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A Study of Roup in the Domesticated Fowl from the Standpoint of Etiology
A STUDY OF ROUP IN THE DOMESTICATED FOWL FROM THE STANDPOINT OF ETIOLOGY C. H. WEAVER Λ.\ΐ) C. A. MITCHELL Canadian Department of Agriculture
1 MooKE, VEKANUS A. A Preliminary Investigation of Diphtheria in Fowls. Bulletin No. 8, U. S. Dept. of Agri., B. A. I. 1895. 2 HABBISON AND STUKIT. Roup. Bulletin 125, Ont. Agri. College 1902. 9
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Moore^ in 1895 investigated Avian d i p h t h e r i a or so called Roup for the United States B u r e a u of A n i m a l I n d u s t r y , and gave a comprehensive p a p e r on the disease as a p p e a r i n g on this continent. I t was shown t h a t r o u p was then widespread, of considerable economic importance, and, while seemingly com municable in n a t u r e was, nevertheless influenced by the en vironment to which the birds were subjected. A n organism, con stantly associated with the disease process, was isolated and found to be pathogenic for laboratory animals, but the disease was not reproduced in fowls although inoculations were made. F r o m this investigation Moore concluded in p a r t as follows :— T h a t " R o u p , diphtheria and influenza resemble each other so closely in their manifestations t h a t they may be considered as belonging to one a n d t h e same disease. The same species of pathogenic bacillus is associated with a p p a r e n t l y different forms or lesions. This bacillus simulates very closely the fowl cholera organism a n d the bacillus of swine plague. The lesions are usually diphtheritic in n a t u r e , but in the beginning are serous or muco-purulent ; the chronic n a t u r e of the disease p e r m i t t i n g more or less modification of the lesions due to introduction of secondary o r g a n i s m s . " I n 1902 H a r r i s o n & Streit- reported on a series of experiments covering four y e a r s ' work in which a bacillus was isolated with M'hich the disease, with all its varied symptoms, Avas reproduced in fowls. The same article states of the disease having been produced by inoculation of other organisms, a n d conclusions are d r a w n to the effect t h a t roup, fowl diphtheria and canker is a complex of s u p p u r a t i v e processes t a k i n g place especially in the head of
10
POULTRY
SCIENCE
ROUP .\S IT OCCURS I N CAN.\U.V
Description of the Disease Roup, in t h e common acceptance of the term, is a disease be ginning as a c a t a r r h a l condition, often associated \vith coughing, 3 SwEKT, CLIFFOBO D. A Study o£ Epithelioma Contagiosum of the Common Fowl. U. of California publication in Zoology, Vol. II, No, 3, Jan. 30, 1913. *WARD AND GALLAGIIEK. Diseases of Domesticated Birds. 5 HAHISG AND KOFOID. Observations Concerning the Pathology of Roup and Chicken-pox. Am. Vet. Rev. Vol. XL, No. 2, 1911-12. 0 HADLEY AND BEACH. Controlling Chicken Pox, Sorehead or Con tagious Epithelioma by Vaccination. Am. Vet. Rev. Vol. XLIV, No. 2, 1913-14.
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fowls. This s u p p u r a t i o n may be caused by different species of bacteria. While investigating epithelioma contagiosum in 1913 Sweet" also a r r a n g e d his studies to determine whether or not a relation ship existed between the disease and roup, a n d he concluded therefrom t h a t the two diseases Λyere entirely independent from each other. W a r d a n d Gallagher^ in their text-book t r e a t m e n t of avian diphtheria and bird pox, characterize the two as internal and external respectively manifested forms of the one disease. Both Avere considered as being due to the filterable virus of cliickenpox. A few p a r a g r a p h s are devoted to the bacteriology of avian diphtheria. H e r e it is pointed out t h a t many bacteriological in vestigations were m a d e previous to the recognition of the role of pox virus in causing diphtheritic lesions. They go on to say t h a t reports of investigations conducted more recently do not contain evidence that pox virus was not concerned in the causa tion of the lesions. Mention is made, however, of the finding of a member of the hemorrhagic septicemia group in diphtheritic lesions in fowls, which observation has been confirmed by a large number of investigators. H a r i n g and Kofoid" r e p o r t i n g upon their observations concern ing the pathology of r o u p a n d chicken pox state t h a t i m m u n i t y to pox does not confer immunity to r o u p and vice versa. Hadley a n d Beach" conclude t h a t it is logical to presume t h a t chicken jwx and roup are the same disease.
ETIOLOGY
Of ROUP
H
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sneezing and noisy breathing. A yellowisli colored caseous mass forms in the nostrils, orbital cavity, sinuses of the head and larynx, and a similar appearing- membrane may appear on the floor of the mouth, or tongue, which Avhen removed leaves a raw bleeding surface. Canker, diphtheria, diplitheritic roup or roup are various terms given to the disease according- as some particular symptom is more prominent. It is more properly characterized as an infectious disease the lesions of which first appear on the mucosa of the nasal passages, the eyes, the mouth, the pharynx and larynx, and may extend to the trachea, bronchi, the airsacs, the intestines and possibly to other abdominal organs. The serous discharge, earlj' associated Avitli tlie inflamed membranes, soon becomes flecked with mucous particles, which gradually increase until the substance becomes entirely mucous in character. This is followed by the formation of caseous yellowish colored masses or membranes according to the location of the disease process. Although the disease commences with a marked suddenness of onset, it, nevertheless, is usually chronic in its course. Average cases under normal conditions will occupy approximately a month's time to recover, about one week of which is required for convalesence. Deviations from the normal course are frequent, both among- individuals comprising a flock unit, and between separate flocks. Extremes are found on the one hand wliere birds appearing- normal at night arc found dead in the morning, the larjmx being filled with exudate causing- death bj- suffocation, and on the other where local lesions make very little if any change for the better, the case dragging on for weeks or even months. A pneumonic form is seen from time to time. With it tiie course is more acute and accompanied by high mortality. As the outbreak begins to wane the lung lesions become less in evidence and the disease settles down to cases with lesions of the usual form. Constitutional disturbances are quite pronounced. Digestive derangement is the rule. The evacuations become more fluid in consistency and take on a greenish color. Weakness, prostra-
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POULTRY
SCIElfCE
Economic Importance The main financial loss incurred from this disease is not due so much to that of wasted flesh through deaths, great as that may be at times, but is the result of shrinkage in production. An indication of this loss is afforded by the following examples : A pen of Single Comb White Leghorn pullets of a given strain, was divided into two pens on November 22, and are designated pens A and B. By the end of the month roup made its appear ance in pen A and increased in virulence throughout the month of December. I t remained at its height during the following two months, and subsided again in March. Pen Β remained free of the disease. There is a marked difference in the production Λ'3ΐυβ8 of these two pens accountable for only by the presence of roup in the one pen and absence of the disease in the other. Egg values for pen A per bird for the three months of December, January and February equal 70 cents. Pen Β egg values per bird for the corresponding period total $2.50 or a difference in favor of the latter of $1.80. For the six Λvinter months the production values are $3.12 and $5.38 respectively or a difference of $2.26 per bird in favor of pen B. Prevalence In geographical distribution roup has become quite general, but it appears more in evidence in sections Avhere poultry farm ing occupies a relativelj^ large part of the agricultural activities. Thus in sections adjacent to the larger urban centers or where henneries thrive for other reasons one may expect to find the disease most prevalent. During the winter season, when weather is changeable or broken, particularly at the beginning or ending of the season,
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tion or a comatose state are symptoms which appear early in the course of the disease. When permanent relief comes early the subject soon revