Brucella suis biovar 2 infection in humans in France: emerging infection or better recognition?

Brucellosis is usually acquired by humans through contact with infected animals or the consumption of raw milk from infected ruminants. Brucella suis biovar 2 (BSB2) is mainly encountered in hares and wild boars (Sus scrofa), and is known to have very low pathogenicity to humans with only two case reports published in the literature. Human cases of brucellosis caused by BSB2 were identified through the national mandatory notification of brucellosis. The identification of the bacterium species and biovar were confirmed by the national reference laboratory. Epidemiological data were obtained during medical follow-up visits. Seven human cases were identified between 2004 and 2016, all confirmed by the isolation of BSB2 in clinical specimens. All patients had direct contact with wild boars while hunting or preparing wild boar meat for consumption. Five patients had chronic medical conditions possibly responsible for an increased risk of infection. Our findings suggest that BSB2 might be an emerging pathogen in hunters with massive exposure through the dressing of wild boar carcasses. Hunters, especially those with chronic medical conditions, should be informed about the risk of BSB2 infection and should receive information on protective measures.

Assessment of the sensitivity of the gamma-interferon test and the single intradermal comparative cervical test for the diagnosis of bovine tuberculosis under field conditions.

In some French départements, the eradication of bovine tuberculosis is incomplete and usual skin tests [single intradermal tuberculin test (SIT) and single intradermal comparative cervical test (SICCT)] have poor specificity due to cross-reactions with non-pathogenic mycobacteria, causing economic losses. In Côte d'Or (Burgundy, France), an experimental serial testing scheme based on the combination of SICCT and gamma-interferon (IFN-γ) tests has been initiated in order to shorten the interval between suspicion and its invalidation in herds with false-positive results to skin tests. Our aim was to assess the scheme's sensitivity and to compare it to the sensitivity of the screening scheme recommended by the European Commission. Our study included 1768 animals from Côte d'Or. The sensitivities of both schemes were estimated using a Bayesian approach. The individual sensitivity of the IFN-γ test [88·1%, 95% credibility interval (CrI) 72·8-97·5] was not significantly different from individual SICCT sensitivity (80·3%, 95% CrI 61·6-98·0) and individual SIT sensitivity (84·2%, 95% CrI 59·0-98·2). The individual specificity of the IFN-γ test was 62·3% (95% CrI 60·2-64·5). No significant difference could be demonstrated between the sensitivities of the serial testing scheme used in Côte d'Or (73·1%, 95% CrI 41·1-100) and the European Union serial testing scheme (70·1%, 95% CrI 31·5-100·0).

Brucellosis in terrestrial wildlife.

The epidemiological link between brucellosis in wildlife and brucellosis in livestock and people is widely recognised. When studying brucellosis in wildlife, three questions arise: (i) Is this the result of a spillover from livestock or a sustainable infection in one or more host species of wildlife? (ii) Does wildlife brucellosis represent a reservoir of Brucella strains for livestock? (iii) Is it of zoonotic concern? Despite their different host preferences, B. abortus and B. suis have been isolated from a variety of wildlife species, whereas B. melitensis is rarely reported in wildlife. The pathogenesis of Brucella spp. in wildlife reservoirs is not yet fully defined. The prevalence of brucellosis in some wildlife species is very low and thus the behaviour of individual animals, and interactions between wildlife and livestock, may be the most important drivers for transmission. Since signs of the disease are non-pathognomonic, definitive diagnosis depends on laboratory testing, including indirect tests that can be applied to blood or milk, as well as direct tests (classical bacteriology and methods based on the polymerase chain reaction [PCR]). However, serological tests cannot determine which Brucella species has induced anti-Brucella antibodies in the host. Only the isolation of Brucella spp. (or specific DNA detection by PCR) allows a definitive diagnosis, using classical or molecular techniques to identify and type specific strains. There is as yet no brucellosis vaccine that demonstrates satisfactory safety and efficacy in wildlife. Therefore, controlling brucellosis in wildlife should be based on good management practices. At present, transmission of Brucella spp. from wildlife to humans seems to be linked to the butchering of meat and dressing of infected wild or feral pig carcasses in thedeveloped world, and infected African buffalo in the developing world. In the Arctic, the traditional consumption of raw bone marrow and the internal organs of freshly killed caribou or reindeer is an important risk factor.

