Assessing the impact of public education on a preventable zoonotic disease: rabies.

Effective methods to increase awareness of preventable infectious diseases are key components of successful control programmes. Rabies is an example of a disease with significant impact, where public awareness is variable. A recent awareness campaign in a rabies endemic region of Azerbaijan provided a unique opportunity to assess the efficacy of such campaigns. A cluster cross-sectional survey concerning rabies was undertaken following the awareness campaign in 600 households in 38 randomly selected towns, in districts covered by the campaign and matched control regions. This survey demonstrated that the relatively simple awareness campaign was effective at improving knowledge of rabies symptoms and vaccination schedules. Crucially, those in the awareness campaign group were also 1·4 times more likely to report that they had vaccinated their pets, an essential component of human rabies prevention. In addition, low knowledge of appropriate post-exposure treatment and animal sources of rabies provide information useful for future public awareness campaigns in the region and other similar areas.

Search for OIE-listed ruminant mycoplasma diseases in Afghanistan.

BACKGROUND: Little is known about the occurrence of important diseases of ruminants in Afghanistan because of the conflict affecting the country over the last 40 years. To address this discrepancy, ruminant herds in Afghanistan were screened for OIE-listed mycoplasma diseases, contagious bovine (CBPP) and caprine pleuropneumonias (CCPP). RESULTS: Of the 825 samples from 24 provinces tested for serological evidence of CBPP caused by Mycoplasma mycoides subsp.mycoides, 20 (3.4%) had ELISA values greater than the positive threshold of 50% though all were less than 55%. Repeat testing of these suspect sera gave values below 50. A smaller number of sera (330) from cattle in nine provinces were also tested by the rapid latex agglutination test (LAT) for CBPP, 10 of which were considered suspect. However, no positive bands were seen when immunoblotting was carried out on all sera that gave suspect results. Serological evidence of Mycoplasma bovis was detected in half of 28 herds in eight provinces. The cause of CCPP, M. capricolum subsp. capripneumoniae was not detected in any of the 107 nasal swabs and lung tissue collected from goats in seven provinces though sample handling and storage were not optimal. However, strong serological evidence was detected in goat herds in several villages near Kabul some of which were over 50% seropositive by LAT and ELISAs for CCPP; immunoblotting confirmed positive results on a selection of these sera. CONCLUSIONS: The data presented here provide a first assessment of the occurrence of the two OIE listed mycoplasma diseases in Afghanistan. From the results of the testing bovine sera from the majority of provinces there is no evidence of the presence of CBPP in Afghanistan. However the samples tested represented only 0.03% of the cattle population so a larger survey is required to confirm these findings. Serological, but not bacterial, evidence was produced during this investigation to show that CCPP is highly likely to be present in parts of Afghanistan.

Outbreak of encephalitic listeriosis in red-legged partridges (Alectoris rufa).

An outbreak of neurological disease was investigated in red-legged partridges between 8 and 28 days of age. Clinical signs included torticollis, head tilt and incoordination and over an initial eight day period approximately 30-40 fatalities occurred per day. No significant gross post mortem findings were detected. Histopathological examination of the brain and bacterial cultures followed by partial sequencing confirmed a diagnosis of encephalitis due to Listeria monocytogenes. Further isolates were obtained from follow-up carcasses, environmental samples and pooled tissue samples of newly imported day-old chicks prior to placement on farm. These isolates had the same antibiotic resistance pattern as the isolate of the initial post mortem submission and belonged to the same fluorescent amplified fragment length polymorphism (fAFLP) subtype. This suggested that the isolates were very closely related or identical and that the pathogen had entered the farm with the imported day-old chicks, resulting in disease manifestation in partridges between 8 and 28 days of age. Reports of outbreaks of encephalitic listeriosis in avian species are rare and this is to the best of our knowledge the first reported outbreak in red-legged partridges.

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.

Epidemiological characteristics of human and animal rabies in Azerbaijan.

