Characteristics of IPS 82 as standard for biological assay of bacillus thuringiensis H-14 preparations

WORLD HEALTH ORGANIZATION WHO/VBC/84.892 ORGANISATION MONDIALE DE LA SANTE ENGLISH ONLY CHARACTERISTICS OF IPS 82 AS STANDARD FOR"'n!OLOGICAL ASSAY OF BACILLUS THURINGIENSIS H-14 PREPARATIONsl by H. de Barjac2 and I. Larget-Thi~ry2 1. INTRODUCTION Evaluation of the activity of B. thuringiensis (B.t.) preparations is currently carried out by biological titration on a target insect, and compared to an adequate homologous standard (Burgerjon, 1959; Burges, 1967a,b; van der Geest & Wassink, 1972). At present, biological titration is considered as the most reliable method for measuring the insecticidal activity of B.t. preparations, despite other existing techniques based on chemical or inmunological or immuno-enzymatic dosage of the toxic crystal protein (Krywienczyk & Fast, 1980; Fast, 1982). Indeed, a major objection to all these other techniques is the uncertainty regarding the toxic components of the crystals and the mode of extraction before dosage. This, in addition to the questionable final toxicity to which the germination of B.t. spores possibly contributes, makes biological titration the method of choice. For a reliable biological titration method, a major condition is a valid standard, meeting all the requirements of homology, stability, ability to be replicated and availability. As regards B.t. H-14 preparations, a first attempt to develop such a standard was made in 1978 and an air-dried formulated B.t. H-14 (strain ONR-60A) powder, called IPS 78 (Institut Pasteur Standard 1978), was produced (de Barjac, 1979; de Barjac & Larget, 1979). As in the case of the first B.t. standard E 61 (Etalon, 1961), produced by the Institut Pasteur with B.t. serotype H-1 (Bonnefoi et al., 1958; Burgerjon, 1959; Burgerjon & Yamvrias, 196o";" Vago & Burges, 1964), the experimental standard IPS 78 was assigned an arbitrary titre of 1000 international units/mg (I.U./mg) for Aedes aegypti larvae (Bora-Bora strain). Much was accomplished with the wide use of IPS 78 in more than 35 different countries. However, after two years of experimentation, a decrease in larvicidal activity, in addition to the difficulty with and instability of stock suspensions, made it necessary to develop a better product. Thus IPS 80 was prepared, based on the lyophilized pure culture of B.t. H-14 (strain 1884) and stored in sealed penicillin flasks in amounts of about 50 mg. This primary powder, titrated by referring to IPS 78, contained 10 OOO I.U./mg for Ae. aegypti. Extensive research during two other years and by different laboratories showed a perfect stability, ability to be replicated and homology, even in stock suspensions. Unfortunately, only a limited amount was prepared (about 2000 flasks) for it to be considered as an international standard by WHO. This is the reason why a third standard powder, IPS 82, has been elaborated, by the same method as IPS 80, but in a larger amount (100 mg per flask). The titre of IPS 82 is 15 OOO I.U./mg for Ae. aegypti. 