Genomic analysis of seven mycobacteriophages identifies three novel species with differing phenotypic stabilities.

Recently, case studies have been published regarding the application of mycobacteriophage (MP) therapy (MPT) in patients with multi-antibiotic-resistant infections. A major limitation in the development of MPT is the paucity of therapeutically useful MP. As there are approximately 10,000 MP that have yet to be sequenced, it is possible that characterization of this cohort would increase the repertoire of useful MP. This study aims to contribute to such a strategy, by characterizing a cohort of 7 mycobacteriophages. Sequencing analyses revealed that the MP have unique sequences, and subsequent gene annotation revealed differences in gene organization. Notably, MP LOCARD has the largest genome and operons encoding for glycosyltransferases. Taxonomic analysis executed with VIRIDIC, Gegenees and VICTOR revealed that LOCARD belongs to a different genus than the other phages and is the foundational member of one of three novel species identified in this study. LOCARD, LOCV2, and LOCV5 were selected as representative members of their species and subjected to phenotypic analyses to compare their stability under biologically and industrially relevant conditions. Again LOCARD stood out, as it was unaffected by the typical temperatures (37 °C) and salinity (0.9%) experienced in mammals, while the viability of LOCV2 and LOCV5 was significantly reduced. LOCARD was also tolerant to pH 10, low levels of antiviral detergent and was the least impacted by a single freeze-thaw cycle. When all these results are considered, it indicates that LOCARD in particular, has potential therapeutic and/or diagnostics applications, given its resilience towards physiological and storage conditions.

Identification of novel genera and subcluster classifications for mycobacteriophages.

Aim: To identify novel genera amongst mycobacteriophages (MP) and verify a hypothesised correlation between the taxonomy set by the International Committee on Taxonomy of Viruses (ICTV) and the National Centre for Biotechnology Information (NCBI) with that of the Actinobacteriophage Database, which may help formalise subcluster assignment. Methods: A dataset of 721 MP genomes was analysed using VIRIDIC, a nucleotide alignment-based software that predicts genus assignments. Potentially novel genera were analysed using Gegenees and VICTOR, respectively. These genera were then compared to the subclusters assigned by the Actinobacteriophage Database to verify a hypothesis that one genus can be assigned to one subcluster (i.e., the genus-subcluster hypothesis). Results: Initially, when comparing the current genus classifications of the 721 MP dataset to the Actinobacteriophage database subcluster assignments, 83.3% of subclusters supported the genus-subcluster hypothesis. Following the sequential VIRIDIC, Gegenees and VICTOR analyses, a total of 20 novel genera were identified based on a ≥ 70% and ~ 50% similarity threshold for VIRIDIC and Gegenees, respectively, and a monophyletic nature in the VICTOR output. Interestingly, these criteria also appear to support the creation of 13 novel subclusters, which would increase the support for the genus-subcluster hypothesis to 97.6%. Conclusion: The link between genus and subcluster classifications appears robust, as most subclusters can be assigned a single genus and vice versa. By relating the taxonomic and clustering classification systems, they can be easily kept up to date to best reflect MP diversity, which could aid the rapid selection of related (or diverse) phages for research, therapeutic and diagnostic purposes.

Alginate-Encapsulated Mycobacteriophage: A Potential Approach for the Management of Intestinal Mycobacterial Disease.

Encapsulated medication is a common method of administering therapeutic treatments. As researchers explore alternative therapies, it is likely that encapsulation will remain a feature of these novel treatments, particularly when routes of delivery are considered. For instance, alginate-encapsulation is often favoured where gastric digestion poses an obstacle. When exposed to cations (namely Ca2+), alginate readily forms gels that are resilient to acidic conditions and readily dissociate in response to mid-range pH. This action can be extremely valuable for the encapsulation of phages. The efficient delivery of phages to the intestine is important when considering mycobacteriophage (MP) therapy (or MP prophylaxis) for disseminated mycobacterial infections and chronic gastroenteritis conditions. This study presents the design and in vitro validation of an alginate-encapsulated MP capable of releasing phages in a pH-dependent manner. Ultimately, it is shown that encapsulated phages pretreated with simulated gastric fluid (SGF) are capable of releasing viable phages into simulated intestinal fluid (SIF) and thereby reducing the mycobacterial numbers in spiked SIF by 90%. These findings suggest that alginate encapsulation may be a viable option for therapeutic and prophylactic approaches to the management of intestinal mycobacterial disease, such as Johne's disease.

