[Show abstract][Hide abstract] ABSTRACT: Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response.
[Show abstract][Hide abstract] ABSTRACT: A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic
promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter
within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving
from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic
bovine lung (EBL) cells and an attenuated AroA- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the
plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage,
some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the
plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring
RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria
within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P.
multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.
[Show abstract][Hide abstract] ABSTRACT: Vacuolating cytotoxic activity in the mouse macrophage cell line RAW 264 was reported in Pasteurella multocida B: 2 strains associated with haemorrhagic septicaemia in buffaloes and cattle. A putative virulence-related gene mviN was reported in the sequenced genome of P multocida serotype A (avian strain Pm70). MviN is an integral membrane protein that belongs to MATE (multi-antimicrobial extrusion) family. The present study was undertaken to detect the presence of putative virulence-related genes, viz. vacB and mviN among P. multocida B: 2 strains (8). Sequence information for the vacB gene was obtained from the sequenced genome of P. multocida serotype A (avian strain Pm70). Oligonucleotide primers were designed for these genes and synthesized. All the 8 P multocida B: 2 strains, 1 P. multocida type A 3 (bovine) and 1 P multocida type D (pig) strain had both of these genes of the expected size (1.5 Kb for mviN and 2.4 Kb for vacB) as confirmed by PCR. E. coli K12 used as negative control did not show any band. Both these putative virulence-related genes were amplified in a single reaction mixture using multiplex PCR. The expected size (1.5 Kb for mviN and 2.4 Kb for vacB) of both the genes was obtained. This is the first report of the detection of these virulence-related genes among P multocida B: 2 strains. Besides providing additional targets for diagnostic applications, the identification and subsequent analysis of virulence-related genes in P. multocida B: 2 strains may help in improving our understanding of molecular mechanism of adaptation, survival and virulence of P multocida B: 2.
No preview · Article · Jul 2013 · The Indian journal of animal sciences
[Show abstract][Hide abstract] ABSTRACT: Background: Adenylate cyclase toxin (CyaA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and a potential component of acellular pertussis vaccine. Objectives: In the present study the impact of invasive CyaA on oxidative activities of phagocytes was compared with the other form of this molecule to investigate the activity of different parts of molecules on leukocytes. Materials and Methods: The work involved the production of two purified forms of CyaA with different enzymic and invasive properties. They were: the native enzymatically-active, invasive toxin (CyaA), an invasive derivative lacking AC enzymic activity (CyaA*). Different concentrations of CyaA and CyaA* were used to investigate dose-dependent effects of the toxins on oxidative burst in U937 human monoblastic cells, J774.2 mouse macrophage-like cells and fresh human granulocyte cells by Burst Test assay. Results: Significant effects were observed with 0.2 μg protein/mL of CyaA. For instance, there was almost complete (80%) inhibition of phagocytosis by J774.2 cells and 70% inhibition of phagocotosis by human granulocyte cells. The results showed that production of the oxidative burst was significantly impaired by increasing concentrations of CyaA compared to cells treated with PBS. However, there was no significant effect with CyaA* on either cells. Conclusions: The results of the study showed that both enzymatic and invasive functions were required for the oxidative burst effects of adenylate cyclase toxin in leukocytes.
[Show abstract][Hide abstract] ABSTRACT: Image processing algorithms were developed and compared with visual assessment from 12 volunteers to quantify the temporal morphological structure of a single Euglena gracilis organism. Representative images of E. gracilis, showing different morphological characteristics from ovate to cylindrical and elongate, were captured with a bright-field video microscopy system. These images were ranked by the volunteers in order from ovate to elongate. The images were analyzed in the spatial and spatial frequency domain, and the order of the images from each analysis was ranked against the visual assessment. The assessment methods agreeing with the volunteer's preferred sequence were an eccentricity measurement (major axis over the sum of the minor axis at three points), the cross correlation of the image without high pass filtering or edge detection, and cross correlation of the power spectral density.
Full-text · Article · Jul 2012 · Microscopy and Microanalysis
[Show abstract][Hide abstract] ABSTRACT: A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.
Full-text · Article · Mar 2012 · Microbial Pathogenesis
[Show abstract][Hide abstract] ABSTRACT: A protein designated Bap-5 (GenBank accession no. AF081494) or BapC (GenBank accession no. AJ277634) has been identified as a member of the Bordetella pertussis autotransporter family and the present work suggests that this protein, like the previously characterised BrkA, is a Bvg-regulated serum resistance factor and virulence determinant. B. pertussis bapC and brkA, bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA, bapC mutants, like the brkA mutant, were found to be more sensitive to the antimicrobial peptide cecropin P1 than the parent strains. In the genome sequence of B. pertussis strain Tohama, bapC is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5' end of the gene which creates a truncated BapC protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms but it appeared that all had an ORF that would be able to produce BapC.
No preview · Article · Sep 2011 · Microbial Pathogenesis
[Show abstract][Hide abstract] ABSTRACT: Although laser sterilisation has been well studied in dentistry and medicine, there have been few studies within the food industry. UV radiation has been used for sterilisation of surfaces and water. The killing effect of microwave radiation has been investigated on many bacteria in food and there has been much controversy over its killing mechanism. In this study, the killing effect of laser, microwave and UV radiation was studied on E. coli and on some spoilage and pathogenic bacteria. The bacterial suspensions were exposed to the treatment processes in sequence and viable cell counts were made before and after each treatment. A difference in the reduction in viable counts was apparent when the sequential treatment was compared with the sum of the individual treatments alone. Similar results were obtained when conventional heating was used in place of microwave radiation. It was found that the order of the treatment processes had a significant influence on killing.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10 kDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.
Full-text · Article · Feb 2011 · Veterinary Microbiology
[Show abstract][Hide abstract] ABSTRACT: The probability of infection during air travel was determined by assuming randomised events (sneezing, coughing, vomiting) and the Well-Riley model for the infectivity rate. The potential of using an Excimer laser operating at 248 nm to decontaminate air was experimentally investigated. Infections spread by air travel is of growing concern in recent times with such outbreaks as SARS and Bird Flu occurring. There are also concerns over how the air is re-circulated through cabins and cleaned of microorganisms and chemical contaminants from the fuel. A mathematical model has been developed to assess the risk factors that may lead to passengers on board becoming infected with airborne respiratory diseases and novel methods of decontaminating air have been investigated. Airborne infections are spread when people are in close proximity for a period of time . Being in an enclosed space such as an aeroplane enhances the risk of infection. Large droplets containing micro-organisms are projected into the air whenever an infected person talks, coughs, sneezes, vomits etc, and these can be intercepted by anyone within a range of a few meters. However smaller lighter particles (droplet nuclei) generated from sneezing for example, can remain suspended in the air for a longer time and have a wider range of infection. In the simulation, the aircraft cabin was divided into four sections/cabins so that the effects of proximity and the air recirculation and mixing were modelled. A mathematical model of the airflow affecting the concentration of infectious agents in the cabin was produced. It is clear from these models and recent press articles that there are potentially serious problems associated with aircraft contamination. Overall, the results demonstrated that lasers could be used successfully to decontaminate air. Exploiting lasers as a means to decontaminate air may therefore provide an efficient, alternative method of cleaning air for improved cabin air quality.
[Show abstract][Hide abstract] ABSTRACT: Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes. As a step towards the identification of individual antigens that may protect against HS, proteins present in a sonicated cell extract (SCE) and outer-membrane protein (OMP) preparation of a wild-type P. multocida serotype B:2 were investigated by immunoblotting with sera from calves which had been protected against challenge with a virulent strain of P. multocida B:2 by vaccination with a live-attenuated aroA derivative of the challenge strain. Five proteins in SCE, of approximately 50, 37, 30, 26 and 16 kDa, were recognised by the sera. In an OMP preparation, two bands, at 37 and 50 kDa, were recognised as strongly immunogenic. Mass spectrometry analysis of proteins corresponding in size to those detected by immunoblotting identified the 37 kDa band as OmpA, but the band at 50 kDa was not identified with certainty. A major 30 kDa OMP, identified as OmpH, was not strongly immunogenic.
No preview · Article · Mar 2009 · Research in Veterinary Science
[Show abstract][Hide abstract] ABSTRACT: The survivability of Pasteurella multocida B: 2 were observed in complement dependent antibody mediated bactericidal assay. The organisms were grown in the presence of specific antibodies against P. multocida B: 2 with and without the supplementation of 2 different sources of complement, viz. fresh bovine serum and guineapig serum. The studies indicated that organisms were not killed and were rather multiplying in any kind of serum, suggesting that complement is not playing a role in the killing of P. multocida B: 2.
[Show abstract][Hide abstract] ABSTRACT: A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.
No preview · Article · Nov 2008 · Microbial Pathogenesis
[Show abstract][Hide abstract] ABSTRACT: Confluent BLEC monolayers were infected with bacterial suspension at a multiplicity of infection (MOI) of 25, 50, 75, 100 or 200 bacteria per cell and centrifuged at 1500 x g for 3 min then the mixture incubated for 2 h at 37 • C in 5% CO 2 . Loosely-bound bacteria were then removed from the cell monolayer by washing with PBS and the cells were detached by trypsin-EDTA. The resulting cell suspension was then lysed with digitonin and the cell-adherent bacteria were enumerated by culture on selective medium. Invasion Bacteria in cell culture medium without antibiotics were mixed with confluent BLEC monolayers or J774.2 cell suspensions (MOI 100:1 or 500:1) and centrifuged as above. They were then incubated for 2 h at 37 • C in a humidified atmosphere containing 5% CO 2 then washed three times and fresh medium containing 50 µg/ml or 350 µg/ml of polymyxin B and gentamicin was added to kill extracellular bacteria. After incubation for 1-2 h, cells were washed two times with antibiotic-free medium, monolayers were treated with trypsin-EDTA for 5 min at 37 • C and lysed by addition of digitonin to release intracellular bacteria for enumeration by plate count. Each assay was carried out in either triplicate or quadruplicate and was repeated independently two or three times. Results are expressed as means of all experiments. Cell viability was routinely evaluated with the trypan blue exclusion test. Intracellular survival After an infection period of 2 h followed by antibiotic treatment as above, the epithelial cells were washed and fresh medium with or without polymyxin B and gentamicin was added. The monolayers were then further incubated for 2 h or 4 h before lysis and enumeration of viable intracellular bacteria as described before. Invasion inhibition Cytochalasin D was dissolved in DMSO and cells were exposed to final concentrations from 1 to 10 µg/mL for 1 h at 37 • C prior to addition of the bacteria at MOI 100:1. The remainder of the invasion assay was done as describe above. Electron microscopy For conventional transmission electron microscopy (TEM) mammalian cells were washed three times in PBS, then fixed in 2.5% glutaraldehyde in 0.05M cacodylate buffer, pH 7.2, at 4 • C for 1 h and washed three times in the same buffer. The specimens were post-fixed with osmium tetroxide (1%) in distilled water and stained en bloc with 0.5% uranyl acetate. Samples were then dehydrated through a graded series of ethanol and embedded in Epon 812 (TAAB). Ultrathin sections were post-stained with uranyl acetate and lead citrate prior to viewing with a Zeiss EM 109 transmission electron microscope operating at 80 kV.
[Show abstract][Hide abstract] ABSTRACT: Tuned cylindrical radial mode ultrasonic horns offer advantages over ultrasonic probes in the design of flow-through devices for bacterial inactivation. This study presents a comparison of the effectiveness of a radial horn and probe in the inactivation of Escherichia coli K12. The radial horn is designed using finite element analysis and the predicted modal parameters are validated using experimental modal analysis. A validated finite element model of the probe is also presented. Visual studies of the cavitation fields produced by the radial horn and probe are carried out using luminol and also backlighting to demonstrate the advantages of radial horns in producing a more focused cavitation field with widely dispersed streamers. Microbiological studies show that, for the same power density, better inactivation of E. coli K12 is achieved using the radial horn and, also, the radial horn offers greater achievable power density resulting in further improvements in bacterial inactivation. The radial horn is shown to be more effective than the probe device and offers opportunities to design in-line flow-through devices for processing applications.
Full-text · Article · Mar 2008 · Ultrasonics Sonochemistry
[Show abstract][Hide abstract] ABSTRACT: Three different recombinant forms of CyaA were used to investigate transcriptional responses of murine bone marrow-derived macrophages (BMMs) using Affymetrix Mouse Genome GeneChips. These forms were enzymically active, invasive CyaA, non-enzymically active, invasive CyaA (CyaA*) and non-enzymically active, non-invasive CyaA (proCyaA*). BMMs, treated with 20 ng/ml of CyaA for 24h, showed over 1000 significant changes in gene transcription compared with control cells. CyaA caused an increase in transcription of many inflammatory genes and genes associated with various signalling cascades such as those involved in cyclic AMP-dependent protein kinase A signalling. Most strikingly, CyaA caused down-regulation of numerous genes involved in cell proliferation. CyaA* at 20 ng/ml significantly up-regulated the transcription of only twelve genes after 24h whereas proCyaA* at this concentration significantly increased the transcription of only two genes.
