spirochete, which, so far as the author is aware, has not been clinical, postmortem, and microbiological examinations had been

Transcription

1 SAPROPHYTIC AND SECONDARY MICROORGANISMS OCCURRING IN THE RESPIRATORY TRACTS OF DOMESTIC FOWLS AND CHICKENS IN HEALTH AND IN DISEASE' CHARLES S. GIBBS Department of Veterinary Science, Massachusetts Agricultural Experiment Station, Amherst, Massachusetts Received for publication, August 7, 1930 This paper is based primarily upon a microbiological study of the larynx, trachea, syrinx, bronchi, and lungs of 56 fowls one year or more of age and 14 chickens about six weeks old. Streaks, from the various portions of the respiratory tract studied, were made on chicken infusion agar, with and without fowl blood, and incubated at 37 C. from twenty-four to forty-eight hours, or until sufficient growth appeared for colony fishing and isolation of bacteria. Most of the micro6rganisms isolated and studied grew best upon chicken infusion agar. In fact only two organisms, both of which proved to be streptococci, were hemophylic. A spirochete, which, so far as the author is aware, has not been reported in the United States before, failed to grow upon any of the culture media used in this study. However, direct smears were made on slides, stained, and studied under the microscope. The birds were divided into the following groups after careful clinical, postmortem, and microbiological examinations had been made: Group I. Infectious trachitis Group II. Pullorum disease... 6 Group III. Chronic laryngitis... 7 Group IV. Avian paralysis Group V. Normal healthy controls Massachusetts Agricultural Experiment Station Contribution No Other names that have been used for this disease are infectious bronchitis, laryngo-trachitis, chicken flu, and Canada flu. 97

2 98 CHARLES S. GIBBS The micro6rganisms isolated from these birds in cultures and in smears fell into groups as follows: Staphylococcus Spirocheta Escherichia Micrococcus... 8 Streptococcus... 5 Eberthella... 5 Alcaligenes... 3 Bacillus... 2 Pseudomonas... 2 Sarcina... 2 Pasteurella... 1 Torula... 1 Saccharomyces...1 INFECTIOUS TRACHITIS According to the preceding general classification, the following microorganisms were isolated from the 42 birds dying of infectious trachitis: Staphylococcus Spirocheta Escherichia Micrococcus... 6 Streptococcus...5 Pseudomonas... 2 Alcaligenes... 2 Eberthella... 2 Bacillus... 2 Pasteurella... 1 Torula... 1 Saccharomyces... 1 On the basis of sub-cultural, morphological, pathological, and chromogenic characteristics, the staphylococci isolated from the birds dying of infectious trachitis were further classified as follows: Staphylococcus albus, Rosenbach Staphylococcus aureus, Rosenbach... 4 Staphylococcus epidermidis, (Welch) Bergey et al... 4 Staphylococcus citreus, Migula... 1 Staphylococcus albus was isolated from 22 of the 42 cases of infectious trachitis, yet in most of the instances studied this

3 SAPROPHYTIC AND SECONDARY MICROORGANISMS organism appeared to be a harmless parasite in the respiratory tract. The most that can be said of it from a pathogenic point of view is that it may have played the role of a secondary invader in acute respiratory diseases of fowls and chickens. In one fowl it was isolated in pure culture from an abscess which had destroyed the right lung, and in six other cases it was present in congestion, inflammation, and pyemia of the lungs of chickens dying of infectious trachitis, complicated with clinical and pathological symptoms of broncho-pneumonia. Staphylococcus aureus, citreus and epidermidis appeared as harmless parasites in the larynx, trachea, and bronchial tubes of nine of the birds studied. In two cases Staphylococcus albus and Staphylococcus aureus were isolated in mixed cultures, but for the most part pure colonies were fished from the plates on which the streaks were made. Spirochetes were found in 21 of the 42 cases of infectious trachitis examined. This was by far the most interesting organism discovered in this study because it was new to the writer. The symptoms of infectious trachitis of fowls resembledbronchospirochetosis of man in the Orient so much that the writer was led to believe for a while that he had discovered the causative agent of infectious trachitis. This organism is a protozoan, closely related to Spirocheta buccalis of higher animals. It was found in the secretions of the beak, throat, pharynx, larynx and trachea of some of the fowls examined. The author first observed it in the trachea of fowls dying of infectious trachitis. The spirochete may have been of some importance as a secondary invader, although it did not penetrate deeper than the epithelium, as the microorganism could not be found in the mucosa, submucosa, or musculature of the larynx, trachea, and bronchi, when stained by Levaditi's technique and examined histologically. The spirochete averaged 2.5,i long and O.1ui wide. The dimensions of the largest spirochete found were 5u long and 1.2u wide. The smallest was 1.5,u long and.08a wide. It was best stained by 2 per cent gentian violet. The smears were air-dried as the microorganism was easily destroyed by heat. It was found that India ink and Romanowsky stains could be used for the demon- 99

