The generation and expression of immunity to Trichinella spiralis in laboratory rodents.
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Animals↗
Biomedical subjects
R G Bell
Publications and source records attributed to R G Bell.
A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes.
Isolates (259) of psychrotrophic Clostridium spp. associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes. No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl. botulinum type B. It is concluded that although the growth of such micro-organisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.
Abattoirs↗
Distribution and sources of microbial contamination on beef carcasses.
Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.
Abattoirs↗
Comparison of aerobic and anaerobic methods for the microbiological monitoring of chilled packaged meat during storage.
Aerobic and anaerobic plate counts were compared for routine monitoring of the microflora, dominated by lactic acid bacteria, developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage. No statistical differences were observed between aerobic and anaerobic enumerations, made on plate count and blood agar plates, of the microflora developing on beef striploins packaged under vacuum or carbon dioxide during 14 weeks' storage at 0 degree C. With both techniques the spoilage microflora development differed between the two packaging regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced by anaerobic plate counts in the routine microbiological examination of the spoilage microflora developing on chilled meats packaged under anoxic modified atmospheres.
Aerobiosis↗
Intestinal transport and catabolism of IgE: a major blood-independent pathway of IgE dissemination during a Trichinella spiralis infection of rats.
Previous work has shown that Trichinella spiralis-infected rats transport IgE from plasma to intestinal tissue and fluids. In this study we quantitate IgE transport to the gut and circulation during T. spiralis infection in rats. Total IgE levels in intestinal fluid from infected rats were elevated by 4 days post-infection (dpi), but were not elevated in serum and lymph until 7 dpi. IgE levels in intestinal fluid ranged from 1 to 6 microg between 10 and 21 dpi, and serum and lymph IgE levels ranged from 100 to 200 ng/ml. Immunoprecipitation of intestinal fluid and enterocyte lysate at 11 dpi showed a protein of 190 kDa that was recognized by mouse anti-rat IgE-MARE-1 in Western blots. This protein was removed from intestinal wash samples with anti-IgE (A2)-Sepharose. The half-life of intact IgE in the intestinal lumen of rats 10 days after infection was 3.25 min. In serum, the half-life of IgE was 5 h. Analysis of IgE production and consumption in 10-day T. spiralis infected rats showed that about 4.67 microg IgE/day entered the serum, while 2570.00 microg IgE/day entered the intestinal lumen. The IgE present in serum 10 days after T. spiralis infection originated in the gut and/or associated lymphoid tissue and was transported to the circulation via thoracic duct lymph. However, most IgE produced in the intestine was transported to the gut lumen at a rate that exceeded transport to plasma by a factor of several-hundredfold.
Animals↗
Psychrotrophic Clostridium spp. associated with 'blown pack' spoilage of chilled vacuum-packed red meats and dog rolls in gas-impermeable plastic casings.
'Blown pack' spoilage of vacuum-packed chilled beef, lamb and venison, and of a cooked meat product, chilled dog rolls packed in an oxygen-impermeable plastic casing, was characterised by sensory, chemical and microbiological analysis. Investigation of the probable causative agents led to the isolation of eight strains of psychrotrophic clostridia. Three strains have been provisionally identified as C. difficile, C. beijerinckii and C. lituseburense; the other five remain unidentified. In inoculation studies only one isolate produced significant amount of gas on meat, causing pack 'blowing'. It is, therefore, possible that 'blown pack' spoilage involves a synergism with one or more other organisms.
Clostridium↗
Association of psychrotrophic Clostridium spp. with deep tissue spoilage of chilled vacuum-packed lamb.
Early spoilage of commercial vacuum-packed chilled lamb legs was manifested as an objectionable 'cheesy', deep tissue odour that became evident when a cut was made into the stifle joint. Investigation of the probable causative agents led to the isolation of two psychrotrophic strains of clostridia. The isolates could not be identified using traditional identification schemes. One isolate was able to produce strong, objectionable 'cheesy' odours in deep tissues of artificially inoculated beef.
Animals↗
IgE, allergies and helminth parasites: a new perspective on an old conundrum.
