Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Real-time PCR detection of Campylobacter spp.: A comparison to classic culturing and enrichment.

The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and broiler caeca in The Netherlands in 2007, were subjected to three different real-time PCR detection methods: one targeting the Campylobacter jejuni hipO gene, one targeting the Campylobacter coli glyA gene, and one generically targeting Campylobacter spp. 16S rDNA sequence. The PCR results from the three different PCR protocols were compared to the work of Nauta et al. (2009) who analyzed the same set of samples collected from 62 broiler flocks by means of enrichment culturing. The results indicate that the generic 16S campylobacter PCR detection is equally reliable but much faster (4 h instead of ≥2 days) than detection by means of culturing. Moreover, PCR detection targeting the hipO and the glyA gene provide the possibility of C. jejuni and C. coli species discrimination. The generic Campylobacter spp. PCR analysis also confirmed the high incidence of Campylobacter spp. in poultry samples (∼90%) and the species specific PCR showed the simultaneous presence of C. jejuni and C. coli in ∼24% of the samples. Furthermore, the results from the three PCR analyses suggested the occurrence of alternative Campylobacter species in almost 10% of the samples. The campylobacter PCR detection methods reported here can replace traditional culturing because of being quicker and more reliable.

In ovo inoculation of chicken embryos with probiotic bacteria and its effect on posthatch Salmonella susceptibility.

The feasibility of establishing probiotic bacteria in the intestine of broiler chickens by in ovo inoculation was investigated, followed by verifying possible subsequent protection against Salmonella Enteriditis infection. In a first study, 7 commercially available probiotics were screened for compatibility with in ovo inoculation. Two of these probiotics, one being a Enterococcus faecium and the other a Bacillus subtilis, were selected for colonizing the chick gut without compromising hatchability. In a second study, these 2 products were administered in ovo and in the feed to chicks reared until 18 d in comparison with noninoculated chicks and with chicks fed an antibiotic. All chicks were orally challenged with Salmonella Enteritidis at 4 d of age. Results showed reduced performance of Salmonella Enteritidis challenged chicks fed no additives compared with challenged chicks fed antibiotic, but no significant differences in mortality was observed. Probiotics offered in ovo or through the diet could only partially recover performance compared with antibiotic-fed chicks. A significant reduction in the number of Salmonella Enteritidis positive chicks was observed when chicks were in ovo inoculated with E. faecium and continued receiving it in the diet. This work establishes standards for future in ovo colonization research and emphasizes its value as a promising method to deliver individual precise dose of probiotics to poultry in mass scale at the earliest possible age based on the competitive exclusion concept. In ovo colonization with probiotic can therefore become an important ally in combination with other approaches to combat Salmonella and other intestinal bacterial infections in poultry.

Ileal microbiota composition of broilers fed various commercial diet compositions.

Microbiota plays a role in the release and absorption of nutrients from feed components, thereby affecting digesta composition and moisture content of the excreta. The objective of the current study was to determine the effects of 5 different diets varying in ingredients (medium-chain fatty acids, nonstarch polysaccharides, and starch) on the microbiota composition of ileal digesta of broiler chickens and excreta DM content. Each treatment was repeated 6 times in cages each containing 18 Ross 308 broilers, with growth performance measured from 0 to 34 d of age and excreta DM and ileal microbiota composition analyzed at 34 d of age. Microbiota composition was evaluated using a novel ribosomal RNA microarray technology containing 370 different probes covering various genera, groups of microbial species, and individual species of the chicken gut microbiota, of which 321 had a signal above the background threshold. Replacing part of the animal fat and soybean oil in the wheat-based diet with medium-chain fatty acids (MCFA; 0.3% C10 and 2.7% C12) improved feed efficiency compared with the other dietary treatments. This coincided with a suppression of gram-positive bacteria belonging to the phylum of the Firmicutes, including Lactobacillus species, and species belonging to the family of the Enterococcaceae and Micrococcaceae, whereas the gram-negative bacteria belonging to the family of the Enterobacteriaceae were promoted. None of the other diets used in the present study notably changed the ileal digesta bacteria composition. Excreta DM content was not affected by dietary treatment. The variation between individual birds per dietary treatment was more pronounced than variation caused by feed composition, with the exception of the digesta microbiota of the birds fed the MCFA diet. It is concluded that a diet with MCFA significantly changes the ileal microbiota composition, whereas the effect of the other diets on the composition of the microbiota and excreta DM content is small in broiler chickens.

