Rough Strains Research Articles

Characterization of prophages in Salmonella Typhimurium definitive type 8

Background Salmonella Typhimurium DT8 has been associated with human outbreaks. Anderson phage typing scheme has been used for more than four decades for subtyping of Salmonella Typhimurium, but it has shown some limitations. Here, we characterize prophages among Salmonella Typhimurium DT8 strains to test the potential use of prophage sequence profiles for subtyping. Method A total of 54 isolates of Salmonella Typhimurium DT8 were selected for this study which was part of an outbreak study by Ashton et al. (2015)*. Twenty-six isolates were associated with an outbreak in the States of Jersey in 2013 and 28 isolates were non-outbreak-associated isolates. Comparative genomics using a range of bioinformatic tools was carried out including SPAdes for de novo assembly of WGS data. PHASTER was used for characterisation of prophages (http://phaster.ca/). Result All DT8 strains are lysogenic for three prophages (RE-2010, ST64B and Gifsy-2). Moreover, the three prophages showed identical sequence among outbreak and non-outbreak isolates. Interestingly, prophage SSU5 was detected in one non-outbreak isolate. Prophage SSU5 is closely related to cryptic plasmid pHMC2 that infects only rough strains of Salmonella Typhimurium. This study is the first to report the presence of prophage SSU5 in Salmonella Typhimurium DT8. Conclusion It is crucial to use accurate, reliable and highly discriminative subtyping methods for epidemiological characterisation and outbreak investigation of Salmonella Typhimurium. Prophage profiling might be unsuitable subtyping method. The emerging genetic analysis should be combined with the conventional method for epidemiological surveillance until WGS-based analysis can be improved and standardized. *https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336196/

Detection of Enterobacteriaceae, antimicrobial susceptibility, and virulence genes of Escherichia coli in canaries (Serinus canaria) in northeastern Brazil

ABSTRACT: This study aimed to verify the presence of members from the Enterobacteriaceae family and determine antimicrobial susceptibility profiles of the isolates in canaries bred in northeastern Brazil; in addition, the presence of diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC) was also verified in these birds. Samples were collected during an exhibition organized by the Brazilian Ornithological Federation in July 2015 in Fortaleza, Brazil. A total of 88 fecal samples were collected and submitted to pre-enrichment step using buffered peptone water, followed by enrichment with the following broths: brain-heart infusion, Rappaport-Vassiliadis, and Selenite-Cystine. Subsequently, aliquots were streaked on MacConkey, brilliant green and salmonella-shigella agar plates. Colonies were selected according to morphological characteristics and submitted to biochemical identification and antimicrobial susceptibility tests with disk-diffusion technique. E. coli strains were evaluated for the presence of eight DEC genes and five APEC genes through conventional polymerase chain reaction (PCR) screening. The most frequent species observed were Pantoea agglomerans (25%), Serratia liquefaciens (12.5%), and Enterobacter aerogenes (9.1%). A single rough strain of Salmonella enterica subsp. enterica was identified in one sample (1.1%). High resistance rates to amoxicillin (78.7%) and ampicillin (75.4%) were identified. Polymyxin B (9.8%), gentamycin (6.6%), and enrofloxacin (6.6%) were the most efficient antibiotics. The total number of multidrug-resistant strains (isolates resistant to more than three antimicrobial classes) was 23 (37.7%). Four E. coli strains were tested for the virulence genes, and two were positive for APEC virulence genes: one strain was positive for iutA and the other for hlyF. In conclusion, canaries in northeastern Brazil participating in exhibitions may present Salmonella spp., Escherichia coli and other enterobacteria in the intestinal microbiota with antimicrobial resistance. These results indicate that, although the E. coli strains recovered from canaries in this study have some virulence genes, they still do not fulfill all the requirements to be considered APEC.

Phagosome Escape of Rough Mycobacterium abscessus Strains in Murine Macrophage via Phagosomal Rupture Can Lead to Type I Interferon Production and Their Cell-To-Cell Spread.

