Pedro Abalos

Evaluation of three Brucella soluble antigens used in an indirect Elisa to discriminate S19 vaccinated from naturally infected cattle

An O-polysaccharide (O-chain) and a hot-water extracted polysaccharide (PS), both obtained from Brucella abortus 1119-3, and a B. melitensis 16M native hapten (NH) were evaluated by indirect enzyme linked immunosorbent assay (ELISA) on three groups of cattle sera. The sera tested were: (a) 75 sera from cows naturally infected with B. abortus; (b) 130 sera from non-infected and non-vaccinated cattle; and (c) 61 sera from non-infected heifers recently vaccinated with B. abortus Strain 19 (S19). Sensitivity (Se), specificity (Sp) and the capability to discriminate vaccinated cattle (ADV) were determined. Using PS antigen, Se was 100% and the Sp was 97.7%, while the highest Sp was obtained by using the O-chain (99.2% ). For the NH antigen, Se was 94.7% and the Sp was 90.0%. The ADV of the three antigens was approximately 85%. Statistical analysis showed significant differences between O-chain/PS and O-chain/NH antigens. The agreement among antigens determined by kappa coefficient was 0.899 for O-chain/PS, 0.845 for O-chain/NH and 0.795 for PS/NH.

Genetic and phenotypic evidence of the Salmonella enterica serotype Enteritidis human-animal interface in Chile

Salmonella enterica serotype Enteritidis is a worldwide zoonotic agent that has been recognized as a very important food-borne bacterial pathogen, mainly associated with consumption of poultry products. The aim of this work was to determine genotypic and phenotypic evidence of S. Enteritidis transmission among seabirds, poultry and humans in Chile. Genotyping was performed using PCR-based virulotyping, pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Pathogenicity-associated phenotypes were determined with survival to free radicals, acidic pH, starvation, antimicrobial resistance, and survival within human dendritic cells. As result of PCR and PFGE assays, some isolates from the three hosts showed identical genotypic patterns, and through MLST it was determined that all of them belong to sequence type 11. Phenotypic assays show diversity of bacterial responses among isolates. When results were analyzed according to bacterial host, statistical differences were identified in starvation and dendritic cells survival assays. In addition, isolates from seabirds showed the highest rates of resistance to gentamycin, tetracycline, and ampicillin. Overall, the very close genetic and phenotypic traits shown by isolates from humans, poultry, and seabirds suggest the inter-species transmission of S. Enteritidis bacteria between hosts, likely through anthropogenic environmental contamination that determines infection of seabirds with bacteria that are potentially pathogenic for other susceptible organism, including humans.

Salmonella enterica in Pinnipeds, Chile

To the Editor: Several wildlife-associated zoonotic agents have played a major role in the emergence of diseases in humans (1). However, diseases can also emerge in wildlife as a result of human activities, such as contamination of the marine environment and its fauna by the disposal of nontreated human sewage. Salmonella enterica is among the agents identified as causing infection in various marine birds and mammals, including pinnipeds, from different geographic regions (2–4). The objective of our study was to determine whether S. enterica infection occurs in pinnipeds from the Chilean coast. During August–December 2010, we obtained samples from 13 South American sea lions (Otaria flavescens) that the sanitary authority found malnourished and stranded at the northern Chilean beaches of Antofagasta (23°40′S; 70°24′W) and Los Vilos (31°54′S; 71°30′W) (Table). The pinnipeds showed no clinical signs or symptoms of disease; however, rectal swab samples were obtained during their stay for rehabilitation at the Buin Marino facilities (Santiago, Chile). After the animals recovered, they were released to their original habitat. Table Characteristics of South American sea lions (Otaria flavescens) tested for infection with Salmonella spp., Chilean coast, August–December 2010* The swab samples were placed in Cary-Blair transport medium (COPAN, Murrieta, CA, USA) for shipment to the laboratory (Laboratory of Infectious Diseases, University of Chile, Santiago). To isolate bacteria, we placed the swab samples into 5 mL of buffered peptone water (Difco APT broth; Becton Dickinson, Franklin Lakes, NJ, USA), incubated them for 24 h at 42°C with agitation, and then aliquots of the suspension were transferred into the following media: modified semisolid Rappaport-Vassiliadis basal medium (Oxoid, Sao Paulo, Brazil) with novobiocin (20 µg/mL), selenite cysteine broth base (Oxoid), and xylose lysine desoxycholate agar (Difco XLD; Becton Dickinson). After the aliquots were incubated at 37°C for 24–48 h, we identified suspected colonies by using biochemical tests and invA gene detection by PCR (5). Results showed that 2 of the 13 animals were infected with S. enterica strains, which were serotyped as S. enterica serotype Newport and S. enterica serotype Havana (Table), according to the Kauffmann-White scheme (6). Testing showed that the strains were susceptible to the following antimicrobial drugs: ampicillin, chloramphenicol, tetracycline, amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefotaxime, nalidixic acid, nitrofurantoin, ciprofloxacin, ceftazidime, and cefoxitin (7). Our results confirm S. enterica infection in pinnipeds from Chile and, more broadly, the South American coast and contrast with previous unsuccessful attempts to detect Salmonella spp. in pinnipeds from Valdivia, 2,200 km to the south (8). This finding suggests geographic variability in the epidemiology of infection; however, this possibility must be confirmed in additional studies with more samples and additional regions. S. enterica is an endemic bacterium in Chile that causes infection in humans and domestic animals. The Chilean sanitary authority includes S. enterica infection among the list of notifiable diseases, but surveillance is not conducted for S. enterica in wildlife. However, consideration should be given to changing this situation, given a report suggesting S. enterica as a priority for active surveillance (9). In addition, S. enterica serotypes Newport and Havana have been detected in Chile’s human population (10), strengthening the necessity for official support for initiatives addressing the need to elucidate the epidemiology of Salmonella in aquatic animals.

