Salmonella spp. infections

Salmonella spp. infections

Salmonellosis in livestock is caused by infection with both host-specific and non-host-specific Salmonella serovars, and results in enteritis, septicaemia or abortion. It is an economically important disease of cattle in many parts of the world, but may also be responsible for serious sporadic outbreaks of disease in sheep, horses and pigs. Carrier animals and contaminated environments are important sources of infection. The most common serovars associated with disease in livestock are listed in Table 1.

The genus Salmonella is classified in the family Enterobacteriaceae, whose members are Gram-negative cocco-bacilli.3 With the exception of Salmonella Gallinarumpullorum, all salmonellae are motile, as they have peritrichous flagella.

The genus Salmonella consists of two species, S. enterica, which comprises six subspecies (enterica, salamae, arizonae, diarizonae, houtenae, and indica), the distinction between subspecies being based on biochemical reactions, and S. bongori. 8, 17–24, 27 Salmonella enterica subsp. enterica contains all the serovars found in warm-blooded animals — those formerly assigned to subgenus 1.

The members of the genus Salmonella are typed into serovars which are differentiated from each other by the combinations of their somatic (O) and flagellar (H) antigens and, to a lesser extent, by their biochemical reactions. The serovars, of which over 2000 have been identified, do not have a species status allocated to them in the current classification of the genus, and the names of the serovars (such as Typhimurium or Dublin) should be used without italicization or underlining, and with the first letter capitalized, e.g. S. enterica subsp. enterica ser. Typhimurium. As it is tedious and impractical to adopt this long, formal nomenclature for everyday use, it is common to refer to serovars as, for example, Salmonella Typhimurium, or as serovar Typhimurium.8, 18

 It should be noted, however, that the use of the shortened nomenclature prohibits abbreviation of the name of the genus (Salmonella = S.) because the abbreviation of the name of a genus is only authorized if it is followed by the name of a species. Nevertheless, as this format is used by many authors, it is also used in this book.

Antigenic composition

The O antigen is part of the lipopolysaccharide component of the cell wall that also contains lipid A and a core portion. The O antigen, or O-specific side chain consists of repetitive oligosaccharide units of which the type, order, and repetition of sugar moieties differ between serovars. These differences and those in the flagella antigens are used to type Salmonella into serovars. At least 67 different O antigens are currently known and they are identified by the Arabic numerals 1 to 67. Some of these occur singly (e.g. 11), while others occur in combination (e.g. 1, 4, 5, 12; and 6, 7) (Table 2).17, 24

Some Salmonella mutants have defects in the synthesis of the oligosaccharide O-specific side chain, with the result that the oligosaccharide is not fully synthesized when cultured. The colonies of such mutant strains have a ground-glass appearance and are therefore referred to as ‘rough’ strains. Colonies of strains which possess the complete oligosaccharide O-specific side chain are smooth and the strains are therefore referred to as ‘smooth’. Rough strains are untypable as they do not agglutinate with O typing antisera.24

Table 1 Most common serovars of Salmonella that infect livestock and the syndromes they induce

SPECIES AFFECTED SEROVARS COMMON SYNDROMES
Cattle S. Dublin Septicaemia, acute and chronic enteritis and abortion
  S. Typhimurium  
  S. Brandenburg  
Sheep S. Typhimurium Septicaemia, typhlocolitis and abortion
  S. Hindmarsh  
  S. Brandenburg Septicaemia and abortion
Pigs S. Choleraesuis Septicaemia and enterotyphlocolitis
  S. Typhimurium  
  S. Typhisuis  
Horses S. Typhimurium Septicaemia, acute colitis and abortion

Table 2 Antigenic composition of common Salmonella serovars

SEROVARS O-ANTIGENS H-ANTIGENS
    Phase 1 Phase 2
S. Abortusovis 4,12 c 1,6
S. Derby 1,4,5,12 f,g 1,2
S. Typhimurium 1,4,5,12 i 1,2
S. Abortusequi 4,12 - e,n,x
S. Choleraesuis 6,7 c 1,5
S. Typhisuis 6,7 c 1,5
S. Bovismorbificans 6,8 r 1,5
S. Typhi 9,12 d -
S. Enteritidis 1,9,12 g,m 1,7
S. Dublin 1,9,12 g,p -
S. Gallinarumpullorum 1,9,12 - -
S. Brandenburg 1,4,12 l,v enz15
S. Hindmarsh 8,20 r 1,5

