This dissertation has been 65—1203 microfilmed exactly as received
MEREDITH, William Edward, 1932- THE IN OVO AND IN VITRO EFFECTS OF POTEN TIATED AND UNPOTENTIATED CHLORTETRA- CYCLINE IN CONTROLLING MYCOPLASMA GAL- L1SEPTICUM.
The Ohio State University, Ph.D., 1964 Bacteriology
University Microfilms, Inc., Ann Arbor, Michigan THE IN OVO AND IN VITRO EFFECTS OF POTENTIATED AND
UNPOTENTIATED CHLORTETRACYCLINE IN CONTROLLING
MYCOPLASMA GALLISEPTICUM
DISSERTATION Presented in Partial Fulfillment of the Requirements for Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By William Edward Meredith, B.Sc., M.Sc.
******
The Ohio State University 1964
Approved by
jsxJ^,-LiyAdviser lAjlXA Department of Microbiology ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to Dr. Harry H. Weiser who freely gave his time and advice during my graduate studies at this university. The author is also grateful to Dr. A. R. Winter for his helpful criticism and interest during the present investigation. A special word of thanks is due Dr. Grant
Stahly for his suggestions during the preparation of this dissertation. Appreciation is also extended to the Department of
Bacteriology, Texas Agriculture and Mechanical College, College Station, Texas, for supplying strains S6 and 801 of Mycoplasma galliseptieum and to Dr. J. Fabricant for supplying strain 293 of Mycoplasma galliseptieum. A debt of gratitude is also extended to Mr. Henry Peck of the
Ohio State Agricultural Experimental Station, Reynoldsburg, Ohio, for supplying a culture of the Infectious Bronchitis
Virus.
ii VITA
November 30, 1932 Born - Dennison, Ohio
1959 ...... B.Sc., Ohio University, Athens, Ohio 1959-1963 . . . Teaching Assistant, Department of Microbiology, The Ohio State University, Columbus, Ohio 1 9 6 1 ...... M.Sc., The Ohio State University, Columbus, Ohio 1963-1964 . . . Research Assistant, Department of Microbiology, The Ohio State University, Columbus, Ohio
FIELDS OF STUDY
Maj‘or Field: Microbiology Studies in Food Microbiology. Professor Harry H. Weiser Studies in Pathogenic Microbiology. Professor Melvin S. Rheins Studies in Serology. Professor Matthew C. Dodd
Studies in Bacterial Physiology. Professor Chester I. Randles Studies in Industrial Mycology. Professor William S. Gray
iii CONTENTS
Page
INTRODUCTION ...... 1
STATEMENT OF PROBLEM...... 5 REVIEW OF LITERATURE ...... 7 MATERIALS AND METHODS ...... 26
RESULTS ...... ^3 DISCUSSION ...... 87
SUMMARY...... 97 BIBLIOGRAPHY ...... 99
iv TABLES
Table Page 1. Quick Reference Chart to Articles on Inhibition of PPLO by Antibiotics ...... 14 2. Carbohydrate Fermentation by 3 Strains of Mycoplasma Gallis epticum ...... 44 3. Chlortetracycline Sensitivity of Mycoplasma Galliseptieum S6 , 801, and 293 as Determined by Pad-Plate Assay ...... 45 4. Sensitivity of Mycoplasma Galliseptieum S6 , 801, and 293 to Chlortetracycline ...... 45 5* 111 Vij^ro Chlortetracycline Sensitivity of Mycoplasma Galliseptieum S6 ...... 48 6 . In Vitro Chlortetracycline Sensitivity of Mycoplasma Galliseptieum 801 ...... 49 7. In Vitro Chlortetracycline Sensitivity of Mycoplasma Galliseptieum. 293 ...... 50 8 . Effect of Chlortetracycline Feed Supplemen tation on Egg Fertility and Embryo Viability . 52
9. Titration of 4 Strains of Mycoplasma Galliseptieum...... 54 10. Titration of Infectious Bronchitis Virus .... 55
11. Mortality of PPLO Control Eggs when CTC was Added by Direct Inoculation ...... 57 12. Mortality of PPLO Control Eggs with CTC Added by Direct Inoculation...... 59 13. Mortality of PPLO Control Eggs with CTC Added by Direct Inoculation...... 6l 14. Mortality of PPLO Control Eggs with CTC Added by Direct Inoculation...... 64 v Table Page 15. Mortality of PPLO Control Eggs when CTC was Added by Direct Inoculation. High PPLO Inoculum...... 65 16. Mortality of PPLO Control Eggs when CTC was Added by Direct Inoculation. High Virus Inoculum...... 67 17. Mortality of PPLO Control Eggs when CTC was Added by Use of Dipping Solutions ...... 68
18. Mortality of PPLO Control Eggs when CTC was Added by Use of Dipping Solutions. High PPLO Inoculum...... 70 19. Mortality of PPLO Control Eggs when CTC was Added by Use of Dipping Solutions. High Virus Inoculum...... 71 20. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement ...... 73 21. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement. High PPLO Inoculum...... 73 22. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement. High Virus Inoculum...... 76 23. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement plus a 400 ppm Dipping Solution ...... 77 24. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement plus a 200 ppm Dipping Solution ...... 79 2 3 . Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement plus a 400 ppm Dipping Solution. High PPLO Inoculum...... 8l 26. Mortality of Embryonated Eggs when CTC was Added as a Feed Supplement plus a 200 ppm Dipping Solution. High PPLO Inoculum...... 82
vi FIGURES
Figure Page 1. Schematic representation of the typical life cycle of Mycoplasma ...... 1° 2. Colonies of M. galliseptieum as seen through the microscope at a magnification of 100X .... 17 3. Colonies of M. galliseptieum as seen through the microscope at a magnification of 100X .... 17 4. Enlarged view of colonies of M. galliseptieum (125X) ...... 18 5. An uninoculated petri dish containing PPLO agar ...... 21 6 . A PPLO agar plate inoculated with M. galliseptieum after 24 hours incubation at 37° C ...... 21 7. A PPLO agar plate inoculated with M. allisepticum after 48 hours incubation at f7'° 0 '. .7.7.7...... 22 8 . Chart showing methods of embryo inoculation .. 31 9. Comparison of 11 day old embryos from a control egg and an egg inoculated with M. galliseptieum S6C ...... 84 10. An 11 day old embryo removed from an egg inoculated with IBV and M. galliseptieum S6C.. 84 11. Contents of a control embryonated egg after 11 days of incubation ...... 83 12. Contents of an embryonated egg inoculated with M. galliseptieum S6C after 11 days incubation...... 85 13. Contents of an embryonated egg inoculated with M. galliseptieum S6C and IBV after 11 days of incubation ...... 86 vii INTRODUCTION
Within the past few years more and more attention has been devoted to a group of microorganisms placed in the family Mycoplasmataceae. These microorganisms have
been popularly called pleuropneumonia-like organisms (PPLO) due to their close morphological and biochemical resemblance to the prototype species, Mycoplasma mycoldes,
the causative agent of bovine pleuropneumonia. The members of this group cause a great number of diseases, and as etiological agents they exhibit a distinct species specificity. Mycoplasma mycoides was found to be the causative agent of bovine pleuropneumonia in 1898 (Nocard and Roux); Mycoplasma pneumoniae the causative agent of primary atypical pneumonia in humans in 1 9 ^ (Eaton et al.); and in 1936 Nelson described in chickens a coryza caused by coccobacilliform organisms. Nelson’s work was confirmed as a PPLO by Smith in 19^8. This agent is called M. galliseptieum.
The most discouraging facet of research on the PPLO is the complete lack of agreement by the investigators that one notes upon scanning the literature on this subject
Even today there is not complete agreement on whether the
PPLO can cause chronic respiratory disease (CRD) in fowl and even on the exact definition of CRD. For years the terms CRD and air-sac disease were used interchangeably and caused a great deal of confusion. In 1953 Van Roekel
suggested using CRD only for the definite poultry disease entity caused by Mycoplasma, and using air-sac disease for
indefinite pathological processes caused by a number of
infectious agents that involved air sacs. Every year thousands of dollars are lost because of
CRD. Common losses occur from lowered egg production, slow growth, condemnation, and poor feed conversion. A large percentage of chicks are born dead each year because of the presence of mycoplasma in the embryonated egg. Investigators have agreed that the principal mode of
transmission of M. galliseptieum is from the hen through the egg to the chick or poult. The rate of transmission
is greatest during the acute infection and decreases with time. Second in importance is contact transmission. Experimental infection under controlled conditions indicated that the bird will not show signs of infection
for a period of time from one to several weeks and, as
expected, this depends on the virulence of the strain used, concentration of the strain used, and route of
exposure. The disease appears to be latent in nature, and contact transmission may occur within a flock without a sign of clinical symptoms. 3 At present there is no commercially produced vaccine effective against CRD. The methods used to produce antigen are varied and the work done by others may not be repeated without considerable difficulty (Hall, 1962).
Control of CRD has met with little or at best questionable success when antibiotics have been used as the controlling agent (Hofstad, 19^9; Olesiuk, 1959)* Work has been done on incorporation of antibiotics into feeds and various dipping procedures to determine weight gains of birds on supplemented feeds and to increase shelf life of eggs. There are three commonly used methods of incorporating antibiotics into fertile eggs. The most frequently used one is by incorporating antibiotics into the feed given the adult bird. This can be done with or without a potentiating agent. When a potentiating agent such as terephthalic acid is used, the detectable antibiotic residue in the egg is two or more times that amount detected when the same amount of antibiotic is incorporated without potentiation (Frye,
1957; Meredith, 1961). Dipping eggs in antibiotic solutions is the next most popular method of antibiotic incorporation. Direct inoculation is the least popular of the three methods and is seldom used because of increased mortality of the embryo which is caused by antibiotic toxicity. The use of antibiotics appears to he the most economical and practical approach to control transovarial passage of the CRD agent. If a method can be shown by which antibiotics can be coupled with good poultry management it should prove to be a big step forward toward the elimination of a poultry disease which causes a substantial loss to poultrymen every year. STATEMENT OF PROBLEM
The three methods of incorporation of antibiotics into the fertile egg and thus to the developing chicken embryo were examined alone and in combination to determine their effectiveness in controlling the transovarial passage of the chronic respiratory disease agent M. galliseptieum.
It was the purpose of this work to determine the effectiveness of chlortetracycline incorporated into the embryo by adding 200 and 400 ppm. of antibiotic to the feed, dipping the eggs into concentrations of 200 and 400 ppm. of CTC, and by injecting various quantities of CTC directly into the embryonated egg. Isolation of the organism was attempted at the end of the observation period
to determine the effect of chlortetracycline (CTC) on three strains of M. galliseptieum. It had not been firmly established whether or not CTC is bactericidal to
M. galliseptieum. Tests were performed to determine the effectiveness
of CTC on M. galliseptieum in vitro and to determine the
effect of potentiation of the antibiotic incorporated into the feed by the use of a 0.5 per cent concentration of terephthalic acid.
5 6
Since combined infections are common in chickens and have a very serious effect on developing embryos, a common viral poultry disease agent, called the Infectious Bronchitis Virus, was used in conjunction with M. galliseptieum to determine the effectiveness of CTC in controlling increased embryo mortality under conditions of individual and combined incorporation of the above disease-producing agents. Four strains of M. galliseptieum of varying virulence were employed throughout -this work. REVIEW OP LITERATURE
Chlortetracycline is one of the most popular antibiotics added to poultry feeds today. At the present time, approximately 95 per cent of all poultry flocks in the United States receive antibiotic fortified feeds. Antibiotic feeds were regarded as drugs by the Food and
Drug Administration (Jester, 1956) but in 1951 they were exempted on the grounds that they were to be used solely to promote growth. It soon became evident that a given antibiotic feed was effective in the prevention or treatment of a specific infectious disease and certain preparations containing prescribed amounts of antibiotics were also made exempt from certification (under section 146.21, January, 1956, of regulations in the Federal Food, Drug, and Cosmetic Act). At the present time the tolerance for feeds supplemented with chlortetracycline is 200 parts per million (ppm) which amounts to approximately 200 grams per ton. The residual tolerance for tissue is 4 ppm for kidney tissue and one ppm in other edible tissues. If the calcium level is maintained below 0.8 per cent the high
concentration antibiotic fortified feed is not to be fed
continuously for more than 5 days (Federal Register, i960).