Re-emergence of brucellosis in cattle in France and risk for human health.

A case of human brucellosis was diagnosed in France in January 2012. The investigation demonstrated that the case had been contaminated by raw milk cheese from a neighbouring dairy farm. As France has been officially free of bovine brucellosis since 2005, veterinary investigations are being conducted to determine the origin of the infection and avoid its spread among other herds. Hypotheses about the source of this infection are discussed.

The first International Standard anti-Brucella melitensis Serum.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.

Brucella suis infection in domestic pigs and wild boar in Croatia.

Between the years 2000 and 2004, 93,107 sera from 1,997 pig herds in 11 regions of Croatia were tested for the presence of antibodies against brucellosis. Positive results were observed in 67 herds from seven regions (mean individual prevalence: approximately 1%; herd prevalence: 3.4%). The herds from all but two of the infected farms were reared outdoors and thus almost certainly came into contact with wildlife. From 2003 to 2004, 424 sera, which were randomly collected from hunted wild boar (Sus scrofa), were also tested and shown to have a mean seroprevalence of 27.6%. Brucella was isolated from 88 out of 151 serologically positive pigs (58.3%) and 7 of the 93 (7.5%) wild boar which were randomly submitted for bacteriological study. All but three isolates were Brucella suis biovar 2; the others being biovar 3. These results suggest that brucellosis is enzootic in Croatian populations of wild boar. These populations represent a potential disease reservoir for free-range pig farms, as they do in other countries of Central and Western Europe. This is the first report of B. suis biovar 3 in swine and wild boar in Europe, which is an issue of serious concern for public health.

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains.

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

Reference laboratory networking: the European experience in brucellosis.

Over the past forty years, a huge effort of standardisation of tests and harmonisation of sanitary policies and requirements has been made in the European Union (EU). The EU has itself greatly evolved in its size and policies. Voluntary uncoordinated actions initially had some success but not enough to attain a level of standardisation compatible with a single market and to achieve the sanitary level foreseen by the community. The creation of a Community Reference laboratory with specific responsibilities and tasks and the provision of adequate financial assistance is expected to lead to better co-ordination of activities and to ensure international harmonisation of tests and procedures.

Prevalence of classical swine fever, Aujeszky's disease and brucellosis in a population of wild boar in Switzerland.

During two survey rounds of a national surveillance system for infectious diseases in wild boar in Switzerland, each lasting four months from November to February, between 2001 and 2003, 1949 blood samples and 62 tissue samples from the spleen and 50 from the reproductive organs were collected from hunted wild boar. The survey was designed so that freedom from infection could be detected with a probability of 95 per cent at a threshold prevalence of less than 1 per cent for classical swine fever and Aujeszky's disease and less than 1.5 per cent for brucellosis. There was no serological evidence of classical swine fever or Aujeszky's disease, but brucellosis due to Brucella suis biovar 2 was confirmed serologically and by bacterial isolation.

Urgent request on avian influenza.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non-previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding-specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses.

Human brucellosis in France in the 21st century: Results from national surveillance 2004-2013.

Brucellosis is a bacterial zoonotic disease mainly transmitted to humans by ruminants. In France, brucellosis has disappeared from ruminants herds. Human brucellosis surveillance is performed through mandatory notification and the national reference center. METHODS: We report the results of human brucellosis surveillance from 2004 to 2013 with regards to epidemiological, clinical and microbiological data. RESULTS: A total of 250 cases were notified, making an annual incidence of 0.3 cases per million inhabitants. Brucella melitensis biovar 3 was the most frequently identified bacterium (79% of isolated strains). In total, 213 (85%) cases had been contaminated abroad in endemic countries. In 2012, an episode of re-emergence of brucellosis in cattle occurred in Haute-Savoie, in the French Alps, and was responsible for 2 human cases. CONCLUSION: Brucellosis has become a disease of travelers in France. However, maintaining a stringent epidemiological surveillance is necessary to be able to early detect any local re-emergence in humans or animals. The multidisciplinary surveillance was implemented in France years ago and is a successful example of the One Health Concept.

