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Studies of Infectious Coryza of Chickens with Special Reference to Its Etiology
Studies of Infectious Coryza of Chickens with Special Reference to Its Etiology O. W. SCHALM AND J. R. BEACH Division of Veterinary Science, University of California (Received for publication, June 23, 1936)
HE isolation from the nasal passages of chickens with infectious colds or coryza of a small bacillus with which a coryza of short duration could be produced in healthy chickens has been reported by de Blieck (1932) in Holland, by Nelson (1932) in New Jersey, by Delaplane and Stuart (1934) in Rhode Island, by Eliot and Lewis (1934) in Maryland, and by Schalm and Beach (1934) in California. Nelson stated that the bacillus failed to grow on open blood agar plates but did so when the plates were sealed with clay. De Blieck, Delaplane and Stuart, and Eliot and Lewis experienced no difficulty in growing their organisms aerobically on blood agar but the latter stated that growth was favored by incubation in an atmosphere containing an excess of carbon dioxide. Schalm and Beach, however, obtained no growth of the organism under aerobic conditions but succeeded when they employed an atmosphere with 10 percent carbon dioxide. Nelson, Delaplane, and the writers exchanged cultures and compared their requirements for growth. Nelson (1935) reported that both Delaplane's and the writers' cultures behaved in his laboratory like his own and that his culture in Delaplane's laboratory grew on aerobic plates in the same manner as Delaplane's cultures. The writers (1936) found that all three cultures colonized not at all or very meagerly on blood agar under aerobic conditions but grew satisfactorily when air was excluded by sealing the plates or tubes with clay or when they were incubated in an atmosphere
of 10 percent carbon dioxide. They also determined that all three cultures required the presence of both the X and the V factors for growth on or in an artificial medium, thus demonstrating that they were identical and that the organism belongs, along with the influenza bacillus of man, in the genus Hemophilus. The name Hemophilus gallinarum, suggested by Eliot and Lewis (1934), was considered more suitable than the name Bacillus haemoglobinophilus coryzae gallinarum which had been proposed by de Blieck (1932), and was adopted. The clinical and pathological manifestations of infectious coryza, the source and method of propagation of the eight strains of the disease studied, the source and method of handling the experiment birds, are described elsewhere (1936). It is the purpose of this paper to discuss the morphology and cultural requirements of Hemophilus gallinarum and present data concerning its relationship to the etiology of the disease. The technic employed for obtaining pure cultures of Hemophilus gallinarum from infected chickens was as follows: Nasal exudate from field cases of the disease was injected into the palatine cleft of healthy chickens. These were chloroformed during the first week of infection and the various pathological exudates streaked on fresh blood agar or boiled blood agar plates and incubated at 37°C. in an atmosphere of 10 percent carbon dioxide. After 24 to 48 hours, Hemophilus gallinarum appeared in the form of pin-point colonies which were either smooth, convex and glistening or flat, dull and rough. On plates streaked with edematous exudate from the subcutaneous tissues of the face
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plate. After 24 to 48 hours at 37 °C. the growth, if pure, was transferred to a fresh blood agar or boiled blood agar slant with broth at the base and maintained as a stock culture. MORPHOLOGY
or wattles a pure growth of Hemophilus gallinarum was obtained but on plates streaked with exudate from the nasal chambers, conjunctivalsacs, trachea, bronchi or air-sacs, colonies of other species of bacteria, principally gram-negative staphylococci, diplococci, and cocco-bacilli, were often present. Single, well-isolated colonies of Hemophilus gallinarum were picked, with the aid of a dissecting microscope, and streaked over a small area of the surface of another blood agar f
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• FIG. 3. Microphotograph of a film of a 16-hour boiled blood agar slant culture of Hemophilus gallinarum showing filament formation, x 1800
FIG. 2. Microphotograph of a film of a 48-hour fresh blood agar plate culture of Hemophilus gallinarum showing the formation of beaded threads, x 1800
filamentous forms were also seen. The latter varied from short beaded threads (Fig. 2) to long filaments which frequently appeared as tangled masses (Fig. 3). Some filaments exceeded 250 microns in length and on two occasions true branching was observed (Fig. 4). After 24 to 48 hours, degeneration frequently began and aberrant forms made their appearance. The rods and threads be-
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FIG. 1. Microphotograph of a methylene blue stained film of exudate showing Hemophilus gallinarum in the form of polar staining rods, x 2700
In film preparations made from the various pathological exudates, the organism appeared as a small rod with rounded ends, which showed a marked tendency toward polar staining (Fig. 1), varied from 1 to 5 microns in length and from 0.3 to 1.0 microns in width, and occurred singly, in pairs or triplets but rarely in longer chains. In cultures on fresh blood agar or boiled blood agar the organism showed marked pleomorphism and a tendency to quickly undergo fragmentation or degeneration. In culture 24 to 48 hours old the most characteristic form was a polar staining rod, but
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1936.
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came swollen and stained faintly and the ends of the polar staining rods became separated and appeared in the culture as swollen faintly staining spheres (Fig. 5), and finally only shapeless, faintly staining remnants were found. Transplants from such cultures gave rise to typical rods and filaments in the subculture. In many cultures small coccoid and cocco-bacillary forms were also observed.
Early in the study it was found that cultures of the hemophilic bacillus must be transferred frequently in order to maintain them on artificial media. To provide more specific information concerning the frequency with which transfer is necessary, the following experiment was conducted. Boiled blood agar plates and slants seeded with California strains I, VI, and VIII, New Jersey strain B5504, and Rhode Island strain N37 were placed in sealed jars containing atmospheres of 10 percent carbon dioxide. The slants were incubated for five days and the plates for'two days at 37° C, after which they were stored at room temperature which averaged 21.5° C.
