Mycobacterium bovis infection in goats from the Northeast region of Brazil

A total of 8,058 male and female mixed-breed goats and 1-4 years of age were slaughtered over a period of 7 months at the public slaughterhouse of Patos city, Paraíba state, in the Northeast region of Brazil; 822 animals were inspected for gross lesions of tuberculosis, and 12 (1.46 percent) had lesions suggestive of tuberculosis in the mammary gland, lungs, liver and mediastinal, mesenteric, submandibular, parotid and prescapular lymph nodes. Presence of granulomatous lesions was confirmed in the submandibular lymph node of one (8.3 percent) goat at the histopathological examination and at the mycobacterium culture the same sample was confirmed positive. Isolate was confirmed as belonging to the M. tuberculosis complex by PCR restriction enzyme analysis (PRA). Spoligotyping identified the isolate into spoligotype SB0295 on the M. bovis Spoligotype Database website (www.mbovis.org), and it was classified as M. bovis. The occurrence of M. bovis in goats in this study suggests that this species may be a potential source of infection for humans and should be regarded as a possible problem in the advancement of control and eradication program for bovine tuberculosis in Brazil.

Diagnóstico e genotipagem do vírus da pseudoraiva por nested-PCR e análise de restrição enzimática / Diagnosis and genotyping of pseudorabies virus by nested-PCR and restriction enzyme analysis

A pseudoraiva (PR) é uma enfermidade viral responsável por consideráveis perdas econômicas na indústria de suínos. O vírus da pseudoraiva (PrV) apresenta apenas um sorotipo, mas, por análise de restrição enzimática, foi classificado em quatro genótipos denominados I, II, III e IV. Os métodos usados para genotipagem dependem do isolamento do vírus, da purificação do DNA viral, da restrição enzimática do genoma completo e da visualização após eletroforese. O objetivo deste trabalho foi estabelecer um método mais rápido e sensível para detectar e genotipar o PrV por nested-PCR e análise de restrição enzimática. Vinte isolados do PrV das regiões Sul e Sudeste do Brasil e a estirpe padrão Shope foram replicadas em células PK-15 e submetidas à nested-PCR para o gene da glicoproteína E. Além desses vírus previamente isolados, foram avaliadas 75 amostras clínicas de cérebro de suíno em um total de 25 animais positivos para a PR no isolamento e na soroneutralização viral e 50 amostras negativas provenientes de animais negativos na soroneutralização viral e de granjas sem histórico de PR. Todas as amostras clínicas tiveram resultados compatíveis com o isolamento e a soroneutralização, e a totalidade das amostras positivas foi classificada como genótipo II. A sensibilidade analítica da nested-PCR foi de 10-1,3 TCID50 mL-1. A combinação da nested-PCR e da restrição enzimática foi capaz de detectar e genotipar o vírus com resultados em um a dois dias, sendo mais rápida que os métodos convencionais de restrição do genoma completo que podem demorar até sete dias.

Pseudorabies is a disease caused by Suid herpesvirus 1 (PrV) and is responsible for considerable economic losses in the swine industry. The PrV has only one serotype, but based on RFLP (restriction fragment length polymorphism) the virus was divided into four genotypes named I, II, III, IV. The classical methods for PrV genotyping usually require virus isolation, DNA purification enzyme restriction analysis and a long electrophoresis. The aim of this research was to describe a faster and more sensitive method to detect and genotype PrV based on nested-PCR and restriction enzyme analysis. Twenty PrV isolates from south and southeast regions of Brazil, and the standard strain Shope were grown in PK-15 cells and submitted to PCR for glycoprotein E gene amplification. Additionally were tested 75 clinical samples (swine brain), with 25 positives for virus isolation and seroneutralization, and 50 negatives from a flock free PR with negative results in seroneutralization test. There was 100 percent of agreement between results of nested-PCR and virus isolation and seroneutralization and all samples detected were classified as genotype II. The nested-PCR, combined with restriction enzyme analysis, was able to detect and genotype PrV in 1-2 days with a sensitivity of 10-1,3 TCID50 mL-1. It was faster than classical methods described in the literature that require at least 7 days to be completed.

Diagnosis of Causative Fungi of Onychomycosis Using Polymerase Chain Reaction and Restriction Enzyme Analysis / 대한의진균학회지

