1975
Nayar & Sauerman: Mosquito blood meal use for survival
103
Acknowledgments: We thank Dr J. D. Edman of the Florida Wildlife 20: 1-61. Medical Entomology Laboratory, Vero Beach, for critical Nath, V., B. L. Gupta & G. S. Bains. 1958. Histochemical reading of the manuscript. and morphological studies of the lipids in oogenesis. V. The egg-follicles of Culex fatigans. Res. Bull. Punjab Univ.
Zool. 148: 135-48. Nayar, J. K. & D. M. Sauerman, Jr. 1971. The effects of Boorman, J. P. T. 1960. Observations on the feeding diet on life-span, fecundity and flight potential of Aedes habits of the mosquito Aedes {Stegomyia) aegypti (Linnaeus): taeniorhynchus adults. J. Med. Ent. 8: 506-13. The loss of fluid after a blood-meal and the amount of 1975. The effects of nutrition on survival and fecundity in blood taken during feeding. Ann. Trop. Med. Parasitol. 54: Florida mosquitoes. Part 1. Utilization of sugar for survival. 8-14. J. Med. Ent. 12: 92-98. Clements, A. N. 1955. The sources of energy for flight in Nayar, J. K. & E. Van Handel. 1971. The fuel for mosquitoes. J. Exp. Biol. 32: 547-54. sustained mosquito flight. J. Insect Physiol. 17: 471-81. Gooding, R. H. 1972. Digestive processes of haematoService, M. W. 1968. Observations on feeding and oviposiphagous insects. 1. A literature review. Quaest. Ent. 8: tion in some British mosquitoes. Ent. Exp. Appl. 11: 277-85. 5-60. Van Handel, E. 1965a. The obese mosquito. J. Physiol. Jeffery, G. M. 1956. Blood meal volume in Anopheles 181:478-86. quadrimaculatus, A. albimanus and Aedes aegypti. Exp. Parasitol. 1965b. Microseparation of glycogen, sugars and lipids. J. 5: 371-75. Anal. Biochem. 11: 266-71. Laurence, B. R. & M. A. Roshdy. 1963. Ovary develop1972. Simple biological and chemical methods to determent in mosquitoes. Nature, Land. 200: 495-96. mine the caloric reserves of mosquitoes. Mosquito News 32: Maciolek, J. A. 1962. Limnological organic analyses by 589-91. quantitative dichromate oxidation. Rept. Bur. Sport Fish. LITERATURE CITED
J. Med. Ent. Vol. 12, no. 1: 103-110
30 April 1975
EVALUATION OF ^-EXOTOXIN OF BACILLUS THURINGIENSIS BERLINER FOR CONTROL OF FLIES IN CHICKEN MANURE By Roy J. Barker1 and William F. Anderson2 Abstract: A calcium and a sodium salt of (3-exotoxin obtained et al. 1973). This structural analog of adenosine from Bakthane-L69® were added to rations supplied to hens triphosphate inhibits nucleic acid biosynthesis and to pullets. This exotoxin was too toxic to be fed to chickens for the control of the house fly, Musca domestica L., in (Mackedonski et al. 1972). A recent review (Faust 1973) on the status of (3-exotoxin as an insecticide droppings. Not more than 3 mg/kg per day could be fed to hens for a week. The oral LDS0 to pullets was ca 1 mg/kg per day. appealed for reports of research on its safety. This Both salts were toxic to chickens, the calcium salt at 20 ppm report concerns such a test conducted 10 years ago. in feed and the sodium salt at 20 ppm in feed or in drinking The ability of B. thuringiensis products to kill fly water. Toxic symptoms were loss of vigor, reduced feeding, and undersized eggs. Reduced feeding was not a consequence of larvae, even after passage through the digestive palatability. The exotoxin caused severe gizzard erosion, then tract, has been useful for fly control; when added enteritis and proventriculitis. The exotoxin degraded when to poultry feed, they have given convenient but manure liquified and lost the ability to kill house fly larvae under such conditions. House flies became resistant to the (3- erratic control of flies in chicken manure (Borgatti & exotoxin. After a 6-month selection on media treated with the Guyer 1962, 1963, Briggs 1960, Burns et al. 1961, calcium salt, the larvae tolerated media containing 600 ppm. Harvey & Brethour 1960, Mechalas & Beyer 1963, Sherman et al. 1962). The insecticidal products of some cultures of Nevertheless, available data on B. thuringiensis Bacillus thuringiensis Berliner are not simply living reveal results which would have raised doubts about biological control agents. (3-exotoxin is a chemical the safety of a synthetic chemical in similar tests. containing adenosylglucose linked to allaric acid Borgatti & Guyer (1962) found Bakthane 3 to be phosphate in a described configuration (Kalvoda toxic to quail even after extraction with ether or 'Formerly with Research Laboratories, Rohm and Haas Company, Spring House, Pa. 19477, U.S.A. Present address: 3231 E. Lester Street, Tucson, Arizona 85716, U.S.A. '(Deceased). Regional Poultry Diagnostic Laboratory, Delaware Valley College of Science and Agriculture, Doylestown, Pa. 18901, U.S.A.
benzene to remove suspected contaminants. They used the same Bakthane Lot no. 3 later (Borgatti & 3
Bakthane® and its exotoxins were produced by a culture of B. thuringiensis selected for high yields of [3-exotoxin. Bakthane has not been produced by Rohm and Haas Co. since 1965.
J. Med. Ent.
104
Guyer 1963) in switch-back tests with no mention of adverse effects. Burns et al. (1961) had chickens which were off feed and which laid fewer eggs. Harvey & Brethour (1960) reported slightly decreased feeding of chickens. Ode & Matthysse (1964) showed a refusal of cows to feed on BakthaneL69. Galichet (1966) found that treated pigs developed anorexia and lost weight. Sebesta et al. (1969) observed that a purified (3-exotoxin inhibited biosynthesis of RNA in mice as well as in insects and thus demonstrated that (3-exotoxin is not specific for insects. Normally, B. thuringiensis var. thuringiensis produces ca 50 mg p-exotoxin/liter supernatant liquid (Faust 1973). If the presence of (3-exotoxin in B. thuringiensis is undesirable, it can be eliminated by appropriate bacterial culture selection or by precipitation with alkaline earths as described by Drake & Smythe (1963). ~~ The objectives of this experiment were to determine the efficacy and safety of (3-exotoxin in feed and in water for control of flies in chicken manure, to establish and to describe early symptoms of overdosage to birds, to check possible long-range toxic effects, and to obtain a rough indication of the ultimate fate of exotoxin. EXPERIMENTAL PROTOCOL
Because it takes chickens about a week to adapt to new cages or rations, we used the same birds in successive tests. First, they were used to establish what level of toxin should be fed to control flies in feces. Next, soluble and insoluble salts were compared. Finally, dosages were increased until a margin of safety between control of flies and harm to birds could be established. On the assumption that it would take 3 days for the composition of feces to reflect the composition of the food, feces were collected for bioassay on
Vol. 12, no. 1
days 4-7. It seemed reasonable to test high dosages on chickens which were adapted on low dosages, rather than to select and adapt new chickens from zero dosage. Furthermore, the chances for detecting cumulative effects with our limited resources were increased by feeding increasing dosages in sequence. We knew the chemical nature of (3exotoxin, so we suspected that interference with nucleotides might give a delayed response. fi-exotoxin source: Two preparations of exotoxin were tested: a water-soluble sodium salt (NaTox) of at least 95% purity, and an insoluble crude calcium precipitate of B. thuringiensis whole culture in which the calcium salt (CaTox) was estimated to be 12% by bioassay with house fly larvae (Drake & Smythe 1963). When NaTox was dissolved in drinking water, the solutions were used within 2 weeks. On the assumption that chickens would consume twice as much water as mash, the concentrations administered in water were 1 /2 of those in mash. CaTox was mixed with feed in 2 kg batches in a V-shaped blender for 20 min. each and then fed within 2 weeks. Hens: Thirty Leghorn layers from 1 flock, age 23 weeks, were individually caged in a battery 4 and were fed laying mash5. Food and water were changed and individual food uptake was measured on days 1, 2, 3-6, and 7 of each week. Eggs were collected daily. Manure was collected individually and weighed on days 3, 4, 5, and 6, and held in polyethylene bags for bioassay. Hens were weighed on day 7 to the nearest 14 g. After attaching identification bands, hens were divided into 5 groups of 6 each. These groups were color coded so that 4
Harford Metal Products, Aberdeen, Md., Model LC8-30 supplied with individual watering cans and feed pans separated with baffles. 5 Ralston Purina Company, St. Louis, Missouri. Purina Eggena Mash. Purina Pullet Developer. CSMA media for house flies.