Establishment and application of test methodology demonstrating the functionality of air purification systems in reducing virus-loaded aerosol in indoor air.

BACKGROUND: The global COVID-19 pandemic has resulted in a greater interest in improving the ventilation of indoor environments in order to remove aerosolized virus and thus reduce transmission. Air purification systems have been proposed as a solution to improve aerosol removal. AIM: The aim was to determine the efficacy of air purification systems in reducing the viral load in the environmental air of a room. METHODS: A containment room equipped with HEPA filter on air intake and exhaust was constructed. It was connected via an inlet with the BSL-2 facility. From the BSL-2, Feline Coronavirus (FCoV)-loaded aerosols were released into the containment room. After nebulization, air sampling was performed to determine the viral load in air prior to assessing the clean air delivery rate of the air purification systems. The infectivity of the captured viruses was also examined. FINDINGS: The air purification systems realized a 97-99% reduction in viral load in air in 1 h. Captured infectious FCoV was reduced by 99.9%-99.99% by use of an ESP technology. CONCLUSIONS: The air purification systems, using ESP technology or HEPA filter, reduce the viral load in air. The ESP purifiers inactivate captured FCoV viruses. Therefore, air purification systems can be used as an adjunctive infection control measure.

Scoping review on the efficacy of filter and germicidal technologies for capture and inactivation of micro-organisms and viruses.

The COVID-19 (SARS-CoV-2) pandemic increased the focus on preventing contamination with airborne pathogens (e.g. viruses, bacteria, and fungi) by reducing their concentration. Filtration, UV or ionization technologies could contribute to air purification of the indoor environment and inactivation of micro-organisms. The aim of this study was to identify the relevant literature and review the scientific evidence presented on the efficacy of filter and germicidal technologies (e.g. non-physical technologies) in air purification applications used to capture and inactivate micro-organisms and airborne viruses (e.g. SARS-CoV-2, rhinovirus, influenzavirus) in practice. A scoping review was performed to collect literature. Adopting exclusion criteria resulted in a final number of 75 studies to be included in this research. Discussion is presented on inactivation efficiencies of ultraviolet germicidal irradiation (UVGI) and ionization applications in laboratory studies and in practice. Specific attention is given to studies relating the use of UVGI and ionization to inactivation of the SARS-CoV-2 virus. Based on the consulted literature, no unambiguous conclusions can be drawn regarding the effectiveness of air purification technologies in practice. The documented and well-controlled laboratory studies do not adequately represent the practical situation in which the purifier systems are used.

Effective ultraviolet C light disinfection of respirators demonstrated in challenges with Geobacillus stearothermophilus spores and SARS-CoV-2 virus.

BACKGROUND: The global COVID-19 pandemic, accompanied by spikes in the number of patients in hospitals, required substantial amounts of respiratory protective devices (respirators), thereby causing shortages. Disinfection of used respirators by applying ultraviolet C (UVC) light may enable safe reuse, reducing shortages. AIM: To determine whether UVC disinfection is applicable to enable repeated safe reuse of respirators. METHODS: The UVC chamber, equipped with low-pressure mercury discharge lamps emitting at 254 nm, was used to determine the sporicidal and virucidal effects. Respirators challenged with spores and viruses were exposed to various UVC energy levels. Deactivation of the biological agents was studied as well as UVC effects on particle filtration properties and respirator fit. FINDINGS: A 5 log10 reduction of G. thermophilus spore viability by a UVC dose of 1.1 J/cm2 was observed. By simulating spores present in the middle of the respirators, a 5 log10 reduction was achieved at a UVC dose of 10 J/cm2. SARS-CoV-2 viruses were inactivated by 4 log10 upon exposure to 19.5 mJ/cm2 UVC. In case UVC must be transmitted through all layers of the respirators to reach the spores and virus, a reduction of >5 log10 was achieved using a UVC dose of 10 J/cm2. Exposure to a six-times higher UVC dose did not significantly affect the integrity of the fit nor aerosol filtering capacity of the respirator. CONCLUSION: UVC was shown to be a mild and effective way of respirator disinfection allowing for reuse of the UVC-treated respirators.