7 One of the advantages of chlortetracycline is that it is relatively unstable and will therefore not maintain detectable residues in foods for long periods of time. Of all the members of the tetracycline family of antibiotics, chlortetracycline is the most unstable. In solutions in which the pH is maintained above J.O the chlortetracycline rapidly forms isochlortetracycline which is not a biologically active substance (Sevick, 1963; Haque, 1958). According to Stephens (195*0 and Pruess (195*0 formation of isochlortetracycline may be a consequence of a repulsion between the chlorine atom and substituents on C-6 since tetracycline is resistant to this cleavage and is much more stable in alkaline solutions. In 1958, Peterson reported that antibiotic residues
resulting from antibiotic supplemented poultry feeds could be increased by incorporating a 0.5 per cent concentration of terephthalic acid into the antibiotic fortified feeds. Peterson tried various other dicarboxylic acids such as malic, fumaric, succinic, arid oxalacetic acid but found these acids to be relatively useless for the purpose of antibiotic potentiation. Many authors have written accounts of the value of antibiotic fortification of poultry feeds and the value of antibiotic incorporation is now unquestioned. Boone and
Morgan (1953) showed that the incorporation of antibiotics into poultry feeds was helpful in controlling poultry diseases such as chronic respiratory disease, blue comb and infectious sinivitis. These same authors also found that egg production from actively laying hens was also
increased. In 1955 > Stokstad and Jukes observed that chicks were stimulated by the addition of CTC. Domermuth and
Johnson (1955) reported that the organism causing CRD does not build up a resistance to antibiotics when used in this manner. Chlortetracycline supplementation in quantities of 50 - 20,000 ppm showed antibiotic levels in chicken serum, tissues and eggs according to Durbin et al. (1953)* Raica et al. (1956) was not able to demonstrate a residue in hens fed under 500 ppm in their feed; however, levels of 500 - 2,000 ppm consistently produced detectable levels of antibiotic. In i960, Boyd et al. observed that terephthalic acid in a 0.5 per cent concentration plus antibiotic resulted in higher residual concentrations of antibiotic
in the serum, liver, white and dark meat. Meredith (1961) observed that chlortetracycline fed to laying hens in concentrations of 200 ppm and 1,000 ppm with and without 0.5 per cent terephthalic acid resulted in detectable residues of the antibiotic in the egg yolks and that this
residue was not always destroyed by mild heat treatments.
Previous work at Ohio State University by Frye (1957) indicated that the largest detectable CTC residues in eggs were found in the yolk of the egg. Calleja (1963) found that detectable CTC residues were found in the embryonated egg after 5 days of incubation. A significant finding concerning the fate of tetracycline residue was reported by Rolle et al. in 1962. She administered tetracycline orally to laying hens and newly hatched chicks in concentrations of 200 and 400 gm/ton of standard feed. Eggs laid by hens after 5 days of tetracycline feeding were incubated and embryos removed and examined at 3» 6 , 9 , 12, and 14 days of incubation. Femurs of the chicken embryos have tetracycline induced fluorescence concurrent with the mineralization of the bone matrix. The bones of newly hatched chicks fed a tetracycline ration for 2 weeks similarly had tetracycline-induced fluorescence of the mineralized bone. When newly hatched chicks were fed the tetracycline ration for 2 weeks and returned to non tetracycline feed for 3 months, all secondary fluorescence disappeared. This was probably due to the rapid bone reorganization taking place during this period of active growth. Inhibition of bone growth or mineralization did not appear to occur during the course of this work.
Effects of Dietary Antibiotics on Infections by Members of the Pleuropneumonia-like Organisms
In 19539 Gross stated that PPLO and Escherichia coli were the only agents involved in air sac disease 11 which were susceptible to antibiotics. He outlined three distinct methods of approach: (1 ) the pathogenic strains °f E. coli could be removed systemically from the infected bird; (2 ) the air sacs could be rendered non-susceptible to the invasion of E. coli by the control of PPLO infection; or (3) E. coli could be removed from the intestinal tract if this should prove to be an important reservoir of infection. In his experiments CTC proved to be the most effective antibiotic for control of the uncomplicated PPLO infection. Erythromycin produced about the same results as CTC against uncomplicated PPLO infection. It would probably be of special value against CTC resistant PPLO strains. The nitrofurans were found to be the most effective against E. coli but they were not as effective against PPLO as the antibiotics. The tetracycline, streptomycin, and neomycin antibiotics were found to be of value in reducing the coliform reservoir in the intestine. The majority of workers believe that streptomycin, dihydrostreptomycin, chlortetracycline, oxytetracycline, erythromycin, chloramphenicol, carbomycin, and viridogrisein are effective against isolates of PPLO from infected fowl. Treatment by these drugs is believed to result in antibiotic- resistant strains of PPLO which may be produced by prolonged antibiotic therapy. Erythromycin appears to be the exception to this rule; however, it is not as effective as 12 the other antibiotics against avian PPLO. Strains of PPLO resistant to the tetracycline group of antibiotics appear to be produced only under the unusual circumstance of prolonged antibiotic therapy. It has also been shown that erythromycin, carbomycin, and oxytetracycline are of value in the treatment of those outbreaks of infectious sinusitis in which a strain resistant to dihydrostreptomycin is involved. A single injection of dihydrostreptomycin has been shown to induce resistant PPLO strains (Osborn, i960). Spiramycin permitted normal weight gains and prevented mortality in birds infected with M. gallinarium when it was incorporated into the feed at concentrations of 400 ppm down to 50 ppm according to Kiser et al. (i960). This group also found that chlortetracycline fed at 400, 200, and 100 ppm gave good protection from mortality and gave weight gains as good as those of an uninfected control group of chickens. Tetracycline was found to be almost as good at controlling the infection when 400 ppm were employed as chlortetracycline was at 200 ppm. Tetracycline at 200 ppm was found to be
ineffective. Chlortetracycline at 2.5, 1.25, or O .63 mg/bird/day for 10 days gave significantly better growth than the untreated infected birds and prevented mortality.
Van Roekel and Olesiuk (1953) used a lesion score as the criterion of activity, supplemented in some
instances by data on weight gains. Their results showed 13 that carbomycin or oxytetracycline appeared to be more effective than chlortetracycline. Their doses of chlortetracycline were small* the period of treatment was for only 7 days, and their results were not uniform. In 1958, Price et al. showed that oxytetracycline at 500 gm/ton of feed suppressed mortality and maintained a normal weight gain in birds infected with PPLO. Penicillin at comparable doses was ineffective.
Duerre and Carlson (1957) reported that birds infected with PPLO and given chlortetracycline at 50 mg/pound of feed (100 ppm) made better weight gains than infected, untreated birds or uninfected, untreated birds, but not as good gains as uninfected birds treated with chlortetracycline. An article by White - Stevens and
Zeibel in 195^ showed that 50 or 100 ppm of chlortetra cycline maintained growth efficiency of broilers with a subclinical infection if the drug was fed continuously, but that 400 ppm CTC in feed or 250 mg/gallon of drinking water was required to maintain or restore growth efficiency when a clinical outbreak occurred.
Hamby et al. (1957) treated 20 large turkeys with 100 mg of erythromycin/bird and reported that 18 recovered from clinical infectious sinusitis.
Pabricant and Chalquest (1959) observed that dipping eggs into antibiotic solutions of varying concentrations of oxytetracycline and erythromycin was 14
TABLE 1
QUICK REFERENCE CHART TO ARTICLES ON INHIBITION OF PPLO BY ANTIBIOTICS
Author Type of Research
Leberman et al., 1950, 1952 In vitro antibiotic Keller and Morton, 1952 testing in liquid media Helen, 1952 Hamby et al., 1957 Robinson et al., 1952 In vitro antibiotic Hachness and“Bushby, 1954 testing on solid media Blyth, 1958
Hitchener, 1949 In vivo or in ovo Grumbles and Boney, 1950 testing of antibiotics Olson, 1951 Gross, 1953 V/hit e-St evens, 1954 Yamamoto, 195° Duerre, 1957 Hamby, 1957 Olesiuk, 1957 Price, 1958 Chalquest and Fabricant, 1959 Kiser et al., i960 Nasemann and Rockel, i960 Olsen et al., i960 Osborn et al., i960 found to reduce the number of reisolations. It was also determined that PPLO inoculated into fertile eggs before the dipping process resulted in fewer successful reisolations. All of this work was performed by dipping warm eggs into cold antibiotic solution.
Characterization of the Pleuropneumonia like Organisms
The organisms of the pleuropneumonia and pleuropneumonia-like group are found in the class Schizomycetes. Within the class Schizomycetes they are placed into the order Mycoplasmatales and defined as organisms which are non-motile, highly pleomorphic, very delicate, and possess filterable stages. The genus
Mycoplasma, according to Bergey's Manual (5)» defines their differentiating characteristics as small, spherical bodies, 150 to 300 millimicrons in diameter, which germinate to form filaments approximately 0.2 microns wide and from 2 to 30 microns long. In later stages of growth endomycelial corpuscles develop in the filaments by a process of successive condensation and constriction. Resulting from this are the spherical bodies which are released by fragmentation. These organisms are highly resistant to penicillin and sulfathiazole. Their colonies on agar have a dense granulated central area which 16
Mycelial stage
1 Elementary Body Intermediate Cell
\J/ Large cell with inclusions 4r Large Cell
Large cell reproduction without formation of elementary bodies
Figure 1. Schematic representation of the typical life cycle of Mycoplasma. Figure 2. Colonies of M. galliseptieum as seen through the microscope at a magnification of 100X.
Figure 3* Colonies of M. galliseptieum as seen through the microscope at a magnification of 100X. 18
Figure 4. Enlarged view of colonies of M. gallisepticum (125X). penetrates into the agar and which is surrounded by a translucent flat peripheral zone. This is termed a "fried egg colony." Fifteen species are described in Bergey's
Manual (5) and are separated by pathogenicity, host specificity, cultural and biochemical characteristics.
Colonies of PPLOfs resemble the L-forms closely but L-form colonies are usually more opaque and more heavily marked on the surface. The L-forms do not require cholesterol, tend to revert to normal bacterial form, are non-pathogens, are more difficult to subculture and react biochemically the same as the parent organisms. The agar-to-agar transfer of colonies of PPLO is unique in that it requires blocks of agar to be cut from one agar plate and transferred to another plate and then be maneuvered over the surface of the new plate. A subculture is seldom obtained using a loop or needle due to the tendency of the growth to imbed itself into the medium. Culturing is difficult because the organisms die quickly at incubator temperatures. In order to transfer colonies from agar to broth a section of the agar must be
cut from the agar plate and be placed into the broth. Transfer from broth to broth can be accomplished in the usual manner. Colonies of PPLO in broth produce little or no turbidity and on agar the colonies must be magnified from 50-100 times before they are clearly visible
(Klieneberger-Nobel, i960). 20
The PPLO can be divided into two groups on the basis of carbohydrate utilization. The organisms in the fermentative group, although unrelated serologically, are similar in the fermentation of carbohydrates and metabolism of glucose. Glycogen, starch, dextrin, maltose, sucrose, and hexose sugars are the only carbohydrates attacked according to Tourtellotte and Jacobs (1959)* They also indicated that the fermentation, or lack of it, of a particular carbohydrate cannot be used as a diagnostic tool in the differentiation between strains of PPLO, especially if the isolate has been carried on media for a long time. Among the most unique of nutritional requirements for microorganisms is the requirement of the pathogenic PPLO's for cholesterol. This sterol requirement is not unique in nature, since some protozoa and the larval or pupal stages of several insects display a similar require ment. In the case of most insects the sterol requirement is lost upon development to the adult stage. The function of the sterol in insects appears to be to supply a precursor for synthesis of hormone-type steroids. The function of the sterol in protozoa and PPLO's remains obscure. In 1951* Edward and Fitzgerald reported that cholesterol was active in promoting growth and could be replaced with either cholestanol or stigmasterol, whereas ergosterol, coprostanol, and certain cholesterol esters were inactive. Numerous mechanisms for cholesterol have Figure 5* An uninoculated petri dish containing PPLO agar.
Figure 6 . A PPLO agar plate inoculated with M. gallisepticum after 24 hours incubation at 37°C. 22
gallifloptlcua 1*8 vftovrr* incubation
Figure 7. A PPLO agar plate inoculated with M. gallisepticum after 48 hours incubation at 37° C.
i 23 been postulated and among them are detoxification of fatty acids, provision of an oxidizable substrate, maintenance of structural integrity of the organism and participation in the permeation of substrates into the cells. All of these mechanisms are discussed in detail by Smith (i960).
The PPLO are extremely small in some stages of growth and quite large in others. A life cycle has been postulated for M. mycoides which establishes the smallest unit of reproduction as the elementary body with a size of 0.12 to 0.12 microns in diameter and the shape of a sphere. The next largest unit is the elementary body which goes on to form a large cell. At this point the large cell may form inclusions which break off to become elementary bodies or the large cells may reproduce by fission and then form inclusions which become elementary bodies. At the present time there is no evidence concerning whether or not a life cycle exists for M. gallisepticum (Morowitz, 1962). There are many substances which exhibit an inhibitory effect on the PPLO’s. Edward in 19^-7 tested a number of inhibitory agents and found the PPLO's to be susceptible to various concentrations of potassium tellurite, brilliant green and gentian violet. He found that the PPLO's were quite resistant to thallium acetate at concentrations as high as 1:1000. Thallium acetate is highly bacteriostatic for aerobic spore formers and gram 24 negative bacteria and is not influenced by the protein content of the media. The tetracycline antibiotics are quite effective against the PPLO group. Other antibiotics which are also effective are chloramphenicol, streptomycin, spiromycin, neomycin, and erythromycin (Leberman, 1950). It has been shown that PPLO resistance to streptomycin may increase when they are cultivated in the presence of this antibiotic. Tetracycline resistance has been shown to slightly increase under the same conditions. In 1956, Lynn and Morton observed that some batches of agar are inhibitory to certain strains of PPLO. The strains of PPLO which appeared to be most fastidious were observed to be the most likely to be inhibited by unsatisfactory lots of agar. Starch was added to reduce the toxicity of the agar but it appeared to have little influence in reducing the inhibitory property of the agar. In 1962, Fabricant tested a large group of com monly used PPLO media for their effectiveness as a primary isolation medium. In no case would any of the 39 isolates used in these experiments grow on all of the 8 media tested and in some cases only 1 out of the 8 media used was successful as a primary isolation medium.
