First confirmed reports of the isolation of Brucella ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca.

Brucella ceti has been recovered from a number species of cetaceans worldwide over the last 25 yr. Here we report, for the first time, the recovery of B. ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca. Recovery from an abdominal mass in the dolphin provides further evidence of the systemic pathogenic potential for B. ceti infection in cetaceans. The isolation of B. ceti ST23 (porpoise cluster) from a killer whale from a group known to eat other marine mammals raises the possibility of infection via ingestion. This report takes the number of cetacean species in UK coastal waters from which B. ceti has been isolated to 11 and highlights the value of routine, comprehensive and specific screening for significant pathogens such as Brucella sp. by strandings networks.

Meningoencephalitis in a common minke whale Balaenoptera acutorostrata associated with Brucella pinnipedialis and gamma-herpesvirus infection.

Fatal marine Brucella infections with histologic lesions specific to the central nervous system (CNS), known as neurobrucellosis, have been described in 5 species of odontocete cetaceans in the UK: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus, short-beaked common dolphins Delphinus delphis, long-finned pilot whale Globicephala melas and Sowerby's beaked whale Mesoplodon bidens. To date, these CNS lesions have only been associated with Brucella ceti ST26 and not with B. pinnipedialis, which is rarely isolated from cetaceans and, although commonly found in various seal species, has never been associated with any pathology. This paper describes the first report of neurobrucellosis in a common minke whale Balaenoptera acutorostrata which was associated with the isolation of Brucella pinnipedialis ST24 and co-infection with Balaenoptera acutorostrata gamma-herpesvirus 2. This is the first report of neurobrucellosis in any species of mysticete and the first report of Brucella pinnipedialis in association with any pathology in any species of marine mammal, which may be due to co-infection with a herpesvirus, as these are known to be associated with immunosuppression.

Neurobrucellosis due to Brucella ceti ST26 in Three Sowerby's Beaked Whales (Mesoplodon bidens).

Fatal meningoencephalitis due to Brucella ceti infection has been described in striped dolphins (Stenella coeruleoalba), Atlantic white-sided dolphins (Lagenorhynchus acutus), short-beaked common dolphins (Delphinus delphis) and long-finned pilot whales (Globicephala melas), which are all within the family Delphinidae. We report B. ceti-associated neurobrucellosis in three juvenile male Sowerby's beaked whales (Mesoplodon bidens) that all had typical lesions of lymphocytic meningoencephalitis, which increased in severity from rostral to caudal regions of the brain. In two cases there was loss of ependymal cells lining the cerebral ventricular system, with large numbers of lymphocytes in the underlying neuropil. This finding suggests that B. ceti gains access to, and multiplies in, the cerebrospinal fluid, and confirms that this is the sample of choice for bacteriological recovery of the causative organism. These findings expand the increasing range of cetaceans susceptible to neurobrucellosis to members of the family Ziphiidae.

Prevalence and risk factors associated with bovine brucellosis in herds under extensive production system in southwestern Nigeria.

Using a cross-sectional survey, we determined the prevalence and risk factors associated with bovine brucellosis in herds under extensive production system in southwestern Nigeria. Antibodies to Brucella species in serum samples were tested using the Rose Bengal test (RBT) and competitive enzyme-linked immunosorbent assay (cELISA); for milk, the milk ring test (MRT) and indirect-ELISA (i-ELISA) were used. Questionnaire was administered to cattle herdsmen to determine factors predisposing the animals to bovine brucellosis. Data were analyzed using STATA 12. From 513 serum and 635 milk samples tested among 120 herds, overall animal-level prevalence of 10.1% (95% CI 7.5-12.7%) and 20.2% (95% CI 17.1-23.3%) were recorded by RBT and MRT, respectively; while 9.4% (95% CI 6.9-11.9%) and 17.8% (95% CI 14.8-20.8%) were obtained using cELISA and i-ELISA, respectively. In all, from the 120 herds tested, 29.2% and 43.3% were positive by RBT and MRT, respectively. Multivariable logistic regression revealed that herd location (OR = 8.12, 95% CI 1.68-38.90) and improper disposal of placenta/fetus (OR = 17.33, 95% CI 4.81-62.33) were predictors for a seropositive herd using RBT; while herd location (OR = 5.13, 95% CI 1.27-20.28), large herd size (OR = 2.62, 95% CI 1.15-5.85), and occurrence of abortion for a year or more (OR = 4.62, 95% CI 1.53-13.71) were predictors of seropositivity to antibodies to Brucella spp. using MRT. We found high prevalence of brucellosis in cattle herds under extensive management system in southwestern Nigeria. Urgent and coordinated control strategies are required to mitigate this problem.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Prevalence of bovine brucellosis in slaughtered cattle and barriers to better protection of abattoir workers in Ibadan, South-Western Nigeria.