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FIG. 5. Microphotograph of a film of a 72hour boiled blood agar plate culture of Hemophilus gallinarum showing degeneration, x 2700
for the period. When the cultures were five, ten, and twelve days old they were tested for viability by making transplants to either boiled blood agar plates or slants. The transplants from the five day old cultures contained numerous colonies; those from the ten day old cultures showed a marked decrease in the number of colonies and in the case of one plate culture remained sterile; and all the transplants from the twelve day old cultures remained sterile. This experiment was repeated several times with similar results. We have been successful in maintaining our stock cultures on boiled blood agar slants or plates by transplanting them once a week and keeping them at 37° C. in an atmosphere of 10 percent carbon dioxide between transfers. TEMPERATURES FOR INCUBATION OF CULTURES OF HEMOPHILUS GALLINARUM
FIG. 4. Microphotograph of a 16-hour boiled blood agar slant culture of Hemophilus gallinarum showing true branching of filaments, x 900
Cultures of California strains I and VII, New Jersey strain B5504, and Rhode Island strain N37 were streaked on boiled blood agar slants and plates and incubated for 96 hours in an atmosphere of 10 per-
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VIABILITY OF HEMOPHILUS GALLINARUM ON CULTURE MEDIA
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SCIENCE
TABLE 1.—Results from incubation of cultures of Hemophilus gallinarum at different temperatures Strain of organism Calif. I Calif. 1 VII New Jersey B5504 Rhode Island N37
20 C.
25 C.
30 C.
34 C.
37 C.
42 C.
45 C.
46 C.
47 C.
0
+ + + +
++ ++ ++ ++
+++ +++ +++ +++
+++ +++ ++ + +++
+++ +++ +++ +++
+ + + +
0
0
0
0
0
0
0
0
0 0 0
No growth. Meager growth. Fair growth. Good growth.
cent carbon dioxide at temperatures ranging from 20° C. to 47° C. The results as given in Table 1 indicate that the minimum and maximum temperatures for growth are respectively 25° C. and 45° C , and that the optimum temperature range is from 34° C. to 42° C. RESULTS OF INOCULATION OF FOWLS WITH PURE CULTURES OF HEMOPHILUS GALLINARUM
Ninety-nine chickens have been inoculated, 46 intranasally, 52 intranasally and
FIG. 6. Edema produced by the injection of a culture of Hemophilus gallinarum into the wattles.
intratracheally, and one into the wattles, with 36 different cultures of Hemophilus gallinarum, representing eight different California strains of infectious coryza. The incidence of symptoms and lesions in the 98 birds inoculated intranasally or both intranasally and intratracheally was as follows: Nasal discharge, 94; facial edema, 72; conjunctivitis, 12; edema of the wattles, 6; sinusitis, 4; rales and cough, 15; and inflammation of the air-sacs, 2. Four percent of the birds receiving only an intranasal injection developed infection of both the upper and lower respiratory tracts. The one fowl inoculated in the wattles developed a pronounced, localized inflammation of the wattles (Fig. 6). The duration of infection varied between one and 30 days, averaging six days. The culture-induced infection was identical in every respect with the natural disease or that produced by the inoculation of fowls with exudate except that it was of much shorter duration. (In 28 fowls inoculated with exudate, the duration of infection varied from 25 to 120 days with an average of 50 days.) THE RESISTANCE OF FOWLS RECOVERED FROM CULTURE-INDUCED DISEASE TO REINFECTION WITH CULTURES Eleven birds (Table 2), varying in age from 98 to 180 days, which had recovered from infection with cultures of Hemophilus
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Test temperatures and degree of growth obtained after 96 hours incubation
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THE RESISTANCE OF FOWLS RECOVERED FROM EXUDATE-INDUCED DISEASE TO REINFECTION WITH EXUDATE
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Thirty-three chickens (Table 2 ) , ranging in age from 45 to 365 days, which had recovered from infection with exudate were tested for susceptibility to reinfection by intranasal injection of virulent exudate. The interval of time which elapsed between disappearance of symptoms and the first test for susceptibility varied with different birds from 5 to 86 days. Thirteen of the 33 birds developed coryza after incubation periods varying from 2 to 33 days. The remaining 20 birds were refractory and 17 of them were subjected to a second test. Four of these, injected on the 43rd to 109th day after recovery, developed a coryza after incubation periods varying from 2 to 9 days, while the remaining 13 birds which were inoculated on the 34th to 180th day after recovery were still refractory. Seven birds of this latter group were tested a third time for susceptibility to infection with exudate. Six of them, inoculated on the 101st to 210th day after recovery, were still refractory, while the remaining bird, inoculated on the 111th day after recovery, developed a coryza of 9 days duration after an incubation period of two days. All tests were controlled by inoculation of normal birds which in each instance developed typical symptoms of exudate infection within 72 hours. These results demonstrate that
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gallinarum, in from one to 47 days after the disappearance of symptoms were given a second injection of culture. Nine of the 11 birds were refractory to reinfection while two, both injected on the fifth day after recovery, developed in two and four days respectively a coryza of five and six days duration. All reinfection tests were controlled by the simultaneous injection of normal birds all of which developed the typical culture-disease within 24 hours.