Ciclosporin kinetics in children after stem cell transplantation.

AIMS: To develop a limited sampling strategy to determine ciclosporin systemic exposure [area-under-the-curve(AUC)]. This is meant to be the first step in a future study of the relationship between AUC and the biological effects of ciclosporin. METHODS: The pharmacokinetics of ciclosporin was investigated prospectively following stem cell transplantation (SCT) in 17 children, aged 1.8-16.1 years. Ciclosporin was given twice daily, intravenously over a short infusion of 2 h duration during the early post-SCT period, or orally later on, when oral medication was well tolerated. Parameter estimation was performed using nonlinear mixed effect modelling as implemented in the NONMEM program. Individual empirical Bayes estimates of clearance and distribution volume were correlated with the demographic variables. RESULTS: Pharmacokinetics was described adequately with a two-compartment model with lag time (population estimates: CL = 11.3 l h(-1); V(c) = 16.5 l; V(p) = 59.9 l; t(1/2) absorption = 0.78 h, t(lag) = 0.6 h). The AUCs, determined for the combination of trough level with one time point between 2 and 3 h after dosing, correlated very well with the reference AUC (r(2) = 0.97). No correlation was found between clearance and distribution volume, and the demographic patient variables length, body weight, age and glomerular filtration rate. CONCLUSION: A two-point limited sampling strategy, in combination with a Bayesian fitting procedure using the pharmacokinetic population model described, can adequately determine the AUC of ciclosporin. Since no correlation between clearance and body weight was found, dosing ciclosporin per kg bodyweight is not supported by the results of this study. We suggest starting with a fixed dose, followed by AUC determination and dose adjustment.

HLA-identical haematopoietic stem cell transplantation for acute leukaemia in children: less relapse with higher biologically effective dose of TBI.

To examine relapse, survival and transplant-related complications in relationship to disease- and pre-treatment-related characteristics, we evaluated 132 children, who consecutively received an allogeneic HLA-identical SCT for acute leukaemia in our centre: ALL in first remission (n=24), ALL in second remission (n=53) and AML in first remission (n=55). The source of the stem cells was bone marrow in all but three cases. Most patients (89%) were pre-treated with cyclophosphamide and an age-related dose of TBI. Initially, GVHD prophylaxis consisted of long-course MTX only (n=24), later short-course MTX and CsA (n=102) was given. All patients were nursed in strictly protective isolation and received total gut decontamination to suppress their potentially pathogenic enteric microflora. The 5-year probability of overall survival was 63, 53 and 74% for ALL1, ALL2 and AML1, respectively (median follow-up: 10.6 years). The overall transplant-related mortality was 6%. The incidence of acute GVHD was 17%; 6% was grades II-IV. A higher total biologically effective TBI dose (BED) resulted in a decreased relapse frequency (P=0.034) and increased overall survival. AML patients with acute GVHD got no relapse (P=0.02); this was not the case in ALL patients. Fractionated TBI regimens with higher BED should be evaluated in prospective studies.

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products.

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.

Microbes in food processing technology.

There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-based methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.

Adenovirus infection in paediatric stem cell transplant recipients: increased risk in young children with a delayed immune recovery.

Adenovirus (HAdV) infections are a frequent cause of morbidity and mortality after allogeneic human stem cell transplantation (HSCT). We report a retrospective single-centre study on 328 consecutive paediatric recipients of an allogeneic HSCT. During the first 6 months after HSCT, HAdV infection occurred in 37 children (cumulative incidence 12%). The highest incidence was found in young children up to 5 years of age, transplanted after 1994, with >2 log T-cell depletion of a graft of another than an HLA-genotypically identical related donor (actuarial frequency at 6 months 84%). Persistence of HAdV and spreading of the virus over multiple sites showed a trend towards the development of HAdV disease or death, but did not reach significance. Recovery of immunity after HSCT, that is, serum concentrations of IgM and peripheral blood counts of T cells and subsets, was delayed in children with an HAdV infection compared with noninfected children. In seven out of seven patients with HAdV DNA in serum and decreasing lymphocyte counts, the infection had a fatal course. Manipulation of cellular immunity either by tapering of immunosuppression, infusion of donor lymphocytes or immunotherapy using HAdV-specific T cells should be considered in graft recipients at risk for a severe HAdV infection.

Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity.

