Development of a multiplex-PCR for rapid detection of the enteric pathogens Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli in porcine faeces

AIMS: To develop an assay to simultaneously detect Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig faeces. METHODS AND RESULTS: A multiplex-polymerase chain reaction (M-PCR) was designed to amplify a 655-base pair (bp) portion of the L. intracellularis 16S rRNA gene, a 354-bp portion of the B. hyodysenteriae NADH oxidase gene, and a 823-bp portion of the B. pilosicoli 16S rRNA gene. Specificity was assessed using 80 strains of Brachyspira spp. and 30 other enteric bacteria. Bacterial DNA was extracted from faeces using the QIAamp DNA Stool Mini Kit. The M-PCR was tested in parallel with culture and/or PCR on 192 faecal samples from eight piggeries. Faeces also were seeded with known cell concentrations of the three pathogenic species, and the limits of detection of the M-PCR tested. The M-PCR was specific, with limits of detection of 10(2)-10(3) cells of the respective species per gram of faeces. CONCLUSIONS: The M-PCR is a rapid, sensitive and specific test for detecting three important enteric bacterial pathogens of pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of a new diagnostic M-PCR will allow rapid detection and control of three key porcine enteric pathogens.     

Identification and antimicrobial susceptibility of porcine bacteria that inhibit the growth of Brachyspira hyodysenteriae in vitro

Aims: To identify bacilli, lactic acid bacteria and bifidobacteria that inhibit the growth of Brachyspira hyodysenteriae. Methods and Results: A total of 80 isolates were obtained from various porcine intestinal compartments using selective conditions and grouped into 15 similarity clusters based on whole‐cell protein profiles. Random amplified polymorphic DNA PCR patterns identified 24 genotypes. 16S rDNA sequencing assigned all genotypes, except eight aerobes, to established species (Bacillus subtilis, Enterococcus faecium, Lactobacillus salivarius, Lactobacillus mucosae, Lactobacillus reuteri, Lactobacillus amylovorus, Bifidobacterium thermophilum). According to their minimum inhibitory concentrations, four strains (Ent. faecium, Lact. reuteri, Lact. amylovorus, Bif. thermophilum) were susceptible to all clinically relevant antibiotics. Two lactobacilli showing multiresistance harboured the erm(B) determinant. A cross‐section of eight representative strains was examined for growth suppression of two strains of Brach. hyodysenteriae, the aetiological agent of swine dysentery, and compared with intestinal strains derived from other animal sources. The Brachyspira strains were inhibited by strains of Lact. salivarius, Bif. thermophilum, Ent. faecium and B. subtilis. Conclusions: Three porcine strains of Ent. faecium, Bif. thermophilum and B. subtilis were found to be suitable as probiotic candidates because of their well‐established identity, antibiotic susceptibility and antagonistic activity. Significance and Impact of the Study: For the first time, antagonistic activity of well‐characterized porcine strains against Brach. hyodysenteriae is presented. These findings suggest that certain intestinal strains might have a potential as probiotic feed additives for prevention of swine dysentery.     

Evaluation of the use of recombinant Bhlp29.7 in immunoblotting with pig serum as a means to identify herds infected with Brachyspira hyodysenteriae

Aims: Aim of the study is to evaluate the use of recombinant Bhlp29.7 in immunoblotting with sera as a means to detect pig herds infected with Brachyspira hyodysenteriae. Methods and Results: Sera samples from 789 sows and rectal swabs from 838 pigs of various categories on 22 farms of different size (median 450 animals), production type and history of swine dysentery (SD) were examined. Sera from 378 sows from farms with previous SD history were examined via immunoblotting. Specific antibodies were detected in 79 of these (20·9%). Examination of 411 serum samples from sows and gilts taken on 11 farms without previous history of SD detected specific antibodies in 13 sows and gilts (3·2%). These 13, however, had come from farms where the presence of B. hyodysenteriae was confirmed or SD status was not known. Seroprevalence in herds with previous SD history ranged from 2·5 to 35·7%. B. hyodysenteriae was confirmed on six (27·3%) of 22 monitored farms. Conclusions: Immunoblotting using recombinant antigen Bhlp29.7 in conjunction with culturing B. hyodysenteriae proved to be a valuable tool for detecting swine herds latently infected with B. hyodysenteriae. Significance and Impact of the Study: The use of immunoblotting with recombinant Bhlp29.7 should prove to be a useful adjunct to detecting herds with SD, and hence, it will assist in controlling this important disease.     

