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Studies of the Effect of Some Antibacterial and Antifungal Agents on Growth and Egg Production of Chickens
Studies of the Effect of Some Antibacterial and Antifungal Agents on Growth and Egg Production of Chickens R. DAM 1 AND L. C. NORRIS 2
Department of Potdtry Husbandry and Graduate School of Nutrition, Cornell University, Ithaca, New York (Received for publication March IS, 1961)
INTRODUCTION
1
Present address: Department of Biochemistry and Nutrition, College of Agriculture, University of Nebraska, Lincoln 3, Nebraska. 2 Present address: Department of Poultry Husbandry, University of California, Davis, California.
penicillin or fish meal supplementation can be obtained in clean quarters when both substances are present in the diet. No response was obtained to either alone. These workers suggested, "It is possible that under clean conditions the growth potential of the basal diet is not reduced by bacterial action. The addition of fish meal may increase the growth potential of the feed (by supplying unidentified nutrients or other factors) but this increased potential may be masked by bacterial action. The addition of antibiotics to the fish meal containing ration under these conditions may then result in the increased growth potential being realized." Combs, Arscott and Jones (1954) have shown that chicks fed rations containing no antibiotics do not respond uniformly in successive experiments to unidentified growth factor supplements. They found that in one-fourth of all the trials conducted in their laboratories no improvement in chick growth resulted from various unidentified factor supplements. They suggested that differences in responses may be due in part to factors which may influence the bacterial flora already established in the tract or which become established in the tract. Combs, Romoser and Bishop (1954) observed that orally administered arsanilic acid increased the dietary requirement of chicks for unidentified growth factors present in fish products and dried whey. Growth response of chicks to these factors was significantly greater in the presence of arsanilic acid. Again, these workers thought that the increased growth rate of 78
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I
N WORK conducted in this laboratory a variation in response to unidentified factor supplements has been noted over a yearly period. The response is usually smaller in the fall, at the time that pullet breeders are used, and gradually increases to the period of June or early July. After this time the response of chicks from these hens to unidentified factor supplements usually decreases. Similar experiences have been reported by Waibel, Morrison and Norris (1955), Barnett and Bird (1956), and Morrison (1956). It has been thought that this response is due in part to seasonal changes in the microbial population in the experimental laboratories. Many studies have been conducted which demonstrate or attempt to explain the influence of environmental microbial populations on chick growth rate in the presence of growth promoting substances. The work of Coates el al. (1951) and others, for instance, indicates that the response to antibiotics is greater in quarters in which the bacterial population is higher. Hill and Kelly (1953) have shown, however, that antibiotics will stimulate growth even in clean quarters (i.e., quarters having a relatively low microbial population) if high enough concentrations are fed. Hill and Kelly (1954, 1955) also found that growth stimulation due to
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
In contrast to this work, Wisman et al. (1956) were able to obtain a growth response to fish solubles in old or new quarters in the presence or absence of varying amounts of terramycin and in the presence or absence of droppings of older birds. As a consequence of the inconclusive findings reported previously, several studies were conducted in an attempt to exclude the possibility of interference by microorganisms with chick growth response to unidentified factors, either via contamination of the drinking water or via microbial influence in the gastrointestinal tract. Zephiran chloride,* a commercial mixture of high molecular, alkyl dimethyl benzyl ammonium chloride, was added to the drinking water to effect its steriliza* Product of Winthrop Laboratories, New York 18, New York.
tion. Mycostatin,f a commercial antifungal antibiotic, sulfasuxidine and streptomycin were added to the diet in attempts to reduce the microbial population of the gut. EXPERIMENTAL White Plymouth Rock chicks of the Cobb strain were used in all experiments. All chicks were obtained from hens maintained at the Department of Poultry Husbandry, Cornell University. These hens, and the hens used in experiment 4, were fed a simplified diet composed essentially of yellow corn meal, soybean oil meal (50% protein), corn gluten meal and necessary vitamin and mineral additions. All chicks were identified with numbered wingbands and placed on experiment at approximately one day of age. The chicks were weighed by lot at the start and individually each week thereafter until the experiments were terminated. The duration of the experiments was 4 weeks. The chicks were housed in electrically heated zinc-coated battery brooders equipped with wire-mesh floors to prevent coprophagy. Feed and water were supplied ad libitum. Water was changed and waterers washed daily. In experiment 3, glass waterers were used and daily water consumption per lot measured by weight difference. Evaporation loss was accounted for by means of separate waterers stationed throughout the laboratory. The feed consumed by each lot of chicks was recorded at the time of weighing. Duplicate lots of chicks were subjected to each treatment. In experiment 1, nine males and ten females were used per lot; in experiment 2, 11 males and nine females per lot; and in experiment 3, ten males and ten females per lot. The composition of the basal diet fed f Trade name for nystatin, product of E. R. Squibb & Sons, New Brunswick, N. J.
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chicks fed supplemented diets containing arsanilic acid was due to an effect of arsanilic acid on the microbial population of the intestinal tract. Barnett and Bird (1956) have also reported attempts to control environmental conditions in order to increase the response of chicks to unidentified factor supplements. They had found a gradual decrease in response to unidentified factor supplements over a three-year period. They believed that the loss of response was in part due to changes in the microbial population of the laboratory. It was found that inclusion in the diet of 1% feces from hens on built-up litter led to a reduction in basal growth that could be overcome by supplementation with 3 % fish solubles. As the procedure was continued, a progressive decrease in basal growth occurred so that eventually poor growth resulted either with or without feces in the diet. This indicated a maximum level of contamination had been reached in the laboratory.
79
80
R. DAM AND L. C. NOREIS
TABLE 1.—Composition of basal diet for chicks Amount
Glucose monohydrate 1 Soybean protein 2 Hydrogenated oil3 Cellulose4 DL-Methionine Glycine Mineral mixture 6 Vitamin mixture 6
61.38 25.47 3.00 3.00 0.70 0.30 S. 43 0.72
%
1
Cerelose. Drackett Assay C-l. Hydora. 4 Solka Floe. 6 The grams per 1,000 gm. of Analytical Reagent Grade chemicals in the mineral mixture were as follows: 396.1 CaHP0 4 ,274.8 CaC0 3 ,159.7 KH 2 P0 4 110.5 NaCl, 46.0 MgS0 4 , 6.1 FeS0 4 -7H 2 0, 6.1 MnS0 4 -H 2 0, 0.31 CuS0 4 -5H 2 0, 0.18 ZnCI2, 0.052 Na 2 Mo0 4 -2H 2 0, 0.048 KI, 0.031 CoCl 2 '6H 2 0. 6 The grams and units per 1,000 gm. of vitamins and other substances in the vitamin mixture were as follows: 208.3 choline chloride, 34.7 inositol, 6.9 niacin, 2.8 (/-calcium pantothenate, 2.8 o-tocopheryl acetate, 1.4 thiamine-HCl, 1.4 riboflavin, 0.63 pyridoxin-HC1, 0.56 folic acid, 0.069 menadione, 0.028 biotin, 0.0028 vitamin Bi2, 6,940 IU vitamin A, 520 ICU vitamin D 3 , 740.3 vitamins A, D 3 and E diluents and glucose. 2
3
the chicks is given in Table 1. The soybean protein used in the diets was purified by repeated washings with tap water at the isoelectric point (pH 4.6) with a final washing with demineralized water, after which it was pressed and dried in a forced draft electrically heated oven at a temperature of 65°C. In experiments 1 and 3, a standard source of unidentified growth factors (UGF) was used. This composite was prepared by mixing two parts corn distillers dried solubles, one part fish solubles, and one part dried whey together, drying, grinding to 20 mesh, and recompositing the material. This standard source of UGF was included in the diet at a level of 10%. When these supplements were added to the diet, an amount of protein (% NX6.25) equal to that supplied by the supplement was removed from the diet, thereby maintaining the diets isonitrogenous. Antibacterial compounds
In experiments 1, 2, 3 total numbers of bacteria and yeasts present in the drinking water and fecal contents were determined. The medium used for the determination of "total counts" of bacteria was Thioglycollate Medium (BBL).* The original commercial mixture was modified by adding 14.3 grams of agar per liter for plating purposes. Yeast counts were made using Malt Agar (BBL) which was brought to pH 3.5. Fecal samples represented a composite slurry of a one or two hour collection from the particular group of hens being studied. Approximately one gram of this slurry was transferred to a sample bottle and sufficient sterile \ strength Ringer's solution added to make a dilution of 1:100. Further serial dilutions were made from this dilution. In the case of the drinking water, the original sample was taken as the initial dilution and serial dilutions made from this. All dilutions were made in \ strength * (BBL), Baltimore Biological Laboratories, Baltimore, Md.