Mycobacterium abscessus complex (MAB) is a rapidly growing mycobacterium(RGM) whose clinical significance as an emerging human pathogen has been increasing worldwide. It has two types of colony morphology, a smooth (S) type, producing high glycopeptidolipid (GPL) content, and a rough (R) type, which produces low levels of GPLs and is associated with increased virulence. However, the mechanism responsible for their difference in virulence is poorly known. By ultrastructural examination of murine macrophages infected, we found that MAB-R strains could replicate more actively in the macrophage phagosome than the S variants and that they could escape into cytosol via phagosomal rupture. The cytosolic access of MAB-R strains via phagosomal rupture led to enhanced Type I interferon (IFN) production and cell death, which resulted in their cell-to-cell spreading. This behavior can provide an additional niche for the survival of MAB-R strains. In addition, we found that their enhancement of cell death mediated cell spreading are dependent on Type I IFN signaling via comparison of wild-type and IFNAR1 knockout mice. In conclusion, our data indicated that a transition of MAB-S strains into MAB-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading. This result provides not only a novel insight into the difference in virulence between MAB-R and -S variants but also hints to their treatment strategy.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis

ABSTRACTBrucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

Antimicrobial susceptibility and diarrheagenic diagnosis of Escherichia coli and Salmonella enterica isolated from feral pigeons (Columba livia) captured in Fortaleza, Brazil

ABSTRACT: This study aimed to isolate Escherichia coli and Salmonella enterica from captured feral pigeons in Fortaleza, Brazil, and, in addition to evaluate the antimicrobial susceptibility profiles and diagnose diarrheagenic E. coli strains. Pigeons were captured in four public locations in Fortaleza with three techniques. Individual cloacal swab samples were collected and submitted to bacterial isolation, biochemical identification and antimicrobial susceptibility test. Disk diffusion technique was used with twelve antibiotics. E. coli strains were submitted to DNA extraction followed by PCR to diagnose five diarrheagenic pathotypes. A total of 124 birds were captured. One bird was positive for Salmonella enterica (0.81%) and 121 (97.58%) were positive for E. coli. Among these, 110 isolates were submitted to antimicrobial susceptibility test and 28.18% (31/110) presented resistance to at least one antibiotic. Resistance to azithromycin was the most frequent (21.82%), followed by tetracycline (10.91%) and sulfamethoxazole with trimethoprim (8.9%). Multidrug resistance, calculated as a resistance to at least 3 antimicrobial classes, was identified in 3.64% (4/110) of strains. The maximum number of antimicrobial classes to which one strain was resistant was seven. Results demonstrated nine different resistance profiles and the most frequent was tetracycline and sulfamethoxazole with trimethoprim (4 strains), followed by chloramphenicol, azithromycin, tetracycline and sulfamethoxazole with trimethoprim (3 strains). Amoxicillin with clavulanic acid and tobramycin presented lowest levels of antimicrobial resistance, to which none of the tested strains were resistant. A single strain was positive for the eltB gene, which is a diagnostic tool to identify the Enterotoxigenic E. coli (ETEC) pathotype. None of the other investigated genes (stx1, stx2, estA, eaeA, ipaH, aatA and aaiC) were identified. The single isolate of S. enterica was a rough strain of Salmonella enterica subsp. enterica, but serotype identification was not possible. However, this isolate presented resistance to amoxicillin, amoxicillin with clavulanic acid, tetracycline and sulfamethoxazole with trimethoprim. Therefore, captured feral pigeons of Fortaleza presented a low prevalence of S. enterica and diarrheagenic E. coli. Considering the investigated pathogens, our results suggest a good health status and a low public health risk. However, important antimicrobial resistance profiles were identified.

