Isolation and molecular characterization of Mycobacterium bovis from Kafue lechwe (Kobus leche kafuensis) from Zambia

Bovine tuberculosis (BTB) is a chronic bacterial disease caused by Mycobacterium bovis. Infections due to M. bovis, which serves as a stable reservoir, can pose serious challenge to control and eradicate in both wildlife and livestock at the interface. This study aimed at isolating and characterizing M. bovis from Kafue lechwe (Kobus leche kafuensis) and black lechwe (Kobus leche smithemani) at the animal/human interface in Zambia. The samples with lesions compatible with BTB collected during the hunting seasons of 2009 and 2010 were cultured for isolation of mycobacteria using Stonebrink with pyruvate (BD Diagnostics, MD, USA) and Middlebrook 7H10 (BD Diagnostics) slants. Isolated mycobacteria were identified using IS6110 polymerase chain reaction and deletion analysis. Molecular characterization of the isolates was performed using spoligotyping and mycobacteria interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR) with nine loci. Data was analyzed using BioNumerics software 6.1. Out of the 39 samples, acid fast bacilli were detected in 27 (69.2 %) based on smear microscopy. Seven isolates were found to belong to Mycobacterium tuberculosis complex, and all were identified as M. bovis based on deletion analysis. All seven isolates were identical on spoligotyping as belonging to the SB0120 (SIT 482). MIRU–VNTR differentiated the isolates into five different patterns. This study has confirmed that M. bovis circulates in the Kafue lechwe, and non-tuberculous mycobacteria were detected in the black lechwe in Zambia which represents a wildlife reservoir, with a potential to spillover to cattle and humans. Isolates of M. bovis from lechwe antelopes are much conserved as only one spoligotype was detected. The study has shown that three loci differentiated fairly well. This option is cheap and less laborious, and hence a better option in resource-strained country like Zambia. The study further showed that some of the loci recommended by the European Reference Laboratory are not suitable for typing M. bovis in Zambia.     

Comparison of Multistage Versus One-Stage Destabilization of a Type II External Fixator Used to Stabilize an Oblique Tibial Osteotomy in Dogs

OBJECTIVE: To compare the biomechanical effects of multistage versus one-stage destabilization of a type II external skeletal fixator (ESF) used to stabilize an oblique unstable tibial osteotomy in dogs. STUDY DESIGN: In vitro, in vivo, and ex vivo experimental study. ANIMAL POPULATION: Twelve healthy adult dogs. METHODS: The biomechanical characteristics of the type II ESF used in this study were determined. This fixator was applied to both tibiae of two groups of 6 dogs to stabilize a 2-mm-wide oblique osteotomy. One fixator on each dog remained unchanged throughout the 11-week study (control group). The fixator on the opposite limb was destabilized late and acutely in one group of dogs (single-stage) and early and progressively in the other (multistage). Clinical examination, radiographic examination, and force-plate analysis were used to evaluate the results. All dogs were euthanatized at 11 weeks. All tibiae were scanned to determine the cross-sectional area of the callus in the center of the osteotomy and subjected to biomechanical tests to determine mean pull-out strength of pins and callus strength and stiffness. RESULTS: Stiffness of the type II ESF used in this study was 578 N/mm in axial compression, 0.767 Nm/deg in torsion, 261 N/mm in medio-lateral bending, and 25 N/mm in cranio-caudal bending. Peak vertical forces of the hindlimbs were significantly lower at 2.5 and 5 weeks than before surgery. Peak vertical forces of the hindlimbs did not change before and after destabilization. No significant differences could be detected between the two destabilization sequences or between all control tibiae and pooled destabilized tibiae with regards to radiographic evaluation of the healing osteotomy, cross-sectional periosteal callus area, mean pull-out strength of transfixation pins, callus strength, and callus stiffness. CONCLUSIONS AND CLINICAL RELEVANCE: Bone healing of unstable osteotomies stabilized with a type II ESF is not significantly enhanced by staged destabilization of the fixation as performed in this study.     