This indicates the fastidious nature of these organisms and points out the necessity for using a variety of media in the primary isolation of mycoplasmas. The amount of moisture and various atmospheric environments have been shown to greatly affect the rate of growth of the members of the PPLO group (Fabricant, 1962). MATERIALS AND METHODS
Cultures The cultures used in this series of experiments were obtained from 4 sources. Mycoplasma galliseptlcum
S6 and 801 were obtained from Texas Agriculture and Mechanical College, College Station, Texas. These strains were adapted for growth on laboratory media and were able to be cultured immediately on the media used in this laboratory. Strain 293 was also a laboratory media adapted culture and was obtained from Dr. J. Fabricant of
Cornell University Veterinary School. Dr. J. Kiser of American Cyanamid supplied the S6C strain of M. gallisepticum. This culture was adapted only to growth in
the yolk sac of an actively developing embryonated egg and could not be successfully adapted to growth on laboratory media during this series of experiments. The culture of Infectious Bronchitis Virus (IBV) was supplied by Mr. Henry Peck of the Ohio Agricultural
Experimental Station located at Reynoldsburg, Ohio. This virus was originally isolated at the University of New Hampshire and was designated IBV-NH-22. The Infectious
Bronchitis Virus was rapidly inactivated at room 26 27 temperature and "because of this all handling of this virus was done in a refrigerated water bath at 1° C. The S6 strain of M. gallisepticum and the Infectious Bronchitis Virus were sufficiently virulent to be titered using an ELD^q as the end point. The method used to calculate this titer is the method of Reed and
Meunch (8 ) and is calculated as follows: # mortality next above 50# - 50# . . % mortality next above 50# - “ proportionate # mortality next below 50# distance Log lower dilution (dilution in which # mortality next above 50#) plus proportionate distance (proportionate distance plus log of dilution factor) = ELD^q The strains of M. gallisepticum which were only slightly virulent were used undiluted after initial attempts to determine their titer revealed that they were not sufficiently virulent to be titered. In order to prepare an adequate inoculum, these strains (S6 , 801, and 293) were blind passed for 10 consecutive passages by inoculation into the yolk sac of a 7 day old embryonated egg. To do this the embryonated eggs were inoculated initially with a 24 hour broth culture of each mycoplasma strain. This inoculum, containing 0.1 ml of the culture, was placed into the yolk sac of a 7 day old embryonated egg. After 3 days incubation the yolk sac material was harvested and this material was used to Inoculate another 7 day old embryonated egg yolk sac. This procedure was repeated for 10 consecutive passages with each of the three strains and on the tenth passage the yolk sac material was collected for each strain respectively, pooled, mixed thoroughly, and placed into screw top test tubes in 2.0 ml amounts and stored in a dry ice chest at approximately -65° C. All of these 3 strains were tested for the ability to grow on laboratory media after the tenth passage and were found to grow well on the laboratory media used. The S6C strain was stored and harvested in the same manner and grown only in the yolk sac of the embryonated egg. The Infectious Bronchitis Virus inoculum was obtained by a similar method. A 0.1 ml inoculum from the culture obtained from Mr. Peck was inoculated into the allantoic cavity of 10 day old embryonated eggs. During incubation, the inoculated eggs were carefully watched and harvested within 4 days. The embryonated eggs had to be
carefully watched as the virus can only propagate on living embryo tissue. Due to the rapid inactivation rate
of this virus the harvested allantoic fluid will be void
of active virus if the embryo is allowed to remain in the incubator over 10-12 hours after the embryo has died. At this stage every attempt was made to harvest and eventually pool this allantoic cavity fluid as soon as possible after death of the embryo. 29
Method of egg inoculation Fertile eggs for the experiments were obtained from 2 sources. The source of all fertile eggs from hens on antibiotic fortified feeds with and without terephthalic acid (TPA) were obtained from the poultry farm of The Ohio State University. The chickens were White Leghorns. The hens were housed and fed as follows:
Pens 1 - 3 200 ppm CTC Pens 4 - 6 200 ppm CTC +0.5# TPA Pens 7 - 9 400 ppm CTC Pens 10 - 12 400 ppm CTC +0.5# TPA
Pens 13 - 15 No CTC, No TPA The source of fertile eggs for mycoplasma and virus harvesting, direct antibiotic inoculation experiments, and some egg dipping experiments was Cobb's Pedigreed Chicks, Goshen, Indiana. The eggs were obtained from flocks of White Rock hens which were carefully grown and classified as PPLO control flocks. The hens had been checked period ically to insure that no mycoplasma could be cultured from eggs or tissues of the birds and that they were serologically free of any antibody for mycoplasma organisms. All birds used in the experiments, which were obtained from The Ohio State University flocks, were artificially inseminated. All feeds for the birds were prepared by a technician of the Poultry Science Department. 30 Eggs from both sources were incubated, as soon as they arrived in the laboratory, in a David Bradley egg incubator (600 egg capacity). The incubation temperature was 99-3/4° P. At the end of 7 days of incubation the eggs were candled and all infertile or dead embryos were discarded. If PPLO cultures were to be used in the experiment at this point the eggs were prepared for inoculation and inoculated as follows. After candling the eggs were placed in egg racks with the air cell end of the egg up. The air cell end of the egg was then scrubbed with a 70 per cent ethyl alcohol solution. As soon as the alcohol had evaporated this area of the egg was swabbed with a 5 per cent iodine solution. The iodine solution was prepared by dissolving 5 grams of iodine in 100 ml of a 50 per cent ethyl alcohol solution. When the iodine solution was thoroughly dry a small hole was placed in the uppermost portion of the air cell end of the egg with an egg punch. The actual inoculation was made with a 1.0 ml tuberculin syringe with a 24 gauge 1.0 inch or 1.5 inch needle. The 1.0 inch needle was used on White Leghorn eggs and the 1.5 inch needle was used on yolk sac inoculation of White Rock eggs due to the increased size of the White Rock egg. All PPLO inoculations were from pooled yolk sac material harvested from previously yolk sac inoculated eggs. The inoculum was delivered on the seventh day of incubation in 0.1 ml quantities in all cases. 31
Figure 8 . Chart showing methods of embryo inoculation. 32
Inoculum of Infectious Bronchitis Virus was always in 0.1 ml quantities and was always delivered on the tenth day of incubation. The inoculum, was placed into the allantoic cavity of the developing embryonated egg. The preparation for inoculation was the same as described for yolk sac inoculation. The point of inoculation was determined by holding the embryonated egg before an egg candler and placing a small "X" approximately 4 mm above the line which marked the boundary of the air cell. The mark was made on the side of the egg on which the major portion of blood vessels were located and great care was
taken to avoid placing the mark over a blood vessel. If the inoculation mark was to be made over a blood vessel,
rapid death of the embryo occurred due to extensive hemorrhage of the blood vessel. All allantoic cavity inoculations were made with a 1.0 ml tuberculin syringe and a 0.5 inch 25 gauge needle. Figure 8 shows various methods and approximate distances used in common procedures for both yolk sac and allantoic cavity inoculations.
An excess number of eggs receiving yolk sac inoculations were used in each experiment so that 10
viable embryos were available, 5 for combined inoculation with both PPLO and virus and 5 containing only the yolk sac inoculated PPLO. In this manner all embryo deaths
caused by trauma from yolk sac inoculation were omitted. 33 All deaths on the eleventh day of embryo growth, 4 days after yolk sac Inoculation, were considered to be caused by trauma due to allantoic sac inoculation. This was true only for embryonated eggs receiving inoculations with both virus and PPLO. Embryo death on the eleventh day was considered to be caused by PPLO if the embryonated egg received inoculation with only the PPLO. All eggs were candled daily after the seventh day of incubation whether they were inoculated or not. If the eggs were not inoculated they were discarded if the embryo had died, and if they had received an inoculation of either virus or PPLO, the date of death was marked and all observations were recorded on the seventeenth day of incubation. Eggs were not allowed to hatch due to lack of facilities and space for handling the large number of resulting chicks.
Isolation attempts were made on the seventeenth day by sampling yolk sac material or opening the trachea of the embryo and swabbing the trachea with a sterile swab and placing the yolk sac material or trachea swab into a 10.0 ml PPLO broth medium. A 0.5 ml quantity of the broth was immediately removed after a thorough mixing with a Virtis
Junior Mixer and placed on a PPLO agar plate. The PPLO agar plate was then incubated at 37° C and observed for growth over a 5 day period. A positive re-isolation was
indicated by the presence of growth on the agar plate. Culture of mycoplasma cultures on laboratory media Due to the difficulty involved in growing mycoplasma cultures on laboratory media, a number of varieties of media were employed before one particular medium was chosen to be employed for all portions of this work involving in vitro antibiotic sensitivity. The medium chosen for in vitro antibiotic sensitivity was prepared from the following formula: Difco PPLO Broth w/o CV 21.00 grams
BBL Trypticase 0.05 grams BBL Soluble Starch 0.50 grams Distilled Water 1,000.00 milliliters
Thallium acetate was added to attain a final concentration of 1:2000 and the medium was autoclaved for 20 minutes at
15 lbs. pressure. The medium was removed from the autoclave and allowed to cool to approximately 60° C and penicillin G was added to a final concentration of 200 oxford units/ml. Horse serum was then added to a final concentration of 10 per cent or Difco PPLO serum extract was added to a final concentration of 3 per cent. The medium was distributed aseptically in 9*0 ml or 10.0 ml quantities into sterile culture tubes. Quantity used was determined by future use of the PPLO broth.
If PPLO agar was desired, 10.0 grams of Difco agar were added before autoclaving. This resulted in a 35 1.0 per cent agar concentration and allowed the PPLO colonies to form more rapidly than the standard 1.5 per cent agar concentration. When horse serum was used in preparation of laboratory media to be used for cultivation of PPLO a fresh supply was obtained periodically. If the horse serum was over 30 days old it caused a precipitate to form on the surface of the agar plate making identification of PPLO colonies virtually impossible.
Thallium acetate crystallized in the agar if the plates were stored for more than 2 weeks. The crystal lization did not impair identification of the PPLO colonies. It was distinguished by a white, star shaped, crystalline precipitate. When a primary isolation was desired, the following medium was employed. This medium was adapted for mycoplasma cultivation by Dr. R. Chanock (7).
PPLO Yeast Extract
Add 250.0 gms. Fleischmanns type 20-40 dry yeast to 1 liter of distilled water. Heat to boiling. Stir. Filter through 2 sheets No. 1 filter paper. Use more than
1 set-up since it filters through very slowly. Add NaOH to pH 8.0. The extract should be crystal clear. Dispense
in 10-12 ml aliquots. Autoclave for 15 minutes at 15 lbs. pressure. Freeze in dry ice chest. A precipitate will form after autoclaving. Avoid using this part of the extract.
PPLO Broth and PPLO Agar
Dissolve 21.0 gms. of Difco PPLO Broth w/o CV in
1 liter of distilled water. Dispense in 70.0 ml amounts in 125.0 ml bottles. Autoclave 15 minutes at 15 lbs. pressure. Store at 4° C until ready for use. When ready to use add: a) 100,000 units of penicillin G b) 2.5 ml of a 1:50 thallium acetate solution c) 1.0 ml Amphotericin B (0.5 mg/ml) d) 10 ml of yeast extract described above
e) 20 ml of Cappel horse serum (do NOT inactivate) To make PPLO agar, prepare as above and add 10.0 gms. of Difco agar. All sugar determinations and broth antibiotic sensitivity tests were performed in Phenol Red Broth Base medium adapted for use in mycoplasma cultivations. It was prepared by adding: Phenol Red Broth Base 16.0 gms. Carbohydrate to be tested 5.00 gms.
Trypticase 0.05 gms. Thallium acetate to 1:2000 concentration This medium was autoclaved at 15 lbs. pressure for 10 minutes, removed from the autoclave, cooled to 60° C and penicillin G added to achieve a concentration of 200 units/ ml. Difco PPLO serum extract was added to achieve a 3 per cent concentration. The medium was then dispensed aseptically in desired quantities into sterile test tubes and stored at refrigerator temperature until used. Soluble starch was omitted since it can be utilized by the mycoplasma organisms as a source of energy and is therefore undesirable in sugar fermentation tests. The PPLO's cannot be treated as bacteria since they cannot be transferred from agar plate to agar plate by the use of a bacteriological inoculation needle or loop. Sections of agar 10.0 mm square containing confluent growth were out from 1 agar plate and transferred to another agar plate containing PPLO agar. The agar square containing confluent growth was moved over the surface of the fresh agar plate thus inoculating the fresh plate. Transfer of PPLO's from agar plates to broth tubes was accomplished by cutting out a 5 mm square of agar containing confluent growth of PPLO and placing the agar square into a test tube containing PPLO broth. The transfer of PPLO cultures from broth to broth was accomplished in the usual manner by pipetting desired quantities of broth containing PPLO from 1 tube to another. During the entire course of this series of experiments the cultures were incubated in a bacteriological incubator at 37° C.