The microbial toxicity of quaternary ammonium ionic liquids is dependent on the type of lipopolysaccharide

In the present study the toxicity of theophylline-based quaternary alkylammonium ionic liquids and their precursors was investigated in relation to smooth and rough Escherichia coli strains. We assumed that the microbial toxicity of the studied amphiphilic compounds was affected by the composition of the lipopolysaccharide (LPS) of Gram-negative bacteria. Our hypothesis was verified using K-12 (smooth) and R1−R4 (rough) strains which are characterised by differences in LPS, especially in the O-antigen region. Interactions between ionic liquids and DNA were also investigated. Digestion by Fpg protein was performed to evaluate DNA damage following both indirect action of ionic liquids and via lipid peroxidation. The results revealed differences in sensitivity to the tested compounds between smooth and rough strains, as well as differences in peroxidase activity and lipid peroxidation. The ionic liquids did not interfere with DNA in vitro, and intracellular DNA damage that occurred after treatment was probably caused by lipid peroxidation and oxidative stress.

Structural definition of hSP-D recognition of Salmonella enterica LPS inner core oligosaccharides reveals alternative binding modes for the same LPS.

The crystal structures of a biologically and therapeutically active recombinant homotrimeric fragment of native human SP-D (hSP-D) complexed with the inner core oligosaccharide of the Salmonella enterica sv Minnesota rough strains R5 and R7 (rough mutant chemotypes Rc and Rd1) have been determined. The structures reveal that hSP-D specifically and preferentially targets the LPS inner core via the innermost conserved Hep-Kdo pair with the flexibility for alternative recognition when this preferred epitope is not available for binding. Hep-Kdo binding is achieved through calcium dependent recognition of the heptose dihydroxyethyl side chain coupled with specific interactions between the Kdo and the binding site flanking residues Arg343 and Asp325 with evidence for an extended binding site for LPS inner cores containing multiple Kdo residues. In one subunit of the R5-bound structure this preferred mode of binding is precluded by the crystal lattice and oligosaccharide is bound through the terminal inner core glucose. The structures presented here thus provide unique multiple insights into the recognition and binding of bacterial LPS by hSP-D. Not only is it demonstrated that hSP-D targets the highly conserved LPS proximal inner core Hep-Kdo motif, but also that hSP-D can recognise either terminal or non-terminal sugars and has the flexibility and versatility to adopt alternative strategies for bacterial recognition, utilising alternative LPS epitopes when the preferred inner core Hep-Kdo disaccharide is not available for binding.

Evidence of ovine brucellosis due to Brucella ovis and Brucella melitensis in Karnataka, India

Ovine brucellosis is often neglected contagious bacterial disease causing enormous economic losses to sheep industry. India is recognized as geographical hotspot for brucellosis and there is only one seroprevalence report of Brucella ovis infection in sheep. Ovine brucellosis is caused by both rough and smooth strains of Brucella sp. In the present study, 300 sheep serum samples collected from 9 different districts of Karnataka by 3 stage random sampling approach were screened by antibody and DNA detection tests (RBPT and iELISA) and PCR. Antibodies specific to B. melitensis and B. ovis infection were 8.67% and 5.34%, respectively. In the same set of samples, 5% (15/300) showed Brucella DNA amplification by bcsp31 and IS711 genus specific PCRs. In B. melitensis and B. ovis species specific PCRs, 3.34% and 1.67%, respectively were detected positive indicating presence of both Brucella species in sheep population. Among the 7 districts, Yadgir, Tumkur, Raichur and Bagalkot showed higher prevalence of antibodies against rough and smooth strains of Brucella. This emphasizes the need for sensitization of national system in designing surveillance and control strategies for both B. ovis and B. melitensis infections in sheep.

