Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid.

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

Characterisation of Listeria monocytogenes isolates from cattle using a bovine caruncular epithelial cell model.

Listeria monocytogenes is an important foodborne pathogen in human and veterinary health, causing significant morbidity and mortality including abortion. It has a particular tropism for the gravid uterus, however, the route of infection in reproductive tissues of ruminants (i.e. placentome), is much less clear. In this study, we aimed to investigate a bovine caruncular epithelial cell (BCEC) line as a model for L. monocytogenes infection of the bovine reproductive tract. The BCEC infection model was used to assess the ability of 14 different L. monocytogenes isolates to infect these cells. Lysozyme sensitivity and bacterial survival in 580 µg lysozyme/ml correlated with attenuated ability to proliferate in BCEC (p = 0.004 and p = 0.02, respectively). Four isolates were significantly attenuated compared to the control strain 10403S. One of these strains (AR008) showed evidence of compromised cell wall leading to increased sensitivity to ß-lactam antibiotics, and another (7644) had compromised cell membrane integrity leading to increased sensitivity to cationic peptides. Whole genome sequencing followed by Multi Locus Sequence Type analysis identified that five invasive isolates had the same sequence type, ST59, despite originating from three different clinical conditions. Virulence gene analysis showed that the attenuated isolate LM4 was lacking two virulence genes (uhpT, virR) known to be involved in intracellular growth and virulence. In conclusion, the BCEC model was able to differentiate between the infective potential of different isolates. Moreover, resistance to lysozyme correlated with the ability to invade and replicate within BCEC, suggesting co-selection for surviving challenging environments as the abomasum.

Diversity of Lactobacillus Species of Stilton Cheese Relates to Site of Isolation.

This study has characterized the dominant non-starter Lactobacillus species isolated from different sites in a Stilton cheese to establish its diversity, stress-tolerance, anti-microbial activity and potential contribution to quality of cheese. Fifty-nine Lactobacillus isolates were cultured from the outer crust, blue veins and white core of the cheese and were speciated phenotypically and by 16S rDNA sequence analysis. Lactobacillus plantarum was the dominant species detected with only two isolates identified as Lactobacillus brevis. Strains were typed by pulse-field gel electrophoresis (PFGE) using the enzyme NotI to examine their genomic diversity. Cluster analysis of PFGE patterns produced five major clusters which associated isolates with their sites of isolation within the cheese. One L. plantarum isolate from each cheese site was selected and evaluated for salt, acid, relative humidity, and heat tolerance to determine whether stress conditions within the isolation site selected their phenotype. D 72°C values were 6, 13, and 17 s for strains from the crust, veins and core, respectively, suggesting strains on the crust may not have been able to survive pasteurization and therefore had been added post-pasteurization. All strains recovered from heat injury within 24-48 h at 4°C. pH values of 3, 3.5, and 4 suppressed growth but strains showed a varying ability to grow at pH 4.5 and 5; isolates from the core (which has the lowest pH) were the most acid-tolerant. All strains grew at 3.5 and 5% salt but were suppressed at 10%; those from the crust (which has a lower water activity) were the most halo-tolerant, growing at 8% salt whereas strains from the core were sensitive to this salt concentration. All 57 L. plantarum isolates were examined for antimicrobial activity and variable activity against Lactobacillus pentosus and other genera was demonstrated; plantaricin EF genes were present in 65% of strains. It was concluded that there are varied phenotypes and genotypes of Lactobacillus in a Stilton cheese according to site of isolation. Occurrence of different L. plantarum genotypes could contribute to variation in the cheese quality from batch to batch and provides criteria for selecting isolates as potential adjunct cultures.

CONSERVATION CHALLENGES: THE LIMITATIONS OF ANTEMORTEM TUBERCULOSIS TESTING IN CAPTIVE ASIATIC LIONS (PANTHERA LEO PERSICA).

Genetic diversity of captive wild animals can be enhanced by moving those individuals with valuable genes between collections and through introduction of a new pair from a range country. This requires movement of animals, which is inherent with disease risks, such as the introduction of pathogenic Mycobacterium sp. (MTBC) into a zoological collection. Decisions need to be made based on the outcome of perimovement disease screening using an array of tests, the majority of which are unvalidated in the species. A pair of endangered Asiatic lions (Panthera leo persica) imported from India to the United Kingdom were screened for MTBC using the comparative intradermal tuberculosis (TB) test, the feline interferon-γ blood test, and the experimental bacteriophage assay. Reactions on all three tests prompted screening of the three resident Asiatic lions using the same tests, all of which were negative for MTBC. Based on these test results, the decision had to be made to exclude the genetically valuable pair from the current collection. MTBC could not be identified using further tests, including culture and PCR on a bronchoalveolar lavage, on feces, or on postmortem tissues. This case series highlights the usefulness of a control group when interpreting unvalidated test results for detection of MTBC, the value of training big cats for conscious blood sampling, and the practical implications of placing the comparative intradermal TB test in the eyelids, when dealing with a species that requires a general anesthetic for most hands-on interventions.

A Novel, High-sensitivity, Bacteriophage-based Assay Identifies Low-level Mycobacterium tuberculosis Bacteremia in Immunocompetent Patients With Active and Incipient Tuberculosis.

The haematogenous dissemination of Mycobacterium tuberculosis (Mtb) is critical to the pathogenesis of progressive tuberculous infections in animal models. Using a novel, phage-based blood assay, we report the first concordant evidence in well-characterized, immunocompetent human cohorts, demonstrating associations of Mtb bacteremia with progressive phenotypes of latent infection and active pulmonary tuberculosis.

