Arve Lund

Pathogenic Escherichia coli found in food

The bacteria constituting the species Escherichia coli are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains might induce disease, and E. coli should therefore be regarded as a potential pathogenic organism. The pathogenic strains can cause distinct disease syndrome as different diarrheal diseases, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome and immunological diseases such as reactive and rheumatoid arthritis. Several different groups of diarrhea-inducing strains are known. The enterotoxigenic E. coli (ETEC) strains produce one or more of toxins from the heat-labile and the heat-stable enterotoxin families. These strains possess specific adhesion fimbria for intestinal attachment and colonization. Some enteropathogenic E. coli strains (EPEC) produce one or more of the cytotoxins, but adhere also to intestinal cells interfering with the electrolyte transport system. The group of strains possessing invasive properties are designated enteroinvasive E. coli (EIEC). Recently, the enterohemorrhagic E. coli (EHEC) strains have been identified and shown to produce one or more of the cytotoxins (vero-cytotoxins, shiga-like toxins). Food originating from warm-blooded animals may be contaminated with E. coli, but contamination from human sources are more common for food involved in outbreak of disease. In general, strains causing disease in animals do possess other colonization factors than those found on human pathogenic strains. EIEC strains are, like Shigella, only known to induce disease in man. However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumption of meat or meat products. Cheeses have been the source of outbreaks of both ETEC and EIEC in Europe and the USA, while water seems to be a major source for the different diarrheic E. coli strains affecting children and tourists in the 3rd world. Strains causing non-enteric disease are less known as being transmitted to humans with food as a vector, but the importance of some of these diseases, should implicate further research on what role food plays in spreading these organisms. The recipient of the potential pathogenic E. coli through food, the humans, are also of different risk of contracting diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

Mycobacteria causing human cervical lymphadenitis in pastoral communities in the Karamoja region of Uganda.

Mycobacteria from lymph node biopsies of patients with cervical lymphadenitis reporting for tuberculosis treatment in Matany and Moroto Hospitals in the transhumant areas of Karamoja, Uganda were isolated and characterized. The AccuProbe culture identification kits for Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC) and M. avium were used to identify the isolates. Spoligotyping, IS901 PCR and IS1311 and IS1245 restriction fragment length polymorphism (RFLP) were used to characterize the isolates. Of the 43 biopsies, ten M. avium, seven M. tuberculosis, three M. bovis, and two M. intracellulare were isolated. Two isolates could not be identified with AccuProbe and from 19 samples no mycobacteria could be isolated. Three isolates with the Beijing spoligotype were identified from the seven M. tuberculosis isolates. The spoligopatterns of the M. bovis isolates had previously been detected in cattle in Uganda. Isolation of members of the MAC group reflects the complex interaction between the transhumant communities, water sources and their cattle. None of the M. avium isolates harboured IS901, and all showed several bands on IS1311 and IS1245 RFLP, in accordance with M. avium subsp. hominissuis. Composite dendrograms of IS1311 and IS1245 RFLP showed that the isolates were similar and identical patterns were found. The isolation of M. bovis confirms the human infection with zoonotic mycobacteria in areas where consumption of raw milk and meat is routine. Isolation of environmental mycobacteria also confirms their increasing role in human disease and the occupational risk of infection in the transhumant ecosystem in the absence of safe drinking water and environmental contamination.

Characterisation of mycobacteria isolated from slaughter cattle in pastoral regions of Uganda

BackgroundBovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates.ResultsOf the 61 lesioned organs and tissues cultured, 19 isolates were identified as M. bovis, 3 as M. avium subsp.hominissuis, 1 as M. intracellulare, 1 as a mixed culture of M. bovis and M. avium sp. and 1 as M. avium sp. and unidentified mycobacteria. Eleven other mycobacteria outside the tuberculosis and avium complex groups were also isolated. Ten new spoligopatterns grouped into three clusters were identified from M. bovis isolates. Two of the three M. avium subsp.hominissuis isolates showed similar patterns on the IS1311 RFLP but all were different on the IS1245 RFLP.ConclusionThe isolation of M. bovis confirms the ongoing infection with spoligotypes unique to Uganda. Isolation of environmental mycobacteria could explain the high avian or non specific tuberculin reactor patterns commonly observed in pastoral cattle and suggests their pathogenic or opportunistic role in the infection of cattle with disseminated bovine tuberculous lesions.

