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African horse sickness (AHS) is a viral disease of equids, caused by a virus of the genus Orbivirus, family Reoviridae. The African horse sickness virus (AHSV) genome is made up of ten double-stranded RNA (dsRNA) segments that together code for seven structural and four nonstructural proteins. AHS is endemic in sub-Saharan countries. The efficacy and safety of inactivated AHS vaccines containing all nine serotypes, produced at the Central Veterinary Research Laboratory (CVRL) in Dubai, United Arab Emirates have been proven in the past. All nine AHSV serotypes were isolated from 102 samples collected in the last 20 years from horse fatalities in seven different area of Kenya, Africa. CVRL inactivated AHS vaccines are used in a few African countries defining the importance of this present study to compare the genome sequences of the nine AHSV serotypes isolated from horse fatalities in Kenya and nine AHSV serotypes isolated in South Africa. The hypothesized serotypes of the newly sequenced AHSV field strains from Kenya were likewise confirmed in this investigation, and they show substantial sequence homologies with recently isolated AHSV field strains.

Little is known regarding the molecular epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) circulating in dromedaries outside Saudi Arabia. To address this knowledge gap, we sequenced 10 complete genomes of MERS-CoVs isolated from 2 live and 8 dead dromedaries from different regions in the United Arab Emirates (UAE). Phylogenetic analysis revealed one novel clade A strain, the first detected in the UAE, and nine clade B strains. Strain D998/15 had a distinct phylogenetic position within clade A, being more closely related to the dromedary isolate NRCE-HKU205 from Egypt than to the human isolates EMC/2012 and Jordan-N3/2012. A comparison of predicted protein sequences also demonstrated the existence of two clade A lineages with unique amino acid substitutions, A1 (EMC/2012 and Jordan-N3/2012) and A2 (D998/15 and NRCE-HKU205), circulating in humans and camels, respectively. The nine clade B isolates belong to three distinct lineages: B1, B3 and B5. Two B3 strains, D1271/15 and D1189.1/15, showed evidence of recombination between lineages B4 and B5 in ORF1ab. Molecular clock analysis dated the time of the most recent common ancestor (tMRCA) of clade A to March 2011 and that of clade B to November 2011. Our data support a polyphyletic origin of MERS-CoV in dromedaries and the co-circulation of diverse MERS-CoVs including recombinant strains in the UAE.

International audience; We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 (‘Qinghai’ lineage) in 2005.

Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted.Emerging MicrobesInfections (2015) 4, e74; doi:10.1038/emi.2015.74; published online 2 December 2015.

The recent discovery of Middle East Respiratory Coronavirus and another novel dromedary camel coronavirus UAE-HKU23 in dromedaries has boosted interest in search of novel viruses in dromedaries. In this study, fecal samples of 203 dromedaries in Dubai were pooled and deep sequenced. Among the 7330 assembled viral contigs, 1970 were assigned to mammalian viruses. The largest groups of these contigs matched to Picobirnaviridae, Circoviridae, Picornaviridae, Parvoviridae, Astroviridae and Hepeviridae. Many of these viral families were previously unknown to dromedaries. In addition to the high abundance of contigs from Circoviridae (n=598 with 14 complete genomes) and Picobirnaviridae (n=1236), a high diversity of contigs from these two families was found, with the 14 Circoviridae complete genomes forming at least five clusters and contigs from both genogroup I and genogroup II potentially novel picobirnaviruses. Further studies comparing the incidence of these viral families in healthy and sick dromedaries will reveal their pathogenic potential., Highlights • The viromes of fecal samples from dromedaries in the Middle East were analyzed. • Circoviridae and Picobirnaviridae are most abundant among the mammalian viruses. • A high diversity of contigs from Circoviridae and Picobirnaviridae was observed.

Working donkeys (n = 395) originating from three different districts of the Oromia region of Ethiopia were examined October 2009 and April 2010 for the prevalence of equine piroplasmosis between. Apart from hematological and serological examination, tick vectors were collected and determined. Intraerythrocytic stages of Theileria equi and Babesia caballi were detected in 12.2% and 1.8% of examined stained blood smears, respectively. The seroprevalence of T. equi and B. caballi was 55.7% and 13.2%, respectively. The majority of active infection coincided with anemia. Only 16 donkeys were infested with four species of hard ticks: Rhipicephalus turanicus, Rhipicephalus evertsi evertsi, Rhipicephalus pulchellus, and Amblyomma variegatum.