The complete O antigen is not only the major immunogen of bacteria in this genus, but it also possesses virulence properties (see Virulence factors, below). It elicits both humoral and cellular immune responses to infections by salmonellas. Strains with incomplete O antigens (rough strains) are therefore used for the production of live attenuated vaccines, such as the calf paratyphoid (Salmonella Dublin) vaccine.

The H antigens are heat-labile and are an integral part of the flagella in those serovars which possess them. The antigens are designated by a combination of letters of the alphabet and numerals (e.g. a to z, z1 to z32 and 1 to 7).8 Two antigenic forms (also referred to as ‘phases’) of the flagella may occur in culture. A culture may therefore contain cells in which the flagella are all in the same phase, or cells which possess flagella of both phases. Most of the serovars contain flagella of two phases, but in some (e.g. S. Dublin) the flagella occur in only one phase (Table 2).

In order to establish the complete antigenic composition of any Salmonella serovar, antigens of both flagellar phases must be known, as well as the O antigens.17 This is done by testing suspensions of the bacteria against antisera produced in rabbits against individual O and H antigens by means of a series of slide agglutination tests.8 For identification purposes the results from these agglutination tests are then compared with known antigenic formulae contained in the Kauffmann-White diagnostic scheme. In this scheme the antigenic formula has three parts: the O antigens, the phase-1 H antigens, and the phase-2 H antigens. As new serovars of Salmonella are constantly emerging, the Kauffmann-White scheme is updated annually.8, 18, 25 The antigenic composition of some of the more common Salmonella serovars is given in Table 2.

Virulence factors

The biological effects of the virulence factors of salmonellas are interrelated and are responsible for enteric and systemic clinical signs and lesions of salmonellosis.

  • Lipopolysaccharide (LPS): Apart from the fact that the O-specific side chain of the LPS is immunogenic and is recognized by the host’s immune system, it has also been associated with invasiveness of salmonellas and enterotoxin production.23 For example, Salmonella strains with incomplete O-specific side chains (rough mutants) are not as invasive as those strains with a complete O-specific side chain, and are also less successful in avoiding phagocytosis and lysis by phagolysosomes after invasion.
  • Endotoxin-mediated effects (see Escherichia coli infections) in clinically affected animals are attributed to the lipid A portion of the LPS. Vasculitis and thrombosis, which may be present in the intestine, liver, spleen and other tissues of affected animals, are thought to be the result of absorption of large amounts of endotoxin through the damaged intestinal mucosa, or of endotoxin that is released locally in infected tissues. The cytotoxin may also be involved in the pathogenesis of the vascular lesions.1, 5, 6, 11, 14, 16, 21, 23, 28-33
  • Enterotoxin: Several serovars of Salmonella produce an enterotoxin similar to the heat-labile toxin of Escherichia coli and the cholera toxin produced by Vibrio cholerae5, 23 (see Escherichia coli infections). The enterotoxin binds to intestinal epithelial cells and stimulates increased intracellular cyclic adenosine monophosphate, which leads to the net secretion of CI, HCO3, Na+ and water into the intestinal lumen, resulting in diarrhoea.
  • Cytotoxin: Some serovars of Salmonella produce a cytotoxin5, 23 that causes increased permeability of intestinal epithelial cellmembranes. The cytotoxin appears to chelate cations, such at Ca++ and Mg++, which it is thought to cause structural changes in the cell membrane, resulting in rounding of cells and selective leakage of molecules.23
  • Adhesion pili (fimbriae): Salmonellas possess type 1 pili which facilitate the adhesion of bacteria to epithelial cells. The differential expression of various fimbriae by salmonellae is likely to be due to the wide variety of mucosal surfaces that are encountered by various strains, and the host immune system may select for a different expression pattern.9, 11, 23
  • Plasmids: The virulence of the most common serovars (e.g. S. Dublin, S. Typhimurium and S. Choleraesuis) that cause disease in livestock is enhanced by serovar-specific plasmids, which provide these serovars with the ability to survive in macrophages.10, 12, 16 Salmonellas may carry R-plasmids that provide resistance to some antimicrobial drugs.15