This paper analyses the association between infection with helminth parasites, the elevated production of IgE and the expression of allergies. Interpretations of this interaction have taken place in a scientific environment whose most secure element is the immunochemistry of allergic reactions resulting in a substantial body of literature that has sought a biological role for allergic reactivity in protective immunity directed against helminth parasites. While the association between helminth infections and elevated levels of IgE, mast cells and eosinophils is well established, a functional role for allergic reactions in protection against helminths has eluded experimental proof. Instead of this hypothesis, it is proposed that allergic reactivity is rarely present in helminth-infected individuals because allergic reactions do not function to regulate helminth infections. Data from many sources are used to establish that the 'normal' state of all mammals is to be infected with helminth parasites from shortly after birth until well into adulthood. Only in the last 100 years or so have people living in areas of high development with sophisticated water and sewage systems been able to escape helminth infection. Allergies are as conspicuously present in these human populations as they are absent in populations that are still regularly exposed to helminths. Furthermore, in populations with endemic helminthoses there is little overt expression of allergic pathology that could be connected to the acquisition or elimination of helminth parasites. Based on these observations, it is suggested that endemic helminthoses activate the Th2 system, particularly at mucosal surfaces, to provide a different level of immunological homeostasis than currently occurs in developed societies. Under these conditions, mast cells, eosinophils and IgE rarely participate in reactions that we would recognize as 'allergic', although their participation in the control of helminth infections is still envisaged. Allergic reactions are considered to be a purely pathologic consequence of the disruption of this homeostatic mechanism and are not protective at all for the individual expressing them. This interpretation is derived from the immunobiology of the host-parasite interaction rather than the biology of allergies and should lead to new concepts regarding both allergic disease and the role of helminth infections in human and animal populations.
Animals↗
The hygienic efficiency of conventional and inverted lamb dressing systems.
Aerobic plate counts (APC 37 degrees C and APC 25 degrees C) and Escherichia coli enumerations (Petrifilm) were used to determine sources of bacterial contamination during sheep dressing, determine the hygienic efficacy of hand wash and knife 'sterilization' procedures and compare the hygiene efficiency of conventional and inverted sheep dressing systems. The major slaughterline sources of microbial contamination were: fleece > workers' hands > faecal pellets > knife blades. Aerobic plate counts (APC 37 degrees C) exceeding log 4.4 cfu cm-2 were considered indicative of direct fleece contact, whereas E. coli numbers exceeding log 3.3 cfu cm-2 were considered indicative of direct faecal contact. A 44 degrees C water hand rinse removed 90% of the microbial contamination from workers' hands, but rinsed hands, particularly those contacting the fleece, still carried a microbial population exceeding log 4.0 cfu cm-2. A 44 degrees C rinse followed by an 82 degrees C water dip reduced the contamination on knife blades to less than log 3.0 cfu cm-2. Inverted dressing systems produced carcasses with a lower contamination level than conventional systems. With both systems little increase in contamination occurred after pelt removal. The areas of highest contamination were the forequarter region with inverted dressing and the hindquarter with conventional dressing. In both cases these regions are the sites where cuts are made through the skin. With both systems contamination around these cuts was entirely consistent with direct fleece contact resulting from 'rollback'.
Animals↗
Evidence for an interleukin 4-inducible immunoglobulin E uptake and transport mechanism in the intestine.
Immunoglobulin (Ig) E is the principal Ig involved in immediate hypersensitivities and chronic allergic diseases such as asthma. Helminths are the most potent infectious agents known for their capacity to stimulate IgE production during the course of infection. In rats, the nematode Trichinella spiralis typically elicits a strong parasite-specific IgE response during infection, and this IgE antibody has been shown to be protective against the parasite in passive transfer experiments. The study reported here analyzed the fate of 125I-labeled myeloma IgE (1R162) in normal and T. spiralis-infected rats after intravenous injection. T. spiralis infection induced a capacity for specific binding to the gut wall of 125I-IgE rather than 125I-IgG1, as well as the transport of IgE, but not IgG1, into the gut lumen. Peak intestinal uptake and transport of 125I-IgE occurred during the first and second weeks after injection but was not elevated in the fourth week, that is, after intestinal adult worms had been expelled. Neither 125I-IgE uptake in the gut wall nor transport to the lumen could be ascribed to tissue damage or vascular leakage. Luminal transport occurred in the small intestine and not the liver, which only transports low molecular weight degraded 125I-IgE. Calculations based on the amount of intact IgE in the lumen suggest that, in a 24-h period, up to 20% of injected 125I-IgE can be transported to the gut lumen during the peak transport period, between 6 and 14 d after infection. The intestinal IgE binding and transport response can be adoptively transferred with T. spiralis immune CD4+ OX22- (CD45RC-) lymphocytes, which are protective, but not the nonprotective sister population CD4+ OX22+ (CD45RC+) of lymphocytes isolated simultaneously from thoracic duct lymph of infected rats. The intravenous infusion of recombinant rat interleukin 4 also elicited significant intestinal uptake of 125I-IgE. We also present evidence for the presence of CD23 on rat intraepithelial lymphocytes. These data provide evidence for a novel, inducible, intestine-specific IgE uptake and transport mechanism.
Animals↗
Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22- and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity.