Quantitative determination of numbers of PPLO in laboratory media Quantitative determination of numbers of PPLO on laboratory media was confined to the use of a 24, 48, etc. hour culture of PPLO. More quantitative determinations were considered to be virtually impossible and other authors working with non-hemolytic species of mycoplasma share this viewpoint. Since the colonies must be magnified from 50 to 150 times to be easily observed, the standard plate count was rendered virtually useless. Reduction of dyes was useless because not all species grow at equal growth rates and quantitation of the initial inoculation into the media containing the dye could not be obtained with any large degree of efficiency. Total nitrogen determination was not practical for normal laboratory methods of quantitation because of the extremely small size of the PPLO and the excessive expenditure of time and equipment to perform such a task. The easiest method of counting mycoplasma colonies is by hemolysins synthesized by some species of mycoplasma which produce a zone of
hemolysis around a colony which can be counted in much the same manner as a plaque produced by a virus. The procedure was to grow the PPLO to be counted on the surface of an 39 agar plate until the growth becomes visible by observation at a 100-150 times magnification. A 2 per cent blood agar suspension was then prepared by adding the required quantity of red blood cells to agar cooled to 50° C and then layering the 2 per cent blood agar mixture over the surface of the agar plate containing the PPLO colonies. The plates were then incubated from 1-4 days and observed
daily for hemolysis. This procedure was employed with strains S6 , 801, and 293 of M. gallisepticum. No hemolysis was observed when chick, adult chicken, human,
horse, sheep, ox, duck, turkey, pigeon, guinea pig, and rabbit red blood cells were employed. Cultures of S6C and Infectious Bronchitis Virus were quantitated by use of ELD^q determination as previously described.
In vitro antibiotic sensitivity tests The determination of antibiotic sensitivity in vitro and on agar plates was determined by 1 of 2 methods. The first method employed was to cover the agar surface of a petri dish containing PPLO agar with the culture of PPLO
desired. This surface was allowed to dry for 30 minutes at 37° C. Antibiotic solutions were prepared by weighing out 0.1 gm of pure chlortetracycline and placing it into 10.0 ml of distilled water. Ten fold dilutions were made and
the dilution desired was placed on 0.5 inch filter pad discs in a 0.1 ml volume. The pads were allowed to dry for 10 minutes at room temperature and then placed on the PPLO agar plates. The petri dishes were incubated at 37° C and observed for growth at the end of 48 hours of incubation. The results were then recorded. The second method used involved the incorporation of various quantities of antibiotic into the PPLO agar. A culture of the desired PPLO was placed on the surface of the PPLO agar plate and the plate was incubated at 37° C and observed after 48 hours of incubation. When this method was employed the PPLO agar medium was adjusted to pH 6.9 by addition of 0.1 N HC1 to prevent rapid inactivation of the anti-microbial action of the chlortetracycline as described in the section "review of literature."
When in vitro, chlortetracycline sensitivities of strains S6 , 801, and 293 were determined using a broth medium, the Phenol Red Broth Base medium (as described previously) was employed. This medium was also adjusted to a pH of 6.9 using 0.1 N HC1, and a 0.5 per cent dextrose concentration was employed to provide substrate for further acid production. At a pH of 6.9 this medium was still red and therefore a valid conclusion could be reached when acid production was observed. Antibiotic was added to this medium to achieve the final desired concentrations. All tubes were incubated at 37° C and observed daily for production of a yellow color which was recorded as a 41 negative test indicating a lack of bactericidal or bacteriostatic activity of the antibiotic.
In ovo antibiotic sensitivity testing Antibiotic used as a feed supplement was incorporated into the egg by physiological mechanisms.
These eggs were collected daily and were employed in the experiments within 1 week after collection. Antibiotic incorporated in dipping solutions was prepared by adding pure chlortetracycline to distilled water chilled to 5° C to obtain a final concentration of 400 ppm. Preparation of the 200 ppm dip solution was achieved by doubling the volume of the 400 ppm dip solution by adding chilled distilled water. Eggs to be used for this experiment were placed in the egg incubator for 1 hour, removed, and placed in the dipping solution for 30 minutes. At the end of 30 minutes the eggs were removed and placed back in the incubator and handled routinely thereafter. Determination
of the effect of chlortetracycline by direct addition was accomplished by inoculating the desired quantity of antibiotic solution, prepared in distilled water, directly into the yolk sac of a 7 day old embryonated egg immediately before inoculation of the strain of PPLO to be tested. These eggs were then handled routinely. 42
Test for viability of mycoplasma organisms used All yolk sac suspensions of PPLO were diluted to
1:10 in nutrient broth and 0.1 ml of this dilution was placed on the surface of a PPLO agar plate to determine
the viability of the yolk sac PPLO suspension. Viability of the S6C strain was determined by inoculation of an
undiluted 0.1 ml quantity into the yolk sac of a 7 day old embryonated egg and observing daily for death of the
embryo. RESULTS
Three of the strains of M. gallisepticum used in this work were easily cultivated on laboratory media. Several variations of media were tried before the media described on page 34 were finally chosen to be used in the in vitro testing and routine laboratory culturing of the organisms. Strains S6 , 801, and 293 were observed for rapidity of growth under anaerobic, microaerophilic, and aerobic atmospheric conditions. They were found to grow most rapidly under aerobic conditions. The cultures were antigenically distinct and had been typed and classified by the workers from whom they were obtained. Antigenic classifications of mycoplasma species is extremely difficult to perform without an appropriate antiserum bank. Reliance was placed on the information supplied by the donors as to the antigenic classification of the organisms. In an attempt to differentiate the 3 strains on the basis of carbohydrate reaction, it was found that they reacted identically to all of the 27 different sugars and sugar alcohols (Table 2) on which their biochemical activity was tested. The only commonly occurring phenomena by which the 3 strains could be differentiated was the more rapid growth of the 801 strain as judged by 43 44
TABLE 2 CARBOHYDRATE FERMENTATION BY 3 STRAINS OF MYCOPLASMA GALLISEPTICUM
Control S6 8oi 293 24 48 24 48 24 ' ' 48 24 4fc
Rhamnose + + + + + + Arabinsoe ------Lyxose ------Ribose ------Xylose - - - — - ---
Dextrose + + + + + + Galactose -- - + - + - + Keltose ------Mannose -- + + + + + + Sorbose ------
Cellobiose — + + + + + + Levulose -- + + + + + + Maltose - - + + + + + + Sucrose - - + + + + + + Trehalose - - + + + + + +
Turanose _ + + + _ + Melezlrose -—— _ _ Raffinose -- — — _ Dextrin - — + + + + + + Inulin ------• -
Starch a . + + + + + + Adonitol -_ _ _ _ Dulcitol - — _„ _ Erythritol --* — - _ Inositol ------Mannitol Sorbitol -—— TABLE 3 CHLORTETBACYCLINE SENSITIVITY OF MYCOPLASMA GALLISEPTICUM S6, 801, AND 293 AS DETERMINED BY PAD-FLATE ASSAY
gms CTC/pad ' 2.‘0 --- 1.0 0.1 o.or
S6 ------+ + + + + +
801 ------± ± ± + - +
293 ------± ± ± + + +
+ = growth; - = no growth.
TABLE 4 SENSITIVITY OF MYCOPLASMA GALLISEPTICUM S6 , 801, AND 29I “TO ■'CHEOKTEfKACYCEHffi""
-^fgms CTC/ml 2.0 1.0 0.1 0.01
S6 ------+ + + + + +
801 ______+ + + + + +
293 - - - _ _ « + + + + + +
+ = growth; - = no growth.
Antibiotic was incorporated directly into PPLO agar media adjusted to pH 6 .9 . the time at which colonies of this organism could he observed on PPLO agar plates. All 3 strains were found to be completely inactive with regard to sugar alcohols and 5 carbon carbohydrates. They did, however, ferment the 5 carbon methyl-pentose, rhamnose, which is in actuality a 6 carbon sugar. The polysaccharides dextrin and starch were attacked while inulin was not. Dextrose, galactose,
mannose, cellobiose, and levulose were fermented as well
as maltose, sucrose, trehalose, and turanose. It can be concluded that differentiation of the 3 strains of M. gallisepticum cannot be accomplished on the basis of bio chemical breakdown of the sugars used in the tests. Tables 3 and 4 show the sensitivity of the S6 , 801, and 293 strains of M. gallisepticum to chlortetra- cycline when the test was performed on solid media by a pad-plate technique and when the antibiotic was incorporated into the agar. A comparison of the results shown in the 2 tables indicates that the pad-plate technique may be slightly
less sensitive than the method by which antibiotic is directly incorporated into the agar. This is probably due to the homogenous distribution of the antibiotic when it is incorporated in the agar as opposed to the concentrated quantity of antibiotic placed on the filter pad which tends
to diffuse out into the agar, thus reducing the effective
concentration of antibiotic in the area immediately surrounding the filter pad. Since the medium was adjusted to pH 6.9 in both cases the instability of the CTC should not have been any greater in one test than in the other.
In both cases the petri dishes containing the test material were observed over a 9 6 hour period and the results shown are those which were observed at the end of the 96 hour period. The reason for such a long observation period was to allow for the inherently slow growth rate of the organisms and to take into account any adverse effects the pH of 6.9 might have in slowing down the growth rate of the organisms. In Tables 5> and 7 are seen the results of an in vitro CTC sensitivity test using concentrations of CTC of 0.5 micrograms to 100.0 micrograms of CTC/ml of broth. As in all other in vitro antibiotic sensitivity tests, this medium was also adjusted to pH 6.9 in an attempt to increase the stability of the CTC molecule and prevent its rapid breakdown to the microbiologically inactive isochlor- tetracycline. The broth was exactly the same before and after transfer of the cultures except for the antibiotic added to the original test tubes. It is evident that concentrations of 0.5-100.0 micrograms of CTC/ml contained in a phenol red broth solution were sufficient to retard the growth of strains S6 , 801, and 293 for a period of at least 96 hours. At the end of 48 hours, however, the original tubes were shaken thoroughly in a Virtis Junior 48 TABLE 5 IN VITRO CHLORTETRACYCLINE SENSITIVITY OF MYCOPLASMA GALLISEPTICUM S6
Concentration Incubation Time (hours)______of CTC (-^gm/ml) “54 48 ^ 5 95
0.5 A.T. -H- ++++ -H-H- 1.0 A.T. ++++ ++++ -H-++ 2.0 A.T. ++++ -H-H- ++++
3.0 A.T. -H-H- ++-H- -H-H- 4.0 A.T. 5.0 A.T. 6.0 A.T. 7.0 A.T. 8.0 A.T. 9.0 A.T. 10.0 A.T. 100.0 A.T.
0.0 ++++ ++++ -H-H- ++++ ++++ A.T. ++++ ++-H- ++++ ++++ 4-H-+
+ = fermentation of dextrose in Phenol Red Broth. - = no fermentation.
A.T. = after transfer.
An uninoculated series containing 0.0-100.0 mgs of CTC/ml remained unchanged throughout the 120 hour observation period. 49 TABLE 6 IN VITRO CHLORTETRACYCLINE SENSITIVITY OP MYCOPLASMA GALLISEPTICUM 801
Concentration Incubation Time (hours) of CTC (y«gm/ml) “ 54 48 75 W I5U"
0.5 - A.T. - - ++++ ++++ ++++ 1.0 _____ A.T. - - ++++ ++++ ++++ 2.0 _____ A.T. - - +-H-+ ++++ ++++
3.0 - A.T. ++ 4.0 _____ A.T. _____ 5.0 _ _ _ _ _ A.T. _____
6.0 _____ A.T. _____ 7.0 _____ A.T. - - 8.0 _____ A.T. _____ 9.0 _____ A.T. _____ 10.0 _____ A.T. _____ 100.0 _____ A.T. _____
0.0 ++++ ++++ ++++ ++++ ++++ A.T. ++++ ++++ ++++ ++++ ++-H-
+ = fermentation of dextrose in Phenol Red Broth. - = no fermentation.
A.T. = after transfer. 50
TABLE 7 IN VITRO CHLORTETRACYCLINE SENSITIVITY OF Mycoplasma gallisepticum 293
Concentration Incubation Time(hours) _____ of CTC {s< gm/ml) TO 75 lSo
0.5 A.T. ++++ ++++ -H-++ 1.0 A.T. ++++ 2.0 A.T.
3.0 A.T. 4.0 A.T. 5.0 A.T. 6.0 A.T. 7.0 A.T. 8.0 A.T. 9.0 A.T. 10.0 A.T. 100.0 A.T.
0.0 -H-H- ++++ -H-H- ++++ ++++ A.T. ++++ -H-H- -H-H- -H-H- -H -H
+ = fermentation of dextrose in Phenol Red Broth. - = no fermentation.