INTRODUCTION: Brucellosis is a neglected zoonosis of public health importance. This study was conducted to determine the prevalence and risk factors of brucellosis among slaughtered cattle as well as challenges to the protection of abattoir workers in Nigeria. METHODS: A slaughterhouse study was conducted in a major abattoir in Ibadan from March to August, 2013. To diagnose brucellosis, serum samples from 1,241 slaughtered cattle were tested using Rose-Bengal test (RBT) and competitive enzyme-linked immunosorbent assay (cELISA); again, 57 milk samples were tested with milk ring test (MRT) and indirect ELISA (iELISA). Furthermore, a survey on the usage of personal protective equipment (PPE) and challenges to its use by abattoir workers was done. Data were analysed using Stata 12. RESULTS: Seroprevalence by RBT was 7.8%; 77.3% (75/97) of these were corroborated by cELISA. Prevalence in milk samples by MRT and indirect ELISA were 33.3% and 3.5%, respectively. Sex (OR: 2.5; 95%CI:1.3-4.5) was the factor significantly associated with Brucella seropositivity. None of the abattoir workers used standard protective overalls; while, 99.6% of the meat handlers and 84.1% of the butchers worked barefoot. Most of the workers (75.7%) wore no protective gloves. The respondents agreed that provision of free PPE and sanctions against non-users would encourage its use. CONCLUSION: Our findings indicate moderate prevalence (7.8%) of bovine brucellosis with sex of cattle being a risk factor. A notable barrier to better protection of abattoir workers against brucellosis is perceived inconvenience arising from use of gloves. Therefore, preventive and control measures against brucellosis must include education and use of PPE among abattoir workers.

Evidence of ongoing brucellosis in livestock animals in North West Libya.

Animal brucellosis is thought to be present in small ruminants, cattle, and camels in Libya, particularly in the west coastal strip. Before the system collapsed due to political unrest in 2011, prevalence of the disease did not exceed 0.2% in cattle, 0.1% in camels, 8.3% in sheep, and 14.8% in goats. The aim of this study was to highlight outbreaks of disease that took place during the 18-month period from November 2014 to April 2016. A total of 1612 serum samples, collected opportunistically from 29 herds in 12 different localities in the northwest region of Libya, were investigated for brucellosis. The samples were screened for Brucella antibodies using the Rose Bengal test, and confirmed with either indirect enzyme linked immunosorbent assay in the case of sheep, and/or a serum agglutination test, followed with a complement fixation test, in the case of cattle and camels. Our results showed the highest rates of brucellosis seropositivity in goats (33.4%) and sheep (9.2%). The overall percentage of brucellosis seropositivity was 21%. The high level of brucellosis identified by this study, particularly in small ruminants, strongly suggests re-emergence of the disease in the region. Re-evaluation of intervention measures applied to the control of brucellosis is highly recommended.

Exposure of harbour seals Phoca vitulina to Brucella in declining populations across Scotland.

Since 2000 there has been a major decline in the abundance of Scottish harbour seals Phoca vitulina. The causes of the decline remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n = 150). Serum samples collected from live capture-released seals (n = 343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). In total, 16% of seals cultured had Brucella isolated from one or more tissues, but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results, showing that overall 25.4% of seals were seropositive, with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.

Characterisation of North American Brucella isolates from marine mammals.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Brucella ceti Infection in a Common Minke Whale ( Balaenoptera acutorostrata ) with Associated Pathology.

There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales ( Balaenoptera acutorostrata ) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

Sero-epidemiological survey and risk factors associated with brucellosis in dogs in south-western Nigeria.

INTRODUCTION: In Nigeria, there is limited information on brucellosis particularly in dogs, despite its public health implications. We undertook a sero-epidemiological survey of brucellosis in dogs to determine the prevalence of the disease and associated risk factors for its occurrence in Nigeria. METHODS: A cross-sectional study was conducted to screen dogs in south-western Nigeria for antibodies to Brucella sp using the rapid slide agglutination test (RSA) and Rose Bengal test (RBT), with positive samples confirmed respectively by serum agglutination test (SAT) and competitive enzyme linked immunosorbent assay (cELISA). Data were analyzed with STATA-12. RESULTS: From the 739 dog sera tested, 81 (10.96%) were positive by RSA and 94 (12.72%) by RBT; these were corroborated with SAT (4/81; 4.94%) and cELISA (1/94; 1.06%), respectively. Logistic regression identified location (OR=0.04; 95% CI: 0.02-0.09), breed (OR=1.71; 95% CI: 1.34-2.19), age (OR=0.10; 95% CI: 0.04-0.30) and management system (OR=8.51; 95% CI: 1.07-68.05) as risk factors for Brucella infection by RSA. However, location (OR=10.83; 95% CI: 5.48-21.39) and history of infertility (OR=2.62; 95% CI: 1.41-4.84) were identified as risk factors using RBT. CONCLUSION: Given the 10.96% to 12.72% seroprevalence of brucellosis recorded in this study, we advocate control of the disease in dogs, and public health education for those at risk of infection. Again, further studies are required to elucidate the role of dogs in the epidemiology of brucellosis in Nigeria considering the conducive human-animal interface and ecological factors responsible for the transmission of the disease.