We analysed the cytokine profile of a T cell subset (CD4+ CD45 RC-) that confers protection against Trichinella spiralis infection in rats. These CD4+ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-gamma and functional cytokine secretion for IL-4, IL-5, IFN-gamma, TNF-alpha and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4+ CD45 RC+ or CD8+), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-gamma in the protective CD4+ CD45 RC- cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-gamma or TNF-alpha was secreted by the protective CD4+ CD45- RC- cells. Whereas the non-protective CD4+ CD45 RC+ cells secreted significant levels of IL-4, IFN-gamma, mast cell differentiating activity and TNF-alpha but little IL-5 activity. Non-protective CD8+ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-gamma secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.
Animals↗
Inactivation of enterococci and fecal coliforms from sewage and meatworks effluents in seawater chambers.
Inactivation in sunlight of fecal coliforms (FC) and enterococci (Ent) from sewage and meatworks effluents was measured in 300-liter effluent-seawater mixtures (2% vol/vol) held in open-topped chambers. Dark inactivation rates (kDs) were measured (from log-linear survival curves) in enclosed chambers and 6-liter pots. The kD for FC was 2 to 4 times that for Ent, and inactivation was generally slower at lower temperatures. Sunlight inactivation was described in terms of shoulder size (n) and the slope (k) of the log-linear portion of the survival curve as a function of global solar insolation and UV-B fluence. The n values tended to be larger for Ent than for FC, and the k values for FC were around twice those for Ent in both effluent-seawater mixtures. The combined sunlight data showed a general inactivation rate (k) ranking in effluent-seawater mixtures of meatworks FC > sewage FC > meatworks Ent > sewage Ent. Describing 90% inactivation in terms of insolation (S90) gave far less seasonal variation than T90 (time-dependent) values. However, there were significant differences in inactivation rates between experiments, indicating the contribution to inactivation of factors other than insolation. Inactivation rates under different long-pass optical filters decreased with the increase in the spectral cutoff wavelength (lambda 50) of the filters and indicated little contribution by UV-B to total inactivation. Most inactivation appeared to be caused by two main regions of the solar spectrum--between 318 and 340 nm in the UV region and > 400 nm in the visible region.
Darkness↗
Sunlight inactivation of enterococci and fecal coliforms in sewage effluent diluted in seawater.
Inactivation (loss of culturability) by sunlight of enterococci and fecal coliforms within sewage effluent diluted in seawater was investigated in field experiments. In most experiments, 500-ml flasks of pure silica were used to confine activated sludge effluent diluted to 2% (vol/vol) in seawater. Inactivation of bacteria in these flasks (diameter, 0.1 m) was faster than in either open chambers (depth, 0.25 m) or patches of dyed effluent (depth of order, 1 m), probably because of the longer light paths in the latter two types of experiment, which caused greater attenuation of sunlight. Inactivation of 90% of enterococci generally required 2.3 times the insolation required for 90% inactivation of fecal coliforms, because of both the presence of larger initial shoulders on survival curves and a lower final inactivation rate. Two parameters are required to model inactivation of enterococci, a shoulder constant as well as a rate coefficient. The depth dependence of inactivation rate for both fecal indicators matched the attenuation profile of UV-A radiation at about 360 nm. Inactivation by UV-B radiation (290 to 320 nm), which penetrates much less into seawater, is of minor importance compared with the UV-A and visible radiation in sunlight, contrary to expectations in consideration of published action spectra for bacterial inactivation.
Journal Article↗
Expression of a protective intestinal immune response can be inhibited at three distinct sites by treatment with anti-alpha 4 integrin.
The alpha 4 integrins mediate lymphocyte adhesion to Peyer's patch high endothelial vessels and homing to Peyer's patch, as well as to mesenteric lymph nodes. In rats, immunity to infection with the nematode Trichinella spiralis is known to be mediated by CD4+ OX22- (CD45RC-) cells that home to the intestine in large numbers. These experiments were conducted to determine whether the alpha 4 integrins or LFA-1 were involved in the expression of intestinal immunity to T. spiralis. Injection of the anti-alpha 4 integrin, mAb TA-2, but not anti-LFA-1, mAb TA-3, impaired the expression of immunity. An effect of TA-2 was measured at three distinct sites along the activation pathway leading to the migration of protective CD4+ OX22- cells to the intestine. Injection of TA-2 on the same day as infection prevented normal rejection of the parasite and abrogated the characteristic appearance of blast cells in draining lymph 3 days after infection. A similar effect on the migration of blast cells at day 3 was seen when TA-2 was injected 1 day after infection, and injection 2 days after infection still reduced the number of protective cells entering TD lymph on day 3. The effect of TA-2 and TA-3 on homing of dividing cells to the gut was examined by injecting dividing cells i.v. at the same time as antibody. Under these conditions migration of dividing cells to the gut was reduced by 90 to 95% and their capacity to adoptively transfer worm rejection blocked. Furthermore, TA-2 treatment also inhibited protection when it was injected 12, 18, or 24 h after the transfer of protective cells, when these cells had already entered the gut, but not when TA-2 injection was delayed for 36 h. These results indicate the involvement of alpha 4 integrins at the following points in the generation, dissemination, and function of CD4+ OX22- effectors: 1) initial activation during the first 48 h of infection; 2) migration of protective cells to and extravasation in the gut; 3) a function after entry into gut tissues. The results suggest that entry of dividing cells into the gut is critical for the adoptive transfer of protection and that alpha 4 integrin has multiple roles in the manifestation of intestinal immunity.