4 100 CHARLES S. GIBBS stration of the organism on slides. Two, three, and sometimes four objects resembling vacuoles appeared in smears stained by gentian violet after standing for a month or longer. The spirochetes in old smears degenerated, even when kept in the dark, leaving only faint spiral outlines on the slides. Ap FIG. 1.MICROPHOTOGRAPH OF SPIROCHETES FRom TRACHEA OF DOMESTIc FOWL. DIRECT SMEAR STAINED iby 2 PER CENT GENTIAN VIOLET. MAGNIFIED 1900 DIAMETERS. Th'is spirochete was not primarily pathogenic to fowls and chickens, although it may have been involved in secondary and chronic infections of the respiratory tract, as indicated above. So far as our studies are concerned, this micro6rganism bears no relation to Spirocheta gallinarum which is a blood parasite. The spirochete under discussion was not found in the blood of any of the cases studied. Morphologically, Spirocheta gallinarum is a slender parasite with wavy spirals, while the organism under

5 SAPROPHYTIC AND SECONDARY MICROORGANISMS consideration is comparatively thick and the spirals are acute. The spirochete was found in about half of the adult birds examined, but the other half became carriers after swabbing the larynx and trachea with contaminated secretions. The inoculated birds showed no serious effects from the treatment, although some of them developed chronic laryngitis. FIG. 2. MICROPHOTOGRAPH OF SPIROCHETES FROM TRACHEA OF DOMESTIC FOWL. SAME FIELD AS SHOWN IN FIGURE 1. MAGNIFIED 950 DIAMETERS. The spirochetes are stained by 2 per cent gentian violet. Since the completion of this study, the writer's attention has been called to the fact that tracheal spirochetes were discovered in fowls in Germany (1927). The German species differ somewhat from those described herein. One of the spirochetes described by the German investigators was long, slender, and irregular in regard to spirals, while the other species resembled the one reported in this paper in every respect, except that whip-like flagella were carried at either one or both ends. Flagella were not demonstrated in the spirochetes studied in this investigation. 101

6 102 CHARLES S. GIBBS FIG. 3. MICROPHOTOGRAPH OF SPIROCHETES FROM TRACHEA op DOMESTIC' FOWL, STAINED BY 2 PER CENT GENTIAN VIOLET, AND MAGNIFIED 1900 DIAMETERS. The spirochete in the center has degenerated, leaving a halo of its former outline. FIG. 4. MICROPHOTOGRAPH OF EPITHELIAL CELL FROM TRACHEA OF DOMEsTIC FOWL SHOWING SPIROCHETES WITHIN. MAGNIFIED 1900 DIAMETERS. GENTIAN VIOLET STAIN.

7 SAPROPHYTIC AND SECONDARY MICROORGANISMS Escherichia were found in 12 of the fowls and 2 of the chickens dying from infectious trachitis. These organisms were classified as follows, according to Bergey's Manual of Determinative Bacteriology: Escherichia communior (Durham) Bergey et al... 6 Escherichia coli (Escherich) Castellani and Chambers... 5 Escherichia gastrica (Ford) Bergey et al... 1 Escherichia alkalescens (Ford) Bergey et al... 1 Escherichia pseudocoloide8, Castellani and Chalmers... 1 All of these micro6rganisms normally occur in the alimentary canal of domestic fowls.. It is quite likely that many of them found their way into the respiratory tract from the alimentary canal after inflammation had set in and the ciliated epithelium had been destroyed. While these micro6rganisms did not appear to be directly involved in the inflammatory processes taking place in infectious trachitis, yet they were often found in great numbers in the secretions of the trachea during the febrile stages of the disease. Six cultures of micrococci were isolated from four fowls and one chicken dead of infectious trachitis. These cultures fell into two unidentified groups, as follows: Group I consisted of four cultures of Gram-negative spheres occurring in pairs. The colonies on chicken infusion'agar were isolated, small, circular, grayish white, glistening, and slightly raised in the centers. Gelatin was not liquefied. A turbid opalescent growth appeared in plain broth after twenty-four hours' incubation at 37 C. Acid was slowly formed in litmus milk. The organism curdled milk after three days' growth. Acid was produced in glucose, lactose, sucrose, maltose, and mannitol. The micrococci were aerobic and non-pathogenic for fowls and chickens. Group II consisted of two cultures. The organisms in this group resembled those in Group I, except that both acid and gas were formed in glucose, lactose, maltose, and mannitol. Also, sucrose and dulcitol were made alkaline at first, later turning acid and producing gas. Five cultures of streptococci were isolated in this study from 103