BACKGROUND: Onychomycosis has become one of the common fungal infection. However, highly reliable and sensitive methods of detecting and identifying causative fungi of onychomycosis are not established yet. Polymerase chain reaction (PCR) analysis of clinical specimens including blood, sputum, urine, and cerebrospinal fluid collected from patient systemically infected fungus is known as a sensitive diagnostic method. But it has been questionable whether PCR analysis is also applicable to onychomycosis. OBJECTIVE: The purpose of this study was to develop a DNA-based diagnostic method to improve the sensitivity and specificity of detection and identification of pathogenic fungi of onychomycosis. METHODS: To detect the fungi in the nail, PCR was performed by using 4 sets of primer (TR1 & TR2, NS5 & NS6, B2F & B4R and CA1 & CA2) designed in conserved sequences of the small ribosomal subunit (185-rRNA) genes and restriction enzyme analysis of amplified product by Hae III was done to identify species. Nail specimens were obtained from 19 cases of onychomycosis confirm by fungus culture. RESULTS: 1. Preparation of nail powder, which is necessary for removal of keratin, and composition of lysis buffer with guanidinium thiocyanate, Tris-HCl, and beta -mercaptoethanol are the most proper modalities for isolation of fungal DNA from fungus-infesting nails. 2. Specific fragments of the 18S-rRNA gene of fungi, 581 bp, 308 bp, 688 bp and 1106 bp were amplified respectively. From sequences of 18S-rRNA gene of fungi by universal primers, dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes) and yeast (Candida albicans) yielded identical products. 3. Using Hae III endonuclease, digested patterns of fragment of Trichophyton rubrum and Candida albicans resulted in different pattern. CONCLUSION: This method released enough DNA from fungus-infected nails to result in proper amplification and it can be possible to differentiate dermatophytes, yeasts, and molds using Hae III endonuclease. The present study is the first one to demonstrate the feasibility of this molecular biologic approach to identify fungi in the infected nail. Therefore, precise detection and identification of the causative fungi would be of help in investigating distribution of the causative fungi of onychomycosis as well as appropriate treatment of the disease.

Comparison of serotypes, restriction enzyme analysis of plasmid DNA pattern and PFGE(pulsed-field gel electrophoresis) patterns of Yersinia pseudotuberculosis isolates in Korea / 대한임상병리학회지

BACKGROUND: Yersinia pseudotuberculosis, a member of genus Enterobactericeae, is a main etiologic organism of diarrhea in childhood. Because a mouse and a unchlorinated spring water are main reservoirs of Y. pseudotuberculosis, the strains from a contaminated spring water and mouse could be involved in human epidemic. The purpose of this study was to investigate a clonality between the strains from patients and those from an unchlorinated spring water and a mouse by restriction enzyme analysis of plasmid DNA and pulsed-field gel electrophoresis (PFGE). METHOD: We isolated 15 Y. pseudotuberculosis strains including 8 isolates from patients (S1-S8), 6 isolates from mountain water (W1-W6), 1 isolate from a mouse (M1) in northeast area of Seoul. Plasmid and chromosomal DNA of all strains were analyzed by REAP with Bam H1 restriction and by PFGE with Xba I restriction , respectively. RESULTS: Restriction enzyme analysis of plasmid DNA was classified into type B and type D. All 7 strains of serotype 15 were classified as type B and 8 strains of serotype 4b were classified as type D. PFGE were classified into 6 different types. Among them, strains of PFGE type I, II, III, IV belong to Y. pseudotuberculosis serotype 15 and Y. pseudotuberculosis 4b strains were classified into PFGE type V, VI. S1 and W1 were classified into PFGE type I . S8, W6 and M1 were classfied into PFGE type VI. CONCLUSIONS: PFGE revealed clonality among strains from patients, a water and a mouse. PFGE was more discriminative than REAP to characterize the Y. pseudotuberculosis outbreaks in Korea.

Study on amotile bacteria of positive blood culture in new-born:the analysis of plasmid and restriction enzyme and determination of outer membrane protein / 实用儿科临床杂志

Objective To search for the reasons of high positive rate of amotile bacteria and the diagnosis of septicemia in new-born Methods The blood was drawn from the different site of the new-born with septicemia and carricd out blood culture. The drug sensitivity test had been done by the method of paper stripdiffusion. The plasmids of bacteria were extracted rapidly by medified Birnboim method and the plasmid analyss was carried out. The plasmids's DNA of 35 epidemic strain was cut off by both restriction enzyme of Hind Ⅲ and EcoR Ⅰ. The outer membrane protein (OMP) was determined by SDS-polyacrylamide gel electrophoresis.Results There are 51 patients with positive blood culture amotile bacterium,of them, pollution; 35 cases (68．6%), septicemia: only 16 cases (31．4%),54．8% (57/104) strains bacteria have drug resistance to more of 12 drugs. 87．3% (165/189) strains bacteria have plasmids. They are cut off as 6 DNA fragments (1．9,2,4,5, 8．5 and 18Kb) by Hind Ⅲ restrietion enzyme. and as 5 DNA fragments (2,2．6,3．2, 6．3 and 22 Kb) by EcoR Ⅰrestrietion enzyme, it is showed that they come from a same clone. The epidemic strain include 10 slips OMP, but non-epidemic strain have 11 slip OMP, increase a 25Kd belt. The amotile bacteria with above-mentioned plasmid spectrum, restriction enzyme spectrum and OMP spectrum are only seen in the air, therapeutic dish and syringe needle.Conclusion The pollution is an important reason of amotile bactorium high positiye rate in new-born.Diagnosing septicemia should depend on bacteria culture, plasmid analysis restriction enzyme analysis of plasmid DNA, oMP determination and combining medical history and clinical manifestation.

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice.

Rice long repetitive DNA (9-20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Camr Ampr Tets), only a few were selected randomly for further characterization. The insert size in all these clones was 3-4 kbp. Restriction enzyme analysis showed the absence of EcoRI and BclI sites, presence of a single PstI and PvuII site and multiple sites for AluI in 3 clones namely pRLl, pRL7 and pRL10. The BamHI-PstI fragment of about 0·4 kbp in the pRL7 insert DNA (pRL7-0·4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25S rDNA sequence of rice, however, hybridisation did not indicate any homology.