Using molecular tools to identify the geographical origin of a case of human brucellosis.

Although Malta is historically linked with the zoonosis brucellosis, there had not been a case of the disease in either the human or livestock population for several years. However, in July 2013 a case of human brucellosis was identified on the island. To determine whether this recent case originated in Malta, four isolates from this case were subjected to molecular analysis. Molecular profiles generated using multilocus sequence analysis and multilocus variable number tandem repeat for the recent human case isolates and 11 Brucella melitensis strains of known Maltese origin were compared with others held on in-house and global databases. While the 11 isolates of Maltese origin formed a distinct cluster, the recent human isolation was not associated with these strains but instead clustered with isolates originating from the Horn of Africa. These data was congruent with epidemiological trace-back showed that the individual had travelled to Malta from Eritrea. This work highlights the potential of using molecular typing data to aid in epidemiological trace-back of Brucella isolations and assist in monitoring of the effectiveness of brucellosis control schemes.

Seroprevalence of Brucella abortus and B. canis in household dogs in southwestern Nigeria: a preliminary report.

A preliminary serological study of 366 household dogs in Lagos and Ibadan, southwestern Nigeria, was carried out to determine antibodies due to exposure to Brucella abortus and B. canis, using the rose bengal test (RBT) and the rapid slide agglutination (RSA) test, respectively. Results showed that 5.46 % (20/366) and 0.27 % (1/366) of the dogs screened were seropositive to B. abortus and B. canis, respectively. Of all dogs, 36 had a history of being fed foetuses from cows and 11 (30.6 %) of these tested positive in the RBT. Our findings, although based on a limited sample size and a dearth of clinical details, revealed that dogs in Nigeria may be infected with Brucella spp. given the wide range of risk factors. Further studies are recommended to elucidate the epidemiology of brucellosis in dogs and its possible zoonotic consequences in the country.

Brucellosis in camels, cattle and humans: associations and evaluation of serological tests used for diagnosis of the disease in certain nomadic localities in Sudan.

Brucellosis was studied in 2,225 camels, 20 camel nomads and 33 abattoir workers in certain nomadic localities in Sudan, using serum and milk samples. Lymph nodes, testicular tissues and udder tissues from positive camels and hygroma aspirates from three affected cows were used for isolation of Brucella. Serum samples were examined by Rose Bengal plate test (RBPT), modified RBPT (mRBPT), serum agglutination test (SAT) and competitive enzyme-linked immunosorbent assay (cELISA), and milk by the milk ring test. Overall seroprevalence in camels (milk and serum samples) was 37.5%. The seroprevalence in males was 28.2% and in females 40.1%. Twelve (60%) of the 20 nomads and three (9%) of the 33 abattoir workers had positive antibody titres. Brucella abortus biovar 6 was isolated from two camels and three cows. Two isolates, one from each species, were atypical. The bacteriological findings suggested that camels were infected from cattle, the primary hosts of B. abortus. The mRBPT was suitable for screening camel sera for brucellosis, but the cELISA detected 2.1% more positives. The SAT antibody concentrations ranged between < 13 and 3,282 IU/ml.

Brucellosis in camels (Camelus dromedarius) in Darfur, Western Sudan.

In a field outbreak of brucellosis in 21 camels mixed with cattle, sheep and goats, five camels, three of which showed clinical signs, were serologically positive. In a subsequent abattoir survey of apparently healthy camels, six animals were seropositive, albeit with titres that tended to be lower than those found in the field outbreak. Of the six seropositive slaughtered camels, five were shown to have lymph nodes (prescapular and supramammary) infected with brucellae (Brucella melitensis biovar 3, two camels; Brucella abortus biovar 6, three camels). Infection of camels with B. abortus biovar 6 had not previously been reported. Infection of the supramammary lymph nodes presents a potential hazard to those who consume raw camels' milk, a common practice in nomadic camel owners.

The improved specificity of bovine brucellosis testing in Great Britain.