TABLE 1.'JDosages of (3-exotoxin salts of Bacillus thuringiensis administered to chickens.* DOSAGE GROUPS (PPM ACTIVE)
Red Green Yellow Hens 1 0 0 0 0 0 0 2&3 Ca in mash 0 4 2 8 16 4&5 Ca in mash 0 20 160 40 80 6 Na in mash 40 0 20 160 80 7 Na in water 0 10 20 80 40 Ca in mash 0 400 1600 8&9 200 800 Pullets Ca in mash 1&2 0 4 8 16 2 3 Ca in mash 0 20 40 80 160 Na in water 4,5,6,7 0 10 20 80 40 •The same 6 hens and 6 pullets were used in each dosage group for the 9-week period. Sequent dosages were increased 10 X, or the soluble Na salt was replaced with the insoluble Ca salt. WEEK
EXOTOXIN SALT
White
Blue
1975
Barker & Anderson: p-exotoxin of Bacillus thuringiensis
105
hens, test materials, and equipment were conFood consumption for each group was measured on spicuously segregated by color groups to reduce days 1, 2, 3, and 7 of each week. Manure was errors and contamination, TABLE 1 gives dosages. collected and weighed on day 7. Pullets were Increasing dosages were represented by colors weighed on day 1. TABLE 1 gives dosage schedules. toward the red end of the color spectrum, and white Autopsies were conducted after week 7. indicated the control. Operators were unaware of Both hens and pullets were held in 14-hr photothe code's relationship to dosage. Newly caged periods in an unheated barn. Tests with hens hens ate less feed and laid fewer eggs, of which many began on 30 Mar. and the pullet test began 4 May were soft-shelled. Hens adapted within 1 week. 1965. Two hens which failed to adapt, 1 control (dosage Feces bioassay: Anticipated LD50 for house fly white) and 1 treated (dosage green), were autopsied larvae in manure was 5 ppm for NaTox and 8 ppm after 4 weeks and were found to have visceral for CaTox, based on our bioassay experience with leukosis. Data from these were not included. fresh CSMA fly media5 and reports on use of They were replaced at the start of week 5 by simul- Bakthane-L69 added to manure. taneously caged untreated hens from the same flock. Feces deposited on days 4-7 of each week were Hens were penned with a flock of roosters for 4 hr scraped from dropping pans, collected into polyon the 1st and 5 th day of week 4 and on the 1st ethylene bags, and stored at room temperature until day of week 5. Eggs collected during week 5 were bioassayed. Within 1 week, duplicate 0.5-liter incubated. Autopsies were conducted at the end (pint) jars of each dosage were 1/2 filled with of week 9. manure (ca 100 g). Eggs from the CSMA strain Pullets: Leghorn pullets, 4 weeks old, also were of houseflieswere added, 25/jar, and the subsequent separated into 5 color-coded groups of 6 each. emerging adults were counted. Pullets were not separated into individual cages, as were the hens. Each group was held in a stacking RESULTS AND DISCUSSION unit of a broiler cage 6and was fed pullet developer5. Toxicity to hens: Food uptake (±SD) for control •Kuhl Poultry Equipment Company, Flemington, New hens averaged 116 ± 12 g/day. Hens produced Jersey. Finishing battery brooder No. 305. TABLE 2. Average daily food intake (g/day; of 5 groups of hens fed varied dosages of Ca or Na salts of |3-exotoxin. WEEK
TREATMENT
1 2:3
Adapting* CaTox in mash
4:5
CaTox in mash
6
NaTox in mash
7
NaTox in water
8:9
CaTox in mash
•Mean : 30 hens.
DOSE (PPM)
0 0 2 4 8 16 0 20 40 80 160 0 20 40 80 160 0 10 20 40 80 0 200 400 800 1600
1 76 110:107 112:108 116:102 117:113 104: 96 109: 96 117: 83 115: 97 113: 74 112: 58
122 95 73 75 38 132:129 100: 52 83: 28 117: 9 27: 2
2 78 121:126 111:123 123:132 123:128 107:115 112:106 127: 92 120: 95 120: 89 111: 71 137 121 100 110 87 100 102 76 78 56 117:109 125: 64 51: 36 56: 10 8: 2
3 94 119:110 129:107 122:122 126:121 108:109 115:103 117: 94 108: 95 96: 94 88: 70 138 108 93 108 78 101 114 87 93 59 119:124 91: 63 36: 28 33: 17 6: 4
4-7 106 114:139 116:138 127:141 124:145 106:140 114:115 111:106 106:116 88:102 60: 75 143 125 111 124 84 120 106 82 96 65 103:145 63: 63 32: 23 13: 13 3: 6
Mean+SD 118±6 118±11 123±11 124±10 111±13 109 ±7 106±15 107±10 97±15 81±21 139±3 118±9 101 ±9 14±9 83±5 111±12 104±8 80±6 86±10 54±12 122±13 78±9 40±19 34±37
7±8
J. Med. Ent.
106 TABLE 3.
Manure output, egg production, and weight loss of hens fed salts of 3-exotoxin as indicated in TABLE 1. MANURE (G/DAY]1
DOSE (PPM)
0 2 4 8 10 16 20 40 80 160 200 400 800 1600
Vol. 12, no. 1
CaTox feed 195 106±8
NaTox feed 195
116±6 110±12 109±9 100±20 102±14 92±19 82±16 74±2 58±5 50±6 40±6
EGGS (NO./WK )
NaTox water 195
CaTox feed
NaTox feed
4.0 3.4,3.2 4.0,3.8 4.9,3.5
3.4
74 88 91 96 60
68 68 54
W T . LOSS (G/WK)
NaTox water 4.1
CaTox feed 1.7
NaTox feed 0.6
4.4 4.4,3.0 5.6,2.3 5.5,2.3 5.9,2.5 5.0,1.0 4.3,3.0 1.4,0.7 2.0,0.2 0.1,0.0
195 g/day of manure, which is 1.7 g manure/g food. CaTox at dosages up to 8 ppm in the diet has no adverse effects on feed consumption (TABLE 2). In fact, the trend of the means suggests that chickens eat more when the feed has 2-4 ppm of exotoxin. However, at dosages from 8-1600 ppm of CaTox, food consumption dropped. A linear regression between log dose (X) and food intake (Y) was fitted by the least squares method to give Y = 144.8 — 31.OX. Thus, for each doubling of dosages, food uptake decreased 9.3 g/day. Pearson's correlation coefficient between log dose and weight of food consumed shows that this negative correlation is highly significant (r = 0.831, df = 62). The lower food intake of treated hens does not seem to be a palatabihty effect because consumption was not lower on the beginning day of treatment with either salt (TABLE 2). In addition, day by day comparisons of food uptake for the 1st week and the 2nd week on the same dosage shows that the intake tends to decrease with time on any given dosage. Although the NaTox is more toxic than the CaTox to house flies, no difference was noticeable in chickens. The reduction of food consumption when NaTox was added to drinking water further indicated that reduced food uptake is a consequence of toxicity, not taste. Water uptake could not be measured because the hens played with the water cups. Manure output (TABLE 3) showed the same trends as food consumption. At dosages from 8 to 1600 ppm of CaTox, the regression of grams of manure (Y) on log dosage in ppm (X) was Y = 177.5 — 49.2X. The correlation here also was highly significant (r = 0.8644, df = 78).
3.3 2.5 1.5 0.2
3.5 2.8 0.5
NaTox water 0
2.0 4.8 0.6 0.6 2.8
2.5 0.6 0 0
7.1 0 8.5
Control hens laid 4.0 eggs/week. At each dosage of CaTox, egg production was higher during the 1st of the 2 consecutive weeks. This suggests that effects on egg production are delayed by a few days. Within the time limits of this experiment, dosages greater than 80 ppm of NaTox or CaTox are associated with a decrease in egg production. The effect on egg size was more conspicuous than the effect on numbers laid. Unfortunately, the protocol did not include weighing the eggs. A typical egg from a hen on the "blue" dosages weighed 38 g. Eggs from higher treatments were even smaller. Eggs from normal hens and those in our control weighed ca 60 g. In week 5, eggs were placed in an incubator on the day they were gathered. A week later the incubator failed; embryos in the eggs thus chilled were examined, and the embryonic development appeared to be normal for 1 week. In view of effects on RNA found later by Sebesta et al. (1969), this test should be repeated and the embryos or emerging chicks carefully scrutinized. An understanding of the nature of the delayed toxic effects did not evolve until the conclusion of the experiment—at autopsy. Although 1 hen in the control died of leukosis in week 4, the other 5 were normal. Lung lesions were common, but these were unrelated to dosage. They may have resulted from caging the 30 hens with roosters for mating; the ensuing dust was thick and could have been injurious. On the "blue" dosage schedule, 1 hen had moderate and 5 had severe gizzard erosion, 3 had moderate and 3 had severe enteritis, and 2 of the 6 had regressed ovaries and 1 had receding ovaries. On "yellow" dosages, 1 hen, which had consistently been a heavy feeder and was the largest hen in the lot, had severe visceral leukosis.
