Validation of an antimicrobial susceptibility testing protocol for Brachyspira hyodysenteriae and Brachyspira pilosicoli in an international ring trial.

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

Infection with Brucella ceti and high levels of polychlorinated biphenyls in bottlenose dolphins (Tursiops truncatus) stranded in south-west England.

Eight bottlenose dolphins (Tursiops truncatus) that stranded in Cornwall, south-west England, between June 2004 and December 2007 were examined using standardised postmortem examination and bacteriological methods. Evidence of Brucella species infection was found in four of these dolphins on culture. In addition, of the eight dolphins, four were positive and two were weakly positive for antibodies to Brucella species on serological analyses of pericardial and other fluids using a competitive ELISA and two indirect ELISAs. High or very high levels of the sum of 25 individual chlorobiphenyl congeners (∑25CBs) were also determined in blubber samples from two of the dolphins (45.5 and 446.6 mg/kg lipid weight).

Prevalence of a host-adapted group B Salmonella enterica in harbour porpoises (Phocoena phocoena) from the south-west coast of England.

A monophasic group B Salmonella enterica 4,12:a:- was first isolated in harbour porpoises (Phocoena phocoena) in Scotland in 1991. This paper reports the isolation of the same group B S enterica from harbour porpoise carcases found stranded along the Cornwall and Devon coastlines. Between 1991 and 2002, 80 harbour porpoises were submitted for postmortem examination and subjected to bacteriological examination under the UK Cetacean Strandings Investigation Programme. A total of 28 Salmonella isolates were recovered and subjected to several tests, including biochemical, molecular and serological analysis.

Isolation and characterization of Brucella from the lungworms of a harbor porpoise (Phocoena phocoena).

Adult female nematodes identified as Pseudalius inflexus were collected from the lungs of a juvenile male harbor porpoise (Phocoena phocoena) found dead on a beach in Cornwall, UK. Classic and molecular typing methods, immunologic and electron microscopy immunolabeling techniques, provided evidence of Brucella sp. infection within the uterine tissue of nematodes of this marine mammal. This finding presents further evidence to suggest parasites should be considered as a potential means of transfer of bacterial infection in marine mammals and highlights the zoonotic implications for humans exposed to marine mammals through occupation or leisure.

Characterization of a Brucella sp. strain as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in New Zealand.

Naturally acquired infection of humans with a marine mammal-associated Brucella sp. has only been reported once previously in a study describing infections of two patients from Peru. We report the isolation and characterization of a strain of Brucella from a New Zealand patient that appears most closely related to strains previously identified from marine mammals. The isolate was preliminarily identified as Brucella suis using conventional bacteriological tests in our laboratory. However, the results profile was not an exact match, and the isolate was forwarded to four international reference laboratories for further identification. The reference laboratories identified the isolate as either B. suis or B. melitensis by traditional bacteriological methods in three laboratories and by a molecular test in the fourth laboratory. Molecular characterization by PCR, PCR-restriction fragment length polymorphism, and DNA sequencing of the bp26 gene; IS711; the omp genes omp25, omp31, omp2a, and omp2b; IRS-PCR fragments I, III, and IV; and five housekeeping gene fragments was conducted to resolve the discrepant identification of the isolate. The isolate was identified to be closely related to a Brucella sp. originating from a United States bottlenose dolphin (Tursiops truncatus) and common seals (Phoca vitulina).

Harmonisation of European tests for serological diagnosis of Brucella infection in bovines.

The principal methods for the serological diagnosis of bovine brucellosis are the complement fixation test (CFT), serum agglutination test (SAT), Rose-Bengal test (RBT), indirect enzyme-linked immunosorbent assay (iELISA) and more recently the competitive ELISA (cELISA) and the fluorescent polarisation assay (FPA). Guidelines set by the World Organisation for Animal Health (OIE) describe methods and diagnostic thresholds for each of these tests. Many countries have adopted these methods for the purposes of eradication of brucellosis and have legislated for the use of these tests (the CFT and SAT in particular) for the prevention of the spread of the disease through international trade. Within the European Union (EU) each member state has a National Reference Laboratory which regulates the quality of brucellosis diagnosis and works to the recommendations set by the OIE. This article describes the results from the first three EU ring trials assessing the harmonisation of diagnostic tests between each member state. The general level of harmony for SAT, CFT, and iELISA was found to be good, but issues of standardisation of the RBT, cELISA and FPA remain. The cELISA and FPA in particular need further work to create European harmony. The ring trials also proved successful at providing specific evidence of poor performance in some areas. The decision on whether or not to take action on the basis of these results rested with the individual laboratories concerned. The increase in the number of participants in these trials over time reflected the enlargement of the EU and increased the need for quality assurance.