Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

The Prevalence and Genetic Characterization of Strains of Borrelia Isolated from Ixodes Tick Vectors in Belarus (2012-2019).

Borrelia burgdorferi sensu lato (s.l.) is the most common pathogen of medical significance transmitted by ticks of the family Ixodidae in Belarus. Human infection with B. burgdorferi causes Lyme borreliosis, most commonly referred to as Lyme disease. Currently, 20 species of Lyme disease-associated Borrelia and more than 20 relapsing fever-associated Borrelia species have been identified. These etiologic agents belong to the genus Borrelia in the family Spirochaetaceae. Genetically characterized isolates with specific sequences have proven that these pathogens are endemically transmitted in many European and Asian countries. In addition, joinpoint regression analysis is often applied to characterize infection trends over time and to identify the time point(s) at which the trend significantly changes. In this epidemiological investigation, joinpoint analysis was applied to investigate the temporal trend of B. burgdorferi s.l. infections in 4070 ticks collected between April and October 2012-2019. Detection of Borrelia species in ticks is an important tool to determine temporal and geographic distribution and abundance, and to predict the risk of Lyme disease to people in different regions. Our data provide a basis for further studies to determine the distribution and abundance of B. burgdorferi s.l. species in Belarus.

Overview of spatio-temporal distribution inferred by multi-locus sequence typing of Taylorella equigenitalis isolated worldwide from 1977 to 2018 in equidae.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977-2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

Presence of Antibodies Against Leptospira interrogans Serovar hardjo in Serum Samples from Cattle in Ukraine.

The article presents data on serological studies of 573 sera samples of cattle that were collected from the farms affected by leptospirosis in different regions of Ukraine in the period of 2014-2015. Samples were investigated by the microscopic agglutination test (MAT), which was conducted within eight serological groups of Leptospira and nine serovars: Sejroe (serovars polonica and hardjo), Hebdomadis (serovar kabura), Tarassovi (serovar tarassovi), Pomona (serovar pomona), Grippotyphosa (serovar grippotyphosa), Canicola (serovar canicola), Icterohaemorrhagiae (serovar copenhageni), and Australis (serovar bratislava). The circulation of L. interrogans serovar hardjo among cattle has been observed in all 11 regions of Ukraine investigated within 25.8-60.0% of the leptospirosis-positive serum samples in these regions. Antibodies in the cattle sera against serovar hardjo (serogroup Sejroe) were detected in 139 of the 370 cows reacting positively in MAT. Overall, they were detected in 24.3% animals out of the total of 573 cows investigated. These are the preliminary results, however, in our opinion, they should allow to include the serovar hardjo in a standard panel of strains for MAT in Ukraine.The article presents data on serological studies of 573 sera samples of cattle that were collected from the farms affected by leptospirosis in different regions of Ukraine in the period of 2014­2015. Samples were investigated by the microscopic agglutination test (MAT), which was conducted within eight serological groups of Leptospira and nine serovars: Sejroe (serovars polonica and hardjo), Hebdomadis (serovar kabura), Tarassovi (serovar tarassovi), Pomona (serovar pomona), Grippotyphosa (serovar grippotyphosa), Canicola (serovar canicola), Icterohaemorrhagiae (serovar copenhageni), and Australis (serovar bratislava). The circulation of L. interrogans serovar hardjo among cattle has been observed in all 11 regions of Ukraine investigated within 25.8­60.0% of the leptospirosis-positive serum samples in these regions. Antibodies in the cattle sera against serovar hardjo (serogroup Sejroe) were detected in 139 of the 370 cows reacting positively in MAT. Overall, they were detected in 24.3% animals out of the total of 573 cows investigated. These are the preliminary results, however, in our opinion, they should allow to include the serovar hardjo in a standard panel of strains for MAT in Ukraine.

Badgers as a potential source of bovine tuberculosis - first studies in Poland.

Since 2009, Poland has been recognized as a country officially free of bovine tuberculosis (BTB). However, new outbreaks are each year quoted. In many countries it has been shown that badgers (Meles meles) are a vector of Mycobacterium bovis/caprae (M.bovis/caprae) and a source of bovine tuberculosis for many domestical species, mainly for cattle. The aim of the presented study was to determine, for the first time in Poland, the occurrence of tuberculosis in badgers in areas where the disease occurs in cattle. Tissue samples were examined by classical microbiology methods, mycobacteria growth indicator tube (MGIT), and real time PCR. A total of 155 samples from 31 badgers were examined. In any case Mycobacterium bovis/caprae infection has not been diagnosed. This indicates that badgers probably are not a vector of bovine tuberculosis in Poland.

Seroprevalence of Selected Zoonotic Agents among Hunters from Eastern Poland.

The aim of our study was the collection of seroprevalence data for Toxoplasma gondii, Coxiella burnetii, Trichinella spp., and Francisella tularensis from hunters in Lublin Province. The antibodies against T. gondii and C. burnetii were recorded in 38.5% and 16.2% of the sera, respectively. 4.05% of the sera were seropositive for both T. gondii and C. burnetii. None of the sera tested reacted positively with F. tulariensis or Trichinella spp. Seroprevalence of T. gondii and C. burnetii is common among the hunters from Lublin Province. It seems reasonable to undertake similar research among hunters from other regions of eastern Poland.

Seroprevalence and risk factors for selected respiratory and reproductive tract pathogen exposure in European bison (Bison bonasus) in Poland.