1 This investigation received financial support from UNDP/World Bank/WHO Special Progranane for Research and Training in Tropical Diseases. 2 Lutte Biologique contre les Insectes II, Institut Pasteur, Paris, France. The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles. Ce document ne constitue pas une publication. II ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que leurs auteurs. WHO/VBC/84.892 page 2 In this paper we are reporting the continuous study on the stability of IPS 80 and IPS 82 powders, and according to these results proposing the adoption of IPS 82 as the final B.t. H-14 standard. 2. MATERIALS AND METHODS 2.1 Larval rearing Bioassays on Ae. aegypti need larvae that are as homogeneous as possible. This can be realized by using a standardized rearing method. Five days before a bioassay, dried papers bearing the laid eggs are dipped into dechlorinated tap water to which a total vitaiuin supplement has been added (United States Biochemical Corporation). Thus, at the temperature of 25°C, hatching occurs in less than an hour. These newly hatched larvae are then removed and transferred by pipette to containers with 2 1 dechlorinated tap water, at the rate of 500-600 larvae per container. Larvae are fed dog biscuit food (about 3 g). Five days later, always at 25°C, the larval population usually looks homogeneous and consists mainly of fourth instars. 2.2 Bioassay Young stage IV larvae are used for the bioassay: 100 larvae for each concentration (25 per plastic cup containing 150 ml of suspension). Six concentrations of IPS 80 or IPS 82 near the LCSO level (0.04, 0.03, 0.02, 0.01, 0.005, 0.004 mg/1) are prepared from stock suspensions. Cups containing water only are used as controls. Mortality is read after 24 and 48 hat 25°C, and regression lines are drawn on log-probit paper. When control mortality exceeds 2%, mortality is corrected according to the Abbott formula. If control mortality exceeds 10%, the whole experiment is rejected. 2.2.1 Stability of IPS 80 and IPS 82 stock suspensions IPS 80 stock suspensions at 5 g/1 have been kept at 25°C for various periods up to more than one year. By now IPS 82 has been kept in stock suspension for eight months at 4°C. Activity of these suspensions have been compared with those of fresh stock suspensions. 2.2.2 Stability of IPS 80 and IPS 82 powders after heat exposure We used a method reconunended by WHO. Five sealed flasks of IPS 80 and IPS 82 have been kept at 50°C during four weeks, then compared to five other "control" flasks stored at S°C. Five replicates have been carried out on five different days using different flasks each time. 3. RESULTS AND DISCUSSION Stability of the two standard powders stored at S°C in sealed flasks has been checked periodically, throughout the years. From January 1981 to September 1982, 18 bioassays (at least one per month) were realized with IPS 80 on Ae. aegypti larvae. The mean of all the LCSOs is 0.011 + 0.0041 mg/1 and 0.0081 .:!:_ 0.0024 mg/1, at 24 and 48 h, respectively. With IPS 82 powder, 14 bioassays, carried out from July to December 1982, gave the following mean for the LCSO: 0.0098 + 0.0033 mg/1 (24 h); 0.0~75 .:!:. 