Evaluation of three competitive ELISAs and a fluorescence polarisation assay for the diagnosis of bovine brucellosis.

Bovine brucellosis is an infectious disease of worldwide public health and economic importance. The usual tests for the diagnosis of this disease include the Rose-Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT) and indirect ELISA. New tests such as competitive ELISAs (C-ELISA) and fluorescence polarisation assay (FPA) have been developed. However, C-ELISA may correspond to different protocols and a wide variation may exist in their diagnostic performance. The aim of this study was to evaluate three commercially available C-ELISA kits (C-ELISA1-3) and FPA for the diagnosis of bovine brucellosis and compare test performance with RBT, CFT, indirect ELISA and FPA. Sera submitted to EU laboratories in 2011 from 5111 adult cattle were tested. Individual test sensitivities (Se) and specificities (Sp) were estimated. Threshold assessment using the receiver operating characteristic method was also performed. The most sensitive tests were FPA (99.0%; 95% confidence interval [CI], 97.9-100%), C-ELISA1 (98.4%; 95% CI, 97.0-99.8%) and RBT (97.7%; 95% CI, 95.9-99.3%). The most specific tests were CFT (99.98%; 95% CI, 99.93-100%), SAT (99.98%; 95% CI, 99.93-100%) and RBT (99.89%; 95% CI, 99.79-99.99%). Among the new tests, none of the three C-ELISA kits studied could be recommended as a single screening test because of their low specificity, especially when used in a herd. C-ELISA3 could not be recommended as confirmatory test on individual animals to determine whether false positive serological test results had occurred.

Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus.

A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals such as seals, porpoises, dolphins and a minke whale. These strains were identified as brucellae by conventional typing tests. However, their overall characteristics were not assimilable to those of any of the six currently recognized Brucella species and it was suggested that they comprise a new nomen species to be called Brucella maris. In the present study we analysed DNA polymorphism at the omp2 locus of 33 marine mammal Brucella strains isolated from seals, dolphins, porpoises and an otter. The omp2 locus contains two gene copies (named omp2a and omp2b) coding for porin proteins and has been found particularly useful for molecular typing and identification of Brucella at the species, biovar, or strain level. PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing showed that strains isolated from dolphins and porpoises carry two omp2b gene copies instead of one omp2a and one omp2b gene copy or two similar omp2a gene copies reported in the currently recognized species. This observation was also recently made for a minke whale Brucella isolate. The otter and all seal isolates except one were shown to carry one omp2a and one omp2b gene copy as encountered in isolates from terrestrial mammals. By PCR-RFLP of the omp2b gene, a specific marker was detected grouping the marine mammal Brucella isolates. Although marine mammal Brucella isolates may represent a separate group from terrestrial mammal isolates based on omp2b sequence constructed phylogenetic trees, the divergence found between their omp2b and also between their omp2a nucleotide sequences indicates that they form a more heterogeneous group than isolates from terrestrial mammals. Therefore, grouping the marine mammal Brucella isolates into one species Brucella maris seems inappropriate unless the currently recognized Brucella species are grouped. With respect to the current classification of brucellae according to the preferential host, brucellae isolated from such diverse marine mammal species as seals and dolphins could actually comprise more than one species, and at least two new species, B. pinnipediae and B. cetaceae, could be compatible with the classical criteria of host preferentialism and DNA polymorphism at their omp2 locus.

Humoral immunity in mice mediated by monoclonal antibodies against the A and M antigens of Brucella.

