Draft genome sequences of Mycolicibacterium peregrinum isolated from a pig with lymphadenitis and from soil on the same Japanese pig farm.

OBJECTIVES: Mycolicibacterium peregrinum, a rapidly growing mycobacterial species, can opportunistically infect humans and other animals. Although M. peregrinum infections in animals have been reported, the infection sources are unknown, as is information on its virulence and drug resistant genes, which limits our current understanding of this bacterium. To address this knowledge gap, we obtained draft genome sequences for two M. peregrinum isolates; one from a case of pig lymphadenitis and one from the pig farm's soil. DATA DESCRIPTION: We report here the draft genome sequences of M. peregrinum isolates 131_1 and 138 (6,451,733-bp and 6,479,047-bp). They were isolated from a pig with mesenteric lymph node lymphadenitis and from soil on the Japanese farm where the pig was reared. A sequence alignment identity score of 100% was obtained by in silico DNA-DNA hybridization of the two isolates, while 98.28% (isolate 131_1) and 98.27% (isolate 138) scores were recorded for hybridization with a human isolate. Both isolates carry arr-1, AAC(2')-Ib, RbpA, mtrA and tap drug-resistance genes. Isolates 131_1 and 138 carry 234 and 236 putative virulence genes, respectively. Therefore, environment M. peregrinum is potentially drug resistant and can cause swine lymphadenitis. Our data provides valuable new information for future studies on nontuberculous mycobacteria.

Draft Genome Sequences of Mycolicibacter senuensis Isolate GF74 and Mycobacterium colombiense Isolates GF28 and GF76 from a Swine Farm in Japan.

Several nontuberculous mycobacteria (NTM) occasionally infect humans and animals. Here, we report the draft genome sequences of Mycolicibacter senuensis isolate GF74 (4,792,997 bp) and Mycobacterium colombiense isolates GF28 and GF76 (5,473,554 bp and 5,426,852 bp, respectively) isolated from a swine farm in Japan. These sequences provide further information on NTM research.

Draft Genome Sequence of Mycobacterium virginiense Strain GF75, Isolated from the Mud of a Swine Farm in Japan.

Mycobacterium virginiense, a newly described species of the Mycobacterium terrae complex, is a cause of tenosynovitis and osteomyelitis in the United States. Here, we report the 4,849,424-bp draft genome sequence of M. virginiense strain GF75, isolated from a mud sample taken from a Japanese swine farm.

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

A function for a specific zinc metalloprotease of African trypanosomes.

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi.

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.