0.0031 mg/1 (48 h). IPS 82 seems to be slightly more active than IPS 80. Percentages of variation are around 30%, which is not very important for a biological titration where difficulties related to the physiological state of target larvae and also to their manipulation must be taken into account. The larvicidal activity of IPS 80 and IPS 82 stock suspensions (5 g/1) is given in Table 1. Suspensions of IPS 80 were kept at 25°C during various periods of time ranging from 1-387 days. The results depended more on the larval age than age of the suspension~ With four-day-old larvae, the LCSO was about 0.0055 mg/1, but with five-day-old larvae the LCSO varied around 0.018 mg/1. As can be seen, these data show an excellent stability of IPS 80 stock suspensions at 25°C. Suspensions of IPS 82 have been stored at 5°C. After 256 days, these WHO/VBC/84.892 page 3 suspensions still had a very high activity, possibly with a slight decrease when compared with the mean value of the first bioassays. Heat stability of both standard powders was checked. LCSO and LC90 from IPS 80 flasks exposed at 50°C were compared with those of flasks kept at 5°C (Table 2; Fig. 1). Results with IPS 82 are shown in Table 3, Fig. 2. Mean values of all LC50s and LC90s, for IPS 80 as well as for IPS 82, are very similar. Thus stability at 50°C looks excellent for both powders. In the five bioassays carried out, the per cent. variation of LC50 is about 40% for IPS 80 and 26% for IPS 82. Regression lines at 24 and 48 h (Figs. 1 & 2) are parallel and very similar for non-heated and heated powders, especially in IPS 82 bioassays. Details of data on IPS 82 are given in Tables 4 and 5. 4. CONCLUSION IPS 82, like IPS 80 standard powder, has shown an excellent stability on storage, even after heat exposure according to the WHO requirements. Availability of IPS 82 in an adequate amount (5000 flasks of about 100 mg each) enables it to be delivered to any research laboratory requesting it. These important qualities, as well as its readiness to suspend seem the best arguments for the adoption of IPS 82 as an official B.t. H-14 standard. ACKNOWLEDGEMENT This investigation received financial support from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. REFERENCES Bonnefoi, A., Burgerjon, A. & Grison, P. (1958) Titrage biologique des pr~parations de Bacillus thuringiensis, C.R. Acad. Sci. (Paris), 247, 1418-1420 Burgerjon, A. (1959) Titrage et d~finition d'une unit~ biologique pour les pr~parations de Bacillus thuringiensis, Entomophaga, ~. 