The Caucasus is a region of geopolitical importance, in the gateway between Europe and Asia. This geographical location makes the region equally important in the epidemiology and control of transboundary infectious diseases such as rabies. Azerbaijan is the largest country in the Caucasus, and although rabies is notifiable and considered endemic, there is little information on the burden of human and animal rabies. Here, we describe a cross-disciplinary international collaboration aimed at improving rabies control in Azerbaijan. Partial nucleoprotein gene sequences were obtained from animal rabies cases for comparison with those from surrounding areas. Reported human and animal rabies cases between 2000 and 2010 were also reviewed and analysed by region and year. Comparison of rabies virus strains circulating in Azerbaijan demonstrates more than one lineage of rabies virus circulating concurrently in Azerbaijan and illustrates the need for further sample collection and characterization. Officially reported rabies data showed an increase in human and animal rabies cases, and an increase in animal bites requiring provision of post-exposure prophylaxis, since 2006. This is despite apparently consistent levels of dog vaccination and culling of stray dogs.

Comparison of Biolog GEN III MicroStation semi-automated bacterial identification system with matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S ribosomal RNA gene sequencing for the identification of bacteria of veterinary interest.

Recent advances in phenotypic and chemotaxonomic methods have improved the ability of systems to resolve bacterial identities at the species level. Key to the effective use of these systems is the ability to draw upon databases which can be augmented with new data gleaned from atypical or novel isolates. In this study we compared the performance of the Biolog GEN III identification system (hereafter, GEN III) with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing in the identification of isolates of veterinary interest. The use of strains that had proven more difficult to identify by routine methods was designed to test the systems' abilities at the extremes of their performance range. Over an 18month period, 100 strains were analysed by all three methods. To highlight the importance of identification to species level, a weighted scoring system was devised to differentiate the capacity to identify at genus and species levels. The overall relative weighted scores were 0.869:0.781:0.769, achieved by 16S rRNA gene sequencing, GEN III and MALDI-TOF MS respectively, when compared to the 'gold standard'. Performance to the genus level was significantly better using 16S rRNA gene sequencing; however, performance to the species level was similar for all three systems.

Demonstration of Ornithobacterium rhinotracheale in pheasants (Phasianus colchicus) with pneumonia and airsacculitis.

Outbreaks of respiratory disease were investigated in reared pheasants (Phasianus colchicus) aged approximately 18 to 32 weeks, released into the semi-wild on four shooting estates in southern England. The clinical signs in the affected birds included swelling of the face and eyes, loss of condition, gasping respirations and coughing. The gross pathology findings included sinusitis, airsacculitis, pleural oedema and lung lesions. The histopathological findings in the affected lungs were characterized by a granulomatous pneumonia. Ornithobacterium rhinotracheale (ORT) was isolated from respiratory tract tissues, and 16S rRNA gene sequencing on three isolates revealed two distinct genotypes, one previously associated with some electrophoretic type (ET) 1 strains and the other a novel genotype that clustered among sequences previously associated with ET 3, ET 4, ET 5 and ET 6 isolates. The localization of ORT within the lung tissue was demonstrated by fluorescent in-situ hybridization in the bronchial exudate of three cases, although not within the granulomatous lesions themselves. In each case, ORT was identified as part of a complex of other respiratory agents including avian paramyxovirus type 2, avian coronavirus, Mycoplasma gallisepticum, Mycoplasma synoviae and other Mycoplasma species, Escherichia coli, Pasteurella multocida, other Pasteurellaceae and Syngamus trachea, suggesting synergism with other agents. Exposure to other intercurrent factors, including adverse weather conditions and internal parasitism, may also have exacerbated the severity of disease.

Newly identified variability in Brucella canis fatty-acid content is associated with geographical origin.