478 recovery from infection with exudate was attended by resistance to reinfection with exudate varying from none at all to a solid immunity lasting from one to seven months, the longest time tested. RESISTANCE OF FOWLS RECOVERED FROM CULTURE-INDUCED DISEASE TO INFECTION WITH EXUDATE
HEMOPHILUS GALLINARUM THE ONLY INFECTIVE AGENT IN INFECTIOUS CORYZA EXUDATE
In searching for an explanation of the difference in the severity of the cultureinduced and exudate-induced disease two
SCIENCE
possibilities were considered: One, that a pathogenic agent besides the hemophilic bacillus is present in the exudate, and the other, that the virulence of the hemophilic bacillus is reduced by cultivation on artificial media. Experiments to determine whether a second pathogenic agent is present in the exudate: Pure cultures of the types of bacteria other than Hemophilus gallinarum, mainly gram-negative cocci, which were encountered most frequently in the pathological exudates were tested for pathogenicity by injection of chickens intranasally with them, both alone and in combination with cultures of Hemophilus gallinarum. The injection of such cultures alone gave negative results and when injected in conjunction with Hemophilus gallinarum, the disease produced was no different than that produced by the latter organism alone. Fourteen Berkefeld V filtrates, 3 Berkefeld N filtrates, and 2 Berkefeld W filtrates of virulent infusion broth suspensions of coryza exudate had been tested for infective properties with negative results. However, in view of Shope's findings (1931) that swine influence is caused by the concerted action of a filterable virus and a hemophillic bacillus, the possibility that the same might apply to fowl coryza seemed worthy of investigation. A broth suspension of a 24 hour bloodagar culture of Hemophilus gallinarum and three different bacteriologically sterile Berkefeld V filtrates of a virulent infusion broth suspension of coryza exudate were used. Fowls were inoculated intranasally and intratracheally with the unfiltered suspensions of exudate; with the filtrates of the suspensions of exudate; with the suspension of Hemophilus gallinarum diluted with an equal volume of the broth; or with mixtures of equal parts of the suspension of Hemophilus gallinarum and a filtrate as indicated in Table 3. The re-
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Twenty-eight fowls (Table 2), ranging in age from 42 to 126 days, which had recovered from infection with cultures of Hemophilus gallinarum, were tested for susceptibility to infection with exudate, 4 to 72 days after the disappearance of symptoms. Twenty-five of these 28 birds developed a simple coryza after incubation periods which ranged from 2 to 8 days in the case of 13 birds and from 12 to 38 days in the remaining 12 birds. Three of the 28 birds were refractory and were subjected to a second test. Two of them, inoculated a second time with exudate on the 105th and 115th day after recovery, developed a simple coryza within 48 hours, while the remaining bird, injected on the 82nd day after recovery was refractory, but when subjected to a third test on the 114th day after disappearance of symptoms it developed a simple coryza within 48 hours. The control birds developed a coryza, which in most cases was severe, within 24 to 48 hours. These results demonstrate that fowls after recovery from culture disease for the most part remain susceptible to infection with exudate but, as indicated by the prolonged incubation period and mildness of symptoms, possess a variable degree of resistance to it.
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suits as presented in the table show that the unfiltered suspension of exudate caused a complicated coryza of long duration; that the filtrate alone was not infective; and, that the culture alone and the mixtures of culture and filtrate each caused a mild coryza of short duration and
479
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Increase in the severity of the cultureinduced disease by rapid serial passage through chickens: To test the validity of the idea that the short duration of the culture-induced disease is due to a reduction in the virulence of the organism by cultivation on artificial media, an attempt
TABLE 3.—Results of inoculation offowls withfiltratesalone, culture alone, and mixtures of culture and filtrates Incubation period (hours)
Nasal Discharge
Facial Edema
Rales and Cough
Duration of Symptoms (days)
Unfiltered 1-20 suspension of exudate
48
+
+
+
91
Berkefeld filtrate V-l
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Berkefeld filtrate V-2
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Berkefeld filtrate V-3
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Symptoms
0.25 cc. broth suspension of culture and 0.25 cc. plain broth
15
+
+
0
7
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-l
15
+
0
0
8
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-2
48
+
+
0
6
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-3
15
+
+
0
5
Remarks
Very severe infection.
(1) Dose uniformly 0.5 cc.—inoculated both intranasally and intratracheally.
did not differ in virulence. It is concluded therefore that the nasal exudate of fowls affected with coryza contained no bacteria other than Hemophilus gallinarum nor a filterable virus which acted in conjunction with Hemophilus gallinarum to produce the severe prolonged infection which occurred in the fowl which was inoculated with unfiltered nasal exudate.
was made to restore the virulence of the organism by rapid serial passages through chickens. For this experiment a culture was selected which had been grown on artificial media for a period of thirteen months and which produced, upon injection into chickens, a mild coryza of two days duration. Starting with this mild coryza, the disease was passed to other fowls, by means of exudate, at two day intervals, for the
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Inoculum (1)
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first 33 passages, and the interval between passage was gradually lengthened until, by the fortieth passage, it was seven days, at which it was maintained. At each of the first 35 passages, two birds were inoculated, one of which was destroyed to
SCIENCE
Between the first and 33rd passage, the duration of the infection fluctuated considerably but exceeded seven days only five times, as follows: 8 days at the 11th pas-
TABLE 4.—Result of Serial Passage of a Culture Through Chickens and Test of Recovered Chickens for Resistance to Infection with Virulent Exudate Symptoms Incubation period (days)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 38 46 48 53 63
4 4 1 1 1 1 1 1 1 2
1 2
•
2
1 2 1 1 1 1 1 1 10 2 2 1 1 1 1 1 1 1 1 1 2 1 2 4 2 2 1 2 1
Nasal discharge
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Facial Edema
+ + + +
+
Rales, Cough
+ +
+
+
+ + +
+ + +
Duration (days) 2 2 3 2 4 2 2 4 4 1 8 2 6 4 4 2 6 2 2 5 14 8 17 6 2 3 2 5 1 2 2 3 12 35 27 31 44 42 51 76
Days elapsing between recovery and in- Incubation period oculation with (days) virulent exudate 1 NT NT NT NT NT NT NT NT NT 47 9 3 7 21 19 NT 13 15 NT NT NT NT 34 NT NT NT NT NT NT NT NT NT 30 46 48 27 8 13 16 NT
Duration of symptoms 2
1 3 3 3 9 3
K15 K22 K24 K23 K4 K10
3 4
K10 K9
14
K8
8 0 0 0 0 0 2
4 0 0 0 0 0 17
N T means that fowl was not inoculated with virulent exudate after recovery. K preceding a number indicates the fowl was destroyed on that day of sickness.
obtain exudate for the next passage and the other was allowed to recover in order that the duration of disease might be determined. After the 35th passage, duplicate inoculations were not regularly made. The results are shown in Table 4.
sage, 14 days at the 21st passage, 8 days at the 22nd passage, 17 days at the 23rd passage, and 12 days at the 33rd passage. The duration of the disease lengthened con-
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Serial passage no.