8 104 CHARLES S. GIBBS fowls dying of infectious trachitis. These cultures also fell into two groups when classified according to morphological, physiological, and pathological characteristics. The first group, consisting of three cultures, produced long chains and was neither hemophilic nor hemolytic. The other group, consisting of two cultures, grew best on chicken infusion blood agar and was decidedly hemolytic. While these micro6rganisms were not serologically typed, they were divided into two groups as stated above. The first group of microdrganisms was not named, because it did not correspond to any of the types given in Bergey's Manual of Determinative Bacteriology for the classification of streptococci, but the characteristics of the second group were like those of Streptococcus pyogenes Rosenbach. So far as could be determined by these experiments, the three cultures classified as non-hemolytic streptococci were harmless saprophytes living on the mucous membrane of the respiratory tract of the domestic fowls studied. But the hemolytic strains may have been involved in the hemorrhagic conditions existing in the larynx and trachea of the fowls, from which they were isolated. Two trachitis cases of long standing yielded pure cultures of Pseudomonas aeruginosa (Schr6ter) Migula from green pus forming pockets in the larynx just below the rima glottis. In these cases Pseudomonas aeruginosa was quite likely a secondary invader, as the swabbing of the trachea of healthy birds did not lead to inflammation or disease. In this study two cultures resembling Alcaligenes bronchisepticus (Ferry) Bergey et al., freshly isolated from the trachea of birds dying of infectious trachitis, did not prove to be pathogenic to healthy fowls and chickens when inoculated intratracheally. The identity of these micro6rganisms was not definitely established by animal inoculation. They grew abundantly on chicken infusion agar under aerobic conditions, which differentiates them quite readily from Alcaligenes abortus (Bang) Bergey et al., which has been reported as isolated from the domestic fowl (1929). A culture resembling Pasteurella avicida (Kitt) Trevisan, morphologically and subculturally, was isolated from the trachea of one of the birds dying of infectious trachitis. This culture was

9 SAPROPHYTIC AND SECONDARY MICROORGANISMS not pathogenic for hens and chickens when inoculated by tracheal swab or hypodermically, or when fed in the drinking water. However, this may have been a culture of the fowl cholera organism which had so lost its virulence that several continuous passages through birds would have been necessary in order to restore its pathogenicity. Since the writer was interested only in the possible relation of these organisms to infectious trachitis, the animal passages were not continued beyond the third transfer. Two cultures, one from a fowl and another from a chicken dying of infectious trachitis, were classified as Eberthella septicemia (Riemer) Bergey et al., although the biochemical reactions did not correspond exactly with the descriptions given for this microorganism in Bergey's Manual of Determinative Bacteriology. According to Bergey's Manual, Eberthella septicemia produces acid in glucose only. The organism described herein, as Eberthella septicemia, formed acid in both glucose and maltose. However, the cultures were not pathogenic to either fowls or chickens. So far as could be determined, these organisms were not involved in the inflammatory conditions existing in the disease under investigation. The two spore-forming cultures isolated from the trachea of fowls dying from infectious trachitis belonged to the subtilis group. They occurred either as harmless saprophytes in the birds studied or as contaminations in the culture media used in these experiments. A torula and a yeast culture were isolated from the trachea of two of the fowls dying from infectious trachitis. The torula culture produced acid in glucose, lactose, sucrose, and maltose. The yeast or saccharomyces culture formed acid and gas in glucose and maltose and slight acid in sucrose. These microorganisms were not pathogenic for fowls. It is quite likely that they got into the trachea from the crop, via esophagus, pharynx, and larynx during a spasm of coughing, vomiting, and gasping for breath during the dyspneic stage of infectious trachitis. PULLORUIM DISEASE The microbiological flora of the respiratory tract of six fowls having chronic focal infections of pullorum disease were included 105

10 106 CHARLES S. GIBBS in these experiments for comparative study. The following cultures were isolated from these fowls at autopsy: Escherichia communior (Durham) Bergey et al... 3 Escherichia coli (Escherich) Castellani and Chalmers... 2 Sarcina, unidentified species... 1 All of these cultures, except the sarcina, were the same as those found in birds having infectious trachitis and need no further discussion here. However, it is pertinent to point out that Salmonella pullora was not isolated from the respiratory tracts of the birds studied in this investigation. The sarcina was not pathogenic to domestic fowls and chickens. CHRONIC LARYNGITIS AVIAN PARALYSIS Five of the birds selected for this study were suffering from avian paralysis. The following cultures and smears were studied from this group: Spirocheta... 2 Eberthella bentolensis, Castellani and Chalmers... 2 Staphylococcus albu, Rosenbach... 1 Seven cases of chronic laryngitis were studied. The spirochete described above was found in the secretions of the larynx of all of these birds. Cultures of Staphylococcus albus were isolated from four of the fowls, while two others had contagiouis epithelioma. These micro6rganisms may have been responsible for the chronic condition existing in the larynx, although it is doubtful if they had anything directly to do with the chicken-pox. The spirochete and the culture of Staphylococcus albus were the same as those previously described in this paper and need no further discussion. However, the species of Eberthella differed from the microorganisms found in any of the other birds. Moreover, it is not likely that Eberthella bentolensis had anything to do with the paralysis of these birds, as it appeared to be innocuous to healthy fowls and chickens over a period of six months.