Full-text · Article · Feb 2008 · Microbial Pathogenesis
[Show abstract][Hide abstract] ABSTRACT: Three groups of five calves, namely, V1, V2, and V3, were immunized intramuscularly at 4 and 8 weeks of age with ca. 109, 108, and 107 CFU, respectively, of a derivative of Pasteurella multocida B:2 wild-type strain 85020 containing a deletion in the aroA gene (strain JRMT12). The first and second vaccinations resulted in significantly (P < 0.01) higher rectal temperature responses in groups V1 and V2 than in group V3. Serum immunoglobulin M (IgM) and IgG titers
did not increase in any group until after the second vaccination and were then significantly higher in groups V1 and V2 than
in group V3 (P = 0.001 for both IgM and IgG). All vaccinated groups and three unvaccinated challenge control calves (group CC) were injected
subcutaneously at 10 weeks of age with ca. 107 CFU of strain 85020. Vaccinated calves survived the challenge, but two CC animals developed clinical disease and were killed
for humane reasons. After challenge, mean serum amyloid A concentrations were significantly higher (P < 0.001) in the CC group than in the vaccinated groups. Postmortem examination revealed that calves in the CC group showed
the most extensive range of bacteriologically positive tissues and gross and histopathological lesions. Overall, a clear dose-dependent
response was present, with those receiving a higher vaccine dose being less affected clinically, bacteriologically, and pathologically
by the wild-type challenge. The V2 treatment appeared to give the best combination of high immune response, protection, and
Full-text · Article · Dec 2007 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Plasmids were present in 8 of the 21 isolates tested. There was variation in the number and size of plasmids. Since all the isolates whether harbouring plasmids or not were found pathogenic, so there seems to be no correlation between the presence or absence of plasmids and virulence of Pasteurella multocida organisms.
No preview · Article · Nov 2007 · The Indian journal of animal sciences
[Show abstract][Hide abstract] ABSTRACT: Adenylate cyclase toxin (CyaA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and, in its detoxified form, a potential component of acellular pertussis vaccines. This study reports the application of a novel technology, formulation of CyaA as protein-coated microcrystals (PCMC), to improve the performance of CyaA as a vaccine component. CyaA is normally stored in a high urea concentration to prevent aggregation and to maintain stability of the protein. The aim of the work was to stabilise CyaA on a crystalline support to create a dry powder that could be reconstituted in aqueous buffer, free of urea. CyaA, formulated as PCMC with microcrystals of dl-valine, retained full adenylate cyclase (AC) and cell invasive (cytotoxic) activities after solubilistion in urea buffer. After storage as a dry powder at 37 degrees C for 2 weeks, the AC activity recovered from the CyaA-PCMC was only marginally reduced when solubilised in urea buffer. No AC activity was detected after attempts to solubilise CyaA-PCMC in aqueous buffer alone, in the absence of urea. Inclusion of various ionic, non-ionic or zwitterionic detergents in the aqueous buffer had little effect on recovery of CyaA activities. However, preparation of PCMC with CyaA plus calmodulin (CaM) or bovine serum albumin (BSA) or with both proteins allowed restoration of AC and cytotoxic activities of CyaA upon solubilisation in aqueous buffer. Incorporation of BSA and CaM with CyaA allowed essentially full recovery of AC activity but lower recovery of cytotoxicity. CyaA-CaM-BSA-PCMC, after reconstitution in aqueous buffer, induced a strong serum IgG response to CyaA when injected subcutaneously into mice.
[Show abstract][Hide abstract] ABSTRACT: Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual PTx activity may likely be present because of the limitations of the detoxification processes used. Furthermore, different detoxification procedures have been shown to result in different amino acid side-chain modifications for the resulting PTd. The histamine-sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns. The ADP-ribosylation enzyme activity of PTx is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the ADP-ribosylation activity in different acellular pertussis-based combination vaccine formulations was measured and compared with reactivity in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and a level which would be significant in relation to the reactivity seen in the HIST could not be defined, except for vaccines that contain genetically detoxified PTx, which do not have enzymatic activity nor in vivo toxicity. Different detoxification procedures as well as formulation factors could contribute to this variation. Relying solely on the residual enzyme activity of PTx in vaccines containing chemically detoxified PTd may not fully reflect the in vivo reactivity observed by the HIST. Refinement of the in vitro test to include a step which monitors the B-subunit activity of PTx may provide a better correlation with the in vivo HIST.