Extended Multilocus Sequence Analysis to Describe the Global Population Structure of the Genus Brucella: Phylogeography and Relationship to Biovars.

An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.

First report of Brucella ceti-associated meningoencephalitis in a long-finned pilot whale Globicephala melas.

Fatal Brucella ceti infection with histological lesions specific to the central nervous system has been described in only 3 species of cetaceans: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus and short-beaked common dolphins Delphinus delphis. This paper describes the first report of a B. ceti-associated meningoencephalitis in a long-finned pilot whale Globicephala melas, showing the increasing range of species susceptibility. Brucella was recovered in larger numbers from cerebrospinal fluid than from brain tissue and is the sample of choice for isolation.

Isolation of Brucella ceti from a Long-finned Pilot Whale (Globicephala melas) and a Sowerby's Beaked Whale (Mesoploden bidens).

Brucella ceti is an emerging zoonotic pathogen that has been recovered from several species of cetaceans in the world's oceans over the past 20 yr. We report the recovery of B. ceti from a Sowerby's beaked whale (Mesoploden bidens) and a long-finned pilot whale (Globicehala melas). Recovery from the testis of a long-finned pilot whale provides further evidence of potential for B. ceti infection to impact the reproductive success of cetaceans, many of which are threatened species. The addition of another two cetacean species to the growing number from which B. ceti has been recovered also further emphasizes the concern for human infections with this organism.

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)).

Seroprevalence and risk factors of brucellosis in goats in selected states in Nigeria and the public health implications.

Available reports on brucellosis in Nigeria are largely confined to cattle while it is believed that other ruminants like sheep and goats are equally exposed to the disease. To have an insight into the role of goats in the epidemiology of brucellosis in Nigeria, we conducted a cross-sectional study between June 2011 and May 2013 to determine the seroprevalence of brucellosis in goats in some selected states in Nigeria. Serum samples were collected from goats at different locations and tested for antibodies to Brucella spp using the Rose Bengal Test (RBT), samples positive by RBT were further subjected to Competitive Enzyme Linked Immunosorbent Assay (cELISA). Data collected to determine risk factors were also analysed using chi-square and logistics regression statistics. Out of a total of 2827 samples tested from the different states (Benue = 331; Borno =195; Oyo = 2155; Sokoto = 146), we recorded an overall seroprevalence of 2.83% (Benue = 17.30%; Borno = 2.05%; Oyo = 0.60% and Sokoto = 0.00%) by RBT. The cELISA further supported 9.45% (7/74) of the total RBT positive samples. Logistic regression analysis showed that the location (p = 0.004) and source (p < 0.0001); are probable risk factors to be considered in the epidemiology of brucellosis with sex (p = 0.179); age (p = 0.791) and breed (p = 0.369) not playing any major role. Our findings reveal a relatively low seroprevalence of brucellosis among goats screened except for Benue State. Since most of the goats sampled in the present study were from the abattoirs, further farm level investigations are required to determine the role of goats in the epidemiology of brucellosis in Nigeria since they share common environment with sheep and cattle that are natural hosts of Brucella species which are of major public health threat.

Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

Meningoencephalitis and arthritis associated with Brucella ceti in a short-beaked common dolphin (Delphinus delphis).

Brucella species infection in marine mammal species has been reported to have a global distribution. In 2007, the description of Brucella ceti was published and formally adopted for those isolates originating from cetaceans and pathologic lesions similar to those seen in terrestrial mammals infected with Brucella spp. have been associated with its isolation. Brucella ceti infection specific to the central nervous system has been described in two species of cetacean: striped dolphins (Stenella coeruleoalba) in Europe and Costa Rica and an Atlantic white-sided dolphin (Lagenorhynchus acutus) in the UK. We describe the first report, to our knowledge, of B. ceti-associated meningitis and arthritis in a third species, the short-beaked common dolphin (Delphinus delphis), in an animal that stranded in the UK.

Evaluation of competitive ELISA for detection of antibodies to Brucella infection in domestic animals.

AIM: To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis. METHODS: cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested. RESULTS: Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%. CONCLUSION: cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.