All smooth strains of Brucella bear two lipopolysaccharide (LPS) antigens in a ratio that defines the classification of strains in serovars, A (A greater than M), M (M greater than A) and A.M (A = M). Anti-LPS-A monoclonal antibodies (MAb-A) were previously shown to convey protection to mice against B. abortus (A) strain 544, as shown by lower spleen counts than in controls at days 7 and 21 after challenge. Anti-LPS-M monoclonal antibodies (MAb-M) were obtained and tested for M-specificity with LPS from reference strains by ELISA, by agglutination of LPS-coated latex particles, and by inhibition of this agglutination. Antigens A and M of three strains were quantified by a homologous LPS-latex and MAb agglutination inhibition assay. Protection conferred by MAb-A and MAb-M against three strains, B. abortus 544 (A), B. abortus 292 (M) and B. melitensis H38 (M), was tested at equivalent challenge and MAb doses: intravenous challenge was adjusted to give similar infection at day 7; MAb doses were adjusted to the same specific ELISA titre. Under these conditions, MAb-A and MAb-M conferred both early and late protection, as shown at days 7 and 21, against the strains that bore the homologous major antigen, i.e., strain 544 on one hand and strains H38 and 292 on the other. In contrast, MAb directed against the minor antigen of the challenge strain conferred significant protection at day 7 only with strains 544 and H38 and no or inconsistent protection against strain 292, which expressed the lowest amount of minor antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Non specific serological reactions in the diagnosis of bovine brucellosis: experimental oral infection of cattle with repeated doses of Yersinia enterocolitica O:9.

Eight heifers were orally infected with 4 x 10(9) colony forming units of a field cattle strain of Yersinia enterocolitica O:9 in a capsule, 5 days a week, for about 9 weeks (day 0-day 64 (D0-D64). The faecal shedding of Y. enterocolitica O:9 began on D5 for seven out of the eight challenged cattle with a high level of excretion during the first month, followed by a decrease till the day of slaughter (D76). Y. enterocolitica O:9 was not isolated from organs collected at slaughter. No clinical symptoms were observed. Hyperplasia of intestinal lymph formations was the sole microscopic lesions observed. Five animals showed a serological reaction against Brucella antigens in at least one of the following tests: Rose-Bengal test, complement fixation test, tube agglutination test or indirect ELISA (iELISA) tests. Only one animal showed a high level of serological response and a positive reaction in the dithiothreitol-microagglutination test. The observed variability in terms of individual sensitivity to the Y. enterocolitica O:9 infection is in agreement with the low individual prevalence rate and the transient serological reaction and faecal Y. entercolitica O:9 shedding observed in herds showing false positive serological reactions in brucellosis.

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 I immunoblot analysis of the antibody response to Brucella protein antigens in bovine brucellosis.

Sera from three groups of Brucella abortus infected cattle were examined in immunoblots with the following antigens: sodium dodecyl sulfate/mercapto ethanol (SDS/ME) extracts of two rought B. abortus strains (45/20 and RB51) and rough B. ovis, smooth lipopolysaccharides (SLPS) from B. abortus strain 99 and Y. enterocolitica 0:9, and a cytoplasmic extract from smooth B. abortus strain 19-S. The sera groups were: (1) 26 sera from animals, experimentally infected with B. abortus strain 544, which were all positive in the conventional brucellosis serological tests; (2) 152 sera from naturally infected cattle herds with varying titres in the conventional brucellosis tests, and (3) 30 sera from naturally infected cattle with varying titres in the conventional brucellosis tests and from which B. abortus was cultured. B. abortus strain 99 and Y. enterocolitica serotype 0:9 SLPS staining showed up frequently in all sera groups and correlated well with the strength in the conventional brucellosis tests, confirming the immunodominance of SLPS in B. abortus infections. Another immunodominant component of 50-80 kDa was found in the rough B. abortus 45/20 antigen preparation but not in the B. abortus RB51 and in the B. ovis cell extracts. This component was also recognised by sera from Y. enterocolitica 0:9 infected cattle and is probably a protein-lipopolysaccharide complex. Although many of the sera from B. abortus infected cattle with high titres in the conventional brucellosis tests showed complex protein staining patterns in blots, no protein bands other than the 50-80 kDa bands were found to be immunodominant.

Risk factors for false-positive serological reactions for bovine brucellosis in Saône-et-Loire (France).