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portion to the increase in severity of the coryza until finally it equalled the resistance of birds recovered from exudate-induced and perpetuated coryza. These results lend support to the view that Hemophilus gallinarum is the only pathogenic agent present in infectious coryza exudate and that the mildness of the culture-induced disease is the result of a reduction of virulence of the organism by cultivation on artificial media. SUMMARY Transfer at intervals of less than 14 days is necessary for the maintenance of slant or plate cultures of Hemophilus gallinarum. Stock cultures can be maintained indefinitely my making transplants once a week. The minimum temperature at which Hemophilus gallinarum will grow in culture is 25°C, the maximum temperature is 45 ° C , and the optimum temperature range is from 34°C. to 42°C. The injection of pure cultures of Hemophilus gallinarum into the respiratory tract of fowls produces a coryza with symptoms identical with those of the natural or exudate-induced disease but of much shorter duration. Attempts to determine whether the longer duration of the natural or the exudate-induced disease was due to the presence in the exudate of types of bacteria other than Hemophilus gallinarum or of a filterable virus yielded negative results. A mild culture-induced coryza of only 2 days duration, by rapid serial passage through chickens, increased in virulence and severity until it was of the same character as that produced by exudate obtained from severe field cases. This increase in virulence and severity occurred without the introduction of any pathogenic agent other than the culture of Hemophilus gallinarum with which the series of passages was initiated. The results are regarded as
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siderably after the 33 rd passage and was 35 days at the 34th passage, 27 days at the 35th passage, 31 days at the 38th passage, 44 days at the 46th passage, 42 days at the 48th passage, 51 days at the 53rd passage, and 76 days at the 63rd passage. A nasal discharge was observed in all the birds, and after the 10th passage an occasional bird showed edema of the face and, less frequently, infection of the lower respiratory tract. Films of the exudates, which were made at each passage and stained with methylene blue, contained Hemophilus gallinarum, usually in a pure state. Some of the birds upon recovery were tested for resistance to intranasal infection with virulent exudate. The interval of time which elapsed between the disappearance of symptoms and the injection of exudate varied between 3 and 48 days. The recovered birds of the first 18 serial passages showed very little if any resistance to virulent exudate. The recovered birds of the 23rd and the 33rd passages exhibited a marked resistance to virulent exudate, developing only a simple coryza of a few days duration after an incubation period of 8 to 14 days. The recovered fowls of the 34th, 38th, 46th, and 48th passages were refractory to virulent exudate, while that of the 53rd passage, the last to be tested, developed a simple coryza of 17 days duration after an incubation period of 2 days. The increase in the severity and the duration of symptoms of the disease from 2 days to more than 2 months during the 63 serial passages of the culture through chickens shows that definite increase in the virulence of the culture-induced disease took place without the addition of any infectious agent to that provided by the culture with which the first passage was made. The degree of resistance of recovered birds to virulent exudate increased in pro-
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evidence that the mild character of the culture-induced disease is due to a decrease in virulence of the organism on artificial media and that the virulence can be restored by continued cultivation in the more favorable environment of the nasal passages of susceptible chickens. REFERENCES
1932. de Blieck, L. E. Vet. Jour., 88, 9. 1932. Nelson, J. B. Proc. Soc. Exp. Biol, and Med., 30, 306.
SCIENCE
1934. Delaplane, J. P. and H. 0 . Stuart. Ann Rpt. R. I. Agr. Exp. Sta., 92. 1934. Eliot, C. P., and M. R. Lewis. Amer. Vet. Med. Assn., 37, 878. 1934. Schalm, O. W., and J. R. Beach. Science 79, 416. 1935. Nelson, J. B., Jour. Amer. Vet. Med. Assn., 39, 409. 1936. Schalm, O. W., and J. R. Beach. Jour. Bact, 31, 161. 1936. Schalm, O. W., and J. R. Beach. In press. (Poultry Science.) 1931. Shope, R. E. Jour. Exp. Med., S4, 373.
The Editor wishes to call attention to the fact that if personals are to appear in the News items these must be submitted to him with the pertinent facts. Personals Winton Appointed Senior Poultry Husbandman. Berley Winton (B.S., Kentucky, 1922, M.S., Missouri) has been appointed senior poultry husbandman in the animal husbandry division of the Bureau of Animal Industry, United States Department of Agriculture to succeed Dr. M. A. Jull. He was formerly extension poultry specialist in Kentucky, Tennessee and Missouri and more recently had charge of the administration of the National Poultry Improvement . Plan. Malcolm Lyons (B.S., Kentucky, 1932, M.S., Iowa, 1934) has been transferred from the position of Assistant Chemist in the Kentucky Experiment Station to thr position of Instructor in Poultry Husbandry in the University of Kentucky. Back Issues Wanted We still have a number of unfilled requests for the January 1936 issue of POUL-
TRY SCIENCE. Any person not wanting or needing this issue for permanent use will render a service by sending it to the Editor. The regular price (75c) will be paid for it. We also have a request for the Proceedings of 1917-18 and Proceedings for 191920. Papers for Poultry Science The Editor wishes to call attention to the fact that articles can now be submitted with the assurance of fairly early publication. There are now in hand papers for not more than two more issues.