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Ingredient
were included at the expense of glucose in the diet. Eighteen White Plymouth Rock hens were used in experiment 4. These were individually caged in a galvanized metal battery with wire floors. They were divided into three groups of six hens, each group occupying a tier of the battery. Each group had access to a common water trough. Three hens of each group shared a common feed trough. Daily records were kept of water consumption per group, and individual egg production. Feed consumed was measured four times each week. Water was changed and waterers washed daily. The hens were housed in the battery for a preliminary period of one week before the experiment was started. These hens ranged in weight from 2.25 to 4.66 kg. with an average weight of 3.30 kg.
81
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
RESULTS
Three experiments were conducted with chicks in which Zephiran was added to the drinking water and several antibacterials or growth factor sources were added to the diet. The four-week growth data for two of these experiments are summarized in Table 2. In a preliminary experiment, experiment 1, Zephiran was added to the drinking water to provide a concentration of one part to 4,000. The water was changed daily. At this level no marked effect was noted on growth or feed conversion. Bacterial counts of the drinking water were made at intervals of two or three days during the first two weeks of the experiment and at intervals of six days
TABLE 2.—The effect of zephiran, sulfasuxidine, streptomycin, mycostatin and unidentified factor supplements on chick growth
Treatment
four weeks grams 1
vors, gam four four weeks weeks
Basal +3TJGF -(-antibacterials2
300 430 385
1.93 1.73 1.78
67 35 37
+Zephiran 3 -f-Zephiran and UGF 4
238 282
1.84 1.76
66 29
+Zephiran, antibacterials -j- Zephiran, antibacterials and 3 UGF
217
2.02
62
292
1.80
38
1 Summary of two experiments. Duplicate lots in both experiments. Experiment 2, 11 males and 9 females per lot; Experiment 3, 10 males and 10 females per lot. 2 The amounts of antibacterials added to the diet were 2 % sulfasuxidine, 200 mg./kg. streptomycin and 200 mg./kg. mycostatin. 3 Zephiran chloride was added to the drinking water at a rate of one part to 3,000 parts water. 4 The unidentified growth factors (UGF) consisted of an initial composite mixture of 2 parts corn distillers dried solubles, 1 part fish solubles and 1 part dried whey. This mixture, when dried and ground, was included at a level of 10% in the diet.
or longer thereafter. The drinking water of these chicks remained essentially sterile until approximately the 14th day. Bacterial growth occurred in the drinking water after this time in these treatments, but it was still markedly below the levels in the untreated drinking water. In the second experiment Zephiran was added to the drinking water to provide a concentration of one part to 3,000. The drinking water was changed daily. Mycostatin and streptomycin were included in the diet in amounts of 200 ppm, and sulfasuxidine was also included in the diet to supply a concentration of 2%. In this experiment, Zephiran markedly depressed growth. There was some indication that perhaps the chicks receiving Zephiran were not consuming as much water as the other chicks. The inclusion of antibacterials alone in the diet caused a marked
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Ringer's solution. Bacteria were incubated at 37°C. for 48 hours, and yeasts were incubated at 25°C. for 48-72 hours. Duplicate plates were used with each dilution. In the summarization, if the dilutions were too numerous to count (more than 300 colonies per plate) or too few to count (0 to 30 colonies per plate) for all dilutions, the series was accordingly labeled greater than ( > ) 300 times, or less than ( < ) 30 times, the dilution factor of the greatest dilution showing growth. Data were not used from those series of dilutions that were countable but showed no decrease between dilutions. Data were also not used from those plates which were contaminated or overgrown by a particular colony type. In some instances, data were used from plates containing less than 30 colonies. This was only done in those cases, however, where there was sufficient uniformity in counts and replication to justify their use. The plate counts reported in the tables represent the arithmetic mean of the counts obtained.
82
R. DAM AND L. C. NORRIS TABLE 3.—Total bacteria and yeast in feces and water, experiment 2*
Treatment
Basal
Bacteria yeast per gram
Bacteria yeast per milliliter
1.0X10 7 1.1X10
9
6.2X10 5
1.6X10 6
12
0
S.4X10 6
55
0
0
1.4X10
4
1.2X10 7 4 weeks 9 1.2X10
1.2X10
1.7X10 7
2.4X10 5
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine
8
+Zephiran (1:3,000)
2.8X10
+Mycostatin, Streptomycin, Sulfasuxidine and Zephiran
1.6X10 7
0
2.3X10 0
0 5
5
1.5X10
0 7
1.2X10 3
7.4X10 7
2.0X10*
0
0
0
0
* Average of data from duplicate lots.
growth increase, in contrast to a marked growth decrease when Zephiran was also present in the drinking water. Examination of the drinking water at two and four weeks revealed that Zephiran effectively sterilized it. High bacterial counts and yeast counts existed in the untreated drinking water. The inclusion of the antibacterials in the diet reduced the microbial population of the gut. No further change in fecal bacteria was produced by Zephiran, even though its use was accompanied by marked growth depression. The results of these examinations are presented in Table 3. A third experiment was carried out during which time water consumption was measured, as well as feed consumption and growth. Zephiran was added to the drinking water to provide a concentration of one part to 3,000. The water was changed daily and consumption measured. Mycostatin, streptomycin and sulfasuxidine were again included as supplements in the diets of certain treatments. In addi-
tion the basal, Zephiran, and Zephiran plus antibacterial treatments were further supplemented with sources of unidentified chick growth factors (UGF). These supplements, when added to the basal diet, greatly increased the growth rate and improved feed efficiency. The growth rate was reduced when Zephiran was added to the drinking water. Growth and feed efficiency were improved when the sources of unidentified factors were supplied either with Zephiran or with Zephiran and other antibacterial agents. These improvements, however, were not as great as those obtained in the absence of these antibacterial substances. Total counts of bacteria and yeasts in the feces and drinking water were carried out at the end of four weeks and are shown in Table 4. The data in the first column indicate contrary to the results of experiment 2 that none of the antibacterial agents had any effect upon the bacterial count in the feces at four weeks. Where Zephiran was used, however, the bacterial
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+Zephiran (1:3,000)
Basal
Water
2 weeks 3.7X10 8
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine
+Mycostatin, Streptomycin, Sulfasuxidine and Zephiran
Feces
83
ANTIBACTERIAL AND A N T I F U N G A L A G E N T S
TABLE 4.—Total bacteria and yeast in feces and water, experiment 3* Feces
Water
Bacteria yeast per gram
Bacteria yeast per milliliter
Treatment
Basal (Table 1) +3XJGF -fZephiran
(1:3,000)
4 weeks 2.SX10 6
1.5X10 2
4.4X10 5
2.3X10 4
8.7X10 5
2.6X10 2
2.3X10 7
2.5X10 6
3.4X10 7
0
0
2.4X10 7
0
0
0
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine and Zephiran
2.8X10 8
0
0
0
-fMycostatin, Streptomycin, Zephiran and 3 UGF
2.3X10 8
0
0
0
Sulfasuxidine,
* Average of data from duplicate lots.