Determination of affinity of Pasteurella multocida isolates for porcine respiratory tract mucus, and partial characterization of the receptors

The ability of 25 Pasteurella multocida isolates to adhere in vitro to porcine respiratory tract mucus was examined. Microplate wells were coated with crude mucus preparation, then bacteria were added. After incubation, unbound bacteria were removed by washing, and the number of mucus-bound bacteria was estimated by quantitation of the adherent colony-forming units and by use of an ELISA. Pasteurella multocida had affinity to respiratory tract mucus, although significant differences were not observed in affinity of capsular type-A and type-D isolates. Preliminary characterization, using ultrafiltration, gel filtration chromatography, electrophoresis, and enzymatic treatments, indicated that the receptors may be a class of protein molecules of low molecular weight (less than 25,000). The origin of these receptors, however, is not known at this time.     

Analysis of epidermal lipids in normal and atopic dogs,before and after administration of an oral omega-6/omega-3 fatty acid feed supplement. A pilot study

Alterations of the lipid expression in the skin of human and canine atopic subjects may be one of the key factors in the disease development. We have analyzed the ultrastructure of the clinically uninvolved skin of atopic dogs and compared it with the lipid composition of their tape-stripped stratum corneum (SC). The effect of a 2 month treatment of atopic dogs by food supplementation with a mixture of essential fatty acids was evaluated on skin samples taken before and after the treatment period. Electron microscopy revealed that the non-lesional skin of atopic dogs exhibited an abnormal and largely incomplete structure of the lamellar lipids with little cohesion between the corneocyte strata. The SC of atopic dogs was characterized by a significant decrease in the lipid content when compared to the healthy controls. Following oral supplementation with the mixture of essential fatty acids, the overall lipid content of the SC markedly increased. This feature was observed both with the free and, most importantly, with the protein-bound lipids (cholesterol, fatty acids and ceramides), the latter constituting the corneocyte-bound scaffold for ordinate organisation of the extracellular lipid bi-layers. Indeed, the semi-quantitative electron microscopy study revealed that the treatment resulted in a significantly improved organization of the lamellar lipids in the lower SC, comparable to that of the healthy dogs. Our results indicate the potential interest of long-term alimentary supplementation with omega-6 and omega-3 essential fatty acids in canine atopic dermatitis.     

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.     

Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies

With the growing emergence of antibiotic resistance and rising consumer demands concerning food safety, vaccination to prevent bacterial infections is of increasing relevance. Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. Despite all the research and trials that were performed with A. pleuropneumoniae vaccination in the past, a safe vaccine that offers complete protection against all serotypes has yet not reached the market. However, recent advances made in the identification of new potential vaccine candidates and in the targeting of specific immune responses, give encouraging vaccination perspectives. Here, we review past and current knowledge on A. pleuropneumoniae vaccines as well as the newly available genomic tools and vaccination strategies that could be useful in the design of an efficient vaccine against A. pleuropneumoniae infection.     

Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.     

The ultrastructure of spermatogenic cells and morphological evaluation of testicular development in the silver pomfret (Pampus argenteus)

The silver pomfret (Pampus argenteus) is a widely distributed and economically important marine fish in the Indo-Pacific. In this study, we acquired the second generation of wild P. argenteus by artificial breeding and further studied the testicular development and ultrastructure of spermatogenesis. The results of gonadosomatic index (GSI) showed the spawning period of this marine fish was from April to June. Besides, through morphological analysis, we found that P. argenteus had an anastomosing tubular testis surrounded by a layer of tunica albuginea, in which spermatogenesis occurred in cysts where the synchronous germ cells were completely surrounded by the cytoplasmic projection of Sertoli cells. Meanwhile, based on submicroscopic characteristics, the germ cells are classified into nine different types. During the ontogenesis of testis, both the early stage of spermatogenesis and sperm were observed in P. argenteus. At sperm maturation stage, different types of spermatozoa and activation of sperms occurred non-synchronously in the tubules. Cytoplasmic bridges also were observed among synchronous germ cells within the cysts, suggesting an interrelated and differentiated relationship among these germ cells.