A.T. = after transfer. 51
Mixer and 0.5 ml removed and placed in identical tubes of phenol red broth base media containing no CTC. Organisms removed from the tubes containing 0.5 to 3.0 micrograms of CTC/ml appeared to be viable and capable of breaking down the dextrose in the media to produce acid. This indicated that the antibiotic, chlortetracycline, had only a bacteriostatic effect on these strains of mycoplasma. The effects of chlortetracycline fortified poultry feeds on embryo viability are shown in Table 8 . A 400 ppm CTC concentration plus 0.5 per cent TPA produced much better viability than the other antibiotic mixtures used
in the feeds. The poor viability of the hens in pen 13-15 can be readily explained since no precautions were taken to prevent these hens from coming into contact with any agent causing poultry disease and it is quite reasonable to assume that these birds were infected naturally with a strain of CRD producing mycoplasma. It was possible during
the course of these experiments to isolate mycoplasma from the yolk sac of eggs from these birds. The rate of
isolation was below 0.10 per cent, however, and this is about the common frequency in which the organism is shed into the eggs by the hen. Shedding is most frequent
during the acute phase of the disease and seldom occurs in other stages. These hens were never observed to show any
overt symptoms of infection. When the 5 pens of hens were 52
TABLE 8
EFFECT OF CHLORTETRACYCLINE FEED SUPPLEMENTATION ON EGG FERTILITY AND EMBRYO VIABILITY
Pens 1-3 Pens 4-6 Pens 7-9 17/58 34# 26/62 42# 39/72 54#
31/64 47# 30/72 42# 35/72 49# 36/72 50# 38/72 53# 41/67 61#
27/72 37# 31/72 43# 36/59 61# 19/45 42# 23/45 51# 38/72 53# 29/72 40# 33/72 45# 29/72 4o#
Avg. 4l# 49# 53#
Pens 10-12 Pens 13-15
45/72 63# 12/72 17# Pens 1-3 200 ppm CTC 51/72 70# 26/72 36# Pens 4-6 200 ppm CTC 0.5# TPA 49/66 74# 37/96 39# Pens 7-9 400 ppm CTC 54/72 75# 42/96 44# Pens 10-12 400 ppm CTC 48/72 67# 31/72 43# 0 . 5 % TPA
55/72 76# 25/72 M Pens 13-15 No CTC or TPA Avg. 71# 36#
Number of viable embryos living at the end of 7 days of incubation/total number of fertile eggs used in initial incubation. 53 observed clinically it was easy to tell* by the vigor of the hens, which groups were receiving antibiotic fortified feeds and which were not. It is likely that the higher viability of the embryos in the eggs from hens in pen 10-12 was due to their healthier conditions, brought about by the CTC and TPA in their feed. The birds were larger and more finely feathered and presented a much better appearance than hens from the other pens. Tables 9 and 10 show the results of the series of eggs inoculated to determine the titers of the S6C strain of M. gallisepticum and the Infectious Bronchitis Virus.
The titers are stated in terms of ELD^q * The procedure used to determine the titer is described on page 27. The reason for employing an undiluted yolk sac suspension of strains S6, 801, and 293 is shown in Table 9- It was not possible to obtain an ELD^q for those strains by inoculating the embryonated eggs with the pooled yolk sac suspension. The yolk sac suspension was used undiluted and no titer given. An attempt to quantitate the number of organisms/ml of the yolk sac suspension resulted in such widely separated numerical values as to make them worthless.
There were viable organisms in the yolk sac suspensions when tested at the beginning of each experiment. Their virulence was so low as to be negligible, and it was not possible to obtain a valid ELD^q . This was fortunate as it provided a chance to determine the effect of 54
TABLE 9 TITRATION OF 4 STRAINS OF MYCOPLASMA GALLISEPTICUM
Days of Incubation Dilution 6 7 8 9 10 11 12 13 14
Strain S6
10_1 0/5 1/5 1/5 1/5 1/5 1/5 1/5 1/5 1/5 10-2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 lcr2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10"4 1/5 1/5 1/5 1/5 1/5 1/5 1/5 1/5 1/5 10-5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Strain 801
1 0 '1 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 1/5 10-2 o/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10-3 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10-4 o/5 0/5 0/5 0/5 0/5 o/5 0/5 0/5 0/5 10-5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Strain 293
10-1 0/5 o/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 10-2 o/5 o/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10-3 0/5 0/5 0/5 1/5 1/5 1/5 1/5 1/5 1/5 10-4 o/5 o/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10-5 o/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Strain S6C
i°'i_ n 0/5 2/5 4/5 5/5 5/5 5/5 5/5 5/5 5/5 10-2 0/5 1/5 3/5 3/5 5/5 5/5 5/5 5/5 5/5 1°"2 0/5 0/5 2/5 3/5 3/5 4/5 5/5 5/5 5/5 10Zc 0/5 0/5 1/5 2/5 3/5 3/5 3/5 4/5 4/5 1® g 0/5 0/5 0/5 0/5 1/5 1/5 1/5 2/5 2/5 10“b 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Number of dead embryos/number of fertile eggs used.
e l d 50 = io-4 *5. 55
TABLE 10 TITRATION OP INFECTIOUS BRONCHITIS VIRUS
Days of Incubation Dilution 11 12 13 14 15 i
H o 5/5 5/5 5/5 5/5 5/5 10~2 5/5 5/5 5/5 5/5 5/5 10-3 5/5 5/5 5/5 5/5 5/5 H i o 3/5 V 5 5/5 5/5 5/5 10-5 2/5 2/5 3/5 5/5 5/5
10-6 0/5 0/5 2/5 3/5 3/5
10-7 0/5 0/5 0/5 1/5 1/5 00 1 1—1 o
0/5 0/5 0/5 0/5 0/5 10"9 1/5 1/5 1/5 1/5 1/5 10"10 0/5 0/5 0/5 0/5 0/5
Number of dead embryos/number of fertile eggs used.
e l d 50 = io~6 -2. non-virulent PPLO on the mortality of embryonated eggs and to determine the effect of CTC and CTC + TPA in controlling any enhancement of mortality caused by this low virulence mycoplasma. The effect of direct inoculation of CTC into 7 day old embryonated eggs is given in Table 11. The PPLO strains used were S6, 801, and 293. They were grown for 24 hours in PPLO broth and inoculated directly into the yolk sac. The reisolation attempts were not very successful; however, there was improvement when yolk sac suspensions of the same organisms were used (see Table 12). The results prompted further use of the egg adapted cultures of mycoplasma in yolk sac suspensions. It is believed that the adapted strains, though of low virulence, grew better in the embryonated egg yolk sac and were, therefore, more suitable for use. A 10 fold dilution of the low virulence organisms further reduced their effectiveness as indicated by the reisolation attempts (Tables 11 and 12); therefore, it was decided that undiluted suspensions of the organisms would be of greater value for inoculation.
The results shown in Table 13 indicate that the S6, 801, and 293 strains of mycoplasma did enhance mortality when in combination with a common viral infection agent (IBV). The direct inoculation of 0.2 to 200.0 micrograms appeared to have little effect on the reduction of mortality during combined infection. The addition of 200 micrograms 57 TABLE 11
MORTALITY OP PPLO CONTROL EGGS WHEN CTC WAS ADDED BY DIRECT INOCULATION
Days of Incubation 11 12 13 14 15 16 17 Isolati(
0.2 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 S6 10-1 1/5 1/5 1/5 1/5 1/5 1/5 1/5 3/4
.801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 801 10-1 0/5 0/5 0/5 0/5 0/5 0/5 o/5 2/5 293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 o/5 4/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5
Control 0/5 0/5 0/5 0/5 o/5 0/5 0/5 0/5
2.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5
801 undiluted 0/5 1/5 i/5 1/5 1/5 1/5 1/5 4/5 801 10-1 1/5 1/5 1/5 1/5 1/5 1/5 1/5 4/5 293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
20.0 Micrograms CTC Added
S6 undiluted 1/5 1/5 1/5 1/5 1/5 1/5 1/5 5/5 s6 io-l 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5
801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 10_I 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 $ Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 58 TABLE 11— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
200.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 10-1 2/5 2/5 2/5 2/5 2/5 2/5 2/5 3/5 293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5
Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
0.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1% 801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 801 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5
293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Isolation = number of successful isolations/number of attempts.
0.1 PPLO suspended in PPLO broth used in all inoculations.
0.1 ml antibiotic solution used in all inoculations. Antibiotic solution prepared in distilled water.
PPLO dilution blanks contained PPLO broth as described in the materials and methods section. 59 TABLE 12 MORTALITY OF PPLO CONTROL EGGS WITH CTC ADDED BY DIRECT INOCULATION
Days of Incubation 11 12 13 14 15 16 17 Isolation
0.2 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 10"! 0/5 0/5 n/c; 0/5 0/5 0/5 0/5 0/5n/iz. 5/5 293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 10-1 1/5 1/5 1/5 1/5 1/5 1/5 1/5 3/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
2.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 undiluted 1/5 1/5 1/5 1/5 1/5 1/5 1/5 5/5 801 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 undiluted 2/5 2/5 2/5 2/5 2/5 2/5 2/5 5/5 293 10-1 !/5 1/5 1/5 i/5 1/5 1/5 1/5 5/5
Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 0/5
20.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 10-1 0/5 0/5 1/5 1/5 1/5 1/5 1/5 5/5
801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 10-1 o/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5
293 undiluted 0/5 0/5 1/5 1/5 2/5 2/5 2/5 5/5 293 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 Control 2/5 2/5 2/5 2/5 2/5 2/5 2/5 0/5 6o
TABLE 12— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
200.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 S6 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 10"1 0/5 0/5 0/5 o/5 0/5 0/5 0/5 3/5 293 undiluted 0/5 0/5 o/5 0/5 0/5 0/5 0/5 4/5 293 lO"1 0/5 0/5 0/5 0/5 o/5 0/5 o/5 2/5
Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 0/5
0.0 Micrograms CTC Added
S6 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 lO'l 0/5 0/5 0/5 0/5 o/5 o/5 0/5 5/5 801 undiluted 1/5 1/5 1/5 1/5 2/5 2/5 2/5 5/5 801 10-1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5
293 undiluted 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 10-1 0/5 2/5 0/5 0/5 0/5 0/5 0/5 4/5
Control 0/5 0/5 o/5 o/5 0/5 0/5 0/5 0/5
Isolation = number of successful isolations/number of attempts.
0.1 ml of PPLO suspended in yolk sac material used in all inoculations.
0.1 ml of antibiotic solution used in all inoculations. Antibiotic solution prepared in distilled water.
PPLO dilution blanks contained PPLO broth as described on page 34. 6l
TABLE 13 MORTALITY OP PPLO CONTROL EGGS WITH CTC ADDED BY DIRECT INOCULATION
Days of Incubation 11 12 13 14 15 16 17 Isolation
0.2 Micrograms CTC Added s6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 S6 and IBV 1/5 1/5 1/5 2/5 2/5 3/5 3/5
801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 801 plus IBV 1/5 0/5 0/5 0/5 1/5 1/5 2/5
293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 293 plus IBV 0/5 0/5 0/5 1/5 2/5 2/5 2/5 IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2.0 Micrograms CTC Added
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 S6 plus IBV 1/5 1/5 1/5 1/5 2/5 2/5 2/5
801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 plus IBV 0/5 2/5 2/5 2/5 2/5 2/5 2/5
293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 IBV 2/5 2/5 2/5 2/5 2/5 2/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 20.0 Micrograms CTC Added
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 2/5 2/5
801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 801 plus IBV 1/5 2/5 2/5 2/5 2/5 3/5 3/5 293 !/5 1/5 1/5 1/5 1/5 1/5 1/5 2/5 293 plus IBV 0/5 0/5 0/5 1/5 3/5 3/5 3/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 62
TABLE 13— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
200.0 Micrograms CTC Added
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 2/5 2/5 801 1/5 1/5 1/5 1/5 1/5 1/5 1/5 1/5 801 plus IBV 0/5 1/5 1/5 2/5 2/5 2/5 2/5
293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 293 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0.0 Micrograms CTC Added
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 2/5 2/5
801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 1/5 1/5 1/5 1/5 2/5 2/5 3/5
293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 2/5 3/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Isolation = number of successful isolations/number of attempts.
0.1 ml of undiluted PPLO suspended in yolk sac material used in all inoculations.
0.1 ml of antibiotic solution was used in all inoculations. Antibiotic solution prepared in distilled water.
IBV diluted in nutrient broth (Difco) and inoculated in 0.1 ml quantities. of CTC into the yolk sac appeared to reduce the mortality rate somewhat but was still not adequately effective. Due to the lack of dramatic effectiveness and the labor and expense that would have been involved to perform further inoculations of antibiotic in this manner, it was decided to conduct no further experiments employing this method of CTC incorporation. The direct inoculation of a quantity of CTC contained in 0.1 ml would undoubtedly not have been as homogenously distributed throughout the yolk sac as would antibiotic incorporated physiologically or by a dipping solution. One would therefore expect more erratic results from a direct inoculation of a quantity of antibiotic. A larger quantity of antibiotic might possibly have provided a more adequate distribution of the antibiotic. However, the mortality rate due to trauma and toxicity could also be expected to rise since it is common knowledge that quantities of materials injected into embryos should be kept as small as possible to reduce non-specific mortality. Larger concentrations of antibiotic would begin to have a toxic effect on the embryo.
Different results were obtained (Table l4) when a virulent strain of PPLO was employed. The incorporation of 0.2 to 200.0 micrograms of CTC were not sufficient to eliminate all the effects of a challenge dose of this organism. A very definite trend was shown toward 64
TABLE 14
MORTALITY OF PPLO CONTROL EGGS WITH CTC ADDED BY DIRECT INOCULATION
Days of Incubation 11 12 13 14 15 16 17
0.2 Micrograms CTC Added s6c 0/5 0/5 1/5 1/5 2/5 2/5 2/5 S6C plus IBV 0/5 1/5 1/5 3/5 3/5 4/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 1/5 !/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 2.0 Micrograms CTC Added
S6C 0/5 0/5 0/5 0/5 2/5 2/5 2/5 S6C plus IBV 0/5 2/5 2/5 2/5 2/5 3/5 4/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 20.0 Micrograms CTC Added s6c 0/5 0/5 0/5 1/5 1/5 1/5 2/5 S6C plus IBV 2/5 2/5 2/5 4/5 4/5 5/5 5/5 IBV 2/5 2/5 2/5 2/5 2/5 2/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 200.0 Micrograms CTC Added s6c 0/5 0/5 0/5 1/5 1/5 !/5 1/5 S6C plus IBV 0/5 0/5 0/5 2/5 2/5 3/5 3/5 IBV 1/5 1/5 1/5 1/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0.0 Micrograms CTC Added s6c 0/5 0/5 1/5 1/5 2/5 2/5 2/5 S6C plus IBV 0/5 2/5 2/5 2/5 4/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
10”5 dilution of S6C.