and yeast counts in the drinking water and the yeast counts in the feces were reduced to zero. Data on water consumption for the four-week period are summarized in Table 5. The overall summary of water consumption when related to feed consumption revealed that those chicks receiving the basal diet and Zephiran in the drinking water drank less water per gram of feed consumed than the others. In all other cases those chicks receiving Zephiran in the drinking water either drank as much, or more, water per gram of feed consumed as chicks not receiving Zephiran. When considered on the basis of grams of water consumed per gram of gain, the slower growing basal group receiving Zephiran drank as much water as the faster growing chicks receiving the UGF and no Zephiran. On the other hand the basal control and the fully supplemented chicks receiving Zephiran consumed more water per gram gain than any of the other treatments. Whether or not the use of Zephiran in the drinking water reduced water consumption and thereby reduced feed consumption with the resulting retardation of growth cannot be deter-
mined from the experimental evidence. The possibility exists, however, that the Zephiran eliminated bacteria from the drinking water which synthesize a substance required for chick growth. The parent flock of hens used to produce chicks for these experiments received demineralized water administered in plastic pails. Although the water was changed daily and pails thoroughly TABLE 5.—Evaluation of water
consumption,
experiment 3 Overall consumption four-week period Treatment
Basal + 3 UGF +Zephiran -(-Zephiran and 3 UGF +Zephiran, antibacterials -t-Zephiran, antibacterials and 3 UGF
Grams water/ gram feed consumed
Grams water/gram gam
Means* 2.22 2.06 a 1.84 2.10 a 2.03 a
Means* 4.31 c 3.57 a,b 3.36 a 3.69 a,b 3.80 b
2.38
4.30
c
* Those means followed by the same letter not significantly different at 1% level. Analysis of variance followed by Duncan's multiple range test (1955).
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0
+Zephiran and 3 UGF
84
R. DAM ADN L. C. N O R M S
TABLE 6.-
Week
1 2 3 4 5 6 7 1 2
DISCUSSION
Alfredson et al. (1951) have conducted toxicity studies on alkyl dimethyl benzyl ammonium chloride in rats and dogs. The acute LD 50 of this compound in rats as determined by single oral dosages was found to be 234.3 + 26.5 mg./kg. body
-The effect of zephiran on water consumption, feed consumption and egg production of hens, experiment 4 Water consumption 1 ml./hen/day
Feed consumption gm./hen/day
Egg production % on hen-day t>asis
Groups 2
Groups 2
Groups2
Treatment
All on demineralized water Groups B and C on 1:10,000 Zephiran Groups B and C on 1:7,000 Zephiran Groups B and C on 1:4,000 Zephiran Group B, 1:3,000 and Group C, 1:2,000 Zephiran Same as week 5 Same as week 5
A
B
C
A
B
C
A
B
C
232
237
202
135
124
112
73
73
67
240
271
208
122
135
118
81
74
62
240
260
210
136
149
141
69
76
64
260
228
213
127
130
127
67
71
60
235 303 251
212 248 242
149 159 160
108 137 122
123 134 126
70 60 62
71 62 64
62 76 76
55 52 33
Figures include evaporation loss from waterer. Each group consisted of six White Plymouth Rock hens.
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group C, were reached and maintained for the final three weeks. Water consumption, feed consumption and egg production data for the seven experimental periods are presented in Table 6. Some adverse effects were observed when group C received a concentration of 1:2,000 of Zephiran. Immediate reduction in feed and water consumption was noted. After this initial reduction, feed and water consumption remained fairly constant over the remainder of the final three-week period. Egg production for this group gradually decreased until it was about half that of the previous production. During the third week on the 1:2,000 level of Zephiran, some of the hens of group C exhibited chronic cases of swelling in the soft tissues around the face and corners of the mouth.
scrubbed, by morning bacterial contamination was in excess of one million organisms per milliliter. The water served then as a potential source of organic materials not only from feed and fecal residues but from possible bacterial synthesis. Preliminary study indicated it was possible to keep the water free of bacteria by using a concentration of 1:2,000 of Zephiran. However, abrupt transition to this concentration was not to be desired because of apparent refusal of hens to accept this mixture. In experiment 4, hens were given drinking water containing various concentrations of Zephiran. The experiment consisted of three groups of six hens which were maintained for seven weeks on various levels of Zephiran and demineralized water. Group A served as a control and was kept on demineralized water for the entire seven-week period. Demineralized water containing a Zephiran concentration of 1:10,000 was administered to groups B and C. The concentration of Zephiran was increased over the experimental periods until concentrations of 1:3,000, for group B, and 1:2,000, for
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
In the present study no gross changes were observed on autopsy in chicks which died during the experimental period or in survivors at four weeks. Microscopic tissue examinations were not made. Gross autopsy showed no evidence of dehydration in any of the chicks. No consistent differences could be shown due to treatment with Zephiran, when water consumption was related to feed consumption and weight gain. In addition to the overall summary presented in Table 5, weekly summaries of water consumption data were also analyzed. Again, although significant differences were found, no consistent pattern for these differences, as related to treatment, was observed. Since water consump-
tion could not be related to the presence or absence of Zephiran in the drinking water, to growth rate or to dietary treatment, it would appear that some other factor(s) is involved. It does not appear likely from these data that antibacterials and UGF in the diet would counteract any possible unpalatability caused by the Zephiran, since, if this conclusion is accepted, one might also conclude that addition of UGF to the normal basal caused a significant decrease in palatability of normal drinking water. Therefore while the possibility exists that Zephiran reduced water consumption and thereby reduced feed consumption with resulting retardation of growth, it seems somewhat probable that the inclusion of Zephiran in the drinking water eliminated bacteria which synthesize a substance required for chick growth. Moreover, as can be observed in Tables 3 and 4, even though the drinking water is changed daily, bacterial growth in untreated water is sufficient during a 24-hour period to constitute a potent reservoir of organic materials. Thus the possible synthesis of growth-stimulating compounds by the bacteria and yeasts in the drinking water cannot be overlooked. The fact that the poorer growth of those chicks receiving Zephiran was not completely overcome through supplementation may have been due to the use of too little UGF or the failure to furnish in the diet the factor obtained in the drinking water through fermentation. Although examination of fresh fecal samples from chicks indicated that the use of streptomycin, sulfasuxidine and mycostatin may have had some effect in reducing total numbers of bacteria in experiment 2 (Table 3), the number of bacteria remaining was still extremely high. The opposite situation was encountered in experiment 3 (Table 4). This
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weight. No demonstrable evidence of toxicity was found in a two-year period for alkyl dimethyl benzyl ammonium chloride when fed continuously in concentrations of 0.25 percent or less in the diet of rats. No statistically significant differences were obtained in weight gain and food consumption on levels of 0.25% or less in the diet. Significantly lower gains and food consumption were obtained when a level of 0.5% was fed. Dogs were found to tolerate a dietary level of 0.12% dimethyl benzyl ammonium chloride during a 15-week trial. On higher levels of this compound, decrease in food consumption and significant weight loss was encountered. On the levels of this material found to be detrimental, the gross and microscopic changes observed were similar in both rats and dogs. These changes involved the appearance of acute gastritis involving the glandular portion of the gastric mucosa of the stomach and upper level of the small intestine. Microscopic examination showed a variable amount of superficial necrosis of the mucosa. No pathological changes could be observed in liver or kidney tissue.
