium containing potassium cyanide.

Genus I--Escherichia

Both acid and gas are formed from a wide variety of fermentable carbohydrates, but anaerogenic biotypes occur. Salicin is fermented by the majority of cultures, but adonitol and inositol are utilized infrequently. Lactose is usually fermented rapidly, but some strains utilize it slowly, and some do not ferment lactose at all. Lysine, arginine and ornithine are decarbosylated by the majority of cultures. Acid is formed from sodium mucate, and sodium acetate is utilized as a sole source of carbon.

Gene II--Shigella

Nonmotile. With the exception of certain biotypes of Shigella flexneri, visible gas is not formed from fermentative carbohydrates. Compared to Escherichia, Shigella are less active in their utilization of carbohydrates. Salicin, adonitol and inositol are not fermented. Strains of Shigella sonnei ferment lactose upon extended incubation, but other species do not utilize this substrate in conventional media. Lysine is not decarbosylated. The majority of Shigella do not possess a demonstrable arginine dihydrolase system, and ornithine is decarboxylated only by Shigella sonnei and a few strains of Shigella boydii.

 

Tribe II: EDWARDSIELLEAE

Motile bacteria. Hydrogen sulfide is produced abundantly. Indol is formed, the methyl red test is positive, and the V-P test is negative. Phenylalanine is not delaminated nor is urea hydrolyzed. Gelatin is not liquefied nor is Simmons citrate. Growth does not occur on sodium acetate medium nor in medium containing KCN. Lipase is not formed and arginine dihydrolase is not formed. Esculin is not hydrolyzed, and erythritol and adonitol are not fermented.

Genus I--Edwardsiella

Motile bacteria. Lysine and ornithine are decarboxylated but neither malonate nor mucate is utilized. Glucose and maltose and fermentous promptly, and with rare exception, gas is formed from these substrates. Glycerol is utilized slowly by the majority of strains, but lactose, sucrose, mannitol, dulcitol, salicin, inositol, soruitol, raffinose, rhamnose, xylose, cellbiose, and alpha methyl glucoside are not attacked.

 Tribe III: SALMONELLEAE

Usually motile bacteria. Methyl red positive, V-P negative, indol is not formed, and phenylalanine is not delaminated. Gelatin is not liquefied rapidly in nutrient medium. With few exceptions, H2S is produced abundantly. Growth occurs on Simmons citrate and sodium acetate media, arginine dihydrolase is formed, and gas is formed from fermentable carbohydrates. Esculin is not hydrolyzed, and erythritol and adonitol are not fermented.

Genus I--Salmonella

Usually motile bacteria. Urease is not produced, sodium malonate is not utilized, gelatin is not liquefied, and growth does not occur in media containing KCN. Lysine, arginine and ornithine are decarboxylated. Acid is produced in Jordan's tartrate medium. Dulcitol is fermented, and inositol is utilized by numerous strains. Sucrose, salicin, raffinose and lactose are not fermented.

Genus II--Arizona

Motile bacteria. Urease is not produced, and growth does not occur in medium containing KCN. Lysine, arginine, and ornithine are decarboxylated, malonate utilized, gelatin is liquefied slowly in nutrient medium, and lactose is fermented by the majority of cultures. With few exceptions, acid is not produced in Jordan's tartrate medium. Dulcitol and inositol are not fermented, and salicin is utilized infrequently.

Genus III--Citrobacter

Motile bacteria. Lysine is not decarboxylated and less than 20% of the strains possess ornithine decarboxylase. Urease is produced slowly by the majority of cultures, but the reactions are weak. Growth occurs in medium containing KCN, and acid is produced in Jordan's tartrate medium. Gelatin is not liquefied in nutrient medium. Dulcitol and cellobiose are fermented rapidly by the majority of cultures. Lactose is utilized but the reactions are frequently delayed.

 Tribe IV: KLEBSIELLEA

Motile or nonmotile. Hydrogen sulfide is not produced and urea is not hydrolyzed rapidly, but delayed reactions may occur. With few exceptions, indol is not produced, the methyl red test is negative, and the Voges-Proskauer reaction is positive. Growth occurs in Simmons citrate and in medium containing KCN. Phenylalanine is not delaminated. Sodium alginate is utilized as a sole source of carbon by certain members of only one genus (Klebsiella) and lipase is produced by only species of Enterobacter (Ent. liquifaciens) and by members of the genus Serratia.

Genus I--Klebsiella

Nonmotile. Hydrogen sulfide is not produced, and urea is hydrolyzed. Indol is seldom produced, methyl red test is usually negative, and V-P test is positive. Sodium alginate can be used as a sole source of carbon by some members of this genus, as can malonate. Gelatin is liquefied, and gas is produced from glucose, lactose and sucrose. 