Neutrophil killing of Mycobacterium abscessus by intra- and extracellular mechanisms.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, are increasingly present in soft tissue infections and chronic lung diseases, including cystic fibrosis, and infections are characterized by growth in neutrophil-rich environments. M. abscessus is observed as two distinct smooth and rough morphotypes. The environmental smooth morphotype initiates infection and has a relatively limited ability to activate neutrophils. The rough morphotype has increased virulence and immunogenicity. However, the neutrophil response to the rough morphotype has not been explored. Killing of the smooth and rough strains, including cystic fibrosis clinical isolates, was equivalent. Neutrophil uptake of M. abscessus was similar between morphotypes. Mechanistically, both rough and smooth morphotypes enhanced neutrophil reactive oxygen species generation but inhibition of NADPH oxidase activity did not affect M. abscessus viability. However, inhibition of phagocytosis and extracellular traps reduced killing of the smooth morphotype with lesser effects against the rough morphotype. Neutrophils treated with M. abscessus released a heat-labile mycobactericidal activity against the rough morphotype, but the activity was heat-tolerant against the smooth morphotype. Overall, M. abscessus stimulates ineffective neutrophil reactive oxygen species generation, and key mechanisms differ in killing of the smooth (phagocytosis-dependent, extracellular traps, and heat-tolerant secreted factor) and rough (extracellular traps and a heat-labile secreted factor) morphotypes. These studies represent an essential advancement in understanding the host response to M. abscessus, and help explain the recalcitrance of infection.

Effects of feedstock and co-culture of Lactobacillus fermentum and wild Saccharomyces cerevisiae strain during fuel ethanol fermentation by the industrial yeast strain PE-2

Even though contamination by bacteria and wild yeasts are frequently observed during fuel ethanol fermentation, our knowledge regarding the effects of both contaminants together is very limited, especially considering that the must composition can vary from exclusively sugarcane juice to a mixture of molasses and juice, affecting the microbial development. Here we studied the effects of the feedstock (sugarcane juice and molasses) and the co-culture of Lactobacillus fermentum and a wild Saccharomyces cerevisiae strain (rough colony and pseudohyphae) in single and multiple-batch fermentation trials with an industrial strain of S. cerevisiae (PE-2) as starter yeast. The results indicate that in multiple-cycle batch system, the feedstock had a minor impact on the fermentation than in single-cycle batch system, however the rough yeast contamination was more harmful than the bacterial contamination in multiple-cycle batch fermentation. The inoculation of both contaminants did not potentiate the detrimental effect in any substrate. The residual sugar concentration in the fermented broth had a higher concentration of fructose than glucose for all fermentations, but in the presence of the rough yeast, the discrepancy between fructose and glucose concentrations were markedly higher, especially in molasses. The biggest problem associated with incomplete fermentation seemed to be the lower consumption rate of sugar and the reduced fructose preference of the rough yeast rather than the lower invertase activity. Lower ethanol production, acetate production and higher residual sugar concentration are characteristics strongly associated with the rough yeast strain and they were not potentiated with the inoculation of L. fermentum.

Characterization and zoonotic impact of Shiga toxin producing Escherichia coli in some wild bird species.

Aim:Wild birds are considered silent vectors of some zoonotic water and food borne pathogens of public health significance. Owing to the importance of Shiga toxin producing Escherichia coli (STEC) as the most pathogenic among the emerging diarrheagenic E. coli groups that can infect man; the present study was designed to detect the occurrence of STEC among wild birds in Egypt.Materials and Methods:A total of 177 intestinal content swab samples originating from five wild bird species were investigated for the presence of E. coli and STEC by standard culture methods. Suspect STEC isolates were further characterized by serotyping, random amplified polymorphic DNA polymerase chain reaction (RAPD PCR), antimicrobial resistance pattern and PCR detection of stx1, stx2, and eae genes.Results:A total of 30 suspect STEC isolates from 30 positive birds’ samples were detected and identified on STEC CHROMagar (semi-captive pigeons, 15; house crows, 8; cattle egrets, 3; moorhens, 2; and house teals, 2). 25 isolates were grouped into 13 serogroups (O:20, O:25, O:26, O:27, O:63, O:78, O:111, O:114, O:125, O:128, O:142, O:153, and O:158), while five were rough strains. The distribution of STEC virulence genes among wild birds was as follows: 16 birds carried stx1 gene only (nine pigeons [28.1%], six crows [7.1%], and one cattle egret [5.6%]). Stx1 and stx2 genes together were detected in four birds (one cattle egret [5.6%], two moorhens [6.1%], and one house teal, [10%]). Only one pigeon (3.1%) possessed the three alleles. Disk diffusion test results showed that cefixime was the most effective against STEC serotypes with (93.3%) sensitivity, followed by gentamycin (56.7%), and amoxicillin (50%). On the other hand, all the recovered STEC isolates were resistant to cefotaxime, doxycycline, cephalothin, and sulfisoxazole. RAPD fingerprinting using primers OPA-2 and OPA-9 showed that STEC isolates were heterogeneous; they yielded 30 and 27 different clusters, respectively.Conclusions:Wild birds carry STEC and may add to the contamination of the surrounding environment.