201-206 Burgerjon, A. & Yamvrias, C. (1960) M~thode de titrage biologique des pr~parations A base de Bacillus thuringiensis avec Anagasta kuehniella, Zell, C.R. Congr~s Int. Entomol., Vienne, 840-844 Burges, H. D. (1967a) Standardization of Bacillus thuringiensis products: Homology of the standard, Nature (London), 215, 664-665 Burges, H. D. et al. (1967b) The standardization of products based on Bacillus thuringiensis. In: Insect pathology and microbial control, 314-337, Amsterdam, North-Holland Publ., 360 pp. Fast, P. G. (1982) Chemistry and biochemistry of biocides, Informal consultation on basic biology of microbial larvicides of human vectors, Geneva, 26-27 April 1982 (Working papers) van der Geest, preparations: .!2., 361-365 L. P. S. & Wassink, H. J.M. (1972) Standardization of Bacillus thuringiensis A new bioassay method Pieris brassicae as test insect, J. Invertebr. Pathol., Krywienczyk, J. & Fast, P. G. (1980) Serological relationships of the crystals of Bacillus thuringiensis var. israelensis., J. Invertebr. Pathol., 36, 139-140 Vago, C. & Burges, H. D. (1964) International symposium on the identification and assay of viruses and Bacillus thuringiensis Berliner used for insect control, J. Insect. Pathol., ..§_, 544-547 WHO/VBC/84.892 page 4 TABLE 1. LARVICIDAL ACTIVITY OF IPS 80 AND IPS 82 STOCK SUSPENSIONS KEPT AT 25° OR 5°C Stock LC50 (mg/1) Fourth-ins tar suspensions larvae (days of (age in days) storage) 24 h 48 h IPS 80 (25°c) 0 0.0077 0.0058 4 5 0.0053 0.0045 4 12 0.0047 0.0034 4 55 0.005 4 70 0.005 0.004 4 1 0 .018 0.012 5 6 0.021 0.0025 5 13 0.021 0.017 5 56 0.019 0.015 5 71 0.021 0.016 5 364 0.019 0 .012 5 (5 mm) 366 0.018 0.013 5 (5-6 mm) 385 0.011 5 (5 mm) 387 0 .015 0.0095 5 (5-6 mm) IPS 82 (5°C) 0 0.0071 0.0038 5 (5-6 mm) 218 0.018 0.010 5 (5-6 mm) 246 0.021 0.010 5 (5-6 mm) 0 0.010 0 .0096 6 (5-6 llllll) 219 0.0064 0.0028 6 (5-6 mm) 247 0 .011 0.0079 6 (5-6 mm) 256 0.017 0.010 5 (6 mm) r Date 30.8.82 31.8.82 1. 9 .82 14. 9 .82 15. 9. 82 Average Average WHO/VBC/84.892 page 5 TABLE 2. LARVICIDAL ACTIVITY OF IPS 80 POWDER ON FOURTH-INSTAR AEDES AEGYPTI LARVAE (FIVE DAYS OLD; 5 nun) IPS 80 (5°C) IPS 80 (50°C) LC 24 h 48 h 24 h 48 h mg/1 mg/1 50 0.010 0.0069 0 .011 0.0093 90 0.024 0.016 0.024 0 .017 50 0.0074 0.0050 0.0057 0.0049 90 0.014 0 .010 0.011 0.0076 50 0.0066 0.0055 0.0049 0.0034 90 O.Oll 0.0087 O.Oll 0.0084 50 0.013 0.0088 0.017 0.010 90 0.025 0.017 0.042 0.023 50 0.020 O.Oll 0.015 0 .010 90 0.034 0.019 0.034 0.026 LC 50 0 • 0 ll + 0 • 004 8 0.0074 + 0.0022 0.010 + 0.0048 0.0075 + 0.0028 LC 90 0.021 + 0.0082 0.014 + 0.004 0.024 + 0.012 0.016 + 0.0074 WHO/VBC/84.892 page 6 Date 23.8.82 24.8.82 25.8.82 18.10.82 19 .10.82 Average Average TABLE 3. LARVICIDAL ACTIVITY OF IPS 82 POWDER ON FOURTH-INSTAR AEDES AEGYPTI LARVAE (FIVE DAYS OLD; 5 mm) IPS 82 (5°C) IPS 82 (5o"c) LC 24 h 48 h 24 h 48 h mg/1 mg/1 50 0 .0073 0.0046 0.0139 0.012 90 0.015 0.0097 0.025 0.023 50 0.0094 0.0058 0.0094 0.0065 90 0.026 0.018 0.021 0.017 50 0.008 0.0061 0.0072 0.0054 90 0.021 0.015 0.017 0.012 50 0.0081 0.0036 0.0069 0.0045 90 0.021 0.011 0.015 0.0096 50 0.015 0.014 0.010 0.0097 90 0.024 0.022 0.019 0.017 LC 50 0.0095 + 0.0028 0.0068 + 0.0036 0.0094 + 0.0025 0.0076 + 0.0028 LC 90 0.021 + 0.0037 0.015 + 0.0045 0.019 + 0.0034 0.014 + 0.0046 I. TABLE 4. LARVICIDAL ACTIVITY OF IPS 82 STORED AT 5°C (PERCENTAGE MORTALITY OF FOURTH-INSTAR AE. AEGYPTI LARVAE) IPS 82 23.08.82 24.08.82 25.08.82 