Decentralised system for demand-oriented collection of food waste - Assessment of biomethane potential, pathogen development and microbial community structure.

Enormous amounts of food waste (FW) are produced worldwide, requiring efficient disposal strategies, both economically and ecologically. Anaerobic digestion to produce biomethane is among the most promising strategies, but requires proper solutions for storage and delivery of the waste material. Here, a decentralized system for demand-oriented FW storage and its practical usability was assessed. FW was stored under batch and fed-batch strategies at 5 °C, 20 °C and 30 °C for 28 days. The results showed that FW can be stored without cooling since bacterially produced lactic acid rapidly stabilized the material and inactivated pathogens. While FW storage worked well under all storage conditions and strategies, 16S analysis revealed a distinct microbiota, which was highly characteristic for each storage temperature. Moreover, FW storage had no negative impact on methane yield and stored FW contained readily degradable substances for demand-oriented biogas production.

Heme ladder, a direct molecular weight marker for immunoblot analysis.

Detection methods for immunoblot analysis are often based on peroxidase conjugates. However, molecular weight markers directly detectable for general use in such systems are not available. Here, we describe the preparation of a direct molecular weight marker consisting of heme-tagged proteins, whose enzymatic activities make them detectable simultaneously with the antigen in peroxidase-based immunoblot systems. The peroxidase activity results from the covalent attachment of heme to selected engineered periplasmic proteins, catalyzed by the cytochrome c maturation system of Escherichia coli. The newly designed heme-tagged proteins were combined with a previously constructed heme-tagged maltose-binding protein and cytochrome c. The resulting heme ladder was shown to be suitable as a protein standard for direct molecular weight estimation in immunoblot analysis due to the peroxidase activity of its constituents. The heme ladder consists of proteins between 12 and 85 kDa and can be produced at low cost. The marker was stable when kept at 4, -20, and -80°C for >6 months.

Detection of Mycoplasma conjunctivae in the eyes of healthy, free-ranging Alpine ibex: possible involvement of Alpine ibex as carriers for the main causing agent of infectious keratoconjunctivitis in wild Caprinae.

Mycoplasma conjunctivae is considered the major cause of infectious keratoconjunctivitis (IKC) in Alpine ibex (Capra i. ibex) and chamois (Rupicapra r. rupicapra). While it is known that domestic sheep can act as healthy carriers for M. conjunctivae, this question has not been addressed in wild ungulates so far. In this study, bacteriological investigations and field observations were performed to assess whether free-ranging Alpine ibex can be healthy carriers of M. conjunctivae. Among 136 ibex without clinical signs of IKC, M. conjunctivae was identified 26 times (19.1%) by TaqMan PCR. To assess the potential pathogenicity of M. conjunctivae strains isolated from asymptomatic eyes, strains from three healthy ibex and from 15 IKC-ibex and IKC-chamois were analysed genetically by DNA sequence analysis of the variable part of the lppS gene. No significant differences were observed between strains from asymptomatic and clinically affected animals, reflecting the assumption that healthy ibex may act as carriers for M. conjunctivae strains that may be pathogenic for other individuals. Our results further indicate that development of IKC is associated with M. conjunctivae load in the eyes. In addition, a questionnaire survey revealed that IKC is generally less common in ibex than chamois and that infection in wild ungulates is not necessarily linked to the presence of sheep. These data support the hypothesis that apparently healthy ibex may be important in the epizootiology of IKC and indicate that host predilection may play a role in IKC development.

First report of Mycoplasma conjunctivae from wild Caprinae with infectious keratoconjunctivitis in the Pyrenees (NE Spain).

Frequent outbreaks of infectious keratoconjunctivitis have been reported in wild Caprinae in Europe. While etiologic studies in the Alps indicate that the main etiologic agent is Mycoplasma conjunctivae, there are few reports from other mountain areas, such as the Pyrenees, where M. conjunctivae has never been reported. In 2006 and 2007, five adult Pyrenean chamois (Rupicapra pyrenaica; two males and three females) and one adult male European mouflon (Ovis orientalis musimon) were studied; they exhibited clinical symptoms of infectious keratoconjunctivitis such as blindness, corneal opacity, and ulceration. In three of the five chamois tested, and in the mouflon, Mycoplasma conjunctivae was identified from conjunctival swabs by means of a TaqMan(R) polymerase chain reaction based on the lipoprotein gene lppS. Cluster analysis indicated that the three southern chamois isolates form a cluster that is distinct from the mouflon isolate. This is the first report of M. conjunctivae in Pyrenean chamois, and it supports the hypothesis that M. conjunctivae also could be the main cause of infectious keratoconjunctivitis in areas other than the Alps, such as the Pyrenees.