Erratum The following correction needs to be made in the paper entitled, "Influence of Ovulation Rate on the Tendency of the Fowl to Produce Eggs in Clutches" which appeared in the September 1936 number: Lines 2 and 3 of the first column on page 386 "For this reason the pairs of eggs from the lighted group were sum-" should be followed by the three lines in the first column immediately above the unnumbered table on page 387 as follows: "marized independently of those of the unlighted ones. The results are given as follows: (Unnumbered table on page 387)"
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News and Notes
HE isolation from the nasal passages of chickens with infectious colds or coryza of a small bacillus with which a coryza of short duration could be produced in healthy chickens has been reported by de Blieck (1932) in Holland, by Nelson (1932) in New Jersey, by Delaplane and Stuart (1934) in Rhode Island, by Eliot and Lewis (1934) in Maryland, and by Schalm and Beach (1934) in California. Nelson stated that the bacillus failed to grow on open blood agar plates but did so when the plates were sealed with clay. De Blieck, Delaplane and Stuart, and Eliot and Lewis experienced no difficulty in growing their organisms aerobically on blood agar but the latter stated that growth was favored by incubation in an atmosphere containing an excess of carbon dioxide. Schalm and Beach, however, obtained no growth of the organism under aerobic conditions but succeeded when they employed an atmosphere with 10 percent carbon dioxide. Nelson, Delaplane, and the writers exchanged cultures and compared their requirements for growth. Nelson (1935) reported that both Delaplane's and the writers' cultures behaved in his laboratory like his own and that his culture in Delaplane's laboratory grew on aerobic plates in the same manner as Delaplane's cultures. The writers (1936) found that all three cultures colonized not at all or very meagerly on blood agar under aerobic conditions but grew satisfactorily when air was excluded by sealing the plates or tubes with clay or when they were incubated in an atmosphere
of 10 percent carbon dioxide. They also determined that all three cultures required the presence of both the X and the V factors for growth on or in an artificial medium, thus demonstrating that they were identical and that the organism belongs, along with the influenza bacillus of man, in the genus Hemophilus. The name Hemophilus gallinarum, suggested by Eliot and Lewis (1934), was considered more suitable than the name Bacillus haemoglobinophilus coryzae gallinarum which had been proposed by de Blieck (1932), and was adopted. The clinical and pathological manifestations of infectious coryza, the source and method of propagation of the eight strains of the disease studied, the source and method of handling the experiment birds, are described elsewhere (1936). It is the purpose of this paper to discuss the morphology and cultural requirements of Hemophilus gallinarum and present data concerning its relationship to the etiology of the disease. The technic employed for obtaining pure cultures of Hemophilus gallinarum from infected chickens was as follows: Nasal exudate from field cases of the disease was injected into the palatine cleft of healthy chickens. These were chloroformed during the first week of infection and the various pathological exudates streaked on fresh blood agar or boiled blood agar plates and incubated at 37°C. in an atmosphere of 10 percent carbon dioxide. After 24 to 48 hours, Hemophilus gallinarum appeared in the form of pin-point colonies which were either smooth, convex and glistening or flat, dull and rough. On plates streaked with edematous exudate from the subcutaneous tissues of the face
[473]
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plate. After 24 to 48 hours at 37 °C. the growth, if pure, was transferred to a fresh blood agar or boiled blood agar slant with broth at the base and maintained as a stock culture. MORPHOLOGY
or wattles a pure growth of Hemophilus gallinarum was obtained but on plates streaked with exudate from the nasal chambers, conjunctivalsacs, trachea, bronchi or air-sacs, colonies of other species of bacteria, principally gram-negative staphylococci, diplococci, and cocco-bacilli, were often present. Single, well-isolated colonies of Hemophilus gallinarum were picked, with the aid of a dissecting microscope, and streaked over a small area of the surface of another blood agar f
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• FIG. 3. Microphotograph of a film of a 16-hour boiled blood agar slant culture of Hemophilus gallinarum showing filament formation, x 1800
FIG. 2. Microphotograph of a film of a 48-hour fresh blood agar plate culture of Hemophilus gallinarum showing the formation of beaded threads, x 1800
filamentous forms were also seen. The latter varied from short beaded threads (Fig. 2) to long filaments which frequently appeared as tangled masses (Fig. 3). Some filaments exceeded 250 microns in length and on two occasions true branching was observed (Fig. 4). After 24 to 48 hours, degeneration frequently began and aberrant forms made their appearance. The rods and threads be-
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FIG. 1. Microphotograph of a methylene blue stained film of exudate showing Hemophilus gallinarum in the form of polar staining rods, x 2700
In film preparations made from the various pathological exudates, the organism appeared as a small rod with rounded ends, which showed a marked tendency toward polar staining (Fig. 1), varied from 1 to 5 microns in length and from 0.3 to 1.0 microns in width, and occurred singly, in pairs or triplets but rarely in longer chains. In cultures on fresh blood agar or boiled blood agar the organism showed marked pleomorphism and a tendency to quickly undergo fragmentation or degeneration. In culture 24 to 48 hours old the most characteristic form was a polar staining rod, but
NOVEMBER,
1936.
VOL.
XV,
No.
came swollen and stained faintly and the ends of the polar staining rods became separated and appeared in the culture as swollen faintly staining spheres (Fig. 5), and finally only shapeless, faintly staining remnants were found. Transplants from such cultures gave rise to typical rods and filaments in the subculture. In many cultures small coccoid and cocco-bacillary forms were also observed.
Early in the study it was found that cultures of the hemophilic bacillus must be transferred frequently in order to maintain them on artificial media. To provide more specific information concerning the frequency with which transfer is necessary, the following experiment was conducted. Boiled blood agar plates and slants seeded with California strains I, VI, and VIII, New Jersey strain B5504, and Rhode Island strain N37 were placed in sealed jars containing atmospheres of 10 percent carbon dioxide. The slants were incubated for five days and the plates for'two days at 37° C, after which they were stored at room temperature which averaged 21.5° C.