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne's disease-infected animals.

Here, we describe the development of a method that exploits bacteriophage D29 as a lysis agent for efficient DNA extraction from low numbers of mycobacterial cells. This method (Actiphage® ) used in combination with PCR achieved rapid and sensitive (LOD ≤ 10 cell ml-1 ) detection and identification of viable, pathogenic mycobacteria in blood samples within 6 h. We demonstrate that mycobacteriophage D29 can be used to detect a range of mycobacteria from clinical blood samples including both Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis without the need for culture and confirms our earlier observations that a low-level bacteraemia is associated with these infections in cattle. In a study of M. bovis-infected cattle (n = 41), the sensitivity of the Actiphage® method was 95 % (95 % CI; 0.84-0.99) and specificity was 100 % (95% CI; 0.92-1). We further used Actiphage® to demonstrate viable Mycobacterium avium subsp. paratuberculosis is present in the blood of Johne's infected cattle. This method provides a revolutionary new tool for the study of infections caused by these difficult to grow pathogens.

Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk.

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.

Evidence that Listeria innocua modulates its membrane's stored curvature elastic stress, but not fluidity, through the cell cycle.

This paper reports that the abundances of endogenous cardiolipin and phosphatidylethanolamine halve during elongation of the Gram-positive bacterium Listeria innocua. The lyotropic phase behaviour of model lipid systems that describe these modulations in lipid composition indicate that the average stored curvature elastic stress of the membrane is reduced on elongation of the cell, while the fluidity appears to be maintained. These findings suggest that phospholipid metabolism is linked to the cell cycle and that changes in membrane composition can facilitate passage to the succeding stage of the cell cycle. This therefore suggests a means by which bacteria can manage the physical properties of their membranes through the cell cycle.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Detection of viable Mycobacterium avium subspecies paratuberculosis in powdered infant formula by phage-PCR and confirmed by culture.

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern.

Factors affecting phage D29 infection: a tool to investigate different growth states of mycobacteria.

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.

Detection of Mycobacterium avium subsp. paratuberculosis in bulk tank milk by combined phage-PCR assay: evidence that plaque number is a good predictor of MAP.

Conventional culture and a rapid phage-PCR method were used to detect Mycobacterium avium subsp. paratuberculosis (MAP) in bulk tank milk (BTM) samples. Only two of 225 samples (0.9%) were found to contain MAP by culture whereas 50 (22%) MAP-positive samples were identified using the phage-PCR assay, including both samples that were MAP-culture positive. Results using the phage-based method for independently tested duplicate samples indicated that the assay is very reproducible (r(2)=0.897), especially when low levels of mycobacteria are present. A relationship was established between plaque number and the presence of MAP in a sample. A cut-off value was determined allowing identification of MAP-positive samples based on plaque number alone (90% sensitivity, 99% specificity; area under the curve=0.976). These results indicate that the assay is a robust method for screening BTM, providing results within 24 h.

Diamagnetic levitation enhances growth of liquid bacterial cultures by increasing oxygen availability.

Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to reproduce aspects of weightlessness, on the Earth. We used a superconducting magnet to levitate growing bacterial cultures for up to 18 h, to determine the effect of diamagnetic levitation on all phases of the bacterial growth cycle. We find that diamagnetic levitation increases the rate of population growth in a liquid culture and reduces the sedimentation rate of the cells. Further experiments and microarray gene analysis show that the increase in growth rate is owing to enhanced oxygen availability. We also demonstrate that the magnetic field that levitates the cells also induces convective stirring in the liquid. We present a simple theoretical model, showing how the paramagnetic force on dissolved oxygen can cause convection during the aerobic phases of bacterial growth. We propose that this convection enhances oxygen availability by transporting oxygen around the liquid culture. Since this process results from the strong magnetic field, it is not present in other weightless environments, e.g. in Earth orbit. Hence, these results are of significance and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria.

BACKGROUND: The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria. RESULTS: Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning. CONCLUSION: The residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours.

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.

Phage amplification assay as rapid method for Salmonella detection

A aplicação de métodos rápidos é crucial para a implantação de programas de HACCP em indústrias de alimentos. Neste contexto, o método de amplificação de bacteriófagos é um instrumento de diagnóstico importante porque está baseado na interação dos bacteriófagos com suas células hospedeiras. Este método, usando o bacteriófago P22, foi aplicado para detectar Salmonella em peito de frango. Amostras de 25 g de peito de frango foram diluídas e as diluições apropriadas foram usadas no método de amplificação de bacteriófagos na detecção de Salmonella. Após 3-4 horas de incubação, foi observado uma titulação de partículas virais de, aproximadamente, 104 ufp mL-1 (unidades formadoras de placas virais), indicando a presença de células de Salmonella na carne de frango. A comprovação da presença de Salmonella neste produto foi verificada usando-se plaqueamento direto em ágar XLD e procedimento de enriquecimento convencional. As colônias suspeitas de Salmonella foram sorologicamente testadas e identificadas como pertencendo aos sorogrupos B (grupo de S. typhimurium) e D (grupo de S. enteritidis). Portanto, concluiu-se que este método pode ser aplicado, na detecção de Salmonella em alimentos, porque fornece rápido e conclusivo resultado, reduzindo o tempo de análise e o trabalho laboratorial para 3-4 horas.