Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.     

Feasibility of the use of ELISA in an immunogenicity-based potency test of anthrax vaccines

Complexities of lethal challenge animal models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA was used to measure the antibody response to protective antigen (PA) in mice immunized once with a commercially available (AVA) or a recombinant PA vaccine (rPAV) formulated in-house with aluminum hydroxide. Results from the anti-PA ELISA were used to select a single dose appropriate for the development of a potency test. Immunization with 0.2 mL of AVA induced a measurable response in the majority of animals. This dose was located in the linear range of the vaccine dose–antibody response curve. In the case of rPAV, practical limitations prevented the finding of the best single dose for the potency testing of purified vaccines. In additional immunogenicity experiments neither the magnitude of the response to a single dose of vaccine, nor the estimation of the dose necessary to induce a measurable response were able to consistently detect brief exposure of vaccines to potentially damaging temperatures. However, differences detected for rPAV in the proportion of mice responding to the same dose of treated and untreated vaccine suggested that further assay development to increase the sensitivity of the latter design may be warranted.     

Experimental dicrocoeliasis: the humoral immune response of golden hamsters and rabbits to primary infection with Dicrocoelium dendriticum

The results presented deal with the humoral immune response of golden hamsters to primary experimental infection with D. dendriticum. The development of serum antibodies has been comparatively investigated with three hamster groups (n = 43) harbouring different burdens of adult flukes. The mean numbers of parasites were 11, 30, or 130 per animal. Serum antibody response was studied during an observation period of at least 331 and up to 496 days postinfection. For antibody detection the sensitivities of precipitation test (PTs) (double diffusion test, immuno- and counterimmunoelectrophoresis), of the indirect haemagglutination test (IHAT), the complement fixation test (CFT), and the enzyme linked immuno sorbent assay (ELISA) were compared using aqueous crude fluke antigen and crude egg antigen. CFT and ELISA were most sensitive for the early detection of initial response. Thereafter all the tests employed revealed increasing antibody titres, which in general remained at constant levels and persisted until the end of the observation period with the exception of CF-antibodies. In general fluke antigen was found to be more sensitive than egg antigen. However, in CFT this antigen occasionally has been associated with unspecific inhibition of haemolysis. Comparison of the results shows that ELISA using crude fluke antigen gave the most realistic picture of the actual fluke burden. Also preliminary results on the precipitin response of rabbits (n = 3) after primary experimental exposure to different numbers of metacercariae (500, 1,000, and 3,000 per animal respectively) are reported. Employing the above mentioned PTs a persisting antibody response could be demonstrated only after exposure to at least 3,000 infective larvae. The initial response was found on day 63, the observation period was 550 days.     

Single-dilution assay of new vaccines produced using plant and insect cell substrates

Large numbers of animals are required in multi-dilution assays of vaccines produced in mammalian, plant and insect cell substrates. Animal vaccination and serum sampling require skilled labour, adding to testing costs. More tests are required with homologous reference preparations. Use of a single antigen reference to assay combined formulations is invalid due to synergistic or other effects of multiple antigens. Licensing authorities in Europe recommend use of single-dilution assays for some vaccines, together with homologous reference preparations. Vaccine and reference formulations in a single-dilution assay must have similar compositions to ensure similar dose response relationships. Design and validation of such a single-dilution assay is the main consideration of this article.Multi-dilution in vivo vaccine assays have inherent problems also affecting single-dilution assays. Statistical analysis of data from multi-dilution assays requires significance tests of assay validity, but with a single-dilution assay, some aspects of validity are assumed. These aspects include linearity and parallelism of the dilution-response curves for both reference and test preparations.This article shows that despite inherent problems associated with multi-dilution animal assays, the need to move to a simpler single-dilution model necessitates establishing minimal inherent conditions in the multi-dilution assay for a valid interpretation of the single-dilution assay.     