The brucellosis surveillance scheme in Great Britain includes the serological testing of approximately 1 million bovine samples per year. These are screened by iELISA, positives going forward for confirmatory testing by CFT and SAT. Samples positive by confirmatory testing prompt substantial field investigations and interventions, but the animals involved are usually uninfected. Described below are a series of modifications to the screening method, which have resulted in a 10-fold reduction in false positive results whilst maintaining sensitivity. The key modifications include the introduction of blocking agents, a change in serum test dilution and the introduction of a control that directly defines the positive/negative cut-off. These simple modifications have had a large impact in reducing the cost of the surveillance programme due to reductions in confirmatory test requirements and a knock on effect of reducing costly field intervention.

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.

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.

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA.

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.

5-HT1A-mediated lower lip retraction: effects of 5-HT1A agonists and antagonists.

This study investigated the production of lower lip retraction (LLR) in the rat by the 5-hydroxytryptamine1A (5-HT1A) agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the effect of the putative 5-HT1A antagonists pindolol and (1-(2-methoxy-phenyl)-4-[4-(2-phthalimido)-butyl]-piperazine (NAN190). 8-OH-DPAT (0.125-1.0 mg/kg, IP) caused a dose-related increase in LLR. Pindolol (10-40 mg/kg, IP) and NAN190 (2.5-10 mg/kg, IP) produced a dose-related block of 8-OH-DPAT-induced LLR. Pindolol (10-40 mg/kg, IP) when administered alone was also found to cause LLR, suggesting that pindolol behaves as a partial agonist in this model. This was not the case with NAN190 (2.5-10 mg/kg, IP), which failed to produce LLR; however, NAN190 (2.5-10 mg/kg, IP) produced a dose-related block of the pindolol-induced LLR. These results clearly demonstrate that the LLR model can be used to detect 5-HT1A agonists, partial agonists, and antagonists.

Correlation of postmortem MRI and CT appearances with neuropathology in brain trauma: a comparison of two methods.

Postmortem magnetic resonance (MR) scans were performed on the brains of 12 victims of fatal head injuries. These were compared with neuropathological studies of the entire brain. The first six subjects were imaged with the brain in situ and comparison was also made with antemortem computed tomography (CT). The brains from the subsequent six subjects were removed at autopsy, fixed in formalin and then imaged in a mitre box designed to overcome the problems encountered in the pilot study. Although both CT and MR imaging (MRI) detected all clinically relevant haemorrhagic lesions, many pathologically significant lesions were missed. MRI detected many more lesions than CT, but still failed to visualize areas of non-haemorrhagic axonal injury.

An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions.

Porcine brucellosis is a zoonotic disease of truly global significance because even in countries without the disease the occurrence of false positive serological reactions (FPSRs) creates significant problems. Statutory diagnostic testing is required in many disease free countries or regions and is often a prerequisite for the movement of live animals. Currently this testing is dependent almost entirely on serological assays and these may result in a significant number of FPSRs. The aim of this study was to examine existing and novel serodiagnostic assays to evaluate their diagnostic sensitivity and resilience to FPSRs. The existing assays evaluated were the RBT, smooth lipopolysaccharide (sLPS) indirect (i) ELISA, sLPS competitive (c) ELISA, and the FPA. The novel assays evaluated were the sLPS TR-FRET assay, a rough (r) LPS iELISA, a recombinant protein BP26 iELISA and a cytoplasmic protein extract (Brucellergene™) iELISA. Four populations of sera were evaluated: those from Brucella suis infected swine (n=34), randomly selected samples from non-infected swine (n=161), sera from non-infected swine within herds exhibiting FPSRs (n=132) and sera from swine experimentally infected with Yersinia enterocolitica O:9 (n=4). The results show that all the assays dependent on the sLPS O-polysaccharide (OPS) for their sensitivity (the RBT, sLPS ELISAs, FPA and the sLPS TR-FRET) had significantly reduced diagnostic specificity when applied to the FPSR population, the RBT being most affected. Of the two rapid homogeneous assays, the TR-FRET was diagnostically superior to the FPA in this study. Neither of the protein based iELISAs demonstrated sufficient diagnostic sensitivity to resolve the FPSRs. The rLPS iELISA showed no cross reaction with the FPSRs and had diagnostic sensitivity similar to that of the OPS based assays.

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).