Safety of vaccination against brucellosis with the rough strain in pregnant cattle.

Brucellosis is an infectious and contagious disease that profoundly impacts public health. However, in many countries, disease prevention is restricted to the vaccination of calves, and there is no prophylactic strategy for pregnant heifers and cows. The aim of this study was to evaluate the safety of the rough strain vaccine against brucellosis in pregnant cattle. Crossbred cows (N=96) at three gestational periods (early, mid, or late pregnancy) were randomly allocated into the vaccine treatment group or to the control group. We then compared the percentage of pregnancies reaching full term, live calves 60days after delivery, and seropositive calves. There was no effect of vaccination in any of the gestational periods on the evaluation endpoints. In conclusion, vaccination against brucellosis with the rough strain is safe for pregnant cattle at all gestational periods.

Not a barrier but a key: How bacteriophages exploit host's O-antigen as an essential receptor to initiate infection.

Tailed bacteriophages specific for Gram-negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events that orchestrate phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O-antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O-antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O-antigen specific host attachment must not always come along with O-antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O-antigen but does not degrade it, whereas rough strains or strains lacking O-antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host's O-antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O-antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps.

Structural diversity of Burkholderia pseudomallei lipopolysaccharides affects innate immune signaling.

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines.

Rough brucella strain RM57 is attenuated and confers protection against Brucella melitensis.

Brucella melitensis (B.melitensis) is a facultative intracellular pathogen, which is the main epidemic strain in China. To overcome disadvantages of traditional live attenuated vaccines, in this study a rough mutant RM57 was induced from a B.melitensis isolate M1981. In order to uncover the reason of changes in the LPS of RM57, the nucleotide sequences and transcription levels of all known genes related to LPS synthesis were detected. As LPS plays an important role in outer membrane integrity, the sensitivities of RM57 to SDS and polymyxin B were examined. The results showed that the expression level of LPS genes of RM57 was not significantly changed, and RM57 was sensitive to polymyxin B, compared to its parent strain. In further study, the virulence and protective efficacy of RM57 in mice and guinea pigs were determined, and our data indicated that RM57 was attenuated and had good protection effects, especially in guinea pigs model. Overall, these results demonstrated that the artificially induced rough mutant strain RM57 was an efficacious vaccine candidate against the challenge of virulent B.melitensis. Our data presented here provided additional insight into the mechanism of LPS synthesis of Brucella spp.

Bacteriophage T7 DNA Translocation during Infection is Bolstered by the Host ATP Synthase Complex

Translocation of bacteriophage T7 dsDNA into its host cell is an integral step in infection initiation that is energy dependent and is catalyzed principally by transcription per host and viral RNA polymerases. Here we show utilizing high-throughput cryo-electron tomography, a hexameric ring complex exists below the T7 trans-envelope channel in the host-cell cytoplasm during infection. Using sub-tomogram averaging and extensive classification we isolated T7 in the brief stage of genome translocation in coordination with the ring. This host-cell nanomachine is ∼30nm in diameter, ∼8nm in height with a ∼10nm channel. The complex was not observed in ATP synthase-deficient Escherichia coli mutants, suggesting that the complex functions in an energy-dependent manner. T7 grows in these mutant strains, and thus DNA ejection is not dependent on the complex. The ring is not host-specific; it has also been observed in an infected rough strain of Salmonella typhimurium, which supports the growth of T7. These results, only obtainable by in situ structural analysis, provide details on the essential process of DNA translocation and reveal the ability of T7 to recruit host-cell machinery to aid in the initiation of infection.