Animals↗
Intestinal helminth expulsion.
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Comment↗
Synthesis and characterization of rat interleukin-10 (IL-10) cDNA clones from the RNA of cultured OX8- OX22- thoracic duct T cells.
A cDNA of the complete coding region of rat IL-10 was cloned and sequenced using RNA isolated from a cultured population of thoracic duct T-lymphocytes obtained from Trichinella spiralis infected animals. The OX8- OX22-T-helper cells were stimulated in vitro with Concanavalin A for 24 hours prior to harvest. Reverse transcription of cellular RNA was primed with oligo-dT followed by amplification of IL-10 specific cDNA by polymerase chain reaction with synthetic oligo nucleotide primers chosen from two highly conserved regions of mouse and human IL-10. The sequence of the coding region of the amplified, cloned rat IL-10 cDNA is 90% identical to the mouse and 82% identical to the human IL-10 cDNA coding regions.
Amino Acid Sequence↗
Preparative high-performance liquid chromatographic separation and isolation of bacitracin components and their relationship to microbiological activity.
Bacitracin, a polypeptide antibiotic, is one of the most commonly used antibiotics in the world. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with a high-performance liquid chromatographic (HPLC) method, bacitracin was chromatographed using HPLC with ultraviolet detection and a YMC basic column. Adequate separation of the isomers was obtained to scale up this procedure to preparative HPLC using a Prep HPLC system and a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The individual fractions could be precipitated by adding zinc or methylene disalicylic acid and lowering the pH. The crude fractions were recycled to ensure chromatographic purity. The chromatograms can accurately predict (in minutes) the microbiologically determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.
Anti-Bacterial Agents↗
Trichinella spiralis: evidence that mice do not express rapid expulsion.
The rapid expulsion of Trichinella spiralis by mice of a variety of inbred and F1 mouse strains was examined. Mice were reinfected once with T. spiralis during and immediately after the natural termination of a primary infection and worm rejection was measured less than or equal to 24 hr after the challenge. The results showed that the challenge (super)infection was consistently rejected by all mouse strains before rejection of the adult worms from the primary infection commenced. Rejection of the challenge infection began at different times after the primary infection with NFS (2 days) less than C3H less than or equal to B10.Q approximately B10.BR (greater than 5 days). In all strains, rejection of the challenge infection preceded adult worm rejection from the primary infection by 5-8 days. At its peak, the loss of challenge worms related directly to the strength of the primary rejection process NFS greater than or equal to 98%, C3H 90-98%, and B10 mice 80-90%. Furthermore, loss of the capacity to reject the challenge followed approximately 7 days after the complete loss of the primary infection in each strain examined. Thus, the sooner worms from the primary infection were lost, the earlier the capacity to promptly reject the challenge infection disappeared. B10.Br mice still partially rejected a superinfection 35 days after the primary infection began, whereas NFS mice lost this capacity around 25 days. However, premature termination of the primary infection in B10.BR mice with methyridine at the same time that NFS mice naturally terminated their infection (15 days) abrogated the capacity of B10.BR mice to reject the superinfection at 24 days. Passive transfer of protective rat IgG monoclonal antibody to mice did not lead to rapid expulsion. Transfer of mouse immune serum to intestinally primed rats did result in rapid expulsion, suggesting that mouse antibody responses were adequate. The expression of superinfection rejection was susceptible to the administration in vivo of GK1.5, anti-mouse L3T4 antibody. The data indicate that the principal determinant of the strength, time of initiation, and longevity of rejection of a challenge infection was the response to the primary infection of that individual mouse strain. The genetic determinants of challenge infection rejection were seen to be identical to those that determined rejection of the primary infection. Since no evidence could be found to support the identity of this response with rapid expulsion, as defined in rats, a new term, "associative expulsion," is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