A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. We screened 2,438 dromedary samples from Pakistan, the United Arab Emirates, and 4 African countries. HEV-7 is long established, diversified and geographically widespread. Dromedaries may constitute a neglected source of zoonotic HEV infections.

Background Among all known picornaviruses, only two species, equine rhinitis A virus and equine rhinitis B virus (ERBV) are known to infect horses, causing respiratory infections. No reports have described the detection of ERBV in fecal samples of horses and no complete genome sequences of ERBV3 are available. Methods We performed a molecular epidemiology study to detect ERBVs in horses from Dubai and Hong Kong. Complete genome sequencing of the ERBVs as well as viral loads and genome, phylogenetic and evolutionary analysis were performed on the positive samples. Results ERBV was detected in four (13.8 %) of the 29 fecal samples in horses from Dubai, with viral loads 8.28 × 103 to 5.83 × 104 copies per ml, but none of the 47 fecal samples in horses from Hong Kong by RT-PCR. Complete genome sequencing and phylogenetic analysis showed that three of the four strains were ERBV3 and one was ERBV2. The major difference between the genomes of ERBV3 and those of ERBV1 and ERBV2 lied in the amino acid sequences of their VP1 proteins. The Ka/Ks ratios of all the coding regions in the ERBV3 genomes were all

The activity of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), lactoperoxidase (LPO), lipase (LIP) and leucine arylamidase (LAP) in raw and pasteurised camel milk was studied, in order, to find a heat treatment indicator suitable to verify an effective pasteurisation. The studies have shown that ALP, GGT and LIP are not suitable as marker because residual activities were detected in pasteurised milk. LAP activity in raw camel milk is too low and the data variation is too high for serving as a marker. The LPO results look promising. The enzyme activity in raw camel milk is high and the respective value in pasteurised milk is predominantly below the detection limit of the method.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria (M 2040T, M 1973 and M 1878-SK2), isolated from milk of camels at a camel-milk production farm in the United Arab Emirates, were investigated for their taxonomic allocation. On the basis of 16S rRNA gene sequence similarities, all three strains were shown to belong to the Alphaproteobacteria and were most closely related to Chelatococcus asaccharovorans and Chelatococcus daeguensis (95.1 and 95.2 % sequence similarity to the respective type strains). meso-Diaminopimelic acid was detected as the characteristic peptidoglycan diamino acid. The predominant compound in the polyamine pattern was spermidine, and sym-homospermidine was not detectable. The quinone system was ubiquinone Q-10. The polar lipid profile included the major compounds phosphatidylcholine and diphosphatidylglycerol and moderate amounts of phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid and two unidentified aminolipids. Minor lipids were also detected. The major fatty acid profile, consisting of C19 : 0 cyclo ω8c and C18 : 1 ω7c, with C18 : 0 3-OH as the major hydroxylated fatty acid, was similar to those of the genus Chelatococcus. The results of DNA–DNA hybridization experiments and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolates from described Chelatococcus species. Isolates M 2040T, M 1973 and M 1878-SK2 were closely related on the basis of DNA–DNA reassociation and therefore represent a single novel species. In summary, low 16S rRNA gene sequence similarities of 95 % with Chelatococcus asaccharovorans and marked differences in polar lipid profiles as well as in polyamine patterns support the description of a novel genus and species to accommodate these strains, for which the name Camelimonas lactis gen. nov., sp. nov. is proposed. The type strain of Camelimonas lactis is M 2040T (=CCUG 58638T =CCM 7696T).