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FIG. 5. Microphotograph of a film of a 72hour boiled blood agar plate culture of Hemophilus gallinarum showing degeneration, x 2700
for the period. When the cultures were five, ten, and twelve days old they were tested for viability by making transplants to either boiled blood agar plates or slants. The transplants from the five day old cultures contained numerous colonies; those from the ten day old cultures showed a marked decrease in the number of colonies and in the case of one plate culture remained sterile; and all the transplants from the twelve day old cultures remained sterile. This experiment was repeated several times with similar results. We have been successful in maintaining our stock cultures on boiled blood agar slants or plates by transplanting them once a week and keeping them at 37° C. in an atmosphere of 10 percent carbon dioxide between transfers. TEMPERATURES FOR INCUBATION OF CULTURES OF HEMOPHILUS GALLINARUM
FIG. 4. Microphotograph of a 16-hour boiled blood agar slant culture of Hemophilus gallinarum showing true branching of filaments, x 900
Cultures of California strains I and VII, New Jersey strain B5504, and Rhode Island strain N37 were streaked on boiled blood agar slants and plates and incubated for 96 hours in an atmosphere of 10 per-
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VIABILITY OF HEMOPHILUS GALLINARUM ON CULTURE MEDIA
475
6
476
POULTRY
SCIENCE
TABLE 1.—Results from incubation of cultures of Hemophilus gallinarum at different temperatures Strain of organism Calif. I Calif. 1 VII New Jersey B5504 Rhode Island N37
20 C.
25 C.
30 C.
34 C.
37 C.
42 C.
45 C.
46 C.
47 C.
0
+ + + +
++ ++ ++ ++
+++ +++ +++ +++
+++ +++ ++ + +++
+++ +++ +++ +++
+ + + +
0
0
0
0
0
0
0
0
0 0 0
No growth. Meager growth. Fair growth. Good growth.
cent carbon dioxide at temperatures ranging from 20° C. to 47° C. The results as given in Table 1 indicate that the minimum and maximum temperatures for growth are respectively 25° C. and 45° C , and that the optimum temperature range is from 34° C. to 42° C. RESULTS OF INOCULATION OF FOWLS WITH PURE CULTURES OF HEMOPHILUS GALLINARUM
Ninety-nine chickens have been inoculated, 46 intranasally, 52 intranasally and
FIG. 6. Edema produced by the injection of a culture of Hemophilus gallinarum into the wattles.
intratracheally, and one into the wattles, with 36 different cultures of Hemophilus gallinarum, representing eight different California strains of infectious coryza. The incidence of symptoms and lesions in the 98 birds inoculated intranasally or both intranasally and intratracheally was as follows: Nasal discharge, 94; facial edema, 72; conjunctivitis, 12; edema of the wattles, 6; sinusitis, 4; rales and cough, 15; and inflammation of the air-sacs, 2. Four percent of the birds receiving only an intranasal injection developed infection of both the upper and lower respiratory tracts. The one fowl inoculated in the wattles developed a pronounced, localized inflammation of the wattles (Fig. 6). The duration of infection varied between one and 30 days, averaging six days. The culture-induced infection was identical in every respect with the natural disease or that produced by the inoculation of fowls with exudate except that it was of much shorter duration. (In 28 fowls inoculated with exudate, the duration of infection varied from 25 to 120 days with an average of 50 days.) THE RESISTANCE OF FOWLS RECOVERED FROM CULTURE-INDUCED DISEASE TO REINFECTION WITH CULTURES Eleven birds (Table 2), varying in age from 98 to 180 days, which had recovered from infection with cultures of Hemophilus
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0 + + -f+ -f+
Test temperatures and degree of growth obtained after 96 hours incubation
NOVEMBER,
1936.
VOL.
XV, No. 6
a
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| o
THE RESISTANCE OF FOWLS RECOVERED FROM EXUDATE-INDUCED DISEASE TO REINFECTION WITH EXUDATE
| -| ^
Thirty-three chickens (Table 2 ) , ranging in age from 45 to 365 days, which had recovered from infection with exudate were tested for susceptibility to reinfection by intranasal injection of virulent exudate. The interval of time which elapsed between disappearance of symptoms and the first test for susceptibility varied with different birds from 5 to 86 days. Thirteen of the 33 birds developed coryza after incubation periods varying from 2 to 33 days. The remaining 20 birds were refractory and 17 of them were subjected to a second test. Four of these, injected on the 43rd to 109th day after recovery, developed a coryza after incubation periods varying from 2 to 9 days, while the remaining 13 birds which were inoculated on the 34th to 180th day after recovery were still refractory. Seven birds of this latter group were tested a third time for susceptibility to infection with exudate. Six of them, inoculated on the 101st to 210th day after recovery, were still refractory, while the remaining bird, inoculated on the 111th day after recovery, developed a coryza of 9 days duration after an incubation period of two days. All tests were controlled by inoculation of normal birds which in each instance developed typical symptoms of exudate infection within 72 hours. These results demonstrate that
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gallinarum, in from one to 47 days after the disappearance of symptoms were given a second injection of culture. Nine of the 11 birds were refractory to reinfection while two, both injected on the fifth day after recovery, developed in two and four days respectively a coryza of five and six days duration. All reinfection tests were controlled by the simultaneous injection of normal birds all of which developed the typical culture-disease within 24 hours.