Herd-level factors for Brucella seropositivity in cattle reared in smallholder dairy farms of Zimbabwe.

A cross-sectional study was conducted to investigate factors for Brucella seropositivity in smallholder dairy cattle herds from Gokwe, Marirangwe, Mushagashe, Nharira, Rusitu and Wedza areas located in different agro-ecological regions of Zimbabwe between September 2004 and November 2005. Sera were collected from cattle aged > or = 2 years from 203 herds. Data on herd-level and management variables were collected using a structured questionnaire. Sera were screened for anti-Brucella antibodies using the Rose Bengal test (RBT) and confirmed by competitive ELISA (c-ELISA). A herd was classified as Brucella seropositive if at least one animal tested seropositive on both tests. The herd-level factors for Brucella seropositivity were tested using multivariable logistic model with herd infection status as dependent variable while the levels of exposure in individual animals within-herds were analysed by negative binomial regression using the number of positive animals as the outcome. Of the 203 herds tested, 52 (25; 95% CI: 18.1, 31.9%) tested seropositive for brucellosis. Using the logistic regression model we identified area, with both Rusitu (OR=0.26; 95% CI: 0.07, 1.03) and Wedza (OR=0.07; 95% CI: 0.01, 0.49) having lower Brucella seropositivity compared to Gokwe. Keeping mixed cattle breeds (OR=8.33; 95% CI: 2.70, 25.72) compared to single breed herds, was associated with increased herd seropositivity. The farmers knowledge of brucellosis was associated with lower odds (OR=0.17; 95% CI: 0.05, 0.55) of farms testing seropositive. The odds of Brucella seropositivity were progressively higher with increasing stocking density and herd size. Using the negative binomial regression model we identified area, keeping mixed breed herds, stocking density and herd size as independently associated with increased counts of seropositive cattle in a herd.

Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro.

ABSTRACT Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P < 0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P < 0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

Characterisation of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway

Eighty‐six Staphylococcus aureus isolates from cases of bovine mastitis were characterised biochemically and with respect to serotype, multilocus enzyme electrophoresis genotypes, antibiotic sensitivity, and production of enterotoxins A through D (SEA‐D) and toxic shock syndrome toxin‐1 (TSST‐1). The samples were obtained from 81 different cows from 79 Norwegian dairy herds in 10 different counties in southern Norway. There was an equal representation of isolates from cases of acute, chronic and subclinical mastitis. Multilocus enzyme electrophoresis using 13 genetic loci showed that 69 of 86 isolates had the same electrophoretic type. This common electrophoretic type comprised isolates that differed in the expression of other phenotypical characteristics studied. Fifty‐eight percent of the isolates produced one or more enterotoxins, predominantly a combination of SEC and TSST‐1. Capsular serotyping revealed that 95% of the isolates belonged to serotype 8. No correlation was found between the factors studied and the clinical classification of mastitis. It appears that the majority of S. aureus isolates recovered from cases of bovine mastitis in Norway are genetically closely related and express common phenotypical characteristics.

Staphylococcus aureus capsular polysaccharide type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle

Dairy heifers were immunized subcutaneously with one of four different vaccines which contained preparations of Staphylococcus aureus capsular polysaccharide type 5 (CP5) and a mineral oil adjuvant, or received a placebo containing saline and adjuvant. The vaccine containing a CP5-human serum albumin conjugate (CP5-HSA) and the vaccine with formaldehyde inactivated whole cells expressing CP5, both elicited strong anti-CP5 antibody responses. After two injections three weeks apart and a third injection 10 months later, the mean level and duration of the anti-CP5 antibody response was significantly higher in the whole cell group. No differences were found between the two groups with regard to the relative proportion of IgG subclasses, and the antibody responses to the polysaccharide were composed of both the IgG1 and IgG2. Vaccines containing only free CP5 or CP5 mixed with HSA produced weak and transient humoral immune responses. Only animals vaccinated with the whole cell vaccine or the conjugate vaccine showed responses to CP5 in a lymphocyte proliferation assay conducted one year after the third vaccination. This study indicates that CP5 expressed on the surface of formaldehyde inactivated whole cells, emulsified in an oil adjuvant, gives a strong and long lasting immune response in cattle. The use of conjugation technology, although effective, might not be necessary in order to achieve an immune response against S. aureus CP5 in cattle.