A virus was isolated from aborted dromedary (Camelus dromedarius) fetuses during an abortion storm in Dubai, United Arab Emirates. Laboratory investigations showed the causative agent to be indistinguishable from equine rhinitis A virus (ERAV), a picornavirus. Two pregnant dromedaries experimentally infected with the camel virus isolate both aborted and an identical virus was reisolated from both fetuses, thus confirming the diagnosis. The extremely high prevalence of antibody (>90 %) and the high titres recorded against ERAV in the dromedary herd clearly showed that ERAV does infect dromedaries. Unlike horses, where ERAV targets the upper respiratory tract, in dromedaries the target organ appears to be the genital tract.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154-156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection.

A polymerase chain reaction (PCR) assay targeting the flagellin P ( fliP )-I S 407 A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP -IS 407 A sequence of B. mallei American Type Culture Collection (ATCC) 23344 T allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344 T . B . mallei DNA was also amplified from various tissues of horses with a generalized B . mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.

The present study describes the laboratory investigations after a single right eye drop (3.1 × 109 CFU live bacteria) immunisation of 6 dromedary camels (Camelus dromedarius) with live attenuated B. melitensis Rev 1 vaccine. The experiment was conducted over a period of 5 months. The vaccine strain was isolated for 16 days from only the right eye of the vaccinated dromedaries, but not from the left eye and both nostrils. Similar pattern of results was obtained by polymerase chain reaction. It was negative for the left eye, both nostrils (except for one dromedary) and for EDTA blood and serum. All vaccinated dromedaries seroconverted from day 16 after vaccination until 4 months shown by Rose-Bengal test and slide-agglutination test. No serological reactions were found after 5 months. The complement fixation test remained negative throughout the experiment. Information about the vaccination against brucellosis in camels, the within host disperse of the vaccine strain and the serological response are scarce. The experiment provided basic data about the feasibility of Brucevac conjunctival vaccine in camels. However, to prove if the immunised dromedaries acquired a lifelong immunity against brucellosis, pregnant vaccinated dromedaries need to be challenged with a field B. melitensis strain. We also recommend changing the conjunctival vaccination route to subcutaneous or intramuscular to prevent accidental infection due to B. melitensis vaccine strain excreted by lacrimation.

Embryonic gonadal tissue was dissected individually from Houbara bustard embryos at eight days post-incubation. Houbara bustard gonadal cells containing gonadal primordial germ cells (gPGCs) were injected into the blood stream of White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara bustard PGCs in chimeric chicken testis were assessed by PCR with Houbara bustard species-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara bustard cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germ line chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara bustard eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara bustard and the other was female but died before hatching.

We confirm a natural infection of dromedaries with glanders. Multilocus variable number tandem repeat analysis of a Burkholderia mallei strain isolated from a diseased dromedary in Bahrain revealed close genetic proximity to strain Dubai 7, which caused an outbreak of glanders in horses in the United Arab Emirates in 2004.

To the Editor: In 2012, a novel coronavirus termed Middle East respiratory syndrome coronavirus (MERS-CoV) emerged on the Arabian Peninsula; the virus has been responsible for >800 human cases. Recently, evidence of MERS-CoV infection in dromedaries was obtained from the Canary Islands, the Arabian Peninsula, and Africa (1–3). Viral sequences from dromedaries and from humans infected with MERS-CoV were highly similar, suggesting a prominent role of dromedaries as an animal reservoir of the virus (4). However, the serologic assessment of other animal species has been incomplete. Investigations of domestic animal species have been restricted to goats, sheep, and cattle (3) and a limited study of horses (n = 3) (5). No evidence of recent infection was found in either study.

Summary During the spring of 1995, 1996 and 1997 following tests on six peregrine falcons (Falco peregrinus) and two gyr falcons (Falco rusticolus), Salmonella typhimurium was isolated from liver, spleen and small intestines. Four of the falcons (two peregrines and two gyrs) had also contracted Chlamydia infection, three peregrines a pox infection and one peregrine a Herpesvirus infection. It is believed that this dual infection was fatal for these birds. The disease was marked by anorexia, dehydration and green-coloured droppings. Necropsy of all falcons revealed discolouration of the liver and enlargement of liver and spleen. Miliary necrosis was detected in all livers. A total of 12 salmonella serovars, including S. typhimurium, were cultured from faeces of 48 falcons which showed no clinical signs.