11 SAPROPHYTIC AND SECONDARY MICROORGANISMS.107 NORMAL HEALTHY CONTROLS Ten fowls were kept as healthy controls. Seven of these birds were found to be carriers of micro6rganisms in their respiratory tracts. The remaining three seemed to be free from such parasites as would grow on the culture media or stain with the dyes in direct smears used in these experiments. The following cultures and smears were secured from the control birds: Staphylococcus albus, Rosenbach... 4 Spirocheta : 3 MicrococCU, unidentified species... 2 Escherichia communior (Durham) Bergey et al... 1 Alcaligene8 bronchisepticus (Ferry) Bergey et al... 1 Eberthella septicemia, Bergey et al... 1 Sarcina lutea, Schroter... 1 The Staphylococcus, Micrococcus, Escherichia, Alcaligenes, and Eberthella cultures were the same morphologically and subculturally as have been described already in this paper under infectious trachitis. Sarcina lutea did not occur in any of the diseased birds included in this investigation. It was found in only one of the controls. There was nothing to indicate that this micro6rganism was in any way pathogenic to fowls. SUMMARY 1. Microbiological cultures and smears were made from the larynx, trachea, syrinx, bronchi, and lungs of 56 domestic fowls and 14 chickens. Forty-two of the birds had died of infectious trachitis, six had pullorum infection, seven were suffering from chronic laryngitis, five were affected with avian paralysis, and ten were kept as healthy controls. 2. Staphylococci were isolated from the respiratory tracts of per cent of tl.e birds having trachitis, per cent of those suffering from chronic laryngitis, and per cent of the healthy controls. 3. Spirochetes were found in 50 per cent of the birds dying of infectious trachitis, 100 per cent of the birds having chronic laryngitis, 40 per cent of tlhe fowls suffering from avian paralysis, and 30 per cent of the healthy fowls and chickens.

12 108 CHARLES S. GIBBS 4. Escherichia of intestinal origin were found in the respiratory tracts of per cent of the birds having infectious trachitis, and per cent of the pullorum infected fowls. Only 10 per cent of the normal controls were found harboring Escherichia in the respiratory tract. 5. Alcaligenes cultures were isolated from 4.76 per cent of the trachea of fowls affected with infectious trachitis and 10 per cent of the control birds. 6. Two cultures of hemolytic streptococci were isolated from the hemorrhagic inflammation of the larynx and trachea of two types of so-called bloody infectious trachitis in fowls. 7. Two cultures of Pseudomonas aeruginosa were isolated from pus pockets in the larynx of two birds having prolonged cases of infectious trachitis. 8. A culture resembling Pasteurella avicida morphologically and sub-culturally was isolated from the trachea of one fowl dying from infectious trachitis. The culture might have lost its virulence for it did not produce fowl cholera in either fowls or chickens. 9. In addition to the above, Micrococcus, Bacillus, Eberthella, Sarcina, and one culture each of Torula and Saccharomyces were isolated from the respiratory tracts of the birds studied in this investigation. These cultures were not pathogenic to any of the fowls and chickens tested. CONCLUSION The microbiological flora of the respiratory tract of domestic fowls and chickens is varied in health and in disease. The number and variety of microorganisms are increased in acute respiratory diseases. This increase in microbial flora may be due to the destruction of the ciliated epithelium of the mucous membrane and the increase in secretions and, other inflammatory products which are utilized as food by the invader. While this investigation did not produce sufficient etiological evidence to implicate any of the microorganisms isolated and studied herein as the primary cause of infectious trachitis, nevertheless, pathological indications demonstrated Staphylococcus albus, Streptococcus pyogenes, Pseudomonas aeruginosa, and a spirochete, n. sp., as important secondary invaders.

13 SAPROPHYTIC AND SECONDARY MICROORGANISMS 109 ACKNOWLEDGMENT 'The microphotographs of the spirochetes described in this article are due to the kindly assistance of Dr. William H. Davis of the Department of Botany, Massachusetts Agricultural College. REFERENCES EMMEL, M. W., AND HUDDLESON, I. F J. A. V. M. A. (75), N.S., 28, 578. KRAGE, P., UND WEISGERBER, F Centralb. f. Bakt., Parasit. und Infectionsk., I Abt., 102, 60. Downloaded from on January 22, 2019 by guest