478 recovery from infection with exudate was attended by resistance to reinfection with exudate varying from none at all to a solid immunity lasting from one to seven months, the longest time tested. RESISTANCE OF FOWLS RECOVERED FROM CULTURE-INDUCED DISEASE TO INFECTION WITH EXUDATE
HEMOPHILUS GALLINARUM THE ONLY INFECTIVE AGENT IN INFECTIOUS CORYZA EXUDATE
In searching for an explanation of the difference in the severity of the cultureinduced and exudate-induced disease two
SCIENCE
possibilities were considered: One, that a pathogenic agent besides the hemophilic bacillus is present in the exudate, and the other, that the virulence of the hemophilic bacillus is reduced by cultivation on artificial media. Experiments to determine whether a second pathogenic agent is present in the exudate: Pure cultures of the types of bacteria other than Hemophilus gallinarum, mainly gram-negative cocci, which were encountered most frequently in the pathological exudates were tested for pathogenicity by injection of chickens intranasally with them, both alone and in combination with cultures of Hemophilus gallinarum. The injection of such cultures alone gave negative results and when injected in conjunction with Hemophilus gallinarum, the disease produced was no different than that produced by the latter organism alone. Fourteen Berkefeld V filtrates, 3 Berkefeld N filtrates, and 2 Berkefeld W filtrates of virulent infusion broth suspensions of coryza exudate had been tested for infective properties with negative results. However, in view of Shope's findings (1931) that swine influence is caused by the concerted action of a filterable virus and a hemophillic bacillus, the possibility that the same might apply to fowl coryza seemed worthy of investigation. A broth suspension of a 24 hour bloodagar culture of Hemophilus gallinarum and three different bacteriologically sterile Berkefeld V filtrates of a virulent infusion broth suspension of coryza exudate were used. Fowls were inoculated intranasally and intratracheally with the unfiltered suspensions of exudate; with the filtrates of the suspensions of exudate; with the suspension of Hemophilus gallinarum diluted with an equal volume of the broth; or with mixtures of equal parts of the suspension of Hemophilus gallinarum and a filtrate as indicated in Table 3. The re-
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Twenty-eight fowls (Table 2), ranging in age from 42 to 126 days, which had recovered from infection with cultures of Hemophilus gallinarum, were tested for susceptibility to infection with exudate, 4 to 72 days after the disappearance of symptoms. Twenty-five of these 28 birds developed a simple coryza after incubation periods which ranged from 2 to 8 days in the case of 13 birds and from 12 to 38 days in the remaining 12 birds. Three of the 28 birds were refractory and were subjected to a second test. Two of them, inoculated a second time with exudate on the 105th and 115th day after recovery, developed a simple coryza within 48 hours, while the remaining bird, injected on the 82nd day after recovery was refractory, but when subjected to a third test on the 114th day after disappearance of symptoms it developed a simple coryza within 48 hours. The control birds developed a coryza, which in most cases was severe, within 24 to 48 hours. These results demonstrate that fowls after recovery from culture disease for the most part remain susceptible to infection with exudate but, as indicated by the prolonged incubation period and mildness of symptoms, possess a variable degree of resistance to it.
POULTRY
NOVEMBER,
1936.
VOL.
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No.
suits as presented in the table show that the unfiltered suspension of exudate caused a complicated coryza of long duration; that the filtrate alone was not infective; and, that the culture alone and the mixtures of culture and filtrate each caused a mild coryza of short duration and
479
6
Increase in the severity of the cultureinduced disease by rapid serial passage through chickens: To test the validity of the idea that the short duration of the culture-induced disease is due to a reduction in the virulence of the organism by cultivation on artificial media, an attempt
TABLE 3.—Results of inoculation offowls withfiltratesalone, culture alone, and mixtures of culture and filtrates Incubation period (hours)
Nasal Discharge
Facial Edema
Rales and Cough
Duration of Symptoms (days)
Unfiltered 1-20 suspension of exudate
48
+
+
+
91
Berkefeld filtrate V-l
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Berkefeld filtrate V-2
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Berkefeld filtrate V-3
0
0
0
Susceptible to infection with virulent exudate when inoculated 14 days later
Symptoms
0.25 cc. broth suspension of culture and 0.25 cc. plain broth
15
+
+
0
7
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-l
15
+
0
0
8
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-2
48
+
+
0
6
0.25 cc. broth suspension of culture plus 0.25 cc. of filtrate V-3
15
+
+
0
5
Remarks
Very severe infection.
(1) Dose uniformly 0.5 cc.—inoculated both intranasally and intratracheally.
did not differ in virulence. It is concluded therefore that the nasal exudate of fowls affected with coryza contained no bacteria other than Hemophilus gallinarum nor a filterable virus which acted in conjunction with Hemophilus gallinarum to produce the severe prolonged infection which occurred in the fowl which was inoculated with unfiltered nasal exudate.
was made to restore the virulence of the organism by rapid serial passages through chickens. For this experiment a culture was selected which had been grown on artificial media for a period of thirteen months and which produced, upon injection into chickens, a mild coryza of two days duration. Starting with this mild coryza, the disease was passed to other fowls, by means of exudate, at two day intervals, for the
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Inoculum (1)
480
POULTRY
first 33 passages, and the interval between passage was gradually lengthened until, by the fortieth passage, it was seven days, at which it was maintained. At each of the first 35 passages, two birds were inoculated, one of which was destroyed to
SCIENCE
Between the first and 33rd passage, the duration of the infection fluctuated considerably but exceeded seven days only five times, as follows: 8 days at the 11th pas-
TABLE 4.—Result of Serial Passage of a Culture Through Chickens and Test of Recovered Chickens for Resistance to Infection with Virulent Exudate Symptoms Incubation period (days)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 38 46 48 53 63
4 4 1 1 1 1 1 1 1 2
1 2
•
2
1 2 1 1 1 1 1 1 10 2 2 1 1 1 1 1 1 1 1 1 2 1 2 4 2 2 1 2 1
Nasal discharge
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Facial Edema
+ + + +
+
Rales, Cough
+ +
+
+
+ + +
+ + +
Duration (days) 2 2 3 2 4 2 2 4 4 1 8 2 6 4 4 2 6 2 2 5 14 8 17 6 2 3 2 5 1 2 2 3 12 35 27 31 44 42 51 76
Days elapsing between recovery and in- Incubation period oculation with (days) virulent exudate 1 NT NT NT NT NT NT NT NT NT 47 9 3 7 21 19 NT 13 15 NT NT NT NT 34 NT NT NT NT NT NT NT NT NT 30 46 48 27 8 13 16 NT
Duration of symptoms 2
1 3 3 3 9 3
K15 K22 K24 K23 K4 K10
3 4
K10 K9
14
K8
8 0 0 0 0 0 2
4 0 0 0 0 0 17
N T means that fowl was not inoculated with virulent exudate after recovery. K preceding a number indicates the fowl was destroyed on that day of sickness.
obtain exudate for the next passage and the other was allowed to recover in order that the duration of disease might be determined. After the 35th passage, duplicate inoculations were not regularly made. The results are shown in Table 4.
sage, 14 days at the 21st passage, 8 days at the 22nd passage, 17 days at the 23rd passage, and 12 days at the 33rd passage. The duration of the disease lengthened con-
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Serial passage no.