After the complete extinction from the wild of European bison (Bison bonasus) at the beginning of the twentieth century, the worldwide species population was restored to approximately 5500 individuals, with the species however remaining endangered. Despite numerous studies on the ecology and genetics of European bison, the threats of infectious diseases have been largely unexamined. The aim of this study was to screen the exposure of the world's largest population of European bison to the pathogens, which may influence the condition and development of the endangered species. A total of 240 free-ranging and captive European bison from eight main Polish populations sampled were tested for the presence of specific antibodies against ten different viruses, bacteria or protozoan. The samples were collected from chemically immobilized, selectively culled or found dead animals. Based on serology, the exposure to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BoHV-1), Mycoplasma and Brucella spp. was determined as rather accidental. Using gamma-interferon assay followed by Mycobacterium tuberculosis subs. caprae detection in tissues, diagnosis of bovine tuberculosis was made for 6 out of 78 (7.7%) bison from one captive herd. The highest seroprevalence was found for bovine adenovirus type 3 (BAdV-3) -60.2% and bovine parainfluenza type 3 (PIV-3) -34.0%, while the antibodies against bovine respiratory syncytial virus (BRSV), Toxoplasma gondii and Leptospira spp. were found in 10.4%, 10.4% and 8.7% of samples, respectively. In the multivariable statistical analysis using generalized linear mixed models (GLMMS), the risk factors for PIV-3 seropositivity included population type (free-living/captive), age and health status (apparently healthy/eliminated due to the poor condition). Higher risk of BAdV-3 seropositive result was observed in free-living female European bison. The high BAdV-3 and PIV-3 seroprevalences may suggest involvement of these pathogens in the most frequently observed respiratory disorders in European bison. Moreover, this is the first study demonstrating BAdV-3 exposure in the species.

Detection of Listeria Spp. and Listeria Monocytogenes in Biological Samples by SYBR Green I and TaqMan Probe-based Real-time PCRs.

INTRODUCTION: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood. MATERIAL AND METHODS: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua,L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted. RESULTS: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products. CONCLUSION: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.

Brucella suis biovar 2 isolations from cattle in Poland.

Bovine brucellosis is an infectious disease caused by bacteria of the Brucella genus, primarily by B. abortus, less frequently by B. melitensis, and occasionally by B. suis. In the European Union, brucellosis in cattle has been eradicated in most of the Member States, which are recognized as 'officially free from bovine brucellosis'. Nevertheless, cattle herds continue to be serologically monitored for the potential re-emergence of the disease. The aim of the presented study was to show the results of bacteriological investigations of cattle slaughtered in Poland in years 2002-2011 on account of positive serological reactions for brucellosis. Specimens (sera and tissues) from 176 cows were examined. Sera from the animals were tested using RBT(rose bengal test), SAT (serum agglutination test), CFT (complement fixation test), 2-ME (2-mercaptoethanol test), Coombs (Coombs antiglobulin test) and ELISA (enzyme linked immunosorbant assay). Tissue samples were cultured for Brucella, according to official protocols. All sera were RBT and SAT-positive, 170 of them were CFT- positive, whereas 6 other samples were CFT negative while positive in Coombs and ELISA. In bacteriological examination, B. abortus was not isolated. On the other hand, B. suis biovar 2 was isolated from 5 cows, which had never been reported previously in Poland. Three cows came from the same herd. Conventional, as well as, molecular investigations based on PCR methods, confirmed that the bacteria isolated bacteria belong to the B. suis biovar 2. In Poland, as in many other European countries, wildlife (wild boars and hares) constitutes a huge reservoir of the said biovar. The results of the presented research indicate that B. suis biovar 2 can easily infect cattle, and undoubtedly plays a role in the epidemiology and control of bovine brucellosis.

Microscopic Li self-diffusion parameters in the lithiated anode material Li4 + xTi5O12 (0 < or = x < or = 3) measured by 7Li solid state NMR.

The microscopic Li diffusion parameters in the lithiated spinel Li4 + xTi5O12, which is on its way to become a commercially used anode material in Li ion batteries, are probed for the first time via nuclear magnetic resonance spectroscopy.

Ultraslow Li diffusion in spinel-type structured Li4Ti5O12 - a comparison of results from solid state NMR and impedance spectroscopy.

The cubic spinel oxides Li(1+x)Ti(2-x)O(4) (0 < or =x< or = 1/3) are promising anode materials for lithium-ion rechargeable batteries. The end member of the Li-Ti-O series, Li(4)Ti(5)O(12), can accommodate Li ions up to the composition Li(7)Ti(5)O(12). Whereas a number of studies focus on the electrochemical behaviour of Li insertion into and Li diffusion in the Li intercalated material, only few investigations about low-temperature Li dynamics in the non-intercalated host material Li(4)Ti(5)O(12) have been reported so far. In the present paper, Li diffusion in pure-phase microcrystalline Li(4)Ti(5)O(12) with an average particle size in the microm range was probed by (7)Li solid state NMR spectroscopy using spin-alignment echo (SAE) and spin-lattice relaxation (SLR) measurements. Between T = 295 K and 400 K extremely slow Li jump rates tau(-1) ranging from 1 s(-1) to about 2200 s(-1) were directly measured by recording the decay of spin-alignment echoes as a function of mixing time and constant evolution time. The results point out the slow Li diffusion in non-intercalated Li(4)Ti(5)O(12) x tau(-1) (1/T) follows Arrhenius behaviour with an activation energy E(ASAE) of about 0.86 eV. Interestingly, E(ASAE) is comparable to activation energies deduced from conductivity measurements (0.94(1) eV) and from SLR measurements in the rotating frame (0.74(2) eV) rather than from those performed in the laboratory frame, E(A)(low-T) = 0.26(1) eV at low T.