We studied the pharmacokinetics of intravenous busulfan (Bu) in children in order to further optimize intravenous Bu dosing in relation to toxicity and survival. A total of 31 children undergoing Bu-based conditioning for allogeneic SCT were enrolled in a study. The starting dose was 1.0 mg/kg (age < 4 years) and 0.8 mg/kg (age > or =4 years), four doses per day during 4 days. Dose adjustment was allowed up to a maximum dose of 1.0 mg/kg per dose if the target area under the serum concentration-time curve (AUC) was not reached. Pharmacokinetic studies were performed after the first dose. Donor engraftment was established in 28 out of 31 patients. The average AUC after the first dose was the same in children < 4 years as in children > or =4 years. Mean clearance was higher in children < 4 years than in children > or =4 years. In 35% of all patients, total AUC was within the target AUC. The other children's AUCs were below the target range. No relationships were found between systemic exposure to Bu and toxicity or clinical outcome. We concluded that, in accordance with previous data, within the observed AUCs no clear relationship was observed between Bu AUC and outcome with respect to toxicity, engraftment and relapse.

Allogeneic bone marrow transplantation for juvenile myelomonocytic leukemia: a single center experience of 23 patients.

Juvenile myelomonocytic leukemia (JMML) is a childhood leukemia for which allogeneic BMT is the only curative therapy. At our pediatric stem cell transplantation unit, we performed 26 BMTs in 23 children (age 0.5-12.7 years). Conditioning was CY/TBI based (1980-1996, n=14) or BU/CY/melphalan based (1996-2001, n=9). Donors were HLA-identical siblings (n=11), unrelated volunteers (n=9) or mismatched family members (n=3). A total of 10 patients survive in CR (median follow-up 6.8 years, range 3.1-22.2 years). Relapse or persistent disease was observed in eight and two patients, respectively. Nine of these patients died, one achieved a second remission following acute nonlymphatic leukemia chemotherapy (duration to date 5.3 years). Transplant-related mortality occurred in four patients. Overall survival at 5 and 10 years was 43.5%. Using T-cell-depleted, one-antigen mismatched unrelated donors was the only significant adverse factor associated with relapse in multivariate analysis (P=0.039, hazard ratio 4.9). Together with a trend towards less relapse in patients with graft-versus-host-disease and in patients transplanted with matched unrelated donors, this suggests a graft-versus-leukemia effect of allogeneic BMT in JMML.

[The possible role of microchimerism in the aetiology of autoimmune diseases]. / Mogelijke rol van microchimerisme bij het ontstaan van auto-immuunziekten.

The aetiology of several autoimmune diseases has not yet been elucidated. Microchimerism, the persistence of small numbers ofallogeneic cells in an individual, has been mentioned recently in connection with the occurrence of autoimmune diseases such as systemic sclerosis and juvenile inflammatory idiopathic myopathy. These allogeneic cells can originate from mutual foeto-maternal exchange of blood cells during pregnancy or from a donor after blood transfusion or (organ) transplantation. In some cases, a syndrome then develops that resembles the chronic graft-versus-host reaction after stem-cell transplantation, in which allogeneic cells react with autologous cells. Studies on microchimerism in patients with systemic sclerosis and juvenile inflammatory idiopathic myopathy, compared to controls, sometimes reveal a clearly increased prevalence of microchimerism in patients. However, microchimerism can also be found in healthy individuals. Direct proof of a causal relation between microchimerism and autoimmune diseases does not exist. Additional genetic or environmental factors may be partly responsible for a disturbed balance between tolerance and aggression.

Study of possible correlations between in vitro growth of bone marrow stromal and hematopoietic precursor cells early after allogeneic bone marrow transplantation.