10"7 dilution of IBV. 65 TABLE 15 MORTALITY OF PPLO CONTROL EGGS WHEN CTC WAS ADDED BY DIRECT INOCULATION HIGH PPLO INOCULUM
Days of Incubation 11 12 13 14 15 16 17
0.2 Micrograms CTC Added s6c 0/5 0/5 1/5 1/5 3/5 4/5 4/5 S6C plus IBV 1/5 2/5 4/5 5/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2.0 Micrograms CTC Added s6c 0/5 1/5 1/5 2/5 2/5 4/5 5/5 S6C plus IBV 2/5 2/5 3/5 5/5 5/5 5/5 5/5 IBV 1/5 1/5 1/5 1/5 1/5 1/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 20.0 Micrograms CTC Added s6c 0/5 0/5 0/5 2/5 2/5 3/5 4/5 S6C plus IBV 0/5 1/5 3/5 3/5 4/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 200.0 Micrograms CTC Added s6c 0/5 0/5 0/5 2/5 2/5 2/5 3/5 S6C plus IBV 0/5 1/5 1/5 2/5 3/5 4/5 4/5 IBV 0/5 o/5 0/5 0/5 1/5 1/5 2/5 Control 0/5 o/5 0/5 0/5 0/5 0/5 0/5 0.0 Micrograms CTC Added s6c 1/5 1/5 2/5 4/5 4/5 4/5 5/5 S6C plus IBV 2/5 3/5 3/5 5/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
10“^ dilution of S6C.
10“7 dilution of IBV. 66
an increased mortality rate when a combined infection was present. The PPLO dose behaved as one might expect when the results were compared with those in Table 9. The CTC
appeared to reduce embryo mortality at 200.0 micrograms concentration and thus would be helpful to the poultryman
in producing more living chicks. The antibiotic concentration was less effective at controlling mortality when a very strong dose of a virulent mycoplasma was used as the challenging agent (Table 15). It was observed that the 200.0 microgram concentration
began to decrease mortality unless a combined infection
was present. When a virus was the major infective agent, the incorporation of antibiotic was ineffective (Table 16). The use of antibiotic incorporated into the embryonated egg by means of a dipping solution was
evaluated (Table 17). It can readily be observed that it controlled mortality to some extent when mycoplasma of low virulence was present. The high number of reisolations indicated further that CTC was only a bacteriostatic agent
and not bacteriocidal. Since Rolle (1962) has indicated that CTC is slowly incorporated into developing bone
tissue, an attempt was made to incorporate the antibiotic
by dipping solution using a 7 day old embryonated egg. As any poultryman could have guessed, almost every embryo
died due to the effect of the chilled dipping solution on 67 TABLE 16
MORTALITY OP PPLO CONTROL EGGS WHEN CTC WAS ADDED BY DIRECT INOCULATION HIGH VIRUS INOCULUM
Days of Incubation 11 12 13 14 15 16 17
0.2 Micrograms CTC Added s6c 0/5 0/5 0/5 0/5 1/5 1/5 2/5 S6C plus IBV 1/5 2/5 5/5 5/5 5/5 5/5 5/5 IBV 0/5 2/5 3/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2.0 Micrograms CTC Added s6c 1/5 1/5 1/5 1/5 1/5 1/5 1/5 S6C plus IBV 0/5 0/5 3/5 5/5 5/5 5/5 5/5 IBV 1/5 2/5 5/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 20.0 Micrograms CTC Added s6c 0/5 0/5 0/5 0/5 0/5 1/5 1/5 S6C plus IBV 1/5 1/5 V 5 5/5 5/5 5/5 5/5 IBV 2/5 2/5 5/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 200.0 Micrograms CTC Added
S6C 1/5 1/5 1/5 1/5 1/5 i/5 1/5 S6C plus IBV 0/5 1/5 1/5 4/5 5/5 5/5 5/5 IBV 0/5 2/5 2/5 4/5 5/5 5/5 5/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 0.10 Micrograms CTC Added s6c 0/5 o/5 0/5 1/5 1/5 1/5 2/5 S6C plus IBV 1/5 3/5 5/5 5/5 5/5 5/5 5/5 IBV 1/5 1/5 4/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
10“5 dilution of PPLO. 10"5 dilution of IBV. 68
TABLE 17 MORTALITY OP PPLO CONTROL EGGS WHEN CTC WAS ADDED BY USE OF DIPPING SOLUTIONS
Days of Incubation 11 12 13 14 15 16 17 Isolation
400 ppm CTC Dipping Solution
S6 0/5 0/5 0/5 0/5 0/5 o/5 0/5 3/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 2/5 2/5 801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 2/5 2/5 2/5 2/5 3/5 3/5 3/5 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 s6c 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - S6c plus IBV 0/5 0/5 0/5 1/5 2/5 2/5 3/5 — IBV 0/5 0/5 o/5 0/5 0/5 0/5 0/5 - Control 0/5 0/5 o/5 0/5 0/5 0/5 0/5 - 200 ppm CTC Dipping Solution
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 plus IBV 0/5 0/5 o/5 0/5 1/5 1/5 1/5 801 0/5 0/5 o/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 0/5 0/5 1/5 1/5 1/5 1/5 2/5 293 0/5 0/5 o/5 0/5 0/5 0/5 0/5 3/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 2/5 S6C 0/5 0/5 o/5 0/5 1/5 1/5 1/5 _ S6C plus IBV 0/5 0/5 o/5 3/5 3/5 4/5 4/5 — IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control o/5 0/5 o/5 0/5 0/5 0/5 0/5 - 0 ppm CTC in Dipping Solution
S6 1/5 1/5 1/5 1/5 1/5 2/5 2/5 4/5 S6 plus IBV 0/5 0/5 1/5 1/5 2/5 2/5 2/5 801 o/5 0/5 0/5 0/5 1/5 1/5 1/5 3/5 801 plus IBV o/5 1/5 1/5 1/5 2/5 3/5 3/5 293 o/5 0/5 1/5 1/5 1/5 1/5 1/5 4/5 293 plus IBV 0/5 0/5 0/5 3/5 3/5 3/5 3/5 s6c o/5 0/5 1/5 1/5 2/5 2/5 2/5 S6c;:plus IBV o/5 1/5 1/5 2/5 4/5 5/5 5/5 . IBV o/5 0/5 0/5 0/5 0/5 0/5 0/5 •m Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - 10-5 lii-Mnn of1 Sfif! nil , TTDTn n >4IMAn«Mn undiluted. 10-7 dilution of IBV. 69 the embryo. The dipping solution shown in Tables 17j 18, and 19 was used on fertile eggs which had been incubated for only 1 hour. It is therefore obvious at what age the embryo must be treated if a chilled dipping solution is to be employed. Table 18 illustrates that dipping solutions display a certain degree of effectiveness toward combating mortality when a high concentration of virulent PPLO is used as the challenge dose. The results indicate that dipping solutions are not completely effective against what could be considered an overwhelming dose of virulent S6C strain of M. gallisepticum. A search for a more
effective antibiotic method must therefore be continued. Table 19 shows that dipping solutions are no more effective than direct inoculation against challenge by an
overwhelming concentration of IBV. The results shown in Table 20 indicate that antibiotic application by any method tried thus far fails to reduce drastically the number of successful reisolations of PPLO. The results shown here compare
favorably with those found when antibiotic is incorporated in dipping solutions. The results obtained by feed
fortification and dipping solutions are as good as or better than the results of incorporation by direct
inoculation. This would seem to indicate that antibiotic
feed fortification and dipping solutions would be the 70 TABLE 18
MORTALITY OF PPLO CONTROL EGGS WHEN CTC WAS ADDED BY USE OF DIPPING SOLUTIONS HIGH PPLO INOCULUM
Days of Incubation 11 12 13 14 15 16 17
400 ppm CTC Dipping Solution
S6C 0/5 1/5 1/5 2/5 2/5 3/5 3/5
S6C plus IBV 2/5 2/5 2/5 2/5 3/5 5/5 5/5
IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5
Control 0/5 0/5 0/5 0/5 0/5- 0/5 0/5 200 ppm CTC Dipping Solution
s6c 1/5 2/5 2/5 2/5 3/5 3/5 3/5 S6C plus IBV 1/5 1/5 3/5 3/5 5/5 5/5 5/5 IBV 0/5 1/5 1/5 1/5 1/5 1/5 1/5
Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0 ppm CTC Dipping Solution
s6c 1/5 2/5 2/5 3/5 5/5 5/5 5/5
S6C plus IBV 0/5 4/5 4/5 5/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5
Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
10'k dilution of S6C. 10"7 dilution of IBV. 71 TABLE 19
MORTALITY OF PPLO CONTROL EGGS WHEN CTC WAS ADDED BY USE OF DIPPING SOLUTION HIGH VIRUS INOCULUM
Days of Incubation 11 12 13 14 15 16 17
400 ppm CTC Dipping Solution s6c 0/5 1/5 1/5 1/5 1/5 1/5 1/5
S6C plus IBV 2/5 2/5 3/5 5/5 5/5 5/5 5/5
IBV 0/5 1/5 4/5 5/5 5/5 5/5 5/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 200 ppm CTC Dipping Solution s6c 1/5 1/5 1/5 1/5 1/5 2/5 2/5 S6C plus IBV 0/5 4/5 5/5 5/5 5/5 5/5 5/5
IBV 2/5 3/5 4/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0 ppm CTC Dipping Solution s6c 0/5 0/5 2/5 2/5 2/5 2/5 2/5 S6C plus IBV 1/5 5/5 5/5 5/5 5/5 5/5 5/5
IBV 1/5 2/5 4/5 5/5 5/5 5/5 5/5
Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5
10-5 dilution of S6C.
10"5 dilution of IBV. 72 methods of choice when applying antibiotics to fertile
eggs to combat mycoplasma infection. Pens 10-12 appeared to be the most successful in combating excessive mortality even when high dilutions of virulent PPLO were employed as
in Table 21. In comparing Tables 20 and 21, it is obvious that antibiotic fortification of poultry feeds delay the
onset of embryo mortality thus indicating a degree of effectiveness even though it is not completely effective.
Table 22 serves to substantiate the evidence that CTC supplementation by any method is worthless against a strong viral infection. The combination of dipping solutions and feed fortification appeared to give much better results than any method tried by itself (Table 23). The reisolation did not appear to be substantially reduced but the
mortality rate was lowered. As in the other tables the eggs from pens 10-12 appeared to be most effective. When Tables 23 and 24 are compared, there is little difference
in the end results. It would appear that the use of fortified feed plus a 200 ppm CTC dip would be more economical and less wasteful than employing a 400 ppm CTC dip plus fortified feed.