1975
Barker & Anderson: (3-exotoxin of Bacillus thuringiensis
107
FIG. 1. Typical gizzards of hens treated with (3-exotoxin of B. thuringiensis (left) and untreated (right). The treated hen has petechial lesions in the proventriculus, and the anterior edge of the gizzard lining is sloughing off.
All had severe gizzard erosion, enteritis, and regressed ovaries. One which seemed normal had a 1-cm liver abcess. Every hen on dosage "red" had severe gizzard erosion, enteritis, and regressed ovaries. One had proventriculitis and 1 had mild leukosis. FIG. 1 shows typical damage. In general, the enteritis was confined to the duodenal loop. Gizzard erosion was unrelated to the contents. A dosage of 20 ppm CaTox in mash fed to hens weighing 1 kg and eating 100 g/day represents only 2 mg/kg per day. A dosage of 20 ppm of NaTox in water, the harmful dosage, represents ca 3 mg/kg per day. Toxicity to pullets: Pullets were more susceptible than hens to exotoxin. After 2 weeks on dosages that were not toxic to hens (CaTox in feed at 216 ppm) and 1 week with 20-160 ppm of CaTox in mash, the pullets on 80 and 160 ppm were underweight (TABLE 4), pale, and lethargic. Even 16 ppm dosages gave retarded growth in pullets. At the end of week 3, one pullet from each group was transferred to a cage with untreated food and the
rest were switched to NaTox in the water. Consumption of food remained unaffected by switching the dosage from mash to water (TABLE 5). Pullets (TABLE 6) actually drank less than the predicted 2: 1 ratio of water/feed, so uptake of NaTox in water was lower than anticipated. The ratio of water: food consumed increased with the dosage in water (TABLE 6). Even so, at 80 ppm water consumption was only 55% of that of healthy chickens. This agrees with data on hens and further discounts palatability as a factor. Also in agreement with results from hens was a general decrease in food consumption with increases in dosage. The difference was modified by mortality. Pullets receiving 80 ppm of Natox water were affected severely and 3 of 5 died within 8 days. Data in the tables are from the survivors. Dying pullets were consuming ca 60 ml of water (TABLE 6) containing 80 ppm (0.5mg/day). For a 500-g pullet, this is 1 mg/kg per day as an estimated LD50 for NaTox. TABLE 7 gives results of autopsies at the end of 6 weeks. With pullets, as with hens, the response
J. Med. Ent.
108 TABLE 4.
Mean weights (g) of pullets fed varied dosages of (3-exo toxin. WEEK
TREATMENT GROUP*
970 a 434 a** 699 a White 876 a 435 ab 652 a Blue 868 a 468 ab 673 ab Green 688 435 ab 588 b Yellow 540 388 b 507 Red *Dosages are given in TABLE 1. ** Means in each column followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test. Weights did not differ between treatments at the start of the test.
was gizzard erosion followed at increasing dosages by enteritis and petechial lesions in the proventriculus. Anemia at the highest dosage possibly is caused by malnutrition. Single pullets that were removed from each dosage at the start of week 4 were caged in pairs to reduce adaptation problems. Feed consumption for the pair from lower prior dosage regimens (1 "green" and 1 "blue") did not differ convincingly from those at higher prior dosages (1 "yellow" and 1 "red"). Food consumption on day 1 averaged 21 g/pullet; on day 2 averaged 45 g; on day 3 averaged 54 g. For the next 2 weeks, food intake was comparable to that of pullets in the control group. Autopsies 3 weeks later indicated mild gizzard erosion in the pullet from "yellow." The pullet from "red" was autopsied at the end of 1 week and was found to be normal. Recovery could be rapid. Weight increase during this recovery period (weeks 4-7) was 156 g for "blue," 199 g for "green," and 326 g for "yellow." At the" same time, the gain of 6 untreated controls averaged 168 g. This also suggests rapid recovery without permanent injury. The possibility remained of CaTox being toxic and of NaTox prolonging or synergizing the effects of the calcium salt. To ascertain whether the sodium exotoxin alone was toxic, 2 birds of average weight were selected from controls at the start of TABLE 5. Feed consumption by pullets (g/day±SD) fed CaTox in feed or NaTox in water. 0 2 4 8 10 16
20 40 80 160
CATOX
NATOX
61±5 52±2 56±3 48±7
64±7
55±8 44±4 44±6 43 ±2 32 ±6 27±6
54±9 39±6 27±4
Vol. 12, no. 1
TABLE 6.
Water consumption per pullet during weeks 4, 5, and 6 while NaTox was added to water.
DOSAGE (PPM)
CONSUMPTION/DAY (ML)
WATER/FOOD (WT.)
0 10 20 40 80
100 86 88 70 55
1.5 1.5 1.6 1.8 2.0
week 4. These were given untreated feed and 80 ppm of NaTox in the water. One pullet was autopsied after 10 days, and showed gizzard erosion, enteritis, and anemia. Food consumption during these 10 days showed no time lag but averaged only 32 g of feed and 50 ml of water. This is comparable to consumption of pullets on the "red" test which had been fed CaTox in earlier dosages. The surviving pullet gained only 47 g, which is 28% of the gain in controls. Therefore, both exotoxin salts are toxic. Tissue bioassay: Perhaps (3-exotoxin is not really highly toxic but was accumulated and concentrated at a critical site to produce its delayed effect. This possibility was checked by separate bioassays of lungs, liver, heart, breast muscle, and gizzards of all tested hens and pullets using the black blow fly, Phormia regina (Meigen). Larvae grew normally on all these organs, even on hens that were fed 1600 ppm. Either exotoxin does not exist in high concentrations ( > 1 0 ppm) in these organs, or it is rapidly degraded during putrefaction. Eggs from treated hens were mixed with expanded mica and infested with black blow flies. Eggs from hens fed dosages of 200-800 ppm in feed were found to support the growth of fly larvae. At 1600 ppm, only 1 egg, too tiny to support flies, was available for bioassay. Toxicity of exotoxin in moist poultry rations fed to house
flies: Normally, over 90% of the house fly eggs added to CSMA media produce flies. In triplicated tests, 25 fly eggs were added to 100 g of treated CSMA larval medium plus 170 g of water. No flies survived dosages of 40 ppm or more of either salt. At 10 ppm (dry wt), the emergence was 3, 4 and 4 from NaTox and 9, 4 and 4 from CaTox. Emergence from 20 ppm of NaTox and CaTox was 1,1,2 and 2, 1,2, respectively. When NaTox was added and flies were reared on the laying mash, no flies survived 20 ppm. Emergence from controls (78%) also was lower than emergence from CSMA medium. Fly eggs added to pullet developer mash treated with NaTox gave the following emergence: 0 ppm = 82%, 2 ppm = 24%, 4 ppm = 6%, 8 ppm = 4%, and 16 ppm = 0.