Growth characteristics of stromal cells, assessed as adherent cells in long-term bone marrow cultures, and of hematopoietic progenitor cells, prior to and shortly after allogeneic bone marrow transplantation (BMT), were investigated, more specifically with regard to their possible correlations. The main constituent cells of the bone marrow stroma, that is, endothelial cells, reticular cells/fibroblasts, and monocytes/macrophages, showed an as yet inexplicable increased growth in samples taken from recipients prior to BMT, as compared with the growth in samples from their healthy donors and those taken after BMT. In the third week after BMT the in vitro outgrowth of hematopoietic precursors was severely depressed, but cell numbers in the adherent layer were normal. No relationship between in vitro growth of hematopoietic precursor cells and stromal cells was observed at that time. Probably the precursor cells growing in vitro are committed progenitor cells, relatively independent of stromal influences. In the eight week after grafting, endothelial cell outgrowth in vitro was highly correlated with granulocyte-macrophage colony-forming unit (CFU-GM) colony formation and to a lesser extent with mixed-lineage colony-forming unit (CFU-Mix) colony formation. This may indicate the reappearance of cytokine-mediated influences or the reappearance of a direct interaction, for example, by cell-cell contact between stromal cells and hematopoietic progenitor cells at that time.

Absence epilepsy and the level of vigilance in rats of the WAG/Rij strain.

In man, a relationship exists between sleep-wake states and absence epilepsy. During wakefulness, spike-wave discharges predominantly occur when the level of vigilance is not high, while during sleep they have a preference to occur during slow-wave sleep. During this latter type of sleep, spike-wave discharges prevail in periods where slow-wave sleep is light. In a series of experiments, the WAG/Rij rat model for absence epilepsy was characterized with respect to the relationships between the level of vigilance, sleep-wake states and the occurrence of spike-wave discharges. In the first experiment, continuous recordings were made for a period of 48 h and a clear circadian rhythm was established for the number of spike-wave discharges. A maximum appeared during the middle of the dark period of the rat, whereas a minimum was detected directly after the onset of the light period, the time period during which deep slow-wave sleep predominates. The relationship of spike-wave discharges with states of vigilance was elaborated in a second study. Spike-wave discharges were mainly found during light slow-wave sleep, during passive wakefulness and in transition phases from sleep to wakefulness. During REM sleep no spike-wave discharges were found. In the last three experiments, the level of alertness was enhanced by various procedures as photostimulation, a learning task and deprivation of REM sleep. In all cases, an increase of alertness decreased the amount of epilepsy.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of adenovirus DNA in plasma for management of infection in stem cell graft recipients.

We used a real-time polymerase chain reaction method for quantification of adenovirus to monitor the dynamics of viral DNA load in plasma in pediatric stem-cell graft recipients. Two cases are described to demonstrate that detection and quantification of the adenovirus DNA load at regular intervals may be important to document the stage of adenovirus infection, to make decisions on clinical intervention, and to accurately monitor the response to antiviral therapy.

The detection of cytoplasmic immunoglobulins by immunofluorescence: improvements in techniques and standardization procedures.

Iprovements in the technique of cytoplasmic immunofluorescence on cytocentrifuge slides obtained from cells in suspension are described. Refinement and standardization of the technique enabled us to obtain representative samples from the tissues under study and to determine the relative distribution of cells containing different heavy and light chain Ig determinants, as well as the absolute numbers of Ig-containing cells. The reproducibility of the results was highly satisfactory.

Measurement of primary in vivo IgM- and IgG-antibody response to KLH in humans: implications of pre-immune IgM binding in antigen-specific ELISA.

The antigen Keyhole Limpet Hemocyanin (KLH) is often used to test the primary in vivo antibody response capacity in humans. However, measurement of IgM anti-KLH antibodies in ELISA is complicated by the presence of natural antibodies in human serum. This problem occurs particularly at low antibody levels, i.e. after immunization with low doses of antigen and, under these conditions, it was found to be impossible to assess a dose-response curve by immunizing a series of individuals with different suboptimal doses of KLH. This problem was circumvented by choosing conditions for minimal binding of pre-immune IgM and to correct for such binding. Although signal-to-background ratios were markedly improved by modifying the ELISA conditions, pre-immune IgM still showed binding to KLH due to interaction with polysaccharide determinants. This non-specific binding was correlated with the total IgM content of the samples. When anti-KLH activities before and after immunization were expressed relative to total serum IgM, a significant correction was achieved, resulting in a diminished inter-individual variability with respect to both pre-immune and post-immunization values. As with IgG-class antibodies to KLH, virtually no binding was observed in pre-immune sera. After expression of the anti-KLH response as a ratio between the post-immunization and pre-immunization titres, a dose of 50 micrograms was found to be sufficient to evoke a detectable IgG-antibody response in the 10 subjects tested. To elicit a positive IgM response, a minimal dose of 250 micrograms was required.