Genus II--Enterobacter

Motile. The V-P reaction is positive; gelatin is liquefied slowly by the most commonly occurring forms (Ent. cloacae). Lysine decarbosylase is not produced by Ent. cloacae, but other species of the genus possess this enzyme system. Ornithine decarboxylase is produced. Sodium alginate is not utilized as a sole source of carbon. Gas is not formed from inositol and glycerol by cultures of Ent. cloacae. Acid is produced from sorbitol rhamnose, arabinose and raffinose by the majority of species. Ent. hafniae does not ferment sorbitol or raffinose. Ent. liquefaciens is lysolytic.

Genus III--Pectinobacterium

Motile or nonmotile bacteria. Sodium pectinate medium is liquefied. A minority of cultures produce indol, but the majority yield positive reactions in the methyl red test. Gelatin is liquefied, although the reactions of a minority of strains may be somewhat delayed. Lysine, arginine and ornithine are not decarboxylated. Sodium alginate is not utilized as a sole carbon source. Gas is not formed from inositol or glycerol, and adonitol is not fermented. Sorbitol is fermented only very rarely, but the majority of cultures produce acid from rhamnose, arabinose and raffinose. The optimum growth temperature is about 25oC, and cultures fail to grow or grow poorly at 370C.

Genus IV--Serratia

Motile. A positive V-P test is given by Serratia marcescens subsp. marcescens, but Ser. marcescens subsp. kiliensis gives negative results. Lipase is produced, gelatin is liquefied rapidly, and lysine and ornithine are decarboxylated. Sodium alginate is not utilized as a sole carbon source. When gas is formed from fermentable substrates, the volumes are small (10% or less). Acid is produced from sorbitol, but rhamnose, arabinose and raffinose are not fermented.

 Tribe V: PROTEEAE

Motile bacteria. The methyl red test is positive, and with the exception of occasional strains of Proteus mirabilis, the V-P reaction is negative. Phenylpyruvic acid is produced rapidly and abundantly from phenylalanine. Growth occurs in medium containing KCN, but sodium alginate is not used as a sole carbon source. Gas volumes produced from fermentable substrate by aerogenic cultures are small (a bubble to about 15%). Urea is hydrolyzed rapidly and abundantly only by members of the genus Proteus.

Genus I--Proteus

Motile. Urea is hydrolyzed rapidly. Two species, Proteus vulgaris and Pr. mirabilis, produce H2S rapidly and abundantly, liquefy gelatin and swarm on moist agar media. The majority of cultures of these two species are lysolytic. The other species, Pr. morganii and Pr. rettgeri, do not possess these particular characteristics. Ornithine is decarbocylated by two species, Pr. morganii. Mannitol is fermented by the majority of strains of Pr. rettgeri, but the remaining species fail to produce acid from this substrate.

Genus II--Providencia

Motile. Urea is not hydrolyzed and H2S not produced. Indol is produced and growth occurs on Simmons citrate medium. Gelatin is not liquefied. Lysine, arginine and ornithine are not decarboxylated, and lipase is not produced. With rare exceptions, mannitol is not fermented, and acid is not produced from alpha methyl glucoside, erythritol or esculin.

 

24.8 Disease States and Natural Habitats

Tribe I: ESCHERICHIEAE

 Escherichia

Called the "colon bacillus" because it is a normal, indeed the predominant facultative inhabitant of the lower bowel of warm-blooded animals, it is rare to absent from the intestines of fish and other cold-blooded animals. It is most common in the feces of carnivores and omnivores, while herbivores have rather few.

Infection and colonization occur within the first few hours or days of life by Escherichia and remain for the duration of the individual's life. The various strains harbored by an individual may be classified as resident and transient. Resistant strains persist over long periods of times while transient strains acquired by ingestion, fail to persist.

Diarrheal disease is produced by certain serotypes in very young individuals. The presence of colostrum modifies the disease state as well as certain viruses. There is evidence that certain E. coli serotypes are involved in Edema Disease of older swine (feeder pigs). Escherichia coli is an important pathogen of the bladder and kidneys. Asymptomatic bacteria is common but when the bacterial count is greater than 100,000/ml of urine, bacterial disease of the urinary tract is probably present.

E. coli is also found in peritonitis, appendicitis (in man), the intestines, the gallbladder and biliary tract. It is a common organism of navel infections of the newborn. Infantile diarrhea may become septicemic producing meningitis and/or joint infections. It occurs on the skin of the genitalia and peritoneum, and often infects wounds contaminated by feces or urine.

 Shigella

Natural disease is produced only in man and the higher apes. May be isolated rarely from the intestinal tracts of dogs, but symptoms are slight or nonexistent. The lesions are confined to the terminal ileum and colon and consist primarily of superficial mucosal ulcerations. These ulcerations are covered by pseudomembrane composed of polymorphonuclear leukocytes, cell debris, and bacteria unmeshed in fibrin. As the acute inflammatory process subsides, the ulcerations fill with granulation tissue and heal. Scar formation is unusual. The severe diarrhea may cause electrolyte imbalance and dehydration. This may be particularly severe in infants, small children and debilitated children.

Potent endotoxins are formed, and Sh. dysenteriae produces a protein exotoxin known as shiga neurotoxin. This heat-labile protein (~75,000 MW) when injected parentally in rabbits, mice or guinea pigs, causes paralysis, diarrhea and death. Specific antiserum neutralizes the toxin. The bacteria are non-invasive. Experimental infections can be induced orally in guinea pigs and mice under extremely restricted conditions. Sh. equuli is really an Acintobacillus.

 Tribe II: EDWARDSIELLA

Many of the cultures have come from snakes. Of the 256 cultures reported, 248 were from the intestinal contents of reptiles; two were from seals; and five were from humans with gastroenteritis and/or meningitis. There is a superficial resemblance to E. coli.

 Tribe III: SALMONELLEAE 

Salmonella

There are only three recognized species of Salmonella: Sal. choleraesuis (the first recognized), Sal. typhi (a pathogen primarily of humans) and Sal. enteritidis (all the rest). Proper usage is therefore important, for there are 1400 or more serotypes and bioserotypes within these species. These specific terms are designations, equivalent to and used in lieu of antigenic formulae. Except where used in the most general way, Sal. enteritidis should be accompanied by its proper specific epithet, i.e. Sal. enteritidis ser typhimurium.

Salmonella infections in animals, exclusive of man, may be grouped into two rough categories:

 Host Adapted-

Those diseases whose clinical manifestations are sufficiently characteristic to permit recognition with some degree of certainty. Some are: equine abortion, fowl typhoid, and Sal. pullorum disease.

 Nonspecific

Those diseases in which the symptoms indicate Salmonella

infection. The infection is septicemic in nature and characterized by weakness, recumbency and increased temperature. Pregnant animals may abort. Central nervous system symptoms and convulsions may occur in chickens. Diarrhea is usually a symptom.

Salmonella serotypes occur mainly in two ill-defined types: host adapted and nonspecific, and also tend toward geographic localization.

The species first isolated and described is Sal. choleraesuis. It is host adapted to swine, worldwide in distribution, and causes characteristic lesions (button ulcers) in the company of the hog cholera virus. In the absence of the hog cholera virus, it usually causes mild to moderately severe enteritis. Many swine are inapparent carriers until stressed, Figure 19. (AV-AT Lab).

 Salmonella typhi is the second described Salmonella species. It is host adapted to man and worldwide in distribution. Many human health authorities feel it is the only Salmonella species of note and ignore the public health significance for man and his animals.

 Sal. enteritidis, One of the first was the serotype abortusequi which causes abortion in pregnant mares. The organism has a wide geographic distribution, but is now rare in the United States. 

Salmonella enteritidis paratyphi A, paratyphi B and paratyphi C are human types, as is serotype sendai.

Serotype sublin is a cattle adapted type with a worldwide distribution. It has a very low incidence in this country. Most isolates are from the far west, are found foxes. Man is often secondarily infected by contact with infected or contaminated milk.

Bioserotype pullorum causes bacillary white diarrhea of chicks. It lacks motility. The disease is transmitted by feeding, and more importantly, transovarily (vertically). A female infected chick, if retained for breeding, will lay eggs containing serotype pullorum in the yolk. Many of these infected eggs fail to hatch, but those which do yield infected chicks, which may in turn infect the entire hatch. Infected chicks may live if not stressed, but when stressed produce a foamy white diarrhea and die within a short time, Figure 20 and 21. (AV-AT Lab).

Treatment is impractical. Prevention consists of sanitation and using only serologically negative birds as breeding animals. Serotype gallinarum causes fowl typhoid of chickens and turkeys; man may be secondarily infected.

Among the non-host adapted serotypes, Sal. enteritidis ser typhimurium is by far the most common isolate. Other commonly isolated serotypes are derby, bredeney, moterides, oranienburg, barielly, munchen, newport and anatum. Common sources are poultry, dried processed foodstuffs (powdered eggs and milk), swine, and man.

 Arizona 

Arizona species are closely aligned to the Salmonella. They show similar epidemiologic patterns, and diseases produced closely resemble the nonspecific Salmonellosis. They seem primarily associated with poultry, although they have been isolated from many other species of both warm- and cold-blooded animals. 

Citrobacter 

Citrobacter are best characterized (crudely) as citrate-positive Escherichia-Aerobacter intermediates. Their close biochemical relationship to Salmonellas and Arizonas is not reflected in their pathogenicity. As normal inhabitants of the intestinal tract of many species, they are relative avirulent, functioning only as weak opportunists.

 Tribe IV: KLEBSIELLA

 Klebsiella 

Klebsiella are nonmotile bacteria. Klebsiella pneumoniae causes severe pneumonia in man, where it is often referred to as "Friedlander's bacillus". It characteristically possess large capsules. Its variety genitalium causes abortion and/or metritis in mares. It is spread by coitus or the attendants at the time of breeding. Klebsiella mastitis is severe in dairy animals. The mode of spread in mastitis is not determined, but it is often associated with treatment.

Klebsiella rhinosclermona is isolated from humans with rhinoscleroma. Kl. ozena is the cause of ozena in humans and is very rare in this country.

Enterobacter 

Enterobacter are the group formerly te