Even when an overwhelming challenging dose of PPLO is employed, the results in Tables 25 and 26 indicate that
a combination of CTC fortified feed and dipping solutions 73 TABLE 20
MORTALITY OP EMBRYONATED EGGS WHEN CTC WAS ADDED AS A FEED SUPPLEMENT
Days of Incubation 11 12 13 l4 15 16 17 Isolation Pens 1-3 200 ppm CTC
s6 0/5 0/5 0/5 0/5 1/5 1/5 1/5 3/5 s6 plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 2/5 - 801 0/5 o/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 1/5 1/5 1/5 1/5 1/5 1/5 2/5 - 293 0/5 0/5 0/5 1/5 1/5 1/5 1/5 5/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - s6c 0/5 0/5 0/5 0/5 0/5 2/5 2/5 - S6C plus IBV 0/5 0/5 0/5 1/5 1/5 2/5 3/5 - IBV 0/5 0/5 o/5 0/5 0/5 1/5 1/5 - Control 0/5 0/5 o/5 0/5 0/5 0/5 0/5 - Pens 4-6 200 ppm CTC plus TPA
S6 1/5 1/5 1/5 1/5 2/5 2/5 2/5 3/5 S6 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 2/5 8oi 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 plus IBV 0/5 0/5 1/5 1/5 2/5 2/5 3/5 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 S6C 0/5 0/5 0/5 0/5 1/5 1/5 2/5 - S6c plus IBV 1/5 1/5 1/5 3/5 3/5 3/5 4/5 — IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 — Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 - Pens 7-9 400 ppm CTC plus TPA
s6 0/5 0/5 0/5 o/5 o/5 o/5 0/5 4/5 S6 plus IBV 1/5 1/5 1/5 1/5 1/5 1/5 1/5 801 0/5 0/5 o/5 0/5 0/5 1/5 1/5 5/5 801 plus IBV 0/5 0/5 0/5 o/5 1/5 1/5 2/5 293 0/5 0/5 0/5 o/5 0/5 0/5 0/5 5/5 293 plus IBV 0/5 0/5 o/5 0/5 0/5 1/5 2/5 s6c 0/5 o/5 o/5 1/5 1/5 1/5 1/5 S6c plus IBV 0/5 o/5 o/5 1/5 1/5 3/5 3/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 0/5 o/5 0/5 0/5 0/5 0/5 0/5 _ 74
TABLE 20— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
Pens 10-12 400 ppm CTC plus TPA
S6 0/5 0/5 0/5 0/5 o/5 0/5 0/5 4/5 S6 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 - 801 0/5 0/5 0/5 0/5 0/5 1/5 1/5 5/5 801 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - 293 0/5 o/5 1/5 1/5 1/5 1/5 1/5 3/5 293 plus IBV 1/5 1/5 1/5 1/5 1/5 1/5 1/5 S6C 0/5 o/5 1/5 1/5 1/5 1/5 1/5 - S6C plus IBV 0/5 1/5 1/5 1/5 1/5 2/5 2/5 - IBV 0/5 0/5 0/5 0/5 0/5 1/5 2/5 - Control 0/5 o/5 0/5 0/5 0/5 0/5 0/5 - Pens 13-15 Control
S6 0/5 o/5 0/5 0/5 0/5 1/5 1/5 4/5 S6 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 2/5 - 801 0/5 0/5 0/5 0/5 0/5 2/5 2/5 5/5 801 plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 - 293 0/5 0/5 o/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 1/5 1/5 1/5 2/5 2/5 - s6c 0/5 0/5 0/5 2/5 2/5 2/5 3/5 - S6C plus IBV 0/5 0/5 1/5 1/5 3/5 4/5 4/5 - IBV 1/5 1/5 1/5 1/5 1/5 1/5 1/5 - Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 -
10-5 dilution of S6C. All other PPLO strains were undiluted. 10-7 dilution of IBV. 75 TABLE 21 MORTALITY OF EMBRYONATED EGGS WHEN CTC WAS ADDED AS A FEED SUPPLEMENT HIGH PPLO INOCULUM
Days of Incubation 11 12 13 14 15 16 17
Pens 1-3 200 ppm CTC s6c 1/5 1/5 1/5 1/5 2/5 2/5 2/5 S6C plus IBV 0/5 0/5 1/5 2/5 4/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 4-6 200 CTC plus TPA S6C 0/5 o/5 2/5 2/5 3/5 3/5 4/5 S6C plus IBV 0/5 0/5 1/5 3/5 3/5 5/5 5/5 IBV 0/5 o/5 0/5 1/5 1/5 1/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 7-9 400 ppm CTC s6c 0/5 o/5 0/5 2/5 2/5 3/5 4/5 S6C plus IBV 0/5 1/5 1/5 1/5 4/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 Pens 10-12 400 ppm CTC plus TPA s6c 0/5 0/5 0/5 1/5 1/5 2/5 S6C plus IBV 0/5 0/5 2/5 2/5 2/5 3/5 1% IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 13-15 Control s6c 0/5 0/5 2/5 3/5 3/5 5/5 5/5 S6C plus IBV 0/5 1/5 4/5 5/5 5/5 5/5 5/5 IBV o/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5
lO-2*- dilution of S6C. 10“? dilution of IBV. 76 TABLE 22 MORTALITY OF EMBRYONATED EGGS WHEN CTC WAS ADDED AS A FEED SUPPLEMENT HIGH VIRUS INOCULUM
Days of Incubation 11 12 13 14 15 *16 17
Pens 1-■3 200 ppm CTC s6c 0/5 0/5 0/5 0/5 1/5 1/5 2/5 S6C plus IBV 1/5 3/5 5/5 5/5 5/5 5/5 5/5 IBV 0/5 2/5 4/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 4-■6 200 ppm I3TC plus TPA s6c 1/5 1/5 1/5 2/5 2/5 2/5 2/5 S6C plus IBV 2/5 3/5 5/5 5/5 5/5 5/5 5/5 IBV 1/5 3/5 3/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 7-•9 400 ppm CTC s6c 0/5 0/5 0/5 0/5 1/5 1/5 1/5 S6C plus IBV 0/5 1/5 4/5 5/5 5/5 5/5 5/5 IBV 0/5 1/5 1/5 4/5 5/5 5/5 5/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 Pens 10-12 400 ppm CTC plus TPA s6c 0/5 0/5 0/5 0/5 1/5 1/5 1/5 S6c plus IBV 1/5 3/5 4/5 5/5 5/5 5/5 5/5 IBV 0/5 2/5 3/5 4/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 13-15 Control S6C 1/5 1/5 1/5 2/5 2/5 2/5 3/5 S6C plus IBV 1/5 4/5 5/5 5/5 5/5 5/5 5/5 IBV 0/5 1/5 3/5 5/5 5/5 5/5 5/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 10"5 dilution of S6c. 10"5 dilution of IBV. 77 TABLE 23 MORTALITY OF EMBRYONATED EGGS WHEN CTC WBS ADDED AS A FEED SUPPLEMENT PLUS A 400 PPM DIPPING SOLUTION
Days of Incubation 11 12 13 14 15 16 17 Isolat:
Pens 1-•3 200 ppm CTC S6 1/5 1/5 1/5 1/5 1/5 1/5 1/5 5/5 S6 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 2/5 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 - s6c 0/5 0/5 0/5 0/5 1/5 1/5 1/5 - S6c plus IBV 0/5 0/5 1/5 1/5 1/5 2/5 2/5 - IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - Pens 4-6 200 ppm CTC plus TPA S6 0/5 0/5 0/5 0/5 0/5 0/5 1/5 4/5 S6 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 801 1/5 1/5 1/5 1/5 1/5 1/5 1/5 3/5 801 plus IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 293 0/5 0/5 o/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 o/5 0/5 0/5 1/5 1/5 s6c 0/5 0/5 o/5 0/5 0/5 1/5 1/5 _ s6c plus IBV 0/5 0/5 o/5 1/5 1/5 1/5 2/5 _ IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 _ Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - Pens 7-■9 40C> ppm CTC s6 0/5 0/5 0/5 0/5 0/5 o/5 0/5 4/5 S6 plus IBV 0/5 0/5 o/5 0/5 0/5 o/5 1/5 801 0/5 0/5 0/5 0/5 0/5 o/5 0/5 5/5 801 plus IBV 0/5 0/5 o/5 1/5 1/5 1/5 2/5 293 1/5 1/5 1/5 1/5 1/5 1/5 1/5 2/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 s6c 0/5 0/5 0/5 0/5 0/5 1/5 1/5 s6c plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 IBV 0/5 o/5 0/5 0/5 0/5 1/5 1/5 Control 0/5 0/5 o/5 0/5 0/5 0/5 0/5 - 78 TABLE 23— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
Pens 10-12 0 0 ppm CTC plus TPA
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 plus IBV 0/5 0/5 o/5 0/5 0/5 1/5 1/5 801 0/5 o/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 0/5 1/5 1/5 1/5 1/5 1/5 1/5 - 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 1/5 1/5 1/5 1/5 2/5 2/5 2/5 - S6c 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - S6C plus IBV 0/5 0/5 0/5 0/5 2/5 2/5 2/5 - IBV 1/5 1/5 1/5 1/5 1/5 2/5 2/5 - Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - Pens 13-■15 Control
S6 0/5 o/5 0/5 0/5 0/5 0/5 0/5 5/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 plus IBV 0/5 0/5 1/5 1/5 1/5 1/5 2/5 293 0/5 0/5 0/5 0/5 1/5 1/5 1/5 5/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 0/5 1/5 S6C 0/5 o/5 1/5 2/5 2/5 2/5 2/5 - S6C plus IBV 1/5 1/5 2/5 3/5 4/5 5/5 5/5 - IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 — Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 -
10 ^ dilution of S6C; all other PPLO strains were undiluted.
10~7 dilution of IBV. 79 TABLE 24 MORTALITY OF EMBRYONATED EGGS WHEN CTC WAS ADDED AS A FEED SUPPLEMENT PLUS A 200 PPM DIPPING SOLUTION
Days of Incubation 11 12 13 14 15 16 17 Isolation
Pens 1-3 200 ppm CTC S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 S6 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 - 801 0/5 1/5 1/5 1/5 1/5 1/5 1/5 5/5 801 plus IBV 0/5 0/5 0/5 1/5 2/5 2/5 2/5 - 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 0/5 1/5 - S6C 0/5 0/5 0/5 0/5 1/5 1/5 1/5 - S6C plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 2/5 - IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - Control o/5 0/5 0/5 o/5 0/5 0/5 0/5 - Pens 4-•6 200 ppm CTC plus TPA S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 S6 plus IBV 1/5 1/5 2/5 2/5 2/5 3/5 801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 293 1/5 1/5 1/5 1/5 1/5 1/5 1/5 4/5 293 plus IBV 0/5 1/5 1/5 1/5 1/5 2/5 2/5 - s6c 0/5 0/5 0/5 0/5 0/5 0/5 1/5 - S6c plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 - IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - Pens 7-9 400 ppm CTC S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 S6 plus IBV 1/5 1/5 1/5 1/5 1/5 1/5 2/5 801 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4/5 801 plus IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 293 2/5 2/5 2/5 2/5 2/5 2/5 2/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 S6C 0/5 0/5 0/5 0/5 0/5 0/5 0/5 — S6C plus IBV 0/5 0/5 1/5 1/5 1/5 1/5 2/5 _ IBV 0/5 0/5 0/5 1/5 1/5 2/5 2/5 — Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 - 80 TABLE 24— Continued
Days of Incubation 11 12 13 14 15 16 17 Isolation
Pens 10-12 400 ppm CTC plus TPA
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 S6 plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 - 801 0/5 0/5 0/5 0/5 0/5 1/5 1/5 4/5 801 plus IBV 0/5 0/5 1/5 1/5 1/5 2/5 2/5 - 293 1/5 1/5 1/5 1/5 1/5 1/5 1/5 4/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - S6C 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - S6C plus IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 - IBV 0/5 0/5 o/5 0/5 0/5 1/5 1/5 - Control o/5 0/5 0/5 0/5 0/5 0/5 0/5 - Pens 13-15 Control
S6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3/5 S6 plus IBV o/5 0/5 0/5 0/5 1/5 1/5 1/5 801 0/5 0/5 o/5 0/5 0/5 0/5 0/5 5/5 801 plus IBV 0/5 1/5 1/5 1/5 2/5 2/5 2/5 293 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5/5 293 plus IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 - S6C 0/5 0/5 o/5 1/5 3/5 5/5 5/5 — S6C plus IBV 0/5 1/5 1/5 4/5 4/5 5/5 5/5 - IBV 0/5 0/5 0/5 0/5 0/5 2/5 2/5 - Control 0/5 0/5 o/5 0/5 0/5 0/5 0/5 —
10"5 dilution of S6C; all other PPLO strains were undiluted. 10-7 dilution of IBV. 8l TABLE 25 MORTALITY OP EMBRYONATED EGGS WHEN CTC WHS ADDED AS A PEED SUPPLEMENT PLUS A 400 PPM DIPPING SOLUTION HIGH PPLO INOCULUM
Days of Incubation 11 12 1314 15 16 17
Pens 1-■3 200 ppm CTC s6c 0/5 0/5 1/5 2/5 2/5 4/5 4/5 S6C plus IBV 0/5 2/5 2/5 3/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 2/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 4-•6 200 ppm (3TC plus TPA s6c 0/5 0/5 2/5 2/5 2/5 3/5 3/5 S6C plus IBV 0/5 0/5 3/5 5/5 5/5 5/5 5/5 IBV 0/5 o/5 1/5 1/5 1/5 1/5 0/5 Control 0/5 o/5 0/5 0/5 0/5 0/5 0/5 Pens 7--9 400 ppm CTC s6c 0/5 0/5 1/5 2/5 2/5 2/5 3/5 S6C plus IBV 0/5 1/5 1/5 3/5 3/5 4/5 4/5 IBV 0/5 1/5 1/5 1/5 1/5 2/5 •2/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5 Pens 10-12 400 ppm CTC plus TPA s6c 0/5 0/5 o/5 0/5 2/5 2/5 2/5 S6C plus IBV 0/5 0/5 0/5 1/5 3/5 3/5 3/5 IBV 0/5 0/5 0/5 0/5 1/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 = Pens 13- 15 Control s6c 0/5 0/5 1/5 3/5 4/5 5/5 5/5 S6C plus IBV 0/5 2/5 2/5 5/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 1/5 1/5 1/5 1/5 Control 0/5 o/5 0/5 0/5 0/5 0/5 0/5
10“^ dilution of S6C.
10"? dilution of IBV. 82 TABLE 26 MORTALITY OF EMBRYONATED EGGS WHEN CTC WAS ADDED AS A FEED SUPPLEMENT PLUS A 200 PPM DIPPING SOLUTION HIGH PPLO INOCULUM
Days of Incubation 11 12 13 14 15 16 17
Pens 1-•3 200 ppm CTC s6c 1/5 1/5 1/5 2/5 2/5 3/5 3/5 S6C plus IBV 0/5 0/5 1/5 3/5 3/5 5/5 5/5 IBV 0/5 0/5 0/5 1/5 1/5 1/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 4-•6 200 ppm CTC plus TPA S6C 0/5 1/5 1/5 2/5 2/5 2/5 3/5 S6C plus IBV 0/5 0/5 0/5 3/5 3/5 4/5 4/5 IBV 0/5 0/5 0/5 0/5 0/5 1/5 2/5 Control 0/5 1/5 1/5 1/5 1/5 1/5 1/5 Pens 7-•9 400 ppm CTC s6c 0/5 1/5 1/5 1/5 3/5 3/5 3/5 S6C plus IBV 0/5 0/5 0/5 2/5 2/5 2/5 3/5 IBV 1/5 1/5 1/5 1/5 2/5 2/5 2/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 10-12 400 ppm CTC plus TPA S6c 0/5 0/5 0/5 0/5 1/5 2/5 2/5 S6C plus IBV 1/5 1/5 2/5 2/5 2/5 3/5 3/5 IBV 0/5 0/5 0/5 0/5 0/5 1/5 1/5 Control 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Pens 13-15 Control S6C 0/5 1/5 2/5 2/5 2/5 4/5 5/5 S6C plus IBV 0/5 0/5 0/5 3/5 5/5 5/5 5/5 IBV 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control 1/5 1/5 1/5 1/5 1/5 1/5 1/5
10"5 dilution of S6C.