The effect of slow-release milk replacer feeding on health and behaviour parameters in dairy breed calves.

The aim of this research paper was to evaluate the effect of a slow-release milk replacer on health and behaviour of neonatal dairy calves. This was done with the potential benefits to welfare during transport in mind. A total of 15 calves were randomly divided into three groups of 5, namely, a control group fed twice in 24 h with 3 l of a conventional milk replacer, a slow-release group fed once in 24 h with 2 l of conventional milk replacer and 1 litre of a specialised micro-encapsulated feed and an enriched-replacer group fed once in 24 h with 3 l of milk replacer enriched with micellar casein. Blood samples were taken before feeding and 6, 12, 18 and 24 h after and analysed for acid-base parameters, electrolytes, glucose, haemoglobin, cortisol, insulin, cholecystokinin and adiponectin. Calf behaviour was recorded between 6 and 14 h after feeding. There was a significant increase in blood pH 6 h after feeding in all groups, but the glucose, HCO3 - and base excess increased significantly in the slow-release group only, whereas sodium increased significantly in the enriched group only. Glucose levels remained significantly higher in the slow-release group, relative to the control, at 6, 12, and 18 h after feeding. Insulin levels changed significantly over time in the enriched and control group but remained constant in the slow-release group. Insulin levels were significantly higher in the control group when compared to the slow-release group after feeding. Adiponectin changed significantly over time after feeding in the control group only, but no significant changes were observed between the feeding groups. Behavioural patterns were similar in control and slow release groups but less favourable (less lying time, more vocalisations) in the enriched group. In conclusion, once-daily feeding of slow-release milk replacer demonstrated favourable patterns of blood variables related to satiety and hunger as well as behavioural patterns that did not differ from conventional twice-daily feeding.

Alternatives to antibiotics in veterinary medicine: considerations for the management of Johne's disease.

Antibiotic resistance has become a major health concern globally, with current predictions expecting deaths related to resistant infections to surpass those of cancer by 2050. Major efforts are being undertaken to develop derivative and novel alternatives to current antibiotic therapies in human medicine. What appears to be lacking however, are similar efforts into researching the application of those alternatives, such as (bacterio)phage therapy, in veterinary contexts. Agriculture is still undoubtedly the most prominent consumer of antibiotics, with up to 70% of annual antibiotic usage attributed to this sector, despite policies to reduce their use in food animals. This not only increases the risk of resistant infections spreading from farm to community but also the risk that animals may acquire species-specific infections that subvert treatment. While these diseases may not directly affect human welfare, they greatly affect the profit margin of industries reliant on livestock due to the cost of treatments and (more frequently) the losses associated with animal death. This means actively combatting animal infection not only benefits animal welfare but also global economies. In particular, targeting recurring or chronic conditions associated with certain livestock has the potential to greatly reduce financial losses. This can be achieved by developing novel diagnostics to quickly identify ill animals alongside the design of novel therapies. To explore this concept further, this review employs Johne's disease, a chronic gastroenteritis condition that affects ruminants, as a case study to exemplify the benefits of rapid diagnostics and effective treatment of chronic disease, with particular regard to the diagnostic and therapeutic potential of phage.

An assessment of the impact of Raman based glucose feedback control on CHO cell bioreactor process development.

Process analytical technology (PAT) tools such as Raman Spectroscopy have become established tools for real time measurement of CHO cell bioreactor process variables and are aligned with the QbD approach to manufacturing. These tools can have a significant impact on process development if adopted early, creating an end-to-end PAT/QbD focused process. This study assessed the impact of Raman based feedback control on early and late phase development bioreactors by using a Raman based PLS model and PAT management system to control glucose in two CHO cell line bioreactor processes. The impact was then compared to bioreactor processes which used manual bolus fed methods for glucose feed delivery. Process improvements were observed in terms of overall bioreactor health, product output and product quality. Raman controlled batches for Cell Line 1 showed a reduction in glycation of 43.4% and 57.9%, respectively. Cell Line 2 batches with Raman based feedback control showed an improved growth profile with higher VCD and viability and a resulting 25% increase in overall product titer with an improved glycation profile. The results presented here demonstrate that Raman spectroscopy can be used in both early and late-stage process development and design for consistent and controlled glucose feed delivery.

Differences in Faecal Microbiome Taxonomy, Diversity and Functional Potential in a Bovine Cohort Experimentally Challenged with Mycobacterium avium subsp. paratuberculosis (MAP).

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants, a chronic enteritis which results in emaciation and eventual loss of the animal. Recent advances in metagenomics have allowed a more in-depth study of complex microbiomes, including that of gastrointestinal tracts, and have the potential to provide insights into consequences of the exposure of an animal to MAP or other pathogens. This study aimed to investigate taxonomic diversity and compositional changes of the faecal microbiome of cattle experimentally challenged with MAP compared to an unexposed control group. Faecal swab samples were collected from a total of 55 animals [exposed group (n = 35) and a control group (n = 20)], across three time points (months 3, 6 and 9 post-inoculation). The composition and functional potential of the faecal microbiota differed across time and between the groups (p < 0.05), with the primary differences, from both a taxonomic and functional perspective, occurring at 3 months post inoculation. These included significant differences in the relative abundance of the genera Methanobrevibacter and Bifidobacterium and also of 11 other species (4 at a higher relative abundance in the exposed group and 7 at a higher relative abundance in the control group). Correlations were made between microbiome data and immunopathology measurements and it was noted that changes in the microbial composition correlated with miRNA-155, miR-146b and IFN-É£. In summary, this study illustrates the impact of exposure to MAP on the ruminant faecal microbiome with a number of species that may have relevance in veterinary medicine for tracking exposure to MAP.

Evidence for local and international spread of Mycobacterium avium subspecies paratuberculosis through whole genome sequencing of isolates from the island of Ireland.

We describe application of whole genome sequencing (WGS) to a collection of 197 Mycobacterium avium subsp paratuberculosis (MAP) isolates gathered from 122 cattle herds across 27 counties of the island of Ireland. We compare WGS to MAP diversity quantified using mycobacterial interspersed random unit - variable number tandem repeats (MIRU-VNTR). While MIRU-VNTR showed only two major types, WGS could split the 197 isolates into eight major groups. We also found six isolates corresponding to INMV 13, a novel MIRU-VNTR type for Ireland. Evidence for dispersal of MAP across Ireland via cattle movement could be discerned from the data, with mixed infections present in several herds. Furthermore, comparisons of MAP WGS data from Ireland to data from Great Britain and continental Europe revealed many instances of close genetic similarity and hence evidence for international transmission of infection. BEAST MASCOT structured coalescent analyses, with relaxed and strict molecular clocks, estimated the substitution rate to be 0.10-0.13 SNPs/site/year and disclosed greater transitions per lineage per year from Europe to Ireland, indicating transmission into Ireland. Our work therefore reveals new insight into the seeding of MAP infection across Ireland, highlighting how WGS can inform policy formulation to ultimately control MAP transmission at local, national and international scales.

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process.

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.

Evaluation of the optimal strong ion difference concentration of an oral electrolyte and buffering solution for the treatment of neonatal calf diarrhea.

An observational study was conducted to comparatively assess the efficacy of three different oral rehydration and buffering solutions, differentiated by their strong ion difference (SID) concentration, for treatment of neonatal calves with naturally acquired diarrhea. The SID concentrations tested were 100 mM, 170 mM and 230 mM for treatments SID100, SID170 and SID230, respectively. Clinical assessment and blood gas analysis were completed for 18 diarrheic calves once pre- and twice post- (6 and 24 hr after) oral administration with one of the three treatments. A repeated measure mixed model approach was used to analyze (a) the within-group efficacy of each treatment over time and (b) the between-group comparison at each timepoint. SID230 treatment resulted in a significant increase in blood pH, HCO3-, BE, SID and Na+ at 6 and 24 hr after treatment, and a significant decrease in AG and K+ by 24 hr after treatment. There were no significant changes in any of the blood gas parameters after treatment with SID100 and SID170. SID230 treatment also resulted in blood gas parameter changes that were significantly different to the other two groups. These results suggest that the optimum SID concentration for the treatment of calves with diarrhea is likely to be higher than current recommendations.

MAP, Johne's disease and the microbiome; current knowledge and future considerations.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in ruminants. As an infectious disease that causes reduced milk yields, effects fertility and, eventually, the loss of the animal, it is a huge financial burden for associated industries. Efforts to control MAP infection and Johne's disease are complicated due to difficulties of diagnosis in the early stages of infection and challenges relating to the specificity and sensitivity of current testing methods. The methods that are available contribute to widely used test and cull strategies, vaccination programmes also in place in some countries. Next generation sequencing technologies have opened up new avenues for the discovery of novel biomarkers for disease prediction within MAP genomes and within ruminant microbiomes. Controlling Johne's disease in herds can lead to improved animal health and welfare, in turn leading to increased productivity. With current climate change bills, such as the European Green Deal, targeting livestock production systems for more sustainable practices, managing animal health is now more important than ever before. This review provides an overview of the current knowledge on genomics and detection of MAP as it pertains to Johne's disease.

Raman spectroscopic based chemometric models to support a dynamic capacitance based cell culture feeding strategy.

Multiple process analytical technology (PAT) tools are now being applied in tandem for cell culture. Research presented used two in-line probes, capacitance for a dynamic feeding strategy and Raman spectroscopy for real-time monitoring. Data collected from eight batches at the 15,000 L scale were used to develop process models. Raman spectroscopic data were modelled using Partial Least Squares (PLS) by two methods-(1) use of the full dataset and (2) split the dataset based on the capacitance feeding strategy. Root mean square error of prediction (RMSEP) for the first model method of capacitance was 1.54 pf/cm and the second modelling method was 1.40 pf/cm. The second Raman method demonstrated results within expected process limits for capacitance and a 0.01% difference in total nutrient feed compared to the capacitance probe. Additional variables modelled using Raman spectroscopy were viable cell density (VCD), viability, average cell diameter, and viable cell volume (VCV).

Analysis of chemometric models applied to Raman spectroscopy for monitoring key metabolites of cell culture.

The Food and Drug Administration (FDA) initiative of Process Analytical Technology (PAT) encourages the monitoring of biopharmaceutical manufacturing processes by innovative solutions. Raman spectroscopy and the chemometric modeling tool partial least squares (PLS) have been applied to this aim for monitoring cell culture process variables. This study compares the chemometric modeling methods of Support Vector Machine radial (SVMr), Random Forests (RF), and Cubist to the commonly used linear PLS model for predicting cell culture components-glucose, lactate, and ammonia. This research is performed to assess whether the use of PLS as standard practice is justified for chemometric modeling of Raman spectroscopy and cell culture data. Model development data from five small-scale bioreactors (2 × 1 L and 3 × 5 L) using two Chinese hamster ovary (CHO) cell lines were used to predict against a manufacturing scale bioreactor (2,000 L). Analysis demonstrated that Cubist predictive models were better for average performance over PLS, SVMr, and RF for glucose, lactate, and ammonia. The root mean square error of prediction (RMSEP) of Cubist modeling was acceptable for the process concentration ranges of glucose (1.437 mM), lactate (2.0 mM), and ammonia (0.819 mM). Interpretation of variable importance (VI) results theorizes the potential advantages of Cubist modeling in avoiding interference of Raman spectral peaks. Predictors/Raman wavenumbers (cm-1 ) of interest for individual variables are X1139-X1141 for glucose, X846-X849 for lactate, and X2941-X2943 for ammonia. These results demonstrate that other beneficial chemometric models are available for use in monitoring cell culture with Raman spectroscopy.

Raman spectroscopy as a method to replace off-line pH during mammalian cell culture processes.

Raman spectroscopy is a robust, well-established tool utilized for measuring important cell culture process variables for example, feed, metabolites, and biomass in real-time. This study further expands the functionality of in-line Raman spectroscopy coupled with partial least squares (PLS) regression modelling to develop a pH measurement tool. Cell line specific models were developed to enhance the robustness for processes with different pH setpoints, deadbands, and cellular metabolism. The modelling strategy further improved robustness by reducing the temporal complexity of pH shifts by splitting data sets into two time zones reflective of major changes in pH. In addition, models were developed to assess if lactate and partial pressure of carbon dioxide (pCO2 ) could be used in a PLS model for pH. Splitting the data sets into early and late for the process resulted in errors of 0.035 pH and 0.034 pH for the two respective Raman cell lines models which was within acceptance criteria. The lactate and pCO2 PLS model with values provided by Raman models had a further 0.001 pH error reduction. This study illustrates the potential to eliminate off-line samples to correct for in-line measurements of pH and further illustrates the capabilities of Raman to measure additional process variables.

A rapid viability and drug-susceptibility assay utilizing mycobacteriophage as an indicator of drug susceptibilities of Anti-TB drugs against Mycobacterium smegmatis mc2 155.

Background: A rapid in-house TM4 mycobacteriophage-based assay, to identify multidrug resistance against various anti-tuberculosis drugs, using the fast-growing Mycobacterium smegmatis mc2 155 in a microtiter plate format was evaluated, based on phage viability assays. Methods: A variety of parameters were optimized before the study including the minimum incubation time for the drugs, phage and M. smegmatis mc2 155 to be in contact. An increase in phage numbers over 2 h was indicative that M. smegmatis mc2 155 is resistant to the drugs under investigation, however when phage numbers remained static, M. smegmatis mc2 155 found to be sensitive to the drug. Results: The study confirmed that the data are statistically significant and that M. smegmatis mc2 155 is, in fact, sensitive to isonazid, iifampicin, pyranzaimide, and ethambutol as phage numbers doubled over 2 h (P = 0.015, 0.018, 0.014, and 0.020). The study also confirmed that M. smegmatis mc2 155 is resistant to the drugs ampicillin, erythromycin, amoxicillin streptomycin as phage numbers remain static over the same 2 h period (P = 0.028, 0.052, 0.049, and 0.04). This drug-susceptibility profiling of eight different drugs against M. smegmatis mc2 155 was detected in as little as 1½ days with a cost of ~ one euro and fifteen cent to test four drugs. Conclusion: This test is rapid to perform and will have widespread applications in drug-susceptibility testing of other members of the mycobacterial genus. In addition, the platform could also be used as a tool for high-throughput screening of novel antimycobacterial drugs. The main assets of this assay include its relatively cheap cost, versatility, and quick turnaround time.

Pectobacterium atrosepticum Phage vB_PatP_CB5: A Member of the Proposed Genus 'Phimunavirus'.

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal⁻arrest⁻release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

Novel N4-Like Bacteriophages of Pectobacterium atrosepticum.

Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control.

Comparison of Staphylococcus Phage K with Close Phage Relatives Commonly Employed in Phage Therapeutics.

The increase in antibiotic resistance in pathogenic bacteria is a public health danger requiring alternative treatment options, and this has led to renewed interest in phage therapy. In this respect, we describe the distinct host ranges of Staphylococcus phage K, and two other K-like phages against 23 isolates, including 21 methicillin-resistant S. aureus (MRSA) representative sequence types representing the Irish National MRSA Reference Laboratory collection. The two K-like phages were isolated from the Fersisi therapeutic phage mix from the Tbilisi Eliava Institute, and were designated B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1). The sequence relatedness of B1 and JA1 to phage K was observed to be 95% and 94% respectively. In terms of host range on the 23 Staphylococcus isolates, B1 and JA1 infected 73.9% and 78.2% respectively, whereas K infected only 43.5%. Eleven open reading frames (ORFs) present in both phages B1 and JA1 but absent in phage K were identified by comparative genomic analysis. These ORFs were also found to be present in the genomes of phages (Team 1, vB_SauM-fRuSau02, Sb_1 and ISP) that are components of several commercial phage mixtures with reported wide host ranges. This is the first comparative study of therapeutic staphylococcal phages within the recently described genus Kayvirus.

Synthesis and In Vitro Antimycobacterial Activity of Novel N-Arylpiperazines Containing an Ethane-1,2-diyl Connecting Chain.

Novel 1-(2-{3-/4-[(alkoxycarbonyl)amino]phenyl}-2-hydroxyethyl)-4-(2-fluorophenyl)-piperazin-1-ium chlorides (alkoxy = methoxy to butoxy; 8a-h) have been designed and synthesized through multistep reactions as a part of on-going research programme focused on finding new antimycobacterials. Lipophilic properties of these compounds were estimated by RP-HPLC using methanol/water mobile phases with a various volume fraction of the organic modifier. The log kw values, which were extrapolated from intercepts of a linear relationship between the logarithm of a retention factor k (log k) and volume fraction of a mobile phase modifier (ϕM), varied from 2.113 (compound 8e) to 2.930 (compound 8h) and indicated relatively high lipophilicity of these salts. Electronic properties of the molecules 8a-h were investigated by evaluation of their UV/Vis spectra. In a next phase of the research, the compounds 8a-h were in vitro screened against M. tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794), M. kansasii CNCTC My 235/80 (identical with ATCC 12478), a M. kansasii 6 509/96 clinical isolate, M. avium CNCTC My 330/80 (identical with ATCC 25291) and M. avium intracellulare ATCC 13950, respectively, as well as against M. kansasii CIT11/06, M. avium subsp. paratuberculosis CIT03 and M. avium hominissuis CIT10/08 clinical isolates using isoniazid, ethambutol, ofloxacin, ciprofloxacin or pyrazinamide as reference drugs. The tested compounds 8a-h were found to be the most promising against M. tuberculosis; a MIC = 8 µM was observed for the most effective 1-(2-{4-[(butoxycarbonyl)amino]phen-ylphenyl}-2-hydroxyethyl)-4-(2-fluorophenyl)piperazin-1-ium chloride (8h). In addition, all of them showed low (insignificant) in vitro toxicity against a human monocytic leukemia THP-1 cell line, as observed LD50 values > 30 µM indicated. The structure-antimycobacterial activity relationships of the analyzed 8a-h series are also discussed.

Is TB Testing Associated With Increased Blood Interferon-Gamma Levels?

The Republic of Ireland reports a relatively low prevalence of Johne's disease (JD) compared to international counterparts. Postulated reasons for this include a lower average herd size and a grass-based production system. Ireland also engages in high levels of bovine tuberculosis (bTB) testing. As interferon-gamma (IFN-γ) is believed to play a key role in protecting against JD, it is our hypothesis that administration of purified protein derivative (PPD), as part of the bTB test, is associated with a systemic increase in IFN-γ production, which may potentially limit clinical progression of the disease. We studied 265 cows (202 Friesian and 63 "Non-Friesian," e.g., JerseyX, Norwegian Red) to assess IFN-γ levels and Mycobacterium avium subspecies paratuberculosis (MAP) antibody response before and after the bTB test. As part of the compulsory annual bTB test, avian and bovine PPD were administered at two separate cervical sites. To assess IFN-γ production, blood samples were taken before and 72 h after PPD administration. MAP antibody response was assessed before and 10 days post-PPD administration. A significant increase in MAP antibody response was identified post-bTB compared to pre-bTB response (p < 0.001). Additionally, IFN-γ production significantly increased at the post-bTB time point (p < 0.001) compared to the pre-bTB test readings. This may indicate a beneficial effect of bTB testing in controlling JD.