Since 1990, unusually high rates of false-positive serological reactions (FPSR) in bovine brucellosis screening have been observed in some countries of the European Union. The aim of this survey was to describe this phenomenon in a highly affected French Department, and to evaluate the links between some individual or herd factors and the occurrence of these FPSR. Before 1990, low backgrounds of FPSR were recorded (individual prevalence rate: less than 0.5 per 10,000). The phenomenon burst during the 1990-91 screening campaign, reached a peak in 1992-93 (50.5 per 10,000), and then decreased until the last studied campaign, 1995-96 (9.1 per 10,000). The phenomenon was transient and sporadic within a herd. At the herd-screening level, four assumed risk factors were isolated: (i) the probability of a herd-screening to be positive was closely and positively linked with the herd screening size; (ii) during a given screening campaign, the prevalence of FPSR decreased from December to November; (iii) the presence of at least one goat on the premises increased the risk for the 1992-93 and 1993-94 screening campaigns; and (iv) a previous FPSR in a given herd appeared to be a weak but significant risk factor. At the individual-animal level, herd size, sex and breed did not seem to be linked with FPSR appearance, while young animals were significantly more affected than older ones. However, global variations in herd or individual prevalences remained unexplained. The lack of link between FPSR and brucellosis is strengthened. The hypothesis of a widely spread causal agent with a low individual host susceptibility and/or a low probability of detecting FPSR animals can be supported by these results.

Statistical procedures for calculating the residual virulence of Brucella abortus strain 19 (S19) and Brucella melitensis strain Rev 1 vaccines in mice: theoretical basis and practical applications.

Regular control of the biological quality of live Brucella abortus strain 19 (S19) and B. melitensis strain Rev 1 vaccines is essential for the successful management of ruminant brucellosis in affected countries. The reference procedures recommended by the OIE (World organisation for animal health) and the European Pharmacopoeia include the determination of residual virulence, expressed as the recovery time 50 (RT50), of the tested (problem) vaccine in a reference mouse model compared with the RT50 of the corresponding reference strains in the same assay. The underlying statistical procedure applied is based on a parallel line assay and a classical probit model. In practice, the currently recommended procedure for calculating the RT50 is based on a graphical method which has never been described in detail. This paper provides a full description of this graphical method with the aim of making the technique comprehensible and accessible to all interested biologists. The procedure is somewhat cumbersome and very few laboratories apply the OIE and European Pharmacopoeia recommendations on a regular basis. Moreover, since this reference graphical method shows some statistical inconsistencies, a dedicated internet interface has been developed to perform RT50 calculations and is now available free of charge on the web (www.afssa.fr/interne/rev2.html).

[Pathogenic organisms in milk and milk products: the situation in France and in Europe]. / Les germes pathogènes dans le lait et les produits laitiers: situation en France et en Europe.

Milk and dairy products harbour a natural microbial flora and/or other micro-organisms, which vary within the wide range of products available on the French market. The origin of contamination by pathogenic bacteria varies with the type of product and the mode of production and processing. Contamination of milk and dairy products by pathogenic micro-organisms can be of endogenous origin, following excretion from the udder of an infected animal. Contamination may also be of exogenous origin, through direct contact with infected herds or through the environment (e.g. water, personnel). Treatment and processing of milk can inhibit or encourage the multiplication of micro-organisms. The authors describe the relevant aspects of bacterial physiology and ecology, the occurrence of bacteria in dairy products, and the public health significance for each of the principal micro-organisms found in such products. Bacteria most frequently involved are mycobacteria, Brucella sp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella). At present, systems of testing and surveillance are required for the control of pathogenic bacteria in milk and dairy products, as specified by regulations currently being developed for all countries in the European Union. Preventive measures should take into account the well-established facts concerning the potential microbiological impact of pathogenic bacteria on milk and dairy products. There should be increased recourse to risk analysis methods to assess the threat to the consumer with regard to the presence of pathogenic bacteria in food.

Isolation of Brucella melitensis biovar 3 from a chamois (Rupicapra rupicapra) in the southern French Alps.

Systemic brucellosis caused by Brucella melitensis biovar 3 was identified in a chamois (Rupicapra rupicapra) near the Parc National des Ecrins in the southern French Alps (France). Clinical signs included orchiepididymitis, polyarthritis, blindness and various neurological signs; necropsy findings included numerous calcified foci in testis, epididymis, kidney, subcutaneous connective tissue and brain. Brucella sp. were identified in brain by indirect immunofluorescence and B. melitensis biovar 3 was isolated from testis, kidney, eye, lung and joints. This report describes the first case of brucellosis and Brucella sp. isolation in chamois in France and the first case of B. melitensis isolation in this host species.