85
86
R. D A M AND L. C. N O R M S
is extremely puzzling. The possibility of a seasonal bacterial population effect, March as compared to M a y , or simply greater laboratory contamination in M a y are suggested explanations. Asepsis of the gut was definitely not achieved. Yeasts were eliminated, however, from the gut. SUMMARY
The drinking water of chicks and hens was effectively sterilized with Zephiran. Its inclusion in the drinking water of chicks at a concentration of 1:3,000 resulted in reduced growth. Whether or not the reduced water consumption, accompanying the use of Zephiran, m a y have caused the retardation of growth could not be determined from the experimental evidence. T h e results favored the hypothesis t h a t Zephiran eliminated bacteria which synthesize a substance required for chick growth. A growth response was still obtained with unidentified factor supplements in the presence of Zephiran in the drinking water, or with Zephiran in the drinking water and streptomycin, sulfasuxidine and mycostatin in the diet. Microbiological examination of freshly voided feces revealed t h a t streptomycin, sulfasuxidine and mycostatin, when fed to chicks, had no pronounced effect on total numbers of bacteria. In one case the inclusion of antibacterials in the diet resulted in a 10-100 fold decrease of total
REFERENCES Alfredson, B. V., J. R. Stiefel, F. Thorp, Jr., W. D. Batons and M. L. Gray, 1951. Toxicity studies on alkyl dimethyl benzyl ammonium chloride in rats and dogs. J. Am. Pharmaceutical Assoc, Scientific Ed. 40: 263-267. Barnett, B. D., and H. R. Bird, 1956. Standardization of assay for unidentified growth factors. Poultry Sci. 35: 705-710. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, S. H. Cummins and W. F. J. Cuthbertson, 1951. Mode of action of antibiotics in stimulating growth of chicks. Nature, 168: 332. Combs, G. F., G. M. Arscott and H. L. Jones, 1954. Unidentified growth factors required by chicks and poults. 3. Chick studies involving practicaltype rations. Poultry Sci. 33: 71-79. Combs, G. F., G. L. Romoser and R. W. Bishop, 1954. Influence of arsanilic acid on dietary requirement of chicks for certain unidentified growth factors. J. Nutrition, 53: 511-522. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Hill, C. H., and J. W. Kelly, 1953. The effect of antibiotics on the growth of chicks raised in new quarters. J. Nutrition, 51: 463-466. Hill, C. H., and J. W. Kelly, 1954. The effect of fish meal on the response of chicks to high levels of penicillin. Poultry Sci. 33: 657-658.
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Several studies were conducted in an a t t e m p t to exclude the possibility of interference b y microorganisms with chick growth response to unidentified factors, either via contamination of the drinking water or via microbial influence in the gastrointestinal tract. Alkyl dimethyl benzyl a m m o n i u m chloride (Zephiran) was used to sterilize the drinking water, and streptomycin, sulfasuxidine and mycostatin were used to reduce the microbial population of the gut.
bacteria, while in a second case an apparent 10-100 fold increase resulted. Even when a reduction in numbers did occur, the number of bacteria remaining was of the order of one to several million. However, in all b u t one case, yeasts were completely eliminated. Hens tolerated a level of 1:3,000 Zephiran for a three-week period without ill effect. A reduction in egg production, as well as reduced feed and water consumption, was noted when hens received a level of 1:2,000 of Zephiran in the drinking water. At this level, chronic swelling was apparent in the soft tissues around the face and mouth after prolonged administration. Gross autopsy performed at the conclusion of the experimental period revealed no visible lesions in the soft tissues of the mouth, nor in the digestive tract, liver or kidney.
ANTIBACTERIAL AND A N T I F U N G A L A G E N T S
Hill, C. H., and J. W. Kelly, 1955. The effect of fish meal and antibiotics on the growth of chicks fed equal amounts of feed. Poultry Sci. 34: 1030. Morrison, A. B., 1956. Studies on an unidentified mineral required by the chick. Ph.D. Thesis, Cornell University. Waibel, P. E., A. B. Morrison and L. C. Norris,
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1955. Production of depleted chicks by feeding maternal diets deficient in unidentified growth factors. Poultry Sci. 34:1322-1329. Wisman, E. L., C. E. Holmes and R. W. Engel, 1956. Chick growth response to condensed fish solubles and varying levels of terramycin. Poultry Sci. 35: 457-462.
M . M I T R I V I C , * P . H. M A T I S H E C K AND L. C. L Y N C H
Research Division, Dr. Salsbury's Laboratories, Charles City, Iowa (Received for publication March 20, 1961)
S
E V E R A L immunizing agents for the prevention of fowl cholera in chickens and turkeys have been reported in the literature. Their efficacy, however, has very often been subjected to controversy. Heddleston and Hall (1958) reported on a fowl cholera emulsified type bacterin which in comparative trials to other types of bacterins appeared to be superior. Heddleston and Resinger (1959) outlined a method for preparation of emulsified bacterin and further reported studies on the duration of i m m u n i t y in chickens and turkeys. These reports stimulated our interest in the subject of fowl cholera immunization. In a series of preliminary experiments conducted in chickens with this type of bacterin, the local tissue reaction characterized b y swelling a t the site of inoculation, attracted our attention. The subject of the present paper, therefore, is to report our studies on the local tissue reaction and on the efficacy of several adjuvants in enhancing the anti* Present address: Department of Veterinary Science, The Pennsylvania State University, University Park, Pa.
genicity of fowl cholera emulsified bacterins in chickens. MATERIALS AND METHODS Detection of bacterin irritating component.—To detect the irritating component of emulsified bacterin for local tissues the following experiment was conducted. A total of 100 Peterson X Nichols 108 chickens, five weeks old, were used. The birds were divided into five different groups, 20 birds per group, kept in holding batteries and fed a complete broiler ration free of medicaments. Each group of birds was injected with a single bacterin component equivalent to the amount present in 0.5 ml. of the finished product. The adjuvant oil was Eurika white mineral oil. All injections were given subcutaneously on the lower dorsal portion of the neck. T w o days following the first injection and thereafter at two day intervals for ten consecutive times, two birds from each group were electrocuted and examined for local tissue reactions. The results are reported in Table 1. Comparative local tissue reactions of mineral and vegetable oils.—To s t u d y the
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Studies on Local Tissue Reaction and Adjuvant Effect on the Antigenicity of Fowl Cholera Emulsified Bacterins in Chickens
Department of Potdtry Husbandry and Graduate School of Nutrition, Cornell University, Ithaca, New York (Received for publication March IS, 1961)
INTRODUCTION
1
Present address: Department of Biochemistry and Nutrition, College of Agriculture, University of Nebraska, Lincoln 3, Nebraska. 2 Present address: Department of Poultry Husbandry, University of California, Davis, California.
penicillin or fish meal supplementation can be obtained in clean quarters when both substances are present in the diet. No response was obtained to either alone. These workers suggested, "It is possible that under clean conditions the growth potential of the basal diet is not reduced by bacterial action. The addition of fish meal may increase the growth potential of the feed (by supplying unidentified nutrients or other factors) but this increased potential may be masked by bacterial action. The addition of antibiotics to the fish meal containing ration under these conditions may then result in the increased growth potential being realized." Combs, Arscott and Jones (1954) have shown that chicks fed rations containing no antibiotics do not respond uniformly in successive experiments to unidentified growth factor supplements. They found that in one-fourth of all the trials conducted in their laboratories no improvement in chick growth resulted from various unidentified factor supplements. They suggested that differences in responses may be due in part to factors which may influence the bacterial flora already established in the tract or which become established in the tract. Combs, Romoser and Bishop (1954) observed that orally administered arsanilic acid increased the dietary requirement of chicks for unidentified growth factors present in fish products and dried whey. Growth response of chicks to these factors was significantly greater in the presence of arsanilic acid. Again, these workers thought that the increased growth rate of 78
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I
N WORK conducted in this laboratory a variation in response to unidentified factor supplements has been noted over a yearly period. The response is usually smaller in the fall, at the time that pullet breeders are used, and gradually increases to the period of June or early July. After this time the response of chicks from these hens to unidentified factor supplements usually decreases. Similar experiences have been reported by Waibel, Morrison and Norris (1955), Barnett and Bird (1956), and Morrison (1956). It has been thought that this response is due in part to seasonal changes in the microbial population in the experimental laboratories. Many studies have been conducted which demonstrate or attempt to explain the influence of environmental microbial populations on chick growth rate in the presence of growth promoting substances. The work of Coates el al. (1951) and others, for instance, indicates that the response to antibiotics is greater in quarters in which the bacterial population is higher. Hill and Kelly (1953) have shown, however, that antibiotics will stimulate growth even in clean quarters (i.e., quarters having a relatively low microbial population) if high enough concentrations are fed. Hill and Kelly (1954, 1955) also found that growth stimulation due to
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
In contrast to this work, Wisman et al. (1956) were able to obtain a growth response to fish solubles in old or new quarters in the presence or absence of varying amounts of terramycin and in the presence or absence of droppings of older birds. As a consequence of the inconclusive findings reported previously, several studies were conducted in an attempt to exclude the possibility of interference by microorganisms with chick growth response to unidentified factors, either via contamination of the drinking water or via microbial influence in the gastrointestinal tract. Zephiran chloride,* a commercial mixture of high molecular, alkyl dimethyl benzyl ammonium chloride, was added to the drinking water to effect its steriliza* Product of Winthrop Laboratories, New York 18, New York.
tion. Mycostatin,f a commercial antifungal antibiotic, sulfasuxidine and streptomycin were added to the diet in attempts to reduce the microbial population of the gut. EXPERIMENTAL White Plymouth Rock chicks of the Cobb strain were used in all experiments. All chicks were obtained from hens maintained at the Department of Poultry Husbandry, Cornell University. These hens, and the hens used in experiment 4, were fed a simplified diet composed essentially of yellow corn meal, soybean oil meal (50% protein), corn gluten meal and necessary vitamin and mineral additions. All chicks were identified with numbered wingbands and placed on experiment at approximately one day of age. The chicks were weighed by lot at the start and individually each week thereafter until the experiments were terminated. The duration of the experiments was 4 weeks. The chicks were housed in electrically heated zinc-coated battery brooders equipped with wire-mesh floors to prevent coprophagy. Feed and water were supplied ad libitum. Water was changed and waterers washed daily. In experiment 3, glass waterers were used and daily water consumption per lot measured by weight difference. Evaporation loss was accounted for by means of separate waterers stationed throughout the laboratory. The feed consumed by each lot of chicks was recorded at the time of weighing. Duplicate lots of chicks were subjected to each treatment. In experiment 1, nine males and ten females were used per lot; in experiment 2, 11 males and nine females per lot; and in experiment 3, ten males and ten females per lot. The composition of the basal diet fed f Trade name for nystatin, product of E. R. Squibb & Sons, New Brunswick, N. J.
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chicks fed supplemented diets containing arsanilic acid was due to an effect of arsanilic acid on the microbial population of the intestinal tract. Barnett and Bird (1956) have also reported attempts to control environmental conditions in order to increase the response of chicks to unidentified factor supplements. They had found a gradual decrease in response to unidentified factor supplements over a three-year period. They believed that the loss of response was in part due to changes in the microbial population of the laboratory. It was found that inclusion in the diet of 1% feces from hens on built-up litter led to a reduction in basal growth that could be overcome by supplementation with 3 % fish solubles. As the procedure was continued, a progressive decrease in basal growth occurred so that eventually poor growth resulted either with or without feces in the diet. This indicated a maximum level of contamination had been reached in the laboratory.
79
80
R. DAM AND L. C. NOREIS
TABLE 1.—Composition of basal diet for chicks Amount
Glucose monohydrate 1 Soybean protein 2 Hydrogenated oil3 Cellulose4 DL-Methionine Glycine Mineral mixture 6 Vitamin mixture 6
61.38 25.47 3.00 3.00 0.70 0.30 S. 43 0.72
%
1
Cerelose. Drackett Assay C-l. Hydora. 4 Solka Floe. 6 The grams per 1,000 gm. of Analytical Reagent Grade chemicals in the mineral mixture were as follows: 396.1 CaHP0 4 ,274.8 CaC0 3 ,159.7 KH 2 P0 4 110.5 NaCl, 46.0 MgS0 4 , 6.1 FeS0 4 -7H 2 0, 6.1 MnS0 4 -H 2 0, 0.31 CuS0 4 -5H 2 0, 0.18 ZnCI2, 0.052 Na 2 Mo0 4 -2H 2 0, 0.048 KI, 0.031 CoCl 2 '6H 2 0. 6 The grams and units per 1,000 gm. of vitamins and other substances in the vitamin mixture were as follows: 208.3 choline chloride, 34.7 inositol, 6.9 niacin, 2.8 (/-calcium pantothenate, 2.8 o-tocopheryl acetate, 1.4 thiamine-HCl, 1.4 riboflavin, 0.63 pyridoxin-HC1, 0.56 folic acid, 0.069 menadione, 0.028 biotin, 0.0028 vitamin Bi2, 6,940 IU vitamin A, 520 ICU vitamin D 3 , 740.3 vitamins A, D 3 and E diluents and glucose. 2
3
the chicks is given in Table 1. The soybean protein used in the diets was purified by repeated washings with tap water at the isoelectric point (pH 4.6) with a final washing with demineralized water, after which it was pressed and dried in a forced draft electrically heated oven at a temperature of 65°C. In experiments 1 and 3, a standard source of unidentified growth factors (UGF) was used. This composite was prepared by mixing two parts corn distillers dried solubles, one part fish solubles, and one part dried whey together, drying, grinding to 20 mesh, and recompositing the material. This standard source of UGF was included in the diet at a level of 10%. When these supplements were added to the diet, an amount of protein (% NX6.25) equal to that supplied by the supplement was removed from the diet, thereby maintaining the diets isonitrogenous. Antibacterial compounds
In experiments 1, 2, 3 total numbers of bacteria and yeasts present in the drinking water and fecal contents were determined. The medium used for the determination of "total counts" of bacteria was Thioglycollate Medium (BBL).* The original commercial mixture was modified by adding 14.3 grams of agar per liter for plating purposes. Yeast counts were made using Malt Agar (BBL) which was brought to pH 3.5. Fecal samples represented a composite slurry of a one or two hour collection from the particular group of hens being studied. Approximately one gram of this slurry was transferred to a sample bottle and sufficient sterile \ strength Ringer's solution added to make a dilution of 1:100. Further serial dilutions were made from this dilution. In the case of the drinking water, the original sample was taken as the initial dilution and serial dilutions made from this. All dilutions were made in \ strength * (BBL), Baltimore Biological Laboratories, Baltimore, Md.
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Ingredient
were included at the expense of glucose in the diet. Eighteen White Plymouth Rock hens were used in experiment 4. These were individually caged in a galvanized metal battery with wire floors. They were divided into three groups of six hens, each group occupying a tier of the battery. Each group had access to a common water trough. Three hens of each group shared a common feed trough. Daily records were kept of water consumption per group, and individual egg production. Feed consumed was measured four times each week. Water was changed and waterers washed daily. The hens were housed in the battery for a preliminary period of one week before the experiment was started. These hens ranged in weight from 2.25 to 4.66 kg. with an average weight of 3.30 kg.
81
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
RESULTS
Three experiments were conducted with chicks in which Zephiran was added to the drinking water and several antibacterials or growth factor sources were added to the diet. The four-week growth data for two of these experiments are summarized in Table 2. In a preliminary experiment, experiment 1, Zephiran was added to the drinking water to provide a concentration of one part to 4,000. The water was changed daily. At this level no marked effect was noted on growth or feed conversion. Bacterial counts of the drinking water were made at intervals of two or three days during the first two weeks of the experiment and at intervals of six days
TABLE 2.—The effect of zephiran, sulfasuxidine, streptomycin, mycostatin and unidentified factor supplements on chick growth
Treatment
four weeks grams 1
vors, gam four four weeks weeks
Basal +3TJGF -(-antibacterials2
300 430 385
1.93 1.73 1.78
67 35 37
+Zephiran 3 -f-Zephiran and UGF 4
238 282
1.84 1.76
66 29
+Zephiran, antibacterials -j- Zephiran, antibacterials and 3 UGF
217
2.02
62
292
1.80
38
1 Summary of two experiments. Duplicate lots in both experiments. Experiment 2, 11 males and 9 females per lot; Experiment 3, 10 males and 10 females per lot. 2 The amounts of antibacterials added to the diet were 2 % sulfasuxidine, 200 mg./kg. streptomycin and 200 mg./kg. mycostatin. 3 Zephiran chloride was added to the drinking water at a rate of one part to 3,000 parts water. 4 The unidentified growth factors (UGF) consisted of an initial composite mixture of 2 parts corn distillers dried solubles, 1 part fish solubles and 1 part dried whey. This mixture, when dried and ground, was included at a level of 10% in the diet.
or longer thereafter. The drinking water of these chicks remained essentially sterile until approximately the 14th day. Bacterial growth occurred in the drinking water after this time in these treatments, but it was still markedly below the levels in the untreated drinking water. In the second experiment Zephiran was added to the drinking water to provide a concentration of one part to 3,000. The drinking water was changed daily. Mycostatin and streptomycin were included in the diet in amounts of 200 ppm, and sulfasuxidine was also included in the diet to supply a concentration of 2%. In this experiment, Zephiran markedly depressed growth. There was some indication that perhaps the chicks receiving Zephiran were not consuming as much water as the other chicks. The inclusion of antibacterials alone in the diet caused a marked
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Ringer's solution. Bacteria were incubated at 37°C. for 48 hours, and yeasts were incubated at 25°C. for 48-72 hours. Duplicate plates were used with each dilution. In the summarization, if the dilutions were too numerous to count (more than 300 colonies per plate) or too few to count (0 to 30 colonies per plate) for all dilutions, the series was accordingly labeled greater than ( > ) 300 times, or less than ( < ) 30 times, the dilution factor of the greatest dilution showing growth. Data were not used from those series of dilutions that were countable but showed no decrease between dilutions. Data were also not used from those plates which were contaminated or overgrown by a particular colony type. In some instances, data were used from plates containing less than 30 colonies. This was only done in those cases, however, where there was sufficient uniformity in counts and replication to justify their use. The plate counts reported in the tables represent the arithmetic mean of the counts obtained.
82
R. DAM AND L. C. NORRIS TABLE 3.—Total bacteria and yeast in feces and water, experiment 2*
Treatment
Basal
Bacteria yeast per gram
Bacteria yeast per milliliter
1.0X10 7 1.1X10
9
6.2X10 5
1.6X10 6
12
0
S.4X10 6
55
0
0
1.4X10
4
1.2X10 7 4 weeks 9 1.2X10
1.2X10
1.7X10 7
2.4X10 5
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine
8
+Zephiran (1:3,000)
2.8X10
+Mycostatin, Streptomycin, Sulfasuxidine and Zephiran
1.6X10 7
0
2.3X10 0
0 5
5
1.5X10
0 7
1.2X10 3
7.4X10 7
2.0X10*
0
0
0
0
* Average of data from duplicate lots.
growth increase, in contrast to a marked growth decrease when Zephiran was also present in the drinking water. Examination of the drinking water at two and four weeks revealed that Zephiran effectively sterilized it. High bacterial counts and yeast counts existed in the untreated drinking water. The inclusion of the antibacterials in the diet reduced the microbial population of the gut. No further change in fecal bacteria was produced by Zephiran, even though its use was accompanied by marked growth depression. The results of these examinations are presented in Table 3. A third experiment was carried out during which time water consumption was measured, as well as feed consumption and growth. Zephiran was added to the drinking water to provide a concentration of one part to 3,000. The water was changed daily and consumption measured. Mycostatin, streptomycin and sulfasuxidine were again included as supplements in the diets of certain treatments. In addi-
tion the basal, Zephiran, and Zephiran plus antibacterial treatments were further supplemented with sources of unidentified chick growth factors (UGF). These supplements, when added to the basal diet, greatly increased the growth rate and improved feed efficiency. The growth rate was reduced when Zephiran was added to the drinking water. Growth and feed efficiency were improved when the sources of unidentified factors were supplied either with Zephiran or with Zephiran and other antibacterial agents. These improvements, however, were not as great as those obtained in the absence of these antibacterial substances. Total counts of bacteria and yeasts in the feces and drinking water were carried out at the end of four weeks and are shown in Table 4. The data in the first column indicate contrary to the results of experiment 2 that none of the antibacterial agents had any effect upon the bacterial count in the feces at four weeks. Where Zephiran was used, however, the bacterial
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+Zephiran (1:3,000)
Basal
Water
2 weeks 3.7X10 8
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine
+Mycostatin, Streptomycin, Sulfasuxidine and Zephiran
Feces
83
ANTIBACTERIAL AND A N T I F U N G A L A G E N T S
TABLE 4.—Total bacteria and yeast in feces and water, experiment 3* Feces
Water
Bacteria yeast per gram
Bacteria yeast per milliliter
Treatment
Basal (Table 1) +3XJGF -fZephiran
(1:3,000)
4 weeks 2.SX10 6
1.5X10 2
4.4X10 5
2.3X10 4
8.7X10 5
2.6X10 2
2.3X10 7
2.5X10 6
3.4X10 7
0
0
2.4X10 7
0
0
0
+200 mg./kg. Mycostatin, 200 mg./kg. Streptomycin, 2 % Sulfasuxidine and Zephiran
2.8X10 8
0
0
0
-fMycostatin, Streptomycin, Zephiran and 3 UGF
2.3X10 8
0
0
0
Sulfasuxidine,
* Average of data from duplicate lots.