Risk factors for Brucella spp. infection in smallholder household herds.

Risk factors for Brucella infection, the association and impact of Brucella seropositivity on abortions were investigated in cattle (n=1291) reared in smallholder household herds (n=203) from six geographical areas of Zimbabwe between September 2004 and 2005. Data on management, abortion and herd structure were collected. Sera were tested for Brucella antibodies using the Rose Bengal test and a competitive enzyme-linked immunosorbent assay. Data were analysed by generalized estimating equation and logistic regression models. Brucella antibodies were estimated at 5·5% and 22·9% for individual cattle and herds, respectively. Abortions were reported in 3·2% of cows and 22·0% herds. The age of cows and Brucella seropositivity predicted abortion. For herds, Brucella seropositivity, geographical area, purchase of cattle and large herd size were independently associated with increased odds of abortion. Exposure to Brucella had a significant impact on abortion. These results highlight the important risk factors for Brucella spp. infection in smallholder herds. Thus, brucellosis control programmes which take these factors into consideration will be beneficial.

Imaging the surface of Staphylococcus aureus by atomic force microscopy

The surfaces of four strains of Staphylococcus aureus, which differed in their expression of capsular polysaccharides, were examined using atomic force microscopy. The images show that it is possible to get information about surface characteristics of S. aureus using atomic force microscopy (AFM) following simple preparation. Strains Smith Diffuse (serotype 2), Reynolds (serotype 5), Wood‐46 (capsule negative) and JL243 (capsule negative) were grown on medium known to promote the expression of capsular polysaccharides. The bacteria were air‐dried prior to being imaged using tapping‐mode AFM. Differences in the appearance of the bacterial surfaces were evident between the strains. The two capsule‐negative strains exhibited a smooth regular surface, as opposed to the mucoid appearance of the two strains having polysaccharide capsules. Moreover, comparison of images of the heavily encapsulated serotype 2 strain and the serotype 5 strain indicates that a type 2 capsule can be distinguished from a type 5 microcapsule.

Evaluation of sensitivity and specificity of RBT, c-ELISA and fluorescence polarisation assay for diagnosis of brucellosis in cattle using latent class analysis.

The sensitivity (Se) and specificity (Sp) of the Rose Bengal test (RBT), competitive ELISA (c-ELISA), serum (sFPA) and blood (bFPA) fluorescence polarisation assay for brucellosis were evaluated using latent class analysis using sera and whole blood collected from infected cattle reared in smallholder dairy farms of Zimbabwe. The latent class model allowed estimation of Se and Sp in the absence of a gold standard test. The c-ELISA had the highest Se (99.0%; 95% credible posterior interval (CPI): 94.8; 100%), while the RBT and sFPA had the highest Sp (99.0%; 95% CPI: 98.0; 99.6%). The bFPA had the lowest Se (71.3%; 95% CPI: 56.2, 83.5%), while its Sp (96.3%; CPI: 93.9; 98.0%) was marginally higher than that of the c-ELISA (95.4% CPI: 93.7; 96.8%). Therefore based on these data, test regimen using the RBT and c-ELISA could be suitable for diagnosis of brucellosis in smallholder dairies in Zimbabwe. Based on cost and ease of performance, the sFPA may be adopted as a confirmatory test, but its performance may be optimised by altering cut-off points to suit the Zimbabwean conditions. Thus, latent class models provide an alternative method for evaluating Se and Sp of diagnostic tests, which could be used to optimise test performance in different cattle populations.