Report on the international workshop on alternative methods for Leptospira vaccine potency testing: State of the science and the way forward

Routine potency testing of Leptospira vaccines is mostly conducted using a vaccination–challenge test that involves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their international partners organized a workshop to review the state of the science of alternative methods that might replace, reduce, and refine the use of animals for veterinary Leptospira vaccine potency testing and to identify ways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate or continue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacements for potency testing of four common Leptospira serogroups. Participants discussed the potential for eliminating the back-titration procedure in the hamster challenge assay, which could reduce animal use by 50% for each individual potency test. Further animal reduction may also be possible by using cryopreserved Leptospira stock to replace continual passaging through hamsters. Serology assays were identified as a way to further reduce and refine animal use but should be considered only after attempting in vitro assays. Workshop participants encouraged consideration of analgesics and use of earlier humane endpoints when the hamster vaccination–challenge potency assay is used. International harmonization of alternative potency methods was recommended to avoid duplicative potency testing to meet regionally different requirements.     

The use of bootstrap resampling to assess the variability of Draize tissue scores

The acute dermal and ocular effects of chemicals are generally assessed by performing the Draize skin and eye tests, respectively. Because the animal data obtained in these tests are also used for the development and validation of alternative methods for skin and eye irritation, it is important to assess the inherent variability of the animal data, since this variability places an upper limit on the predictive performance that can be expected of any alternative model. The statistical method of bootstrap resampling was used to estimate the variability arising from the use of different animals and time-points, and the estimates of variability were used to determine the maximal extent to which Draize test tissue scores can be predicted.     

Development, preparation and safety testing of a Clostridium welchii type C toxoid. II. Laboratory evaluation of potency.

The results obtained with four laboratory tests on four candidate formulations of Clostridium welchii type C vaccine for use in man have been compared with clinical responses to the same vaccines. Quantal response assays in mice appeared to reflect the ranking of the four vaccines in human subjects better than did the guinea pig tests. They also enabled the potency of the vaccine preparations to be related to an existing International Reference Preparation. Mouse assays in which the animals received two spaced doses of vaccine prior to challenge yielded marginally more satisfactory results in terms of precision and reflection of human responses than did assays involving a single dose of vaccine.     

Development of passive haemagglutination (PHA) and haemagglutination inhibition (HAI) technique for potency estimation of Cobra Antisnake Venom Serum (ASVS).

Antibodies against snake venom or antivenom potency are assayed quantitatively by in-vivo neutralization test in mice, which requires large number of laboratory animals. In potency assays of biological substances such as antivenoms, it is highly desirable to avoid suffering and death of animals by substituting in-vivo method with in-vitro methods, provided such methods measure life-saving capability with precision similar to that of in-vivo method. The in-vitro tests determine the neutralizing power of antivenom by permitting the evaluation of a particular biological activity of the venom and its neutralization after mixing the venom with the antivenom [Theakston RDG, Reid HA. Development of simple standard assay procedures for the characterization of snake venom. Bull WHO 1983;61:949-956; Gutierrez JM, Rojas G, Lomonte B, Gene JA, Chaves F, Alvarado J, et al. Standardizing of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1127-1129; Theakston R.D.G. Comments on letter of Gutierrez et al. on standardization of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1131-1132; Harvey AL, Barfaraz A, Thomson E, Faiz A, Preston S, Harris JB. Screening of snake venom for neurotoxic and myotoxic effects using simple in-vitro preparation from rodents and chicks. Toxicon 1994;32:257-265; World Health Organization Progress in characterization of venom and standardization of anti-venoms. Geneva: WHO offset publication; 1981. p. 58.]. Hence, the ideal requirements for an assay in detecting venom and venom antibody include high level of sensitivity, specificity (ability to differentiate between venom and venom antibody produced by closely related species of snakes), reproducibility and simplicity. A new in-vitro procedure for quantitative analysis of potency of ASVS by passive haemagglutination (PHA) and haemagglutination inhibition (HAI) has been explored. The methods described are simple, rapid, economical, reproducible and useful in replacing the more expensive in-vivo neutralization assays. Moreover, it also eliminates the use of laboratory animals.     