Barker & Anderson: p-exotoxin of Bacillus thuringiensis
1975
TABLE 7. Postmortem observations on pullets at end of week 7. (TABLE 1 gives dosages for each group during each of the 7 weeks.) TEST
White
Blue
Green
Yellow
Red
WEIGHT (O)
ABNORMALITIES
None Leukosis; breast blister 1080 Pale liver Pale marrow 1020 910 Moderately pale marrow 1020 Gizzard erosion; enteritis; pale liver 940 Leukosis 800 Gizzard erosion 770 Gizzard erosion 850 Very mild gizzard erosion; pale marrow 910 Gizzard erosion; enteritis; proventriculitis 850 Gizzard erosion; pale liver 850 Gizzard erosion 850 Moderate gizzard erosion; mild anemia Gizzard erosion, pale liver 880 Severe gizzard erosion; proven540 triculitis 820 Gizzard erosion; proventriculitis 600 Gizzard erosion; proventriculitis 710 Mild gizzard erosion; proventriculitis; enteritis 770 Gizzard erosion; proventriculitis; mild enteritis; anemia 660 Severe gizzard erosion; anemia; pale liver 480 Severe gizzard erosion; anemia; pale liver Died, week 4 Gizzard erosion; enteritis Died, week 4 Gizzard erosion; enteritis; severe anemia Died, week 5 Gizzard erosion; enteritis; severe anemia 800 960
109
TABLE 8. Percentages of flies emerged from fly eggs added to manure of chickens fed salts of p-exotoxin. DOSAGE (PPM)
CaTox in feed 0 2 4 8 16 20 40 80 160 200 400 800
1600 NaTox in feed 0 20 40 80 160
HENS
PULLETS
87
87 84 84 79 86 88 76 70 46
82 71 69 59 58 21 25 9 24 0 0 8 91 45 28 16 1
NaTox in water 0 10 20 40
80
72 82 69 61 34
99 66 100
50 18
effect or decomposition of exotoxin in manure. Consequently, sealed polyethylene bags of stored samples were reopened, held on greenhouse benches, and 25 house fly eggs added to each on 8 June. The samples from hens had been collected from 2 Apr. to 24 May. Pullet manure was collected between 5 May and 16 June. On 10 June, survival House fly survival in manure: Emergence from untreated chicken manure was 91% with no differ- was found to be related to viscosity of the feces but not to exotoxin dosage. Some larvae drowned ence between hens in different groups. The when fermenting manure liquified and some bags emergence from manure plus sawdust was 89%, and flies were more difficult to find and count, TABLE 8 were too hot from fermentation for the larvae to survive. These bags were left open and more fly gives results from manure of chickens fed treated eggs were added on 20 June. If the physical conmash. Each percentage represents 4-8 samples of sistency was satisfactory, flies survived at rates 25 flies each. Fly eggs were added 1-10 days after manure was deposited. The 1st tests on hen's comparable to survival on untreated manure. Infestations were repeated on 22 and 25 June with manure suggested that CaTox added to feed had the same results. Even 1600 ppm in feed failed to an LD50 of ca 20 ppm. This was more than control flies whenever there was sufficient manure anticipated, since the LD50 in feed was below 10 and water available. In addition to the artificially ppm. The amount of water added to feed bioassays added house flies, larvae of the black blow fly and was equal to the amount of water added by chickens an unidentified gnat infested feces from treated to produce manure from treated feed (dilution chickens. factor 1.7). In pullet's manure, even 160 ppm in feed failed to control flies in replicated tests. These In prior reports of fly control, fresh feces usually results were obtained in mid-May. Dosages of had been used for bioassay. Borgatti & Guyer 200 to 1600 ppm were then being fed, and the (1963) held samples 1 year at -18°C. Smirnoff & pullets began to get seriously sick. Even so, wild McLeod (1961) counted spores. The tests on flies in the chicken house were breeding in the liquified manure are more nearly those of field droppings. This suggested either a temperature conditions. We also found rapid biodegradability
Vol. 12, no. 1
J. Med. Ent.
110
of exotoxin in Shory's insect medium made from soaked lima beans. Degradation in manure could be influenced by diet as well as microfauna. In contrast to activity of bean enzymes, on a soil surface, exotoxin was stable for at least 4 months (Hitchings 1967). House fly resistance: The low dosage-response slope of exotoxin suggested that flies would readily become resistant to treated manure. Treating larval media has proven to be the most efficient means of selecting an insecticide-resistant strain of flies. From a continual population of ca 2000 adults, a strain of house flies originally resistant to DDT (Rutgers Strain A) was selected at the LD 95 level starting in March. By September (ca 12 generations), the LD 95 dosage in larval media had increased from 80 ppm to 600 ppm. This dosage is 30 X a dosage toxic to chickens and exceeds practical dosage. A lower level of fly resistance to Bakthane-L69 was achieved by Harvey & Howell (1965) in 50 generations with a lower selection level. CONCLUSIONS
The (3-exotoxin was highly toxic to chickens whether added to feed as a calcium or a sodium salt or to water as the sodium salt. Early poisoning symptoms were loss of vigor, reduced feeding, and undersized eggs. Reduced feeding was not a consequence of palatability; exotoxin caused severe gizzard erosion, enteritis, and proventriculitis. A lethal dosage of 20 ppm CaTox in mash fed to hens weighing 1 kg and eating 100 g/day represents 2 mg/kg per day. A dosage of 20 ppm NaTox in water is ca 3 mg/kg per day. Dying pullets were consuming ca 60 ml of water containing 80 ppm NaTox (0.5 mg/day). For a 500-g pullet, the LD 50 is ca 1 mg/kg per day for NaTox. Even the largest experimental dosage of 1600 ppm in feed failed to control flies in manure when conditions for larval growth were otherwise favorable. Larvicidal action was lost when manure fermented. Bioassay of chicken tissues with larvae of Phormia regina (Meigen) furnished further evidence of biodegradability of (3-exotoxin. House flies acquired resistance to (3-exotoxin after 6 months continuous selection from larval exposures. The LD 95 increased 30 times, to the point that larvae grew with 600 ppm of exotoxin in the medium.
Knestrick cared for the chickens and collected manure samples. At Rohm and Haas, D. L. Munchback bioassayed house flies on fresh manure samples and developed the resistant strain. M. Nicoli tested exotoxin in Shory's media. S. L. Satterthwaite photographed the autopsies. LITERATURE CITED
Borgatti, A. L. & G. E. Guyer. 1962. Formulations of Bacillus thuringiensis Berliner found to be contaminated with chlorinated hydrocarbon insecticides. J. Econ. Ent. 55: 1015-16. 1963. The effectiveness of commercial formulations of Bacillus thuringiensis Berliner on house fly larvae. J. Insect
Pathol. 5: 377-84. Briggs, J. D. 1960. Reduction of adult house-fly emergence by the effects of Bacillus spp. on the development of immature forms. J. Insect Pathol. 2: 418-32. Burns, E. C , B. H. Wilson & B. A. Tower. 1961. Effect of feeding Bacillus thuringiensis to caged layers for fly control. J. Econ. Ent. 54: 913-15.
Drake, B. B. & C. V. Smythe. 1963. Process for making pesticidal compositions. U. S. Pat. 3,087,865. Faust, R. M. 1973. The Bacillus thuringiensis (3-exo toxin: Current status. Bull. Ent. Soc. Amer. 19: 153-56. Galichet, P. F. 1966. Administration aux animaux domestiques d'une toxine thermostable secretee par Bacillus thuringiensis Berliner, en vue d'empecher la multiplication de Musca domestica Linnaeus dans les feces. Ann. Zootech., Paris
15: 135-45. Harvey, T. L. & J. R. Brethour. 1960. Feed additives for control of house fly larvae in livestock feces. J. Econ. Ent. 53: 774-77. Harvey, T. L. & D. E. Howell. 1965. Resistance of the house fly to Bacillus thuringiensis Berliner. J. Invert. Pathol. 7:
92-100. Hitchings, D. 1967. Bacillus thuringiensis: A reproduction inhibitor for southern armyworm. J. Econ. Ent. 60: 59697. Kalvoda, L., M. Prystas & F. Sorm. 1973. The structure of the allaric portion of exotoxin from Bacillus thuringiensis. Tetrahedron Ltrs. 1973: 1873-76.
Mackedonski, V. V., N. Nikolaev, K. Sebesta & A. A. Hadjiolov. 1972. Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of Bacillus thuringiensis. Biochim. Biophys. Acta 272: 56-66.
Mechalas, B. J. & O. Beyer.
1963. Production and assay
of extracellular toxins of Bacillus thuringiensis. Dev. Ind. Microbiol. 4 : 142-47.
Ode, P. E. & J. G. Matthysse. 1964. Feed additive larviciding to control face fly. J. Econ. Ent. 57: 637-40. Sebesta, K., K. Horska & J. Vankova. 1969. Inhibition of de novo RNA synthesis by the insecticidal exotoxin of Bacillus thuringiensis var. gelechiae. Collect. Czech. Chem. Commun.
34: 1786-91. Sherman, M., E. Ross & G. H. Komatsu. 1962. Differential susceptibility of maggots of several species to droppings from chickens fed insecticide treated rations. J. Econ. Ent. 55: 990-93. Smirnoff, W. A. & C. F. MacLeod. 1961. Study of the
Acknowledgments: At Delaware Valley College of Science and Agriculture, John C. Harrison, James P. Harner, and Ralph C.
survival of Bacillus thuringiensis var. thuringiensis Berliner in
the digestive tracts and in feces of a small mammal and birds. J. Insect Pathol. 3: 266-70.