Background Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of Brucella in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting Brucella infection in camels. Findings A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that bcsp31 kDa real-time PCR detected Brucella DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species. Conclusion We suggest combining bcsp31 real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.

Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

Staphylococcus aureus is a common cause of mastitis and other diseases in camels. In order to obtain data on population structure as well as on the carriage of toxin genes and resistance markers, a collection of 45 isolates from dromedaries of Dubai, United Arab Emirates, were genotyped. These isolates belonged to clonal complexes CC6 (twenty isolates; 44.44%), CC30 (sixteen isolates; 35.56%), CC188 (five isolates; 11.11%), CC152 (1 isolate, 2.2%) and to a previously un-described sequence type (ST1755: arcc-18, aroe-115, glpf-6, gmk-2 pta-109, tpi-50 and yqil-2; three isolates; 6.67%). Resistance genes proved to be rare. Only three out of 45 isolates (6.67%) carried the beta-lactamase operon. The tetracycline resistance gene tetK was also detected in three isolates (6.67%). Neither the mecA gene, defining MRSA, nor other resistance genes were found. Common virulence markers included leukocidin genes lukD+lukE (in twenty-five isolates; 55.56%), the staphylokinase gene sak (twenty-two isolates; 48.89%), the enterotoxin gene cluster egc (fifteen isolates; 33.33%), and a distinct variant of the enterotoxin A gene (sea-320E, GenBank AY196686.1; thirteen isolates; 28.89%). One CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). This study provides first genotyping data on the population structure and the presence of toxin genes and resistance markers of S. aureus strains in Middle Eastern camels.

Clostridium perfringens de type A a été isolé dans différents organes et dans l'intestin de jeunes dromadaires âgés de 3 à 5 semaines, morts de nécrose du muscle cardiaque. Aucun autre agent pathogène bactérien n'a été isolé. L'isolement de virus sur deux lignées cellulaires différentes, dont une culture de cellules cutanées foetales de dromadaire s'est également avéré négatif. Les souris ayant reçu des injections de filtrats dépourvus de bactéries préparés à partir de contenus intestinaux de jeunes dromadaires autopsiés sont mortes après 1 à 6 h, révélant la présence de toxines clostridiennes. Nos résultats montrent que la nécrose du muscle cardiaque est provoquée par les toxines de Clostridium perfringens de type A.

We report a case of a 64-year-old veterinarian working in a state camel veterinary laboratory who was diagnosed with and treated for acute brucellosis with complicating epididymo-orchitis. Genomic tandem repeat analysis (MLVA-16) revealed identical Brucella strains in patient cultures and from different dromedary milk samples positive for Brucella melitensis, thereby confirming the diagnosis of a laboratory acquired infection. The case illustrates the high (airborne) infectivity of brucellosis in laboratory settings and the need to implement vigorous bio-safety measures in veterinary laboratories handling camel specimen diagnostic veterinary laboratory.

Background: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. Methods: We developed a B. mallei-specific 5′-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Results: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Conclusions: Compared with conventional PCR, our rapid 5′-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.

Background Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology. Methodology/Principal Findings We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA) and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources. Conclusion/Significance High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections., Author Summary Glanders is a disease of antiquity, recognized as a malady of equines by Hippocrates and Aristotle. The causative agent, Burkholderia mallei, is currently feared as a potential biological weapon and has been used as such in the American Civil War and both World Wars to cripple equine military components. In the more economically developed countries, glanders has been eradicated through large scale culling. As a result, our understanding of transmission dynamics and networks is limited. However, regions of endemicity still exist in Asia, the Middle-East, Africa, and South America where it infects solipeds and camels. These areas provide reservoirs for re-introduction of glanders into countries previously listed as glanders-free. Here, we demonstrate the utility of high-resolution genotyping and whole genome sequence analysis in the investigation of a recent outbreak of glanders in horses and camels in Bahrain, a previously declared glanders-free country. Our analyses demonstrate that not one, but two strains likely caused this outbreak, and that these strains probably came from a similar geographic region via importation of infected animals. Even with careful monitoring, the global trade of animals from glanders-endemic regions can re-introduce and possibly re-establish this disease in animal populations of countries that have previously eradicated it.