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Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis.

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.

Animals

Influence of bacteriological media constituents on the reproduction of Salmonella enteritidis bacteriophages.

Three different Salmonella enteritidis phages were isolated and purified from raw sewage by agar-layer technique. The sensitivity of the host organisms toward phages was changed when they were grown on different bacteriological media. The effect of single components of the medium on phage reproduction was determined by the omission of that substance from the medium. CaCl2,MgSO4, and glycerol each had a pronounced stimulatory effect on the phage reproduction, while bile salts had a profound inhibitory effect. The inhibitory effect of bile salts on phage growth was much greater on one strain of Salmonella enteritidis than on the other.

Bile Acids and Salts

Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents.

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.

Antibodies, Bacterial

Biological and physicochemical characteristics of four serotypes of Salmonella enteritidis.

Four serotypes of Salmonella enteritidis, Anatum ATCC 9270, Newbrunswick ATCC 1608, Oranienburg 200 E, and Pullorum RM, were studied to determine biological, chemical, or physical differences which might explain variations in Salmonella virulence as previously reported by McCullough and Eisele (J. Infect. Dis. 88:278-289, 1951; 89:259-265, 1951). These investigators found that serotype Pullorum was significantly less virulent than serotypes Newport, Derby, Barielly, Meleagridis and Anatum when fed to healthy humans. Results of our own experiments showed that serotype Pullorum RM had a generation time approximately twice that of serotype Anatum 9270. The volume of serotype Pullorum was approximately one-half the volume of the other serotypes used (Anatum 9270, Newbrunswick 1608, Oranienburg 200 E, Cubana 12007, and Meleagridis DR). The number of cells required to yield 1 g dry weight was substantially higher for serotype Pullorum RM than for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E. The yield of endotoxin per gram dry weight for serotype Pullorum RM averaged 22 mg/g, whereas yields of endotoxin for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E averaged 32 to 35 mg/g. The relative abundance of the four major fatty acids (measured by gas chromatography) also showed distinct differences among the serotypes. Pullorum RM contained less lauric and 3-hydroxymyristic acids and more myristic and palmitic acids than the other three serotypes. The identity of 3-hydroxymyristate was confirmed by mass spectroscopy. Serotype Pullorum RM required 10 times more lipopolysaccharides (endotoxin) to obtain a 50% lethal dose in mice than the other three serotypes. When the lipid part was separated from the polysaccharide and solubilized with bovine serum, the 50% lethal dose of serotype Pullorum RM was equal to that of the other three.

Endotoxins

Starvation survival of Salmonella enteritidis.

Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.

Buffers

Cross-protection against Salmonella enteritidis infection in mice. III. Delayed hypersensitivity reaction and clearance of the challenge organism.

Mice were immunized with live vaccines and with live vaccines with complete adjuvant incorporating Salmonella enteritidis, Salmonella typhi-murium, Salmonella gallinarum or Salmonella pullorum. On the 21st day after vacination, the hypersensitivity reactions elicited by the mice to extracts of the challenge organism (S. enteritidis 5694 SMR) were assessed. The degree of delayed hypersensitivity reaction was compared with the level of protection induced by the vaccine. The role in protection of delayed hypersensitivity is discussed. Clearance of the challenge organism from the liver of previously vaccinated and unvaccinated mice was assessed quantitatively.

Adjuvants, Immunologic

Temporal reconstruction of a Salmonella Enteritidis ST11 outbreak in New Zealand.