Nayar & Sauerman: Mosquito blood meal use for survival
103
Acknowledgments: We thank Dr J. D. Edman of the Florida Wildlife 20: 1-61. Medical Entomology Laboratory, Vero Beach, for critical Nath, V., B. L. Gupta & G. S. Bains. 1958. Histochemical reading of the manuscript. and morphological studies of the lipids in oogenesis. V. The egg-follicles of Culex fatigans. Res. Bull. Punjab Univ.
Zool. 148: 135-48. Nayar, J. K. & D. M. Sauerman, Jr. 1971. The effects of Boorman, J. P. T. 1960. Observations on the feeding diet on life-span, fecundity and flight potential of Aedes habits of the mosquito Aedes {Stegomyia) aegypti (Linnaeus): taeniorhynchus adults. J. Med. Ent. 8: 506-13. The loss of fluid after a blood-meal and the amount of 1975. The effects of nutrition on survival and fecundity in blood taken during feeding. Ann. Trop. Med. Parasitol. 54: Florida mosquitoes. Part 1. Utilization of sugar for survival. 8-14. J. Med. Ent. 12: 92-98. Clements, A. N. 1955. The sources of energy for flight in Nayar, J. K. & E. Van Handel. 1971. The fuel for mosquitoes. J. Exp. Biol. 32: 547-54. sustained mosquito flight. J. Insect Physiol. 17: 471-81. Gooding, R. H. 1972. Digestive processes of haematoService, M. W. 1968. Observations on feeding and oviposiphagous insects. 1. A literature review. Quaest. Ent. 8: tion in some British mosquitoes. Ent. Exp. Appl. 11: 277-85. 5-60. Van Handel, E. 1965a. The obese mosquito. J. Physiol. Jeffery, G. M. 1956. Blood meal volume in Anopheles 181:478-86. quadrimaculatus, A. albimanus and Aedes aegypti. Exp. Parasitol. 1965b. Microseparation of glycogen, sugars and lipids. J. 5: 371-75. Anal. Biochem. 11: 266-71. Laurence, B. R. & M. A. Roshdy. 1963. Ovary develop1972. Simple biological and chemical methods to determent in mosquitoes. Nature, Land. 200: 495-96. mine the caloric reserves of mosquitoes. Mosquito News 32: Maciolek, J. A. 1962. Limnological organic analyses by 589-91. quantitative dichromate oxidation. Rept. Bur. Sport Fish. LITERATURE CITED
J. Med. Ent. Vol. 12, no. 1: 103-110
30 April 1975
EVALUATION OF ^-EXOTOXIN OF BACILLUS THURINGIENSIS BERLINER FOR CONTROL OF FLIES IN CHICKEN MANURE By Roy J. Barker1 and William F. Anderson2 Abstract: A calcium and a sodium salt of (3-exotoxin obtained et al. 1973). This structural analog of adenosine from Bakthane-L69® were added to rations supplied to hens triphosphate inhibits nucleic acid biosynthesis and to pullets. This exotoxin was too toxic to be fed to chickens for the control of the house fly, Musca domestica L., in (Mackedonski et al. 1972). A recent review (Faust 1973) on the status of (3-exotoxin as an insecticide droppings. Not more than 3 mg/kg per day could be fed to hens for a week. The oral LDS0 to pullets was ca 1 mg/kg per day. appealed for reports of research on its safety. This Both salts were toxic to chickens, the calcium salt at 20 ppm report concerns such a test conducted 10 years ago. in feed and the sodium salt at 20 ppm in feed or in drinking The ability of B. thuringiensis products to kill fly water. Toxic symptoms were loss of vigor, reduced feeding, and undersized eggs. Reduced feeding was not a consequence of larvae, even after passage through the digestive palatability. The exotoxin caused severe gizzard erosion, then tract, has been useful for fly control; when added enteritis and proventriculitis. The exotoxin degraded when to poultry feed, they have given convenient but manure liquified and lost the ability to kill house fly larvae under such conditions. House flies became resistant to the (3- erratic control of flies in chicken manure (Borgatti & exotoxin. After a 6-month selection on media treated with the Guyer 1962, 1963, Briggs 1960, Burns et al. 1961, calcium salt, the larvae tolerated media containing 600 ppm. Harvey & Brethour 1960, Mechalas & Beyer 1963, Sherman et al. 1962). The insecticidal products of some cultures of Nevertheless, available data on B. thuringiensis Bacillus thuringiensis Berliner are not simply living reveal results which would have raised doubts about biological control agents. (3-exotoxin is a chemical the safety of a synthetic chemical in similar tests. containing adenosylglucose linked to allaric acid Borgatti & Guyer (1962) found Bakthane 3 to be phosphate in a described configuration (Kalvoda toxic to quail even after extraction with ether or 'Formerly with Research Laboratories, Rohm and Haas Company, Spring House, Pa. 19477, U.S.A. Present address: 3231 E. Lester Street, Tucson, Arizona 85716, U.S.A. '(Deceased). Regional Poultry Diagnostic Laboratory, Delaware Valley College of Science and Agriculture, Doylestown, Pa. 18901, U.S.A.
benzene to remove suspected contaminants. They used the same Bakthane Lot no. 3 later (Borgatti & 3
Bakthane® and its exotoxins were produced by a culture of B. thuringiensis selected for high yields of [3-exotoxin. Bakthane has not been produced by Rohm and Haas Co. since 1965.
J. Med. Ent.
104
Guyer 1963) in switch-back tests with no mention of adverse effects. Burns et al. (1961) had chickens which were off feed and which laid fewer eggs. Harvey & Brethour (1960) reported slightly decreased feeding of chickens. Ode & Matthysse (1964) showed a refusal of cows to feed on BakthaneL69. Galichet (1966) found that treated pigs developed anorexia and lost weight. Sebesta et al. (1969) observed that a purified (3-exotoxin inhibited biosynthesis of RNA in mice as well as in insects and thus demonstrated that (3-exotoxin is not specific for insects. Normally, B. thuringiensis var. thuringiensis produces ca 50 mg p-exotoxin/liter supernatant liquid (Faust 1973). If the presence of (3-exotoxin in B. thuringiensis is undesirable, it can be eliminated by appropriate bacterial culture selection or by precipitation with alkaline earths as described by Drake & Smythe (1963). ~~ The objectives of this experiment were to determine the efficacy and safety of (3-exotoxin in feed and in water for control of flies in chicken manure, to establish and to describe early symptoms of overdosage to birds, to check possible long-range toxic effects, and to obtain a rough indication of the ultimate fate of exotoxin. EXPERIMENTAL PROTOCOL
Because it takes chickens about a week to adapt to new cages or rations, we used the same birds in successive tests. First, they were used to establish what level of toxin should be fed to control flies in feces. Next, soluble and insoluble salts were compared. Finally, dosages were increased until a margin of safety between control of flies and harm to birds could be established. On the assumption that it would take 3 days for the composition of feces to reflect the composition of the food, feces were collected for bioassay on
Vol. 12, no. 1
days 4-7. It seemed reasonable to test high dosages on chickens which were adapted on low dosages, rather than to select and adapt new chickens from zero dosage. Furthermore, the chances for detecting cumulative effects with our limited resources were increased by feeding increasing dosages in sequence. We knew the chemical nature of (3exotoxin, so we suspected that interference with nucleotides might give a delayed response. fi-exotoxin source: Two preparations of exotoxin were tested: a water-soluble sodium salt (NaTox) of at least 95% purity, and an insoluble crude calcium precipitate of B. thuringiensis whole culture in which the calcium salt (CaTox) was estimated to be 12% by bioassay with house fly larvae (Drake & Smythe 1963). When NaTox was dissolved in drinking water, the solutions were used within 2 weeks. On the assumption that chickens would consume twice as much water as mash, the concentrations administered in water were 1 /2 of those in mash. CaTox was mixed with feed in 2 kg batches in a V-shaped blender for 20 min. each and then fed within 2 weeks. Hens: Thirty Leghorn layers from 1 flock, age 23 weeks, were individually caged in a battery 4 and were fed laying mash5. Food and water were changed and individual food uptake was measured on days 1, 2, 3-6, and 7 of each week. Eggs were collected daily. Manure was collected individually and weighed on days 3, 4, 5, and 6, and held in polyethylene bags for bioassay. Hens were weighed on day 7 to the nearest 14 g. After attaching identification bands, hens were divided into 5 groups of 6 each. These groups were color coded so that 4
Harford Metal Products, Aberdeen, Md., Model LC8-30 supplied with individual watering cans and feed pans separated with baffles. 5 Ralston Purina Company, St. Louis, Missouri. Purina Eggena Mash. Purina Pullet Developer. CSMA media for house flies.