and yeast counts in the drinking water and the yeast counts in the feces were reduced to zero. Data on water consumption for the four-week period are summarized in Table 5. The overall summary of water consumption when related to feed consumption revealed that those chicks receiving the basal diet and Zephiran in the drinking water drank less water per gram of feed consumed than the others. In all other cases those chicks receiving Zephiran in the drinking water either drank as much, or more, water per gram of feed consumed as chicks not receiving Zephiran. When considered on the basis of grams of water consumed per gram of gain, the slower growing basal group receiving Zephiran drank as much water as the faster growing chicks receiving the UGF and no Zephiran. On the other hand the basal control and the fully supplemented chicks receiving Zephiran consumed more water per gram gain than any of the other treatments. Whether or not the use of Zephiran in the drinking water reduced water consumption and thereby reduced feed consumption with the resulting retardation of growth cannot be deter-
mined from the experimental evidence. The possibility exists, however, that the Zephiran eliminated bacteria from the drinking water which synthesize a substance required for chick growth. The parent flock of hens used to produce chicks for these experiments received demineralized water administered in plastic pails. Although the water was changed daily and pails thoroughly TABLE 5.—Evaluation of water
consumption,
experiment 3 Overall consumption four-week period Treatment
Basal + 3 UGF +Zephiran -(-Zephiran and 3 UGF +Zephiran, antibacterials -t-Zephiran, antibacterials and 3 UGF
Grams water/ gram feed consumed
Grams water/gram gam
Means* 2.22 2.06 a 1.84 2.10 a 2.03 a
Means* 4.31 c 3.57 a,b 3.36 a 3.69 a,b 3.80 b
2.38
4.30
c
* Those means followed by the same letter not significantly different at 1% level. Analysis of variance followed by Duncan's multiple range test (1955).
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0
+Zephiran and 3 UGF
84
R. DAM ADN L. C. N O R M S
TABLE 6.-
Week
1 2 3 4 5 6 7 1 2
DISCUSSION
Alfredson et al. (1951) have conducted toxicity studies on alkyl dimethyl benzyl ammonium chloride in rats and dogs. The acute LD 50 of this compound in rats as determined by single oral dosages was found to be 234.3 + 26.5 mg./kg. body
-The effect of zephiran on water consumption, feed consumption and egg production of hens, experiment 4 Water consumption 1 ml./hen/day
Feed consumption gm./hen/day
Egg production % on hen-day t>asis
Groups 2
Groups 2
Groups2
Treatment
All on demineralized water Groups B and C on 1:10,000 Zephiran Groups B and C on 1:7,000 Zephiran Groups B and C on 1:4,000 Zephiran Group B, 1:3,000 and Group C, 1:2,000 Zephiran Same as week 5 Same as week 5
A
B
C
A
B
C
A
B
C
232
237
202
135
124
112
73
73
67
240
271
208
122
135
118
81
74
62
240
260
210
136
149
141
69
76
64
260
228
213
127
130
127
67
71
60
235 303 251
212 248 242
149 159 160
108 137 122
123 134 126
70 60 62
71 62 64
62 76 76
55 52 33
Figures include evaporation loss from waterer. Each group consisted of six White Plymouth Rock hens.
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group C, were reached and maintained for the final three weeks. Water consumption, feed consumption and egg production data for the seven experimental periods are presented in Table 6. Some adverse effects were observed when group C received a concentration of 1:2,000 of Zephiran. Immediate reduction in feed and water consumption was noted. After this initial reduction, feed and water consumption remained fairly constant over the remainder of the final three-week period. Egg production for this group gradually decreased until it was about half that of the previous production. During the third week on the 1:2,000 level of Zephiran, some of the hens of group C exhibited chronic cases of swelling in the soft tissues around the face and corners of the mouth.
scrubbed, by morning bacterial contamination was in excess of one million organisms per milliliter. The water served then as a potential source of organic materials not only from feed and fecal residues but from possible bacterial synthesis. Preliminary study indicated it was possible to keep the water free of bacteria by using a concentration of 1:2,000 of Zephiran. However, abrupt transition to this concentration was not to be desired because of apparent refusal of hens to accept this mixture. In experiment 4, hens were given drinking water containing various concentrations of Zephiran. The experiment consisted of three groups of six hens which were maintained for seven weeks on various levels of Zephiran and demineralized water. Group A served as a control and was kept on demineralized water for the entire seven-week period. Demineralized water containing a Zephiran concentration of 1:10,000 was administered to groups B and C. The concentration of Zephiran was increased over the experimental periods until concentrations of 1:3,000, for group B, and 1:2,000, for
ANTIBACTERIAL AND ANTIFUNGAL AGENTS
In the present study no gross changes were observed on autopsy in chicks which died during the experimental period or in survivors at four weeks. Microscopic tissue examinations were not made. Gross autopsy showed no evidence of dehydration in any of the chicks. No consistent differences could be shown due to treatment with Zephiran, when water consumption was related to feed consumption and weight gain. In addition to the overall summary presented in Table 5, weekly summaries of water consumption data were also analyzed. Again, although significant differences were found, no consistent pattern for these differences, as related to treatment, was observed. Since water consump-
tion could not be related to the presence or absence of Zephiran in the drinking water, to growth rate or to dietary treatment, it would appear that some other factor(s) is involved. It does not appear likely from these data that antibacterials and UGF in the diet would counteract any possible unpalatability caused by the Zephiran, since, if this conclusion is accepted, one might also conclude that addition of UGF to the normal basal caused a significant decrease in palatability of normal drinking water. Therefore while the possibility exists that Zephiran reduced water consumption and thereby reduced feed consumption with resulting retardation of growth, it seems somewhat probable that the inclusion of Zephiran in the drinking water eliminated bacteria which synthesize a substance required for chick growth. Moreover, as can be observed in Tables 3 and 4, even though the drinking water is changed daily, bacterial growth in untreated water is sufficient during a 24-hour period to constitute a potent reservoir of organic materials. Thus the possible synthesis of growth-stimulating compounds by the bacteria and yeasts in the drinking water cannot be overlooked. The fact that the poorer growth of those chicks receiving Zephiran was not completely overcome through supplementation may have been due to the use of too little UGF or the failure to furnish in the diet the factor obtained in the drinking water through fermentation. Although examination of fresh fecal samples from chicks indicated that the use of streptomycin, sulfasuxidine and mycostatin may have had some effect in reducing total numbers of bacteria in experiment 2 (Table 3), the number of bacteria remaining was still extremely high. The opposite situation was encountered in experiment 3 (Table 4). This
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weight. No demonstrable evidence of toxicity was found in a two-year period for alkyl dimethyl benzyl ammonium chloride when fed continuously in concentrations of 0.25 percent or less in the diet of rats. No statistically significant differences were obtained in weight gain and food consumption on levels of 0.25% or less in the diet. Significantly lower gains and food consumption were obtained when a level of 0.5% was fed. Dogs were found to tolerate a dietary level of 0.12% dimethyl benzyl ammonium chloride during a 15-week trial. On higher levels of this compound, decrease in food consumption and significant weight loss was encountered. On the levels of this material found to be detrimental, the gross and microscopic changes observed were similar in both rats and dogs. These changes involved the appearance of acute gastritis involving the glandular portion of the gastric mucosa of the stomach and upper level of the small intestine. Microscopic examination showed a variable amount of superficial necrosis of the mucosa. No pathological changes could be observed in liver or kidney tissue.