Effect of levamisole administration on immunogenic and protective capacity of Brucella abortus RB51

Background: Brucella abortus strain RB51 is a rough strain that was derived after multiple passages of virulent strain 2308. Levamisole, a potent anthelmintic that is widely used in veterinary medicine, is also known as an immune stimulant. Aim and Objectives: Determine the capability of levamisole to enhance the humoral antibody response and cell-mediated immunity as well as protective capacity of RB51 vaccine in mice against challenge with virulent Brucella melitensis strain. Materials and Methods: Six groups of 7–8-week-old female Bulb/C mice were used. Group I was vaccinated I/P with 5 × 108 colony-forming units (CFU) of RB51 strain. Group II was vaccinated I/P with 5 × 108 CFU of RB51 strain simultaneously injected (at 0 day) s/cu with 12.5 mg/Kg levamisole. Group III was vaccinated I/P with 5 × 108 CFU of RB51 strain and injected at 7-day postvaccination with 12.5 mg/Kg levamisole. Group IV was vaccinated I/P with 5 × 108 CFU of RB51 strain and injected at the day of vaccination (0 day) and 7-day postvaccination with 12.5 mg/Kg levamisole. Group V was injected with levamisole alone. Group VI was kept as control group. 8-week postvaccination; all vaccinated as well as control animals groups received I/P challenge of 2 ml dose of 2 × 104 CFU/ml of virulent strain of B. melitensis biovar 3. Results: (Groups III and IV) At the day of vaccination (0 day) and 7-day postvaccination showed the highest significant serum antibody titer measured by ELISA all over the experimental period as well as increase in delayed type hypersensitivity (DTH) response 48 h after elicitation by intradermal inoculation of B. abortus soluble antigen compared to vaccinated control group (Group I). While RB51 vaccinated and simultaneously treated with levamisole (Group II) showed mild elevation in antibody titer as well as the lowest significant increase in DTH response compared to vaccinated control group. Bioassay test of challenged mice groups showed significant improvement in the resistance of vaccinated groups treated with levamisole either 7-day post-vaccination (Group III) or 0 and 7 days postvaccination (Group IV) which showed 10% bacteriologically positive mice, whereas only 30% of the vaccinated control mice (Group I) and vaccinated simultaneously treated with levamisole (Group II), were bacteriologically positive.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781.

We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

Molecular typing of isolates obtained from aborted foetuses in Brucella-free Holstein dairy cattle herd after immunisation with Brucella abortus RB51 vaccine in Egypt

Bovine brucellosis is endemic in Egypt in spite of application of surveillance and control measures. An increase of abortions was reported in a Holstein dairy cattle herd with 600 animals in Damietta governorate in Egypt after immunisation with Brucella (B.) abortus RB51 vaccine. Twenty one (10.6%) of 197 vaccinated cows aborted after 3 months. All aborted cows had been tested seronegative for brucellosis in the past 3 years. B. abortus was isolated from four foetuses. Conventional biochemical and bacteriological identification and polymerase chain reaction (PCR) confirmed two B. abortus biovar (bv.) 1 smooth and two B. abortus rough strains. None of the B. abortus isolates were identified as RB51. Genotyping analysis by multiple locus of variable number tandem repeats analysis based on 16 markers (MLVA-16) revealed two different profiles with low genetic diversity. B. abortus bv1 was introduced in the herd and caused abortions.