TABLE 1.'JDosages of (3-exotoxin salts of Bacillus thuringiensis administered to chickens.* DOSAGE GROUPS (PPM ACTIVE)
Red Green Yellow Hens 1 0 0 0 0 0 0 2&3 Ca in mash 0 4 2 8 16 4&5 Ca in mash 0 20 160 40 80 6 Na in mash 40 0 20 160 80 7 Na in water 0 10 20 80 40 Ca in mash 0 400 1600 8&9 200 800 Pullets Ca in mash 1&2 0 4 8 16 2 3 Ca in mash 0 20 40 80 160 Na in water 4,5,6,7 0 10 20 80 40 •The same 6 hens and 6 pullets were used in each dosage group for the 9-week period. Sequent dosages were increased 10 X, or the soluble Na salt was replaced with the insoluble Ca salt. WEEK
EXOTOXIN SALT
White
Blue
1975
Barker & Anderson: p-exotoxin of Bacillus thuringiensis
105
hens, test materials, and equipment were conFood consumption for each group was measured on spicuously segregated by color groups to reduce days 1, 2, 3, and 7 of each week. Manure was errors and contamination, TABLE 1 gives dosages. collected and weighed on day 7. Pullets were Increasing dosages were represented by colors weighed on day 1. TABLE 1 gives dosage schedules. toward the red end of the color spectrum, and white Autopsies were conducted after week 7. indicated the control. Operators were unaware of Both hens and pullets were held in 14-hr photothe code's relationship to dosage. Newly caged periods in an unheated barn. Tests with hens hens ate less feed and laid fewer eggs, of which many began on 30 Mar. and the pullet test began 4 May were soft-shelled. Hens adapted within 1 week. 1965. Two hens which failed to adapt, 1 control (dosage Feces bioassay: Anticipated LD50 for house fly white) and 1 treated (dosage green), were autopsied larvae in manure was 5 ppm for NaTox and 8 ppm after 4 weeks and were found to have visceral for CaTox, based on our bioassay experience with leukosis. Data from these were not included. fresh CSMA fly media5 and reports on use of They were replaced at the start of week 5 by simul- Bakthane-L69 added to manure. taneously caged untreated hens from the same flock. Feces deposited on days 4-7 of each week were Hens were penned with a flock of roosters for 4 hr scraped from dropping pans, collected into polyon the 1st and 5 th day of week 4 and on the 1st ethylene bags, and stored at room temperature until day of week 5. Eggs collected during week 5 were bioassayed. Within 1 week, duplicate 0.5-liter incubated. Autopsies were conducted at the end (pint) jars of each dosage were 1/2 filled with of week 9. manure (ca 100 g). Eggs from the CSMA strain Pullets: Leghorn pullets, 4 weeks old, also were of houseflieswere added, 25/jar, and the subsequent separated into 5 color-coded groups of 6 each. emerging adults were counted. Pullets were not separated into individual cages, as were the hens. Each group was held in a stacking RESULTS AND DISCUSSION unit of a broiler cage 6and was fed pullet developer5. Toxicity to hens: Food uptake (±SD) for control •Kuhl Poultry Equipment Company, Flemington, New hens averaged 116 ± 12 g/day. Hens produced Jersey. Finishing battery brooder No. 305. TABLE 2. Average daily food intake (g/day; of 5 groups of hens fed varied dosages of Ca or Na salts of |3-exotoxin. WEEK
TREATMENT
1 2:3
Adapting* CaTox in mash
4:5
CaTox in mash
6
NaTox in mash
7
NaTox in water
8:9
CaTox in mash
•Mean : 30 hens.
DOSE (PPM)
0 0 2 4 8 16 0 20 40 80 160 0 20 40 80 160 0 10 20 40 80 0 200 400 800 1600
1 76 110:107 112:108 116:102 117:113 104: 96 109: 96 117: 83 115: 97 113: 74 112: 58
122 95 73 75 38 132:129 100: 52 83: 28 117: 9 27: 2
2 78 121:126 111:123 123:132 123:128 107:115 112:106 127: 92 120: 95 120: 89 111: 71 137 121 100 110 87 100 102 76 78 56 117:109 125: 64 51: 36 56: 10 8: 2
3 94 119:110 129:107 122:122 126:121 108:109 115:103 117: 94 108: 95 96: 94 88: 70 138 108 93 108 78 101 114 87 93 59 119:124 91: 63 36: 28 33: 17 6: 4
4-7 106 114:139 116:138 127:141 124:145 106:140 114:115 111:106 106:116 88:102 60: 75 143 125 111 124 84 120 106 82 96 65 103:145 63: 63 32: 23 13: 13 3: 6
Mean+SD 118±6 118±11 123±11 124±10 111±13 109 ±7 106±15 107±10 97±15 81±21 139±3 118±9 101 ±9 14±9 83±5 111±12 104±8 80±6 86±10 54±12 122±13 78±9 40±19 34±37
7±8
J. Med. Ent.
106 TABLE 3.
Manure output, egg production, and weight loss of hens fed salts of 3-exotoxin as indicated in TABLE 1. MANURE (G/DAY]1
DOSE (PPM)
0 2 4 8 10 16 20 40 80 160 200 400 800 1600
Vol. 12, no. 1
CaTox feed 195 106±8
NaTox feed 195
116±6 110±12 109±9 100±20 102±14 92±19 82±16 74±2 58±5 50±6 40±6
EGGS (NO./WK )
NaTox water 195
CaTox feed
NaTox feed
4.0 3.4,3.2 4.0,3.8 4.9,3.5
3.4
74 88 91 96 60
68 68 54
W T . LOSS (G/WK)
NaTox water 4.1
CaTox feed 1.7
NaTox feed 0.6
4.4 4.4,3.0 5.6,2.3 5.5,2.3 5.9,2.5 5.0,1.0 4.3,3.0 1.4,0.7 2.0,0.2 0.1,0.0
195 g/day of manure, which is 1.7 g manure/g food. CaTox at dosages up to 8 ppm in the diet has no adverse effects on feed consumption (TABLE 2). In fact, the trend of the means suggests that chickens eat more when the feed has 2-4 ppm of exotoxin. However, at dosages from 8-1600 ppm of CaTox, food consumption dropped. A linear regression between log dose (X) and food intake (Y) was fitted by the least squares method to give Y = 144.8 — 31.OX. Thus, for each doubling of dosages, food uptake decreased 9.3 g/day. Pearson's correlation coefficient between log dose and weight of food consumed shows that this negative correlation is highly significant (r = 0.831, df = 62). The lower food intake of treated hens does not seem to be a palatabihty effect because consumption was not lower on the beginning day of treatment with either salt (TABLE 2). In addition, day by day comparisons of food uptake for the 1st week and the 2nd week on the same dosage shows that the intake tends to decrease with time on any given dosage. Although the NaTox is more toxic than the CaTox to house flies, no difference was noticeable in chickens. The reduction of food consumption when NaTox was added to drinking water further indicated that reduced food uptake is a consequence of toxicity, not taste. Water uptake could not be measured because the hens played with the water cups. Manure output (TABLE 3) showed the same trends as food consumption. At dosages from 8 to 1600 ppm of CaTox, the regression of grams of manure (Y) on log dosage in ppm (X) was Y = 177.5 — 49.2X. The correlation here also was highly significant (r = 0.8644, df = 78).
3.3 2.5 1.5 0.2
3.5 2.8 0.5
NaTox water 0
2.0 4.8 0.6 0.6 2.8
2.5 0.6 0 0
7.1 0 8.5
Control hens laid 4.0 eggs/week. At each dosage of CaTox, egg production was higher during the 1st of the 2 consecutive weeks. This suggests that effects on egg production are delayed by a few days. Within the time limits of this experiment, dosages greater than 80 ppm of NaTox or CaTox are associated with a decrease in egg production. The effect on egg size was more conspicuous than the effect on numbers laid. Unfortunately, the protocol did not include weighing the eggs. A typical egg from a hen on the "blue" dosages weighed 38 g. Eggs from higher treatments were even smaller. Eggs from normal hens and those in our control weighed ca 60 g. In week 5, eggs were placed in an incubator on the day they were gathered. A week later the incubator failed; embryos in the eggs thus chilled were examined, and the embryonic development appeared to be normal for 1 week. In view of effects on RNA found later by Sebesta et al. (1969), this test should be repeated and the embryos or emerging chicks carefully scrutinized. An understanding of the nature of the delayed toxic effects did not evolve until the conclusion of the experiment—at autopsy. Although 1 hen in the control died of leukosis in week 4, the other 5 were normal. Lung lesions were common, but these were unrelated to dosage. They may have resulted from caging the 30 hens with roosters for mating; the ensuing dust was thick and could have been injurious. On the "blue" dosage schedule, 1 hen had moderate and 5 had severe gizzard erosion, 3 had moderate and 3 had severe enteritis, and 2 of the 6 had regressed ovaries and 1 had receding ovaries. On "yellow" dosages, 1 hen, which had consistently been a heavy feeder and was the largest hen in the lot, had severe visceral leukosis.