85
86
R. D A M AND L. C. N O R M S
is extremely puzzling. The possibility of a seasonal bacterial population effect, March as compared to M a y , or simply greater laboratory contamination in M a y are suggested explanations. Asepsis of the gut was definitely not achieved. Yeasts were eliminated, however, from the gut. SUMMARY
The drinking water of chicks and hens was effectively sterilized with Zephiran. Its inclusion in the drinking water of chicks at a concentration of 1:3,000 resulted in reduced growth. Whether or not the reduced water consumption, accompanying the use of Zephiran, m a y have caused the retardation of growth could not be determined from the experimental evidence. T h e results favored the hypothesis t h a t Zephiran eliminated bacteria which synthesize a substance required for chick growth. A growth response was still obtained with unidentified factor supplements in the presence of Zephiran in the drinking water, or with Zephiran in the drinking water and streptomycin, sulfasuxidine and mycostatin in the diet. Microbiological examination of freshly voided feces revealed t h a t streptomycin, sulfasuxidine and mycostatin, when fed to chicks, had no pronounced effect on total numbers of bacteria. In one case the inclusion of antibacterials in the diet resulted in a 10-100 fold decrease of total
REFERENCES Alfredson, B. V., J. R. Stiefel, F. Thorp, Jr., W. D. Batons and M. L. Gray, 1951. Toxicity studies on alkyl dimethyl benzyl ammonium chloride in rats and dogs. J. Am. Pharmaceutical Assoc, Scientific Ed. 40: 263-267. Barnett, B. D., and H. R. Bird, 1956. Standardization of assay for unidentified growth factors. Poultry Sci. 35: 705-710. Coates, M. E., C. D. Dickinson, G. F. Harrison, S. K. Kon, S. H. Cummins and W. F. J. Cuthbertson, 1951. Mode of action of antibiotics in stimulating growth of chicks. Nature, 168: 332. Combs, G. F., G. M. Arscott and H. L. Jones, 1954. Unidentified growth factors required by chicks and poults. 3. Chick studies involving practicaltype rations. Poultry Sci. 33: 71-79. Combs, G. F., G. L. Romoser and R. W. Bishop, 1954. Influence of arsanilic acid on dietary requirement of chicks for certain unidentified growth factors. J. Nutrition, 53: 511-522. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Hill, C. H., and J. W. Kelly, 1953. The effect of antibiotics on the growth of chicks raised in new quarters. J. Nutrition, 51: 463-466. Hill, C. H., and J. W. Kelly, 1954. The effect of fish meal on the response of chicks to high levels of penicillin. Poultry Sci. 33: 657-658.
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Several studies were conducted in an a t t e m p t to exclude the possibility of interference b y microorganisms with chick growth response to unidentified factors, either via contamination of the drinking water or via microbial influence in the gastrointestinal tract. Alkyl dimethyl benzyl a m m o n i u m chloride (Zephiran) was used to sterilize the drinking water, and streptomycin, sulfasuxidine and mycostatin were used to reduce the microbial population of the gut.
bacteria, while in a second case an apparent 10-100 fold increase resulted. Even when a reduction in numbers did occur, the number of bacteria remaining was of the order of one to several million. However, in all b u t one case, yeasts were completely eliminated. Hens tolerated a level of 1:3,000 Zephiran for a three-week period without ill effect. A reduction in egg production, as well as reduced feed and water consumption, was noted when hens received a level of 1:2,000 of Zephiran in the drinking water. At this level, chronic swelling was apparent in the soft tissues around the face and mouth after prolonged administration. Gross autopsy performed at the conclusion of the experimental period revealed no visible lesions in the soft tissues of the mouth, nor in the digestive tract, liver or kidney.
ANTIBACTERIAL AND A N T I F U N G A L A G E N T S
Hill, C. H., and J. W. Kelly, 1955. The effect of fish meal and antibiotics on the growth of chicks fed equal amounts of feed. Poultry Sci. 34: 1030. Morrison, A. B., 1956. Studies on an unidentified mineral required by the chick. Ph.D. Thesis, Cornell University. Waibel, P. E., A. B. Morrison and L. C. Norris,
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1955. Production of depleted chicks by feeding maternal diets deficient in unidentified growth factors. Poultry Sci. 34:1322-1329. Wisman, E. L., C. E. Holmes and R. W. Engel, 1956. Chick growth response to condensed fish solubles and varying levels of terramycin. Poultry Sci. 35: 457-462.
M . M I T R I V I C , * P . H. M A T I S H E C K AND L. C. L Y N C H
Research Division, Dr. Salsbury's Laboratories, Charles City, Iowa (Received for publication March 20, 1961)
S
E V E R A L immunizing agents for the prevention of fowl cholera in chickens and turkeys have been reported in the literature. Their efficacy, however, has very often been subjected to controversy. Heddleston and Hall (1958) reported on a fowl cholera emulsified type bacterin which in comparative trials to other types of bacterins appeared to be superior. Heddleston and Resinger (1959) outlined a method for preparation of emulsified bacterin and further reported studies on the duration of i m m u n i t y in chickens and turkeys. These reports stimulated our interest in the subject of fowl cholera immunization. In a series of preliminary experiments conducted in chickens with this type of bacterin, the local tissue reaction characterized b y swelling a t the site of inoculation, attracted our attention. The subject of the present paper, therefore, is to report our studies on the local tissue reaction and on the efficacy of several adjuvants in enhancing the anti* Present address: Department of Veterinary Science, The Pennsylvania State University, University Park, Pa.
genicity of fowl cholera emulsified bacterins in chickens. MATERIALS AND METHODS Detection of bacterin irritating component.—To detect the irritating component of emulsified bacterin for local tissues the following experiment was conducted. A total of 100 Peterson X Nichols 108 chickens, five weeks old, were used. The birds were divided into five different groups, 20 birds per group, kept in holding batteries and fed a complete broiler ration free of medicaments. Each group of birds was injected with a single bacterin component equivalent to the amount present in 0.5 ml. of the finished product. The adjuvant oil was Eurika white mineral oil. All injections were given subcutaneously on the lower dorsal portion of the neck. T w o days following the first injection and thereafter at two day intervals for ten consecutive times, two birds from each group were electrocuted and examined for local tissue reactions. The results are reported in Table 1. Comparative local tissue reactions of mineral and vegetable oils.—To s t u d y the
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Studies on Local Tissue Reaction and Adjuvant Effect on the Antigenicity of Fowl Cholera Emulsified Bacterins in Chickens