18.10.82 19.10.82 Dose mg/1 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h nb L4 nb L4 0.04 99 100 100 100 100 100 100 100 100 100 100 100 0.03 98 99 97 100 100 100 99 100 100 100 100 100 0.02 97 100 80 90 94 99 86 95 98 71 79 99 0.01 73 91 38 63 47 75 46 78 95 7 9 88 0.005 25 60 14 32 11 14 36 67 100 1 1 94 0.003 5 18 19 36 24 32 Slope b 3 .88 4.02 2.86 2.51 3.06 3.28 3.02 2.58 6.92 6.89 S.E. of b 0.26 0.36 0.18 0.18 0.21 0.24 0.24 0.33 0.55 0.57 x 2 5.67 18 .19 37.2 24 52.71 50.7 19.04 3.78 41.07 17.63 nb. f.d. 4 4 4 4 3 3 3 3 3 3 X 2 prob. 1.5 x 10-7 7. 7 x 10-5 1.10-10 2.6 x 10-4 signif. 0.22 0.0011 1.10-10 0.28 6.4 x 10-9 5 .2 x 10-4 Factor g 0.01 0.28 0.3 0.25 0.91 1.0 0.43 0.063 0.9 0.41 LC50 0.0073 0.0046 0.0094 0.0058 0.0080 0.0061 0.0081 0.0036 0.015 0.014 0.0067 ·0.0032 0.0051 0.0031 0.003 0.0030 0.0026 0.0014 0.0094 Range 0.0080 0.0062 0.015 0.0088 0.051 0.012 0.0045 0.030 0.019 LC90 0.015 0.0097 0.026 0.018 0.021 0.015 0.021 0.011 0.024 0.022 ~~ (IQ O (1) ..._ 0.014 0.007 0.30 0.012 0.010 0.013 0.0098 0.016 0 .017 <: ..... t,I Range C"l ---0.018 0.022 0.016 0.053 0.089 0.014 0.047 00 "" 00 "' .., TABLE 5. LARVICIDAL ACTIVITY OF IPS 82 STORED AT 50°C FOR FOUR WEEKS -g ~ (PERCENTAGE MORTALITY OF FOURTH-INSTAR AE. AEGYPTI LARVAE) (IQ O !T) --~ 00 t;j (":) --IPS 82 23.08.82 24.08.82 25.08.82 18 .10 .82 19.10.82 00 f' 00 '° N Dose mg/ 1 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h nb L4 nb L4 nb L4 0.04 97 100 98 100 100 100 98 100 100 100 100 100 100 0.03 97 100 100 100 97 98 100 100 98 100 97 98 100 0.02 81 100 84 100 91 94 97 99 98 100 92 94 100 0.01 19 99 25 99 46 70 66 84 65 89 46 57 100 0.005 1 100 5 98 11 30 19 29 34 58 3 4 90 0.003 1 100 1 100 9 20 16 27 - Slope b 4.87 4.65 3.63 3.07 3.42 3.56 3.55 3.9 5.02 5.02 S.E. of b 0.34 0.31 0.22 0.21 0.28 0.28 0.31 0.53 0.37 0.40 x 2 20.86 12.1 14.67 4.57 17.67 17 5.24 1.25 3.44 4.34 nb. f.d. 4 4 4 4 4 3 3 3 3 3 x 2 prob. signif. 3.3 x 10-4 0.016 0.0054 0.33 0.0014 6.9 x 10-4 0.14 o. 739 0.32 0.22 Factor g 0.20 0.11 0.11 0.018 0.15 0.37 0.03 0.071 0.021 0.024 LC50 0 .0139 0.012 0.0094 0.0065 0.0072 0.0054 0.0069 0.0045 0.010 0.0097 0.010 0.0098 0.0071 0.0058 0.0052 0.003 0.0060 0 .0037 0.0099 0.0089 Range 0.018 0.015 0.012 0 .0072 0.0097 0.008 0.0077 0.0051 0.011 0.010 LC90 0.025 0.023 0.021 0 .017 0.017 0.012 0.015 0.0096 0.019 0.017 0.019 0.018 0.015 0.014 0.012 0.0082 0.014 0.0084 0.017 0.015 Range 0.042 0.033 0.034 0.02 0.031 0.045 0.018 0.011 0.022 0.020 90 >, ,I.J .... 50 .... .,, ,I.J i.. 0 x ~ FIG 1. COMPARISON OF LARVICIDAL ACTIVITY OF IPS 80 ON FOURTH-INSTAR AEDES AEGYPTI LARVAE (MEAN; FIVE EXPERIMENTS) , 11 I PS 80 ilJ 1.11ft. !/!;· • /) 'f. I ·- -24h sooc ,. I 0 ___ 4sh 1 month 1·1 ·--· 24h s~c 0 ____ .4sh i 1• I ~ I 0.00 5 0,01 Q,02 0,04 log (concentration.) -,/1 WHO/VBC/84.892 page 9 WHO/VBC/84.892 page 10 90 50 FIG. 2. COMPARISON OF LARVICIDAL ACTIVITY OF IPS 82 ON FOURTH-INSTAR AEDES AEGYPTI LARVAE (MEAN; FIVE EXPERIMENTS) I p5a2 •··---24h 0 ___ 4ah • 24h so'c O- - 48h 1 month 0,005 0,0 1 0.02 0,04 Log (concentration) mg/1