Use of acid treatment and a selective medium to enhance the recovery of Francisella tularensis from water

Francisella tularensis has been associated with naturally occurring waterborne outbreaks and is also of interest as a potential biological weapon. Recovery of this pathogen from water using cultural methods is challenging due to the organism's fastidious growth requirements and interference by indigenous bacteria. A 15-min acid treatment procedure prior to culture on a selective agar was evaluated for recovery of F. tularensis from seeded water samples. Mean levels of reduction of virulent strains of F. tularensis subsp. holarctica and subsp. tularensis were less than 20% following acid treatment. The attenuated live vaccine strain (LVS) was less resistant to acid exposure. The acid treatment procedure coupled with plating on cystine heart agar with rabbit blood and antibiotics (CHARBab) allowed the isolation of F. tularensis seeded into five natural water samples.     

Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs

AIMS: This project investigated the utility of HP selective medium to isolate H. pylori cells from seawater and from marine molluscs. METHODS AND RESULTS: Nested-PCR was performed to reveal the presence of Helicobacter genus. All samples were cultured in HP selective medium and 16 cultures were initially selected as putative Helicobacter. Helicobacter spp. DNA were detected in 9/16 cultures and three of them had 99-100% homology to H. pylori based on 16S RNA gene sequence. Helicobacter pylori isolation was unsuccessful. On the basis of 16S RNA gene sequences the contaminating organisms were shown to be Proteus mirabilis and Vibrio cholerae. CONCLUSIONS: These results indicate the coexistence of three predominant bacterial genera in the cultures and that HP selective medium can grow other enteric bacteria besides Helicobacter. Additional assays will improve the HP selective medium formulation for marine samples avoiding P. mirabilis and V. cholerae interferents. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows the effectiveness of the selective HP medium for the Helicobacter culture from marine samples.     

基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价

【背景】猪水肿病大肠杆菌引发的疾病造成了很大的危害,但现有培养基存在培养密度低的问题。【目的】研制出高抗原活性猪水肿病大肠杆菌疫苗培养基。【方法】以常用的市售猪水肿培养基为对照,通过单因素试验、爬坡试验(Plackett-Burman, PB)、响应面(Box-Behnken, BB)试验对猪水肿培养基进行响应面优化,得到猪水肿培养基最优配方。以响应面试验得到的培养基培养猪水肿病大肠杆菌,评价不同培养时间点菌株的抗原活性,制作灭活疫苗,进行动物免疫保护试验。【结果】对研制的培养基进行扩大培养验证,发现扩大培养得到的菌株活菌数可达5×10⁹ CFU/mL以上,约为对照组的2倍。制备的灭活疫苗效价可达1:140 000,并在9 h时抗原蛋白效价达到最高。【结论】本研究研制出的疫苗培养基显著提高了猪大肠杆菌菌体密度,并可提高菌体抗原活性,为猪水肿病灭活疫苗的制备提供了技术指引。     

Development of a new medium useful for the recovery of dermatophytes from clinical specimens by minimizing the carryover effect of antifungal agents

Two surface-active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti-dermatophytic activity of the three drugs in a concentration-dependent manner. A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MIC's of BFZ were increased more than 30-fold and those of LCZ and TBF more than 200-fold compared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that the combined deactivators-supplemented medium yielded increased numbers of fungi compared with the basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the combined deactivators-supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.     

Development of larvae of Ascaris suum from the third to the fourth stage in a chemically defined medium

Third stage larvae of Ascaris suum, recovered from the lungs of rabbits 7 days after oral infection with eggs, were cultured to the fourth larval stage in chemically defined, low molecular weight medium. The medium consisted of tissue culture medium 199, supplemented with glucose and glycyl-hystidyl-lysine and gassed with a mixture of N₂-CO₂-O₂ (90:5:5). Growth and development in this medium were similar to that in media supplemented with whole serum or with a serum dialysate.     

Development of a serum-free medium for growth of NS-1 mouse myeloma cells and its application to the isolation of NS-1 hybridomas

In this study, we have devised a hormone-supplemented, lipid-enriched serum-free medium, designated KSLM, that supports the growth of NS-1 mouse myeloma cells and have studied its applicability to the efficient isolation of antibody-producing hybridomas formed from the fusion of NS-1 myeloma cells and spleen cells from immunized mice. Our results show that KSLM medium, when used in conjunction with our hybridization protocol, allowed for the isolation, in a reproducible manner, of antibody-secreting hybridomas. Moreover, the yield of antibody-producing hybridomas was similar in KSLM medium and serum-supplemented medium. Here, we also report on the adaptation of NS-1 myeloma cells to growth in lipid-deficient KSLM medium. The use of the adapted myeloma cells (NS-1-503), instead of NS-1 myeloma cells, in fusion experiments not only permitted the isolation of antibody-secreting hybridomas in lipid-free KSLM medium but also resulted in a higher yield of antibody-producing hybridomas in both complete KSLM medium and serum-supplemented medium.