10“^ dilution of IBV. offer the poultrymen some protection against excessive embryo mortality caused by M. gallisepticum. Figures 8 and 9 reveal the effects of M. gallisepticum S6C and a combination of S6C and IBV on an embryo. A large degree of hemorrhage can be observed when death occurs due to the mycoplasma (Figures 9 and 10). When low dilutions of virus were used in conjunction with
M. gallisepticum the hemorrhaging was less extensive than that observed when a higher virus dilution was used in conjunction with a low PPLO dilution. By this type of observation it might be possible to observe the dead embryo and get an idea as to which organism was primarily responsible for death, since extensive hemorrhaging is characteristic of a mycoplasma infection. 84
Figure 9. Comparison of 11 day old embryos from a control egg and an egg inoculated with M. gallisepticum S6C.
Figure 10. An 11 day old embryo removed from an egg inoculated with IBV and M. gallisepticum S6C. Figure 11. Contents of a control embryonated egg after 11 days of incubation.
Figure 12. Contents of an embryonated egg inoculated with M. gallisepticum S6C after 11 days of incubation. Figure 13. Contents of an embryonated egg inoculated with M. gallisepticum S6C and IBV after 11 days of incubation. DISCUSSION
During the past few years the poultry industry has undergone extensive changes. These changes have resulted in centralization of the poultry industry. It is no
longer profitable for the average farmer to raise 200 or 300 chickens; the poultry producer of today must think in terms of thousands of chickens and not in hundreds. In order to realize a profit the large poultry producer must utilize the capabilities of the scientist advantageously to be able to get rapid growth of the chicken, high egg production and low loss of embryos in the hatchery. The
low market price per pound of chicken has forced him to use good poultry management. It is toward these ends that this series of experiments was dedicated. To be worthwhile, this type of research must show bad poultry
management practices for what they are, or attempt to provide newer methods which are economical and can be practiced in active poultry production. Chlortetracycline
is the most popular antibiotic used in poultry feeds at this time and it has been shown to rapidly increase growth
of the chicken, to increase egg production, and its use has been sanctioned for treating flocks infected with
M. gallisepticum, the CRD agent (Goldberg, 1959)• 88
Many authors have published results on the in vitro effectiveness of CTC against various strains of M. gallisepticum (Table 1). CTC is not stable under the pH conditions found in the majority of microbiological media. Many of the reports on the effectiveness of CTC have been underestimated because this instability was not taken into account. Regardless of the fact that CTC is not bactericidal it is still effective in controlling infections of mycoplasma on a routine level. The majority of PPLO infections taking place under normal conditions would not be as severe as some of those set up in the laboratory. One of the major advantages of an antibiotic such as CTC is that it does control, to a certain degree, in ovo PPLO infections and that it is unstable enough to deteriorate under normal storage conditions so that unduly large residues are not built up in the edible tissues causing problems for the consumer of poultry tissue and poultry products.
The reisolation of the organisms capable of growing on laboratory media and the results of the in vitro broth antibiotic sensitivity tests shown in Tables 5» 6, and 7 indicate that CTC has only a bacteriostatic effect on the organisms used in this series of experiments. There is little reason to assume that this would not be true for the more virulent organisms used here and for other strains of mycoplasma which have also been involved in CRD. There is evidence that certain strains of PPLO are capable of killing the developing embryo. The Infectious Bronchitis Virus has been shown to kill the embryo with ease. It is only natural, therefore, to conclude that an infection with both disease-producing agents would increase the mortality rate of the developing embryo even further than either agent alone. By incorporating antibiotic into the developing embryo by feed fortifi cation, dipping solutions, and by direct inoculation it has been shown that the mortality rate can be significantly lowered. The potentiating effect of terephthalic acid was not very dramatic except when 400 ppm of antibiotic and TPA were used but when a dipping solution of 200 or 400 ppm was used in conjunction with 400 ppm of CTC plus 0.5 per cent TPA incorporated into the feed mixture the mortality rate was controlled quite successfully. It would appear that by using these 2 methods simultaneously the poultryman could make significant reductions in embryo mortality thus bringing a larger number of birds to market. The incorporation of antibiotics into feeds has been shown to result in healthier flocks and evidence has been shown here that viability rates of hens on high antibiotic fortified feeds were higher than those hens receiving no antibiotic in their feeds. Incorporation of 90 antibiotics by direct inoculation will probably never be used commercially since it is too expensive in terms of antibiotic waste and labor. The results shown here also indicate that it is not significantly better than either of the other 2 methods employed. The data indicate that mycoplasma of low virulence are also capable of increasing the mortality rate of the chicken embryo when participating in a combined infection. This is the virulence level that one would expect to find most routinely in the average flock since severe symptoms and death seldom occur in flocks except in the period when acute symptoms of CRD are present. This period seldom occurs unless stress of some type has altered the natural ability of the birds in the flock to control the disease agent. The normal course followed by CRD indicates that it is a latent type of infection.
The work done by Rolle (1962) indicates that a better method might be found to incorporate the tetracycline antibiotics into the embryonated egg. Since the CTC is gradually incorporated into the developing bone
matrix, a method must be developed to add small quantities of antibiotic to the developing embryo so that a
concentration of antibiotic capable of controlling the CRD agent is present throughout the entire egg incubation period. Direct inoculation would again be ruled out for
this purpose because of the increased chance of killing 91 the embryo by non-specific causes as the course of egg incubation proceeds. Dipping the eggs into a chilled antibiotic solution after the first day of incubation will result in almost 100 per cent non-specific mortality
(page 66). By using the dilutions of myocplasma and Infectious
Bronchitis Virus employed throughout this work it was possible to detect the effects of various methods of antibiotic incorporation. The dilutions made it possible to illustrate the borderline CRD case3 the borderline IBV case and the results of combining 2 such cases. The use of large CRD and IBV disease were also used so that one could definitely see that the methods employed separately to combat CRD were minimally effective while combining 2 methods proved much more effective. The useless adminis tration of CTC to combat extensive IBV Infection in ovo was also illustrated dramatically. At the present time the poultryman is able to detect CRD in his flock only by the symptoms of the birds during the acute stage of the disease. It is unfortunate that the CRD agent cannot be isolated with any great degree of regularity. Pabricant (1962) has pointed out clearly the folly of attempting to make a primary isolation of any
PPLO without the use of many different types of media. It
is possible that a flock could exhibit all of the symptoms
of CRD and yet the microbiologist may not be able to make one successful isolation from any bird. During the course of this work, a large number of 2 day old turkey poults were inoculated with the S6 strain of M. gallisepticum.
None of the poults exhibited symptoms of CRD, but a test of their serum using a poly-valent CRD antigen used for
field testing flocks for CRD revealed that all of the birds inoculated had antibody against the S6 strain of M. gallisepticum in their serum. All of the hens used throughout this series were also found to have antibody in
their serum against the S6 strain, but none of them had ever been inoculated with that organism. Isolation of the organism from the yolk sac of eggs from these birds was
only accomplished with a frequency of approximately 0.1 per cent. This agrees with claims by other authors that the shedding of mycoplasma from the hen to the egg occurs
only irregularly. .Here again one must rely only on the ability of the media which is being used to facilitate
growth of these organisms. Perhaps the frequency of isolation would have been greater if other media had been used. A limit to the use of many types of media must be
reached to prevent scientists interested in PPLO's from dedicating their lives to the isolation of a PPLO strain from 1 egg or 1 specimen.
The accurate determination of numbers of mycoplasma in a given suspension still remains a problem. Some
species of avian mycoplasma produce hemolysins and can be counted quite readily by utilizing each zone of hemolysis in the same manner that one counts viral plaques on tissue culture media. The strains of mycoplasma which were employed do not produce a hemolysin for a number of types of red blood cells. Utilization of turbidity in broth tubes for accurate quantitation is unsatisfactory since the mycoplasma will not produce any visible turbity in PPLO broth tubes for periods of incubation up to 96 hours. Broth cultures incubated for this period of time are usually sterile and further incubation is of no value. The standard plate count is also quite useless since the colonies are not visible macroscopically and the degree of error in counting colonies of these organisms is so inherently large that the results obtained by this method are thoroughly unreliable. It is immediately noticeable to the average reader that authors of articles on the PPLO invariably use vague terns to quantitate work done on artificial media. It is only by using titers such as the ELD^q and EID^0 that any reasonable quantitation can be
introduced, but these terms can only be used where adequate virulence or pathological response can be demonstrated.
It appears that the transovarial passage of mycoplasma from hen to chick cannot effectively be controlled by the use of CTC or CTC plus TPA. The
antibiotic does increase the general well being of the flock and it has been shown by previous authors that the shedding of mycoplasma occurs intermittently and the largest incidence of shedding occurs during the acute phase of the disease and drops off sharply as this phase of the disease passes. One can therefore conclude that the mycoplasma cannot be eliminated from the embryonated egg once it gains entrance to the fertile egg but the incidence of transmission can be effectively reduced by maintaining a healthier flock by the proper application of antibiotics combined with good poultry management. It is obvious that addition of CTC by direct inoculation and by means of dipping solutions will incorporate a detectable concentration of CTC residue into the egg. A considerable amount of work has been done at The Ohio State University which shows that a detectable CTC residue can also be incorporated into the egg by means of antibiotic fortified poultry feeds (Frye, 1957; Filson, 1964; Boyd, i960). Frye (1957) has also demonstrated that the largest detectable antibiotic residue is found in the yolk. This is most-advantageous since the site of active multiplication of the avian PPLO is also in the yolk sac.
One is therefore able to place the concentration of residual CTC where it will be most effective.
At the present time federal control over the use of CTC in poultry feeds limits the concentration to be employed at 200 ppm and has never certified the use of 95 terephthalic acid as a potentiating agent. Investigations performed at the Poultry Science Department of The Ohio State University on birds which have received antibiotic supplemented poultry feed and TPA potentiation have shown that TPA feeding for at least 2 years duration has no undesirable side effects on the birds involved. These results were obtained by post mortem examination of the b irds. Aside from combating mycoplasma disease agents and other microbial disease agents (due to a broad spectrum) CTC and other antibiotics appear to increase the overall welfare of the chicken and of other animals. Goldberg (1959) has provided an impressive amount of literature concerning the side effect of antibiotic supplemented feed stuffs. It has been shown that the weight of the chicken intestinal tract is diminished when they are fed antibiotic supplemented feeds. This is believed to be due to the loss of scar tissue being formed in the intestine because of the control the antibiotic exerts over mild bacterial infections in the intestine of the chicken. Goldberg stated that many ideas are available on the subject but the most common ones are that the antibiotic allows the vitamin producing bacteria to grow unrestricted, controls the number and actions of pathogenic microorganisms, prohibits the colon flora from entering the upper intestinal tract and producing an enteritis, and maintains the number of organisms of all types at a lower numerical level in the intestine. SUMMARY
Chlortetracycline has not been shown to he effective in eliminating the transovarial passage of
Mycoplasma gallisepticum when it is employed in dipping solutions, by direct inoculation, or by addition to poultry feeds with or without terephthalic acid. Evidence has been presented that chlortetracycline acts only as a bacteriostatic agent and does not kill the chronic respiratory disease agent in vitro or in ovo. By combining the use of a 200 or 400 ppm dipping solution with incorporation of 400 ppm of CTC plus 0.5 per cent terephthalic acid a substantial reduction in the number of embryo deaths due to the CRD agent alone or in combination with the Infectious Bronchitis Virus was shown. This antibiotic has no effect against the Infectious Bronchitis Virus but permits a reduction in mortality in combined infections by its action on the chronic respiratory disease agent.
A method for testing the in vitro sensitivity of CTC against M. gallisepticum is presented which takes into
account the rapid inactivation of the chlortetracycline molecule in alkaline solutions at incubator temperatures.
By adjusting the pH of the test broth the CTC molecule is
97 prevented from becoming inactive as rapidly as it would at the normal bacteriological media of pH 7.2-7.^-. An attempt was made to use hemolysin production as an aid in counting 3 strains of M. gallisepticum. It was found that chick, adult chicken, human, horse, ox, guinea pig, rabbit, pigeon, duck, and turkey red blood cells were of no value. These strains of mycoplasma did not show signs of producing a hemolysin which is capable of lysing any of these types of red blood cells. When chlortetra cycline was used as a feed supplement at a 400 ppm level, a 0.3 per cent concentration of terephthalic acid appeared to be of value in increasing its effectiveness against mycoplasma. This effect was not as dramatic when 200 ppm of CTC plus 0.5 per cent TPA were incorporated into the poultry feed. BIBLIOGRAPHY
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