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System.

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model.

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.

Optimization of recombinant adeno-associated viral vectors for human beta-globin gene transfer and transgene expression.

Therapeutic levels of expression of the beta-globin gene have been difficult to achieve with conventional retroviral vectors without the inclusion of DNase I-hypersensitive site (HS2, HS3, and HS4) enhancer elements. We generated recombinant adeno-associated viral (AAV) vectors carrying an antisickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer or the erythroid cell-specific human parvovirus B19 promoter at map unit 6 (B19p6) without any enhancer, and tested their efficacy in a human erythroid cell line (K-562) and in primary murine hematopoietic progenitor cells (c-kit(+)lin()). We report here that (1) self-complementary AAV serotype 2 (scAAV2)-beta-globin vectors containing only the HS2 enhancer are more efficient than single-stranded AAV (ssAAV2)-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (2) scAAV2-beta-globin vectors recombine with scAAV2-HS2+HS3+HS4 vectors after dual-vector transduction, leading to transgene expression; (3) scAAV2-beta-globin as well as scAAV1-beta-globin vectors containing the B19p6 promoter without the HS2 enhancer element are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (4) scAAV2-B19p6-beta-globin vectors in K-562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit(+)lin() cells, yield efficient expression of the beta-globin protein. Thus, the combined use of scAAV vectors and the parvovirus B19 promoter may lead to expression of therapeutic levels the beta-globin gene in human erythroid cells, which has implications in the use of these vectors in gene therapy of beta-thalassemia and sickle cell disease.

Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells.

The cytotoxicities of various strains of Mycoplasma mycoides subsp. mycoides small colony type (SC), the agent of contagious bovine pleuropneumonia (CBPP), were measured in vitro using embryonic calf nasal epithelial (ECaNEp) cells. Strains isolated from acute cases of CBPP induced high cytotoxicity in the presence of glycerol, concomitant with the release of large amounts of toxic H2O2 that were found to be translocated into the cytoplasms of the host cells by close contact of the Mycoplasma strains with the host cells. Currently used vaccine strains also showed high cytotoxicity and high H2O2 release, indicating that they are attenuated in another virulence attribute. Strains isolated from recent European outbreaks of CBPP with mild clinical signs, which are characterized by a defect in the glycerol uptake system, released small amounts of H2O2 and showed low cytotoxicity to ECaNEp cells. M. mycoides subsp. mycoides SC strain PG1 released large amounts of H2O2 but was only slightly cytotoxic. PG1 was found to have a reduced capacity to bind to ECaNEp cells and was unable to translocate H2O2 into the bovine cells, in contrast to virulent strains that release large amounts of H2O2. Thus, an efficient translocation of H2O2 into host cells is a prerequisite for the cytotoxic effect and requires an intact adhesion mechanism to ensure a close contact between mycoplasmas and host cells.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects.

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Vector production in an academic environment: a tool to assess production costs.

Generating gene and cell therapy products under good manufacturing practices is a complex process. When determining the cost of these products, researchers must consider the large number of supplies used for manufacturing and the personnel and facility costs to generate vector and maintain a cleanroom facility. To facilitate cost estimates, the Indiana University Vector Production Facility teamed with the Indiana University Kelley School of Business to develop a costing tool that, in turn, provides pricing. The tool is designed in Microsoft Excel and is customizable to meet the needs of other core facilities. It is available from the National Gene Vector Biorepository. The tool allows cost determinations using three different costing methods and was developed in an effort to meet the A21 circular requirements for U.S. core facilities performing work for federally funded projects. The costing tool analysis reveals that the cost of vector production does not have a linear relationship with batch size. For example, increasing the production from 9 to18 liters of a retroviral vector product increases total costs a modest 1.2-fold rather than doubling in total cost. The analysis discussed in this article will help core facilities and investigators plan a cost-effective strategy for gene and cell therapy production.

Using Pulmozyme DNase treatment in lentiviral vector production.