Residual anti-Brucella abortus strain 19 antibodies detected in adult cattle by two indirect-ELISA tests.

DURING the validation process of two indireCt-ELISA (I-ELISA) kits produced by the Joint Food and Agriculture Organization and the International Atomic Energy Agency (FAO/IAEA) for brucellosis serological diagnosis, two groups of sera obtained from adult cows, negative to the Rose Bengal (RB) and the Rivanol (RV) tests (Alton and others 1988) were analysed. One group of sera, named Brucellosis Free Area (BFA), was collected from cattle in a geographically isolated area where brucellosis had never been reported and use of the B abortus strain 19 (S19) vaccine is not permitted. The second group of sera, named Brucellosis Negative Herd (BNH) was collected from herds certified as brucellosis free by the Ministry of Agriculture, Chile, but in which S19 is used traditionally in heifer calves between three and eight months of age for controlling the disease. All ELISA tests were performed using as antigen, a smoothlipopolysaccharide of B abortus diluted 1:1000 in carbonate/ bicarbonate buffer solution (pH 9-6). Polystyrene NUNC 2-69620 microplates were sensitised with the antigen solution by incubation overnight at 4°C. Working volumes during the assay procedure were always 100 jl. After sensitisation, microplates were washed three times in a PBS 0-002 M solution containing 0-05 per cent Tween 20. Test sera and also a strong, a weak and a negative reference control sera were diluted 1:200 in PBS 0.01 M (pH 7.4

Serotype-associated polymorphisms in a partial rpoB gene sequence of Salmonella enterica

Salmonella enterica is a zoonotic bacterium with more than 2500 serotypes, which affect a wide range of hosts and produce diverse clinical outcomes. Strain identification usually involves costly and time-demanding procedures. This paper describes the sequencing of a rpoB hypervariable gene segment (847 bp) that allows identification of serotypes in S. enterica strains isolated from several hosts. The nucleotide similarity values among S. enterica serotypes ranged from 98.23% to 99.88%, with potential usefulness for devising a simple one-step sequencing as a first approach for identification of S. enterica strains. In conclusion, the analysis of polymorphisms in the partial rpoB sequence can discriminate S. enterica strains at the subspecies level.

Brucella infection in marine mammals in Antarctica

Infection with Brucella pinnipedialis and Brucella ceti in marine mammals of the northern hemisphere has been extensively reported. In Chile, the National Antarctic Institute (INACH) and the University of Chile have developed a collaborative study establishing the presence of anti- Brucella

Corynebacterium pseudotuberculosis Infection in Patagonian Huemul (Hippocamelus bisulcus)

Abstract Corynebacterium pseudotuberculosis is an intracellular bacteria and the etiologic agent of caseous lymphadenitis in domestic and wildlife species. We report C. pseudotuberculosis infection in Patagonian huemul (Hippocamelus bisulcus) from the Cerro Castillo National Reserve, Region of Aysen, Chile. Subcutaneous abscesses in the abdominal and pectoral regions from two animals were sampled and bacteriologic isolation was performed. In both cases, we isolated a C. pseudotuberculosis strain belonging to the ovine genotype. In addition, one isolate was resistant to ciprofloxacin and streptomycin. We report that H. bisulcus is a susceptible species to this bacterium, which is transmitted by direct or indirect contact with domestic sheep (Ovis aries) and which represents a potential conservation threat to populations of H. bisulcus. Additional research and prevention efforts should be addressed.

Detection of Salmonella enterica in Magellanic penguins ( Spheniscus magellanicus ) of Chilean Patagonia: evidences of inter-species transmission

Patagonia in southern South America is among the few world regions where direct human impact is still limited but progressively increasing, mainly represented by tourism, farming, fishing and mining activities. The sanitary condition of Patagonian wildlife is unknown, in spite of being critical for the assessment of anthropogenic effects there. The aim of this study was the characterization of Salmonella enterica strains isolated from wild colonies of Magellanic penguins (Spheniscus magellanicus) located in Magdalena Island and Otway Sound, in Chilean Patagonia. Eight isolates of Salmonella were found, belonging to Agona and Enteritidis serotypes, with an infection rate of 0·38%. Resistance to ampicillin, cefotaxime, ceftiofur and tetracycline antimicrobials were detected, and some of these strains showed genotypic similarity with Salmonella strains isolated from humans and gulls, suggesting inter-species transmission cycles and strengthening the role of penguins as sanitary sentinels in the Patagonian ecosystem.

Diagnostic efficiency of Brucella soluble antigens in immunodiffusion tests and ability to differentiate Brucella abortus strain 19 vaccinated cattle

Three soluble antigens were compared by radial immunodiffusion (RID) and agar gel immunodiffusion (AGID) tests: a native haptene (NH) from Brucella melitensis 16M, and a polysaccharide (PS) from B. abortus 1119-3, both obtained by non-hydrolytic methods, and the (O-Chain) polysaccharide extracted also from B. abortus 1119-3 but using an hydrolytic method. Three groups of bovine sera were tested: a) Naturally infected (n = 76); b) Non-infected (n = 130) and c) S-19 vaccinated (n = 61); the sensitivity (Se), the specificity (Sp) and the ability to differentiate vaccinated (ADV) were determined in each group a, b and c respectively. The highest Se in the RID test (84.3%) was achieved by NH; while the three antigens gave 100% Sp. The O-Chain showed 100% ADV in this test. In the AGID test PS antigen showed the best Se (86.6%), and all antigens showed 100% of Sp and ADV. Finally, for its production qualities and efficiency the antigens PS and NH represent a promising alternative for complementary diagnosis of brucellosis.