1975
Barker & Anderson: (3-exotoxin of Bacillus thuringiensis
107
FIG. 1. Typical gizzards of hens treated with (3-exotoxin of B. thuringiensis (left) and untreated (right). The treated hen has petechial lesions in the proventriculus, and the anterior edge of the gizzard lining is sloughing off.
All had severe gizzard erosion, enteritis, and regressed ovaries. One which seemed normal had a 1-cm liver abcess. Every hen on dosage "red" had severe gizzard erosion, enteritis, and regressed ovaries. One had proventriculitis and 1 had mild leukosis. FIG. 1 shows typical damage. In general, the enteritis was confined to the duodenal loop. Gizzard erosion was unrelated to the contents. A dosage of 20 ppm CaTox in mash fed to hens weighing 1 kg and eating 100 g/day represents only 2 mg/kg per day. A dosage of 20 ppm of NaTox in water, the harmful dosage, represents ca 3 mg/kg per day. Toxicity to pullets: Pullets were more susceptible than hens to exotoxin. After 2 weeks on dosages that were not toxic to hens (CaTox in feed at 216 ppm) and 1 week with 20-160 ppm of CaTox in mash, the pullets on 80 and 160 ppm were underweight (TABLE 4), pale, and lethargic. Even 16 ppm dosages gave retarded growth in pullets. At the end of week 3, one pullet from each group was transferred to a cage with untreated food and the
rest were switched to NaTox in the water. Consumption of food remained unaffected by switching the dosage from mash to water (TABLE 5). Pullets (TABLE 6) actually drank less than the predicted 2: 1 ratio of water/feed, so uptake of NaTox in water was lower than anticipated. The ratio of water: food consumed increased with the dosage in water (TABLE 6). Even so, at 80 ppm water consumption was only 55% of that of healthy chickens. This agrees with data on hens and further discounts palatability as a factor. Also in agreement with results from hens was a general decrease in food consumption with increases in dosage. The difference was modified by mortality. Pullets receiving 80 ppm of Natox water were affected severely and 3 of 5 died within 8 days. Data in the tables are from the survivors. Dying pullets were consuming ca 60 ml of water (TABLE 6) containing 80 ppm (0.5mg/day). For a 500-g pullet, this is 1 mg/kg per day as an estimated LD50 for NaTox. TABLE 7 gives results of autopsies at the end of 6 weeks. With pullets, as with hens, the response
J. Med. Ent.
108 TABLE 4.
Mean weights (g) of pullets fed varied dosages of (3-exo toxin. WEEK
TREATMENT GROUP*
970 a 434 a** 699 a White 876 a 435 ab 652 a Blue 868 a 468 ab 673 ab Green 688 435 ab 588 b Yellow 540 388 b 507 Red *Dosages are given in TABLE 1. ** Means in each column followed by the same letter are not significantly different at the 5% level by Duncan's multiple range test. Weights did not differ between treatments at the start of the test.
was gizzard erosion followed at increasing dosages by enteritis and petechial lesions in the proventriculus. Anemia at the highest dosage possibly is caused by malnutrition. Single pullets that were removed from each dosage at the start of week 4 were caged in pairs to reduce adaptation problems. Feed consumption for the pair from lower prior dosage regimens (1 "green" and 1 "blue") did not differ convincingly from those at higher prior dosages (1 "yellow" and 1 "red"). Food consumption on day 1 averaged 21 g/pullet; on day 2 averaged 45 g; on day 3 averaged 54 g. For the next 2 weeks, food intake was comparable to that of pullets in the control group. Autopsies 3 weeks later indicated mild gizzard erosion in the pullet from "yellow." The pullet from "red" was autopsied at the end of 1 week and was found to be normal. Recovery could be rapid. Weight increase during this recovery period (weeks 4-7) was 156 g for "blue," 199 g for "green," and 326 g for "yellow." At the" same time, the gain of 6 untreated controls averaged 168 g. This also suggests rapid recovery without permanent injury. The possibility remained of CaTox being toxic and of NaTox prolonging or synergizing the effects of the calcium salt. To ascertain whether the sodium exotoxin alone was toxic, 2 birds of average weight were selected from controls at the start of TABLE 5. Feed consumption by pullets (g/day±SD) fed CaTox in feed or NaTox in water. 0 2 4 8 10 16
20 40 80 160
CATOX
NATOX
61±5 52±2 56±3 48±7
64±7
55±8 44±4 44±6 43 ±2 32 ±6 27±6
54±9 39±6 27±4
Vol. 12, no. 1
TABLE 6.
Water consumption per pullet during weeks 4, 5, and 6 while NaTox was added to water.
DOSAGE (PPM)
CONSUMPTION/DAY (ML)
WATER/FOOD (WT.)
0 10 20 40 80
100 86 88 70 55
1.5 1.5 1.6 1.8 2.0
week 4. These were given untreated feed and 80 ppm of NaTox in the water. One pullet was autopsied after 10 days, and showed gizzard erosion, enteritis, and anemia. Food consumption during these 10 days showed no time lag but averaged only 32 g of feed and 50 ml of water. This is comparable to consumption of pullets on the "red" test which had been fed CaTox in earlier dosages. The surviving pullet gained only 47 g, which is 28% of the gain in controls. Therefore, both exotoxin salts are toxic. Tissue bioassay: Perhaps (3-exotoxin is not really highly toxic but was accumulated and concentrated at a critical site to produce its delayed effect. This possibility was checked by separate bioassays of lungs, liver, heart, breast muscle, and gizzards of all tested hens and pullets using the black blow fly, Phormia regina (Meigen). Larvae grew normally on all these organs, even on hens that were fed 1600 ppm. Either exotoxin does not exist in high concentrations ( > 1 0 ppm) in these organs, or it is rapidly degraded during putrefaction. Eggs from treated hens were mixed with expanded mica and infested with black blow flies. Eggs from hens fed dosages of 200-800 ppm in feed were found to support the growth of fly larvae. At 1600 ppm, only 1 egg, too tiny to support flies, was available for bioassay. Toxicity of exotoxin in moist poultry rations fed to house
flies: Normally, over 90% of the house fly eggs added to CSMA media produce flies. In triplicated tests, 25 fly eggs were added to 100 g of treated CSMA larval medium plus 170 g of water. No flies survived dosages of 40 ppm or more of either salt. At 10 ppm (dry wt), the emergence was 3, 4 and 4 from NaTox and 9, 4 and 4 from CaTox. Emergence from 20 ppm of NaTox and CaTox was 1,1,2 and 2, 1,2, respectively. When NaTox was added and flies were reared on the laying mash, no flies survived 20 ppm. Emergence from controls (78%) also was lower than emergence from CSMA medium. Fly eggs added to pullet developer mash treated with NaTox gave the following emergence: 0 ppm = 82%, 2 ppm = 24%, 4 ppm = 6%, 8 ppm = 4%, and 16 ppm = 0.