In the production of lentiviral vector for clinical studies the purity of the final product is of vital importance. To remove plasmid and producer cell line DNA, investigators have incubated the vector product with Benzonase, a bacterially derived DNase. As an alternative we investigated the use of Pulmozyme, a U.S. Food and Drug Administration-approved human DNase for the treatment of cystic fibrosis, by comparing the efficiency of DNA removal from lentiviral vector preparations. A green fluorescent protein-expressing lentiviral vector was prepared by transient calcium phosphate transfection of HEK 293T cells and DNA removal was compared when treating vector after harvest or immediately after transfection. The effectiveness of DNase treatment was measured by quantitative PCR using primers for vesicular stomatitis virus glycoprotein G viral envelope plasmid. When treating the final product, 1-hr incubations (37°C) with Pulmozyme at 20 U/ml reduced plasmid DNA to undetectable levels. Longer incubations (up to 4 hr) did not improve DNA removal at lower concentrations and the effectiveness was equivalent to or better than Benzonase at 50 U/ml. Attempting to use Pulmozyme immediately after transfection, but before final medium change, as a means to decrease Pulmozyme concentration in the final product provided a 2-log reduction in DNA but was inferior to treatment at the end of production. Pulmozyme, at concentrations up to 100 U/ml, had no measurable effect on infectious titer of the final vector product. The use of Pulmozyme is likely to increase the cost of DNase treatment when preparing vector product and should be considered when generating clinical-grade vector products.

A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes.

Human ex vivo gene therapy protocols have been used successfully to treat a variety of genetic disorders, infectious diseases, and cancer. Murine oncoretroviruses (specifically, gammaretroviruses) have served as the primary gene delivery vehicles for these trials. However, in some cases, such vectors have been associated with insertional mutagenesis. As a result, alternative vector platforms such as lentiviral vectors (LVVs) are being developed. LVVs may provide advantages compared with gammaretroviral vectors, including the ability to transduce large numbers of nondividing cells, resistance to gene silencing, and a potentially safer integration profile. The aim of this study was to develop a simplified process for the rapid production of clinical-grade LVVs. To that end, we used a self-inactivating bicistronic LVV encoding an MART (melanoma antigen recognized by T cells)-1-reactive T cell receptor containing oPRE, an optimized and truncated version of woodchuck hepatitis virus posttranslational regulatory element (wPRE). Using our simplified clinical production process, 293T cells were transiently transfected in roller bottles. The LVV supernatant was collected, treated with Benzonase, and clarified by modified step filtration. LVV produced in this manner exhibited titers and a biosafety profile similar to those of cGMP (current Good Manufacturing Practices) LVVs previously manufactured at the Indiana University Vector Production Facility in support of a phase I/II clinical trial. We describe a simple, efficient, and low-cost method for the production of clinical-grade LVV for ex vivo gene therapy protocols.

Flexibility in cell targeting by pseudotyping lentiviral vectors.

Lentiviral vectors have become an important research tool and have just entered into clinical trials. As wild-type lentiviruses engage specific receptors that have limited tropism, most investigators have replaced the endogenous envelope glycoprotein with an alternative envelope. Such pseudotyped vectors have the potential to infect a wide variety of cell types and species. Alternatively, selection of certain viral envelope glycoproteins may also facilitate cell targeting to enhance directed gene transfer. We describe the method for generating pseudotyped vector and provide information regarding available pseudotypes and their respective target tissues.

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant.

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Unmarked insertional mutagenesis in the bovine pathogen Mycoplasma mycoides subsp. mycoides SC: characterization of a lppQ mutant.

Mycoplasma mycoides subspecies mycoides small colony (SC) is the aetiologic agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease causing important losses in cattle production. The publication of the genome sequence of M. mycoides subsp. mycoides SC should facilitate the identification of putative virulence factors. However, real progress in the study of molecular mechanisms of pathogenicity also requires efficient molecular tools for gene inactivation. In the present study, we have developed a transposon-based approach for the random mutagenesis of M. mycoides subsp. mycoides SC. A PCR-based screening assay enabled the characterization of several mutants with knockouts of genes potentially involved in pathogenicity. The initial transposon was further improved by combining it with the transposon gammadelta TnpR/res recombination system to allow the production of unmarked mutations. Using this approach, we isolated a mutant free of antibiotic-resistance genes, in which the gene encoding the main lipoprotein LppQ was disrupted. The mutant was found to express only residual amounts of the truncated N-terminal end of LppQ. This approach opens the way to study virulence factors and pathogen-host interactions of M. mycoides subsp. mycoides SC and to develop new, genetically defined vaccine strains.

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type.

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.