Outbreaks caused by Salmonella Enteritidis are commonly linked to eggs and poultry meat internationally, but this serovar had never been detected in Aotearoa New Zealand (NZ) poultry prior to 2021. Locally designated genomic cluster Salmonella Enteritidis_2019_C_01, was implicated in a 2019 outbreak associated with a restaurant in Auckland. Four Enteritidis_2019_C_01 sub-clusters have since been identified, two retrospectively, in the Auckland region. Authorities initiated a formal outbreak investigation after genomically indistinguishable S. Enteritidis was isolated from the NZ poultry production environment. This study analysed 231 S. Enteritidis genomes obtained from the outbreak using Bayesian phylodynamic tools to gain insight into the outbreak's dynamics and origin. We used Bayesian integrated coalescent epoch plots to estimate the change of the Enteritidis ST11 population size over time and marginal structured coalescent approximation to estimate transmission between poultry producers. We investigated human and poultry isolates to elucidate the time and location of the most recent common ancestor of the outbreak and transmission pathways. The median most recent common ancestor was estimated to be February 2019. We found evidence of amplification and spread of strain Enteritidis_2019_C_01 within the poultry industry, as well as transmission events throughout the production chain. The intervention by the public health and food safety authorities coincided with a drop in the effective population size of the S. Enteritidis ST11 as well as notified human cases. This information is crucial for understanding and preventing the transmission of S. Enteritidis in NZ poultry to ensure poultry meat and eggs are safe for consumption.

Salmonella enteritidis

[Introduction of an additional H-agglutinating test antigen for the differentiation of specific positive reactions in pullorum disease-fowl typhoid in poultry experimentally infected with Salmonella gallinarum-pullorum and Salmonella enteritidis].

Explored were the possibilities to differentiate the specific positive reactions in case of pullorum disease in experimentally infected (Salmonella gallinarum-pullorum and Salmonella enteritidis) birds with the use of a typhoid test antigen and two H test antigens, obtained by the routinely employed method with formalin from Salmonella enteritidis and Salmonella adeoyo. It was demonstrated that the H-agglutinating antibodies persisted in the peripheral blood of the experimentally infected birds for at least 30 days (as long as the test lasted). The H test antigen of S. enteritidis proved unsuitable to detect the H-agglutinating antibodies only as it retained to a large extent the O antigens. By means of the S. adeoyo H test antigen only the H-agglutinating antibodies in the peripheral blood of S. enteritidis-infected birds could be quickly and precisely demonstrated. A combined serologic examination of serum with the thyphoid test antigen and the S. adeoyo test antigen could be carried out to differentiate the specific positive reactions for establishing pullorum disease from the concomitant agglutination reactions observed in cases of experimental salmonellosis in birds caused by S. enteritidis.

Agglutination Tests

Endocarditis caused by Salmonella enteritidis.

A case of endocarditis caused by Salmonella enteritidis is reported in a 20-year-old African woman. This is only the fourth published case. The development of this rare infection in the patient reported here probably resulted from a reduction in immunity caused by pregnancy and a past splenectomy.

Adult

Salmonella enteritidis infection in the Sudan.

Twenty-one Salmonella enteritidis isolates were recovered from several poultry farms in three states in the Sudan over an eighteen-month period. The infection was disseminated from a distributing company which had imported infected fertilized eggs and parent stock. The Sudan S. enteritidis epidemic which devastated many poultry farms during 1990 was attributable and concurrent to that in Europe in particular and throughout the world in general.

Animals

Resistance of mice to Krebs ascites tumour, sarcoma S180 and PC6 plasmacytoma after immunisation with Salmonella enteritidis 11RX.

Resistance to Krebs ascites tumour, sarcoma S180 (ascitic form) and PC6 plasmacytoma can be induced with prior immunisation with live Salmonella enteritidis 11RX. One thousand to ten thousand more tumour cells are needed to give an LD50 after immunisation. Resistance to PC6 could be recalled 137 days after intravenous immunisation with 11RX protein antigen intraperitoneally as measured by prolonged survival or monitoring the death of 5[131I]-iodo-2'-deoxyuridine labelled tumour cells in vivo. The range of tumours to which resistance can be produced by 11RX immunisation suggests that the mechanism of resistance does not involve antigens common to the tumours and Salmonella enteritidis 11RX.

Animals

A model of salmonella enteritis: the behaviour of Salmonella enteritidis in chick intestine studies by light and electron microscopy.