Barker & Anderson: p-exotoxin of Bacillus thuringiensis
1975
TABLE 7. Postmortem observations on pullets at end of week 7. (TABLE 1 gives dosages for each group during each of the 7 weeks.) TEST
White
Blue
Green
Yellow
Red
WEIGHT (O)
ABNORMALITIES
None Leukosis; breast blister 1080 Pale liver Pale marrow 1020 910 Moderately pale marrow 1020 Gizzard erosion; enteritis; pale liver 940 Leukosis 800 Gizzard erosion 770 Gizzard erosion 850 Very mild gizzard erosion; pale marrow 910 Gizzard erosion; enteritis; proventriculitis 850 Gizzard erosion; pale liver 850 Gizzard erosion 850 Moderate gizzard erosion; mild anemia Gizzard erosion, pale liver 880 Severe gizzard erosion; proven540 triculitis 820 Gizzard erosion; proventriculitis 600 Gizzard erosion; proventriculitis 710 Mild gizzard erosion; proventriculitis; enteritis 770 Gizzard erosion; proventriculitis; mild enteritis; anemia 660 Severe gizzard erosion; anemia; pale liver 480 Severe gizzard erosion; anemia; pale liver Died, week 4 Gizzard erosion; enteritis Died, week 4 Gizzard erosion; enteritis; severe anemia Died, week 5 Gizzard erosion; enteritis; severe anemia 800 960
109
TABLE 8. Percentages of flies emerged from fly eggs added to manure of chickens fed salts of p-exotoxin. DOSAGE (PPM)
CaTox in feed 0 2 4 8 16 20 40 80 160 200 400 800
1600 NaTox in feed 0 20 40 80 160
HENS
PULLETS
87
87 84 84 79 86 88 76 70 46
82 71 69 59 58 21 25 9 24 0 0 8 91 45 28 16 1
NaTox in water 0 10 20 40
80
72 82 69 61 34
99 66 100
50 18
effect or decomposition of exotoxin in manure. Consequently, sealed polyethylene bags of stored samples were reopened, held on greenhouse benches, and 25 house fly eggs added to each on 8 June. The samples from hens had been collected from 2 Apr. to 24 May. Pullet manure was collected between 5 May and 16 June. On 10 June, survival House fly survival in manure: Emergence from untreated chicken manure was 91% with no differ- was found to be related to viscosity of the feces but not to exotoxin dosage. Some larvae drowned ence between hens in different groups. The when fermenting manure liquified and some bags emergence from manure plus sawdust was 89%, and flies were more difficult to find and count, TABLE 8 were too hot from fermentation for the larvae to survive. These bags were left open and more fly gives results from manure of chickens fed treated eggs were added on 20 June. If the physical conmash. Each percentage represents 4-8 samples of sistency was satisfactory, flies survived at rates 25 flies each. Fly eggs were added 1-10 days after manure was deposited. The 1st tests on hen's comparable to survival on untreated manure. Infestations were repeated on 22 and 25 June with manure suggested that CaTox added to feed had the same results. Even 1600 ppm in feed failed to an LD50 of ca 20 ppm. This was more than control flies whenever there was sufficient manure anticipated, since the LD50 in feed was below 10 and water available. In addition to the artificially ppm. The amount of water added to feed bioassays added house flies, larvae of the black blow fly and was equal to the amount of water added by chickens an unidentified gnat infested feces from treated to produce manure from treated feed (dilution chickens. factor 1.7). In pullet's manure, even 160 ppm in feed failed to control flies in replicated tests. These In prior reports of fly control, fresh feces usually results were obtained in mid-May. Dosages of had been used for bioassay. Borgatti & Guyer 200 to 1600 ppm were then being fed, and the (1963) held samples 1 year at -18°C. Smirnoff & pullets began to get seriously sick. Even so, wild McLeod (1961) counted spores. The tests on flies in the chicken house were breeding in the liquified manure are more nearly those of field droppings. This suggested either a temperature conditions. We also found rapid biodegradability
Vol. 12, no. 1
J. Med. Ent.
110
of exotoxin in Shory's insect medium made from soaked lima beans. Degradation in manure could be influenced by diet as well as microfauna. In contrast to activity of bean enzymes, on a soil surface, exotoxin was stable for at least 4 months (Hitchings 1967). House fly resistance: The low dosage-response slope of exotoxin suggested that flies would readily become resistant to treated manure. Treating larval media has proven to be the most efficient means of selecting an insecticide-resistant strain of flies. From a continual population of ca 2000 adults, a strain of house flies originally resistant to DDT (Rutgers Strain A) was selected at the LD 95 level starting in March. By September (ca 12 generations), the LD 95 dosage in larval media had increased from 80 ppm to 600 ppm. This dosage is 30 X a dosage toxic to chickens and exceeds practical dosage. A lower level of fly resistance to Bakthane-L69 was achieved by Harvey & Howell (1965) in 50 generations with a lower selection level. CONCLUSIONS
The (3-exotoxin was highly toxic to chickens whether added to feed as a calcium or a sodium salt or to water as the sodium salt. Early poisoning symptoms were loss of vigor, reduced feeding, and undersized eggs. Reduced feeding was not a consequence of palatability; exotoxin caused severe gizzard erosion, enteritis, and proventriculitis. A lethal dosage of 20 ppm CaTox in mash fed to hens weighing 1 kg and eating 100 g/day represents 2 mg/kg per day. A dosage of 20 ppm NaTox in water is ca 3 mg/kg per day. Dying pullets were consuming ca 60 ml of water containing 80 ppm NaTox (0.5 mg/day). For a 500-g pullet, the LD 50 is ca 1 mg/kg per day for NaTox. Even the largest experimental dosage of 1600 ppm in feed failed to control flies in manure when conditions for larval growth were otherwise favorable. Larvicidal action was lost when manure fermented. Bioassay of chicken tissues with larvae of Phormia regina (Meigen) furnished further evidence of biodegradability of (3-exotoxin. House flies acquired resistance to (3-exotoxin after 6 months continuous selection from larval exposures. The LD 95 increased 30 times, to the point that larvae grew with 600 ppm of exotoxin in the medium.
Knestrick cared for the chickens and collected manure samples. At Rohm and Haas, D. L. Munchback bioassayed house flies on fresh manure samples and developed the resistant strain. M. Nicoli tested exotoxin in Shory's media. S. L. Satterthwaite photographed the autopsies. LITERATURE CITED
Borgatti, A. L. & G. E. Guyer. 1962. Formulations of Bacillus thuringiensis Berliner found to be contaminated with chlorinated hydrocarbon insecticides. J. Econ. Ent. 55: 1015-16. 1963. The effectiveness of commercial formulations of Bacillus thuringiensis Berliner on house fly larvae. J. Insect
Pathol. 5: 377-84. Briggs, J. D. 1960. Reduction of adult house-fly emergence by the effects of Bacillus spp. on the development of immature forms. J. Insect Pathol. 2: 418-32. Burns, E. C , B. H. Wilson & B. A. Tower. 1961. Effect of feeding Bacillus thuringiensis to caged layers for fly control. J. Econ. Ent. 54: 913-15.
Drake, B. B. & C. V. Smythe. 1963. Process for making pesticidal compositions. U. S. Pat. 3,087,865. Faust, R. M. 1973. The Bacillus thuringiensis (3-exo toxin: Current status. Bull. Ent. Soc. Amer. 19: 153-56. Galichet, P. F. 1966. Administration aux animaux domestiques d'une toxine thermostable secretee par Bacillus thuringiensis Berliner, en vue d'empecher la multiplication de Musca domestica Linnaeus dans les feces. Ann. Zootech., Paris
15: 135-45. Harvey, T. L. & J. R. Brethour. 1960. Feed additives for control of house fly larvae in livestock feces. J. Econ. Ent. 53: 774-77. Harvey, T. L. & D. E. Howell. 1965. Resistance of the house fly to Bacillus thuringiensis Berliner. J. Invert. Pathol. 7:
92-100. Hitchings, D. 1967. Bacillus thuringiensis: A reproduction inhibitor for southern armyworm. J. Econ. Ent. 60: 59697. Kalvoda, L., M. Prystas & F. Sorm. 1973. The structure of the allaric portion of exotoxin from Bacillus thuringiensis. Tetrahedron Ltrs. 1973: 1873-76.
Mackedonski, V. V., N. Nikolaev, K. Sebesta & A. A. Hadjiolov. 1972. Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of Bacillus thuringiensis. Biochim. Biophys. Acta 272: 56-66.
Mechalas, B. J. & O. Beyer.
1963. Production and assay
of extracellular toxins of Bacillus thuringiensis. Dev. Ind. Microbiol. 4 : 142-47.
Ode, P. E. & J. G. Matthysse. 1964. Feed additive larviciding to control face fly. J. Econ. Ent. 57: 637-40. Sebesta, K., K. Horska & J. Vankova. 1969. Inhibition of de novo RNA synthesis by the insecticidal exotoxin of Bacillus thuringiensis var. gelechiae. Collect. Czech. Chem. Commun.
34: 1786-91. Sherman, M., E. Ross & G. H. Komatsu. 1962. Differential susceptibility of maggots of several species to droppings from chickens fed insecticide treated rations. J. Econ. Ent. 55: 990-93. Smirnoff, W. A. & C. F. MacLeod. 1961. Study of the
Acknowledgments: At Delaware Valley College of Science and Agriculture, John C. Harrison, James P. Harner, and Ralph C.
survival of Bacillus thuringiensis var. thuringiensis Berliner in
the digestive tracts and in feces of a small mammal and birds. J. Insect Pathol. 3: 266-70.