NOVEMBER,
1936.
VOL.
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No.
481
portion to the increase in severity of the coryza until finally it equalled the resistance of birds recovered from exudate-induced and perpetuated coryza. These results lend support to the view that Hemophilus gallinarum is the only pathogenic agent present in infectious coryza exudate and that the mildness of the culture-induced disease is the result of a reduction of virulence of the organism by cultivation on artificial media. SUMMARY Transfer at intervals of less than 14 days is necessary for the maintenance of slant or plate cultures of Hemophilus gallinarum. Stock cultures can be maintained indefinitely my making transplants once a week. The minimum temperature at which Hemophilus gallinarum will grow in culture is 25°C, the maximum temperature is 45 ° C , and the optimum temperature range is from 34°C. to 42°C. The injection of pure cultures of Hemophilus gallinarum into the respiratory tract of fowls produces a coryza with symptoms identical with those of the natural or exudate-induced disease but of much shorter duration. Attempts to determine whether the longer duration of the natural or the exudate-induced disease was due to the presence in the exudate of types of bacteria other than Hemophilus gallinarum or of a filterable virus yielded negative results. A mild culture-induced coryza of only 2 days duration, by rapid serial passage through chickens, increased in virulence and severity until it was of the same character as that produced by exudate obtained from severe field cases. This increase in virulence and severity occurred without the introduction of any pathogenic agent other than the culture of Hemophilus gallinarum with which the series of passages was initiated. The results are regarded as
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siderably after the 33 rd passage and was 35 days at the 34th passage, 27 days at the 35th passage, 31 days at the 38th passage, 44 days at the 46th passage, 42 days at the 48th passage, 51 days at the 53rd passage, and 76 days at the 63rd passage. A nasal discharge was observed in all the birds, and after the 10th passage an occasional bird showed edema of the face and, less frequently, infection of the lower respiratory tract. Films of the exudates, which were made at each passage and stained with methylene blue, contained Hemophilus gallinarum, usually in a pure state. Some of the birds upon recovery were tested for resistance to intranasal infection with virulent exudate. The interval of time which elapsed between the disappearance of symptoms and the injection of exudate varied between 3 and 48 days. The recovered birds of the first 18 serial passages showed very little if any resistance to virulent exudate. The recovered birds of the 23rd and the 33rd passages exhibited a marked resistance to virulent exudate, developing only a simple coryza of a few days duration after an incubation period of 8 to 14 days. The recovered fowls of the 34th, 38th, 46th, and 48th passages were refractory to virulent exudate, while that of the 53rd passage, the last to be tested, developed a simple coryza of 17 days duration after an incubation period of 2 days. The increase in the severity and the duration of symptoms of the disease from 2 days to more than 2 months during the 63 serial passages of the culture through chickens shows that definite increase in the virulence of the culture-induced disease took place without the addition of any infectious agent to that provided by the culture with which the first passage was made. The degree of resistance of recovered birds to virulent exudate increased in pro-
6
482
POULTRY
evidence that the mild character of the culture-induced disease is due to a decrease in virulence of the organism on artificial media and that the virulence can be restored by continued cultivation in the more favorable environment of the nasal passages of susceptible chickens. REFERENCES
1932. de Blieck, L. E. Vet. Jour., 88, 9. 1932. Nelson, J. B. Proc. Soc. Exp. Biol, and Med., 30, 306.
SCIENCE
1934. Delaplane, J. P. and H. 0 . Stuart. Ann Rpt. R. I. Agr. Exp. Sta., 92. 1934. Eliot, C. P., and M. R. Lewis. Amer. Vet. Med. Assn., 37, 878. 1934. Schalm, O. W., and J. R. Beach. Science 79, 416. 1935. Nelson, J. B., Jour. Amer. Vet. Med. Assn., 39, 409. 1936. Schalm, O. W., and J. R. Beach. Jour. Bact, 31, 161. 1936. Schalm, O. W., and J. R. Beach. In press. (Poultry Science.) 1931. Shope, R. E. Jour. Exp. Med., S4, 373.
The Editor wishes to call attention to the fact that if personals are to appear in the News items these must be submitted to him with the pertinent facts. Personals Winton Appointed Senior Poultry Husbandman. Berley Winton (B.S., Kentucky, 1922, M.S., Missouri) has been appointed senior poultry husbandman in the animal husbandry division of the Bureau of Animal Industry, United States Department of Agriculture to succeed Dr. M. A. Jull. He was formerly extension poultry specialist in Kentucky, Tennessee and Missouri and more recently had charge of the administration of the National Poultry Improvement . Plan. Malcolm Lyons (B.S., Kentucky, 1932, M.S., Iowa, 1934) has been transferred from the position of Assistant Chemist in the Kentucky Experiment Station to thr position of Instructor in Poultry Husbandry in the University of Kentucky. Back Issues Wanted We still have a number of unfilled requests for the January 1936 issue of POUL-
TRY SCIENCE. Any person not wanting or needing this issue for permanent use will render a service by sending it to the Editor. The regular price (75c) will be paid for it. We also have a request for the Proceedings of 1917-18 and Proceedings for 191920. Papers for Poultry Science The Editor wishes to call attention to the fact that articles can now be submitted with the assurance of fairly early publication. There are now in hand papers for not more than two more issues.
Erratum The following correction needs to be made in the paper entitled, "Influence of Ovulation Rate on the Tendency of the Fowl to Produce Eggs in Clutches" which appeared in the September 1936 number: Lines 2 and 3 of the first column on page 386 "For this reason the pairs of eggs from the lighted group were sum-" should be followed by the three lines in the first column immediately above the unnumbered table on page 387 as follows: "marized independently of those of the unlighted ones. The results are given as follows: (Unnumbered table on page 387)"
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