Salmonella enteritidis was injected into isolated "loops" of duodenum, midgut and ileocaeca in anaesthetized 1-day-old chicks. Using light microscopy with conventional staining and fluorescent antibody techniques, the organism were observed in increasing numbers in the epithelium and lamina propria of each region between 2 and 12 h after inoculation. The potential ability of the salmonellas to enter the mucosa at any level of the intestine was thus demonstrated. The penetration of caecal epithelium by the organisms was studied by electron-microscopy. Uptake of salmonellas was associated with evaginations which formed at the luminal surface of the epithelium. In the epithelial cells, the bacteria were enclosed within membrane-bound vacuoles and appeared undamaged by intracellular passage. Surface evaginations almost completely replaced the brush border in regions where large numbers of the organisms were involved. Occasional salmonellas were found in the lamina propria both free and intracellularly. Results of culture and light microscopy following oral and intracloacal inoculation are also presented.

Animals

Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella enteritidis in mice.

Through hemagglutination tests two isogenic strains of Salmonella enteritidis were shown to possess type 1 fimbriae (strain V) and type 1 and type 3 fimbriae (strain A). The two strains bound to human buccal and mouse small intestine epithelial cells. Strain A attached to the epithelial cells more readily and in larger numbers in comparison to strain V. Adherence of both strains were sensitive to the presence of D-mannose and pretreatment of the epithelial cells with tannic acid did not promote D-mannose resistant type binding of strain A S. enteritidis to human buccal and mouse small intestine epithelial cells. Furthermore, results from LD50 study indicated that, when the tests were carried out through oral inoculation of the mice the highly fimbriated stain A appeared to be more virulent. However, when the tests were carried out through intraperitoneal inoculation strain V was more virulent. These results indicate that adherence is a major contributing factor to the virulence of S. enteritidis and both type 1 and type 3 fimbriae contribute to this phenomenon.

Administration, Oral

Cross-protection against Salmonella enteritidis infection in mice.

Mice were immunized subcutaneously with live vaccines and live vaccines with complete adjuvant incorporating Salmonella enteritidis Se 795, Salmonella typhi-murium M206, Salmonella gallinarum 9R or Salmonella pullorum Sp223. They were challenged along with unvaccinated controls with 100 LD50 of virulent S. enteritidis 5694 SMR subcutaneously on the 21st day post-vaccination. The humoral immune response was studied by assessing the sequential level of agglutinins, complete and incomplete somatic antibodies, opsonophagocytic antibodies, cytophilic antibodies and bactericidal antibodies before and after challenge. The level of these antibodies and the protection afforded by the particular vaccine is correlated and the possible involvement of a humoral mechanism is discussed.

Adjuvants, Immunologic

Genetic relationships among strains of Salmonella enteritidis in a national epidemic in Switzerland.

A collection of Salmonella enteritidis strains isolated in Switzerland (1965-90) was characterized. The phage type and plasmid profile of isolates were compared with the copy number and insertion loci of the DNA insertion element IS200. Three clonal lines of S. enteritidis were identified by IS200 profile; the various phage types were subtypes reproducibly associated with one of these lines. All human and poultry isolates contained a 38 Mda plasmid which hybridized with a mouse virulence-associated gene probe. In S. enteritidis, the IS200 profile is a race-specific molecular marker of the chromosome, and may be particularly applicable for studying the epidemiology of less common serovars.

Bacterial Typing Techniques

Salmonellosis in young calves due to Salmonella enteritidis.

The clinical and epidemiological features of an outbreak of salmonellosis due to Salmonella enteritidis in a group of calves are described. The major clinical signs were dullness, pyrexia and diarrhoea. Five of the 15 calves died but deaths were mainly confined to the younger members of the group. The recovery of salmonella organisms from rectal swabs was maximal shortly before four of the five deaths occurred and declined rapidly thereafter. Only two of the surviving 10 calves developed significant flagellar agglutination titres.

Agglutinins