A semi-quantitative serological method to assess the potency of inactivated rabies vaccine for veterinary use

Potency is one of the most important indexes of inactivated vaccines. A number of methods have been established to assay the potency, of which the NIH test and single-dose mouse protection test are the “prescribed methods”. Here, we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine, which uses fewer animals and takes less time to complete. Depending on the quality requirements of a vaccine (e.g. minimum potency), a rabies reference vaccine is, for example, diluted to the minimum potency, and 50 μL of the dilution is taken to inoculate 10 mice. The same amount of the test rabies vaccine is inoculated into another 10 mice. After two weeks, all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization (FAVN) test. By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine, the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality. The reliability of this method was also confirmed in dogs. The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.     

Resource heterogeneity and ungulate population dynamics

It has been suggested that climatic variation has the effect on the dynamics of arid and semi-arid grazing systems of reducing animal numbers below the level at which they have much impact on vegetation or soils, and that spatial heterogeneity in resource availability serves to buffer herbivores against climatic variation. Modelling was used to test these hypotheses and to examine the interacting effects of temporal and spatial variability in plant production on animal population dynamics and defoliation intensity. The model distinguishes areas of the range that are accessible during wet and dry seasons, and examines the effect of seasonal restrictions in foraging area. It was established that the animal population is in long-term equilibrium with dry-season resources, on which it depends for survival; that dry season resource areas and outlying areas thus operate in a source-sink manner; and that the ratio of these areas determines the strength of consumer-resource coupling outside the dry-season range. A high ratio of dry season to wet season resources may support a sufficiently large animal population to impose non-trivial defoliation impacts on the outlying range. Increasing degrees of variability in primary production on areas used by animals for surviving the dry season increased the annual variation in animal abundance and reduced the mean. By comparison with a stable environment, for which the model predicts virtually stable animal numbers and constant, low defoliation intensity, variation in annual rainfall causes wide fluctuations in animal numbers and defoliation intensity. Under climatic variation, animal numbers can build up enough to impose much higher defoliation intensities than under a constant regime. Periodic intense defoliation is a consequence of climatic variability which is likely to make these environments more, not less, prone to ecological change.     

Experimental plague in rock squirrels, Spermophilus variegatus (Erxleben)

Experimental infections with Yersinia pestis were followed in groups of rock squirrels. Development of coagulopathy and pneumonia were observed in 2-4% and 11-12% of the test animals, respectively. Susceptibility to experimental infection was heterogeneous with some animals surviving inoculation with large numbers of organisms and others succumbing after inoculation with small numbers. Production and longevity of serum antibody titers, as measured by passive hemagglutination tests, were variable as well, and apparently unrelated to dose. The data presented attest to the need for care in interpreting serologic tests results for individual animals.     

Filter-paper blood samples for ELISA detection of Brucella antibodies in caribou

We evaluated blood collected on Nobuto filter-paper (FP) strips for use in detecting Brucella spp. antibodies in caribou. Whole blood (for serum) and blood-saturated FP strips were obtained from 185 killed arctic caribou (Rangifer tarandus groenlandicus). Sample pairs (serum and FP eluates) were simultaneously tested in duplicate using competitive enzyme-linked immunosorbent assay (c-ELISA) and indirect ELISA (i-ELISA) for Brucella spp. Prior work based on isolation of Brucella spp. revealed sensitivity (SE) and specificity (SP) of 100% and 99%, respectively, for both these serum assays in caribou. Infection status of the animals in the current study was unknown but recent sampling had revealed clinical brucellosis and >40% Brucella antibody prevalence in the herd. To assess the performance of FP relative to serum in these assays, serum was used as the putative gold standard. On both assays, the findings for duplicate runs (A and B) were similar. For c-ELISA run A, the FP Brucella prevalence (47%) was lower than serum prevalence (52%), with SE 89% (95% confidence interval [CI]: 82-95%) and SP 99% (97-100%). For i-ELISA run A, serum and FP Brucella prevalence rates were identical (43%), and the SE and SP of FP testing were 100% and 99% (97-100%), respectively. The findings suggest better FP test performance with i-ELISA than with c-ELISA; however, i-ELISA does not distinguish cross-reacting antibodies induced by Brucella vaccination or exposure to certain other Gram-negative pathogens. Results for duplicate FP eluates (prepared using separate FP strips from each animal) were strongly correlated for both protocols (r=0.996 and 0.999 for c-ELISA and i-ELISA, respectively), indicating minimal variability among FPs from any individual caribou. Dried caribou FP blood samples stored for 2 mo at room temperature are comparable with serum for use in Brucella spp. c-ELISA and i-ELISA. Hunter-based FP sampling can facilitate detection of disease exposure in remote regions and under adverse conditions, and can expand wildlife disease surveillance across temporospatial scales.     

Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing

Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the “International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward” held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination–challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination–challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified.     

Pasteurella multocida toxin type D serological assay as an alternative to the toxin neutralisation lethality test in mice.

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0.94 and Pearson analysis=0.84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing.     

A Comparison of Two Serological Methods for Detecting the Immune Response after Rabies Vaccination in Dogs and Cats being Exported to Rabies-free Areas

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre of > or = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre < 1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (> or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.     

A comparison of combined diphtheria, tetanus and pertussis vaccines produced by different manufacturers, in laboratory animals and in infants

Three DTP vaccines were investigated for potency and toxicity (reactogenicity) both in laboratory animals and in infants. Animal tests were carried out in conformity with the WHO recommendations. Three- to five-month-old infants were investigated for their specific antibody responses and for local and systemic vaccination reactions. No correlation was found between the potency values of the vaccines as expressed in IUs and the antibody titres of the vaccinated infants. The most striking difference between the human and animal responses to vaccination was observed in the case of the tetanus toxoid. The severity of the vaccination reactions in infants correlated with the toxicity of the vaccines as assessed in the mouse weight gain test (MWGT) carried out in CBA mice. No correlation was found, however when conventional or AKR mice were used in the MWGT.     

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.     

The Pertussis Serological Potency Test. Collaborative study to evaluatereplacement of the Mouse Protection Test.

The Pertussis Serological Potency Test (PSPT)--based on in vitro assessment of the humoral immune response against Bordetella pertussis--was developed as an alternative for the Mouse Protection Test (MPT). A small-scale collaborative study was carried out in five laboratories to evaluate the relevance and reliability of the PSPT. The study has been divided into three separate phases, each with its own objective. A pilot-phase study of the antibody detection assay, the 18323-whole cell ELISA (WCE), was included for training purposes. Significant differences in absorbance and antibody concentrations between the laboratories were found. In the Phase I study, the intra-assay, inter-assay and inter-laboratory precisions of the 18323-WCE were assessed. Although a precision of less than 20% was not always established and significant differences in antibody concentrations were found at random throughout the Phase I study, the ranking of the antibody concentrations corresponded well between the laboratories and should warrant a reliable potency estimation of whole cell vaccines (WCV's) in the PSPT. Phase II was a comparative study of the PSPT and the MPT to evaluate the implementation of the PSPT, to demonstrate correlation and to compare the reproducibility and reliability of both tests. The mean antibody concentrations per vaccine dose in the PSPT and the survival of mice in the MPT differed significantly within and between the laboratories. Nevertheless, the potencies of the vaccines under test estimated in both test models did not differ significantly (P>0.05). The PSPT and MPT correlated well in chi2-test of homogeneity within and between the laboratories. The potencies were similar (overall ratio=0.877), but the PSPT is more reproducible and reduces the chance of re-testing due to the smaller 95% confidence intervals. We have demonstrated that the PSPT is a valid model to estimate the potencies of pertussis WCV's from different manufacturers. Moreover, the 18323-WCE is easy to carry out and the intra-assay precision and antibody ranking warrants a reliable potency testing of pertussis WCV's in the PSPT.