This study compared the fatty-acid profiles of Brucella canis blood culture isolates obtained from infected dogs in the UK, Germany, Japan, South Africa, Peru, Mexico, Colombia, and Argentina, and from a human clinical case in Argentina, to a bank of isolates obtained from canine outbreaks in the USA. Analysis of a total of 42 B. canis isolates and one reference strain found a marked variation within the species. Fatty-acid analysis showed that only the isolates from Argentina, Colombia, and Mexico, which included the human B. canis isolate, contained a specific fatty acid, 19:0 cyclopropane (lactobacillic acid), w8c (cis-11,12-methylene octadecanoic acid), and that this fatty acid, when present, made up a large percentage of overall fatty-acid content. Prior to this study, the cellular fatty-acid 19:0 cyclopropane had been identified in all of the species of Brucella considered to be pathogenic to humans (B. abortus, B. melitensis, B. suis) except for B. canis. Discovering that this fatty acid not only occurs in B. canis, but also that it is only present in some strains of the species provides a new focus for investigations aimed at identifying the cause of reported geographical variability in human B. canis infection, and at finding predictors of biological behaviour and human pathogenicity within this Brucella species.

Association of a lukM-positive clone of Staphylococcus aureus with fatal exudative dermatitis in red squirrels (Sciurus vulgaris).

Fatal exudative dermatitis (FED) is a recently described condition affecting red squirrels (Sciurus vulgaris) on the Isle of Wight and Jersey (Simpson et al., 2010a). Staphylococcus aureus strains isolated from skin lesions in cases of FED were characterised by molecular and phenotypic approaches. The strains were found to belong to a single MLST clonal complex (CC49) representing either ST49 or a novel single locus variant thereof (ST1957), were closely related by other molecular typing approaches, and all possessed the leukotoxin M encoding gene (lukM). In contrast S. aureus was either not isolated from none-FED cases or belonged to distinct and diverse molecular types that, with one exception, did not encode lukM. All isolates from FED cases were susceptible to all antimicrobials tested, including penicillin, and all proved negative for mecA and mecC as well as 14 other staphylococcal toxin genes. As all squirrels affected by FED were infected with S. aureus of the same lineage and encoded the lukM gene, it is possible that strains of this lineage may be involved in the pathogenesis of the dermatitis.

Phenotypic characterisation and 16S rRNA sequence analysis of veterinary isolates of Streptococcus pluranimalium.

Forty-two isolates of Streptococcus pluranimalium were identified from cattle (n=38), sheep (n=2), an alpaca (n=1) and a pheasant (n=1) in the United Kingdom. The isolates were confirmed as S. pluranimalium by 16S rRNA sequence analysis but could not be differentiated reliably from Streptococcus acidominimus by phenotypic characterisation using commercial kits routinely used in veterinary laboratories. The alanyl-phenylalanyl-proline arylamidase reaction could be used to differentiate S. pluranimalium (positive) from Aerococcus urinae (negative).

Camelimonas abortus sp. nov., isolated from placental tissue of cattle.

A gram-negative, rod-shaped, non-spore-forming bacterium, isolated from placental tissue of a cow, was investigated for its taxonomic position. On the basis of 16S rRNA gene sequence similarities, strain UK34/07-5(T) was shown to belong to the class Alphaproteobacteria, closely related to the type strain of Camelimonas lactis (96.0 % sequence similarity). The polyamine pattern showed the major compound spermidine and moderate amounts of putrescine. The major quinone was ubiquinone Q-10. The polar lipid profile was composed of the major compounds phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine and moderate amounts of diphosphatidylglycerol, three unidentified aminolipids and an unidentified phospholipid. The profile of major fatty acids, consisting of C(19 : 0) cyclo ω8c and C(18 : 1)ω7c, with C(18 : 0) 3-OH as the hydroxylated fatty acid, was very similar to that of C. lactis M 2040(T). The results of DNA-DNA hybridization and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolate from C. lactis. The relatively low 16S rRNA gene sequence similarity of 96.0 % to C. lactis M 2040(T) and marked differences in the polar lipid profiles as well as the results of physiological tests and the DNA-DNA hybridization data support the creation of a novel species, for which the name Camelimonas abortus sp. nov. is proposed, with the type strain UK34/07-5(T) ( = CIP 110303(T)  = CCUG 61094(T)  = DSM 24741(T)  = CCM 7941(T)).

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century.

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio- and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

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

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

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

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