For a current review of the virulence traits of the genus Salmonella refer to Fierer & Guiney, 2001.9

Isolation and identification

Salmonellas have simple nutrient requirements and growth in vitro is therefore possible in simple salt-glucose media,4 occurring over a temperature range of 10 to 49 °C.2 At temperatures between 0 and 5 °C the organisms remain viable even though there is no growth.2

Selective procedures are used for the isolation of salmonellae from specimens such as faeces that contain a mixed bacterial flora. Enrichment media, such as a liquid-nutrient broth that contains agents which selectively inhibit the growth of other bacteria, are used for this purpose. Enrichment media commonly used are tetrathionate broth, Rappaport-Vassiliadis medium, and selenite F broth.13 Small pieces of tissue from the spleen, liver, mesenteric lymph nodes and/or the small and large intestines, or small amounts of faeces are each inoculated into the enrichment medium and, after incubation at 37 °C for 48 hours, a loopful of the medium is plated onto a discriminating agar medium such as MacConkey agar, XLD agar, brilliant green agar, Salmonella-Shigella agar or Rambach agar.13 These media differentiate the genera of the family Enterobacteriaceae according to differences in lactose fermentation and other biochemical reactions.3 Serovars of the genus Salmonella are non-lactose fermenting, as aremembers of the genus Proteus, and some strains of Citrobacter and Enterobacter (Table 3). As all these bacteria are morphologically indistinguishable from each other, they are further characterized by the biochemical reactions that they induce in suitably prepared media.17

Salmonellas are facultatively anaerobic and, with a few exceptions, all produce gas from fermentable sugars.17 In culture, nitrates are reduced to nitrites, and hydrogen sulphide (but not indole and urease) is produced.

Glucose and maltose are fermented, dulcitol and inositol are variably used, and sucrose is not fermented. The other non-lactose-fermenting genera of the family Enterobacteriaceae which also produce hydrogen sulphide are Proteus and some strains of Citrobacter. 3The biochemical differentiation of these enterobacteria is given in Table 3. 3

Phage typing

Bacteriophage typing schemes for a number of important Salmonella serovars have been developed, e.g. S. Typhimurium and S. Enteritidis, and are used internationally for epidemiological studies. Certain phage types have been found to be commonly associated with particular animal species, aiding in the identification of potential sources of human infection. Phage types are designated as either PT (e.g. S. Typhimurium PT 135) or DT (definitive type). However, DT is commonly reserved for important epidemic phage types, e.g. multi-antibiotic resistant S. Typhimurium DT 104.13

Factors affecting growth and survival

Salmonellas are killed when exposed to temperatures of 55 °C for one hour or 60 °C for 15 to 20 minutes, and by pasteurisation at 71 °C for 15 seconds or 63 °C for 30 minutes.2 The cooking of food will destroy salmonellas if the internal temperature of the food reaches 74 to 77 °C.2, 4 Salmonellas in animal feeds and feed ingredients are killed by a process where the feed is heated to boiling point in steam-jacketed agitating cookers (110 °C for 2,5 to 3,5 hours).4, 22, 26 There is a marked reduction in the number of salmonellas during freezing and long-term frozen storage, but not all are destroyed.4

Salmonellas are able to grow in media with a pH range of 4,5 to 9,0 (optimum 6,5 to 7,5),4, 26 lower or higher pH values cause them to die.

Table 3 Biochemical differentiation of non-lactose-fermenting enterobacteria. (Adapted from Brenner3 )

GENUS BIOCHEMICAL REACTION
  Lactose fermentation H2S production Urease production Indole production Lysine production
Salmonella + +
Proteus + + +/–
Enterobacter +/– +/– +/–
Citrobacter +/– +/– +/– +/–

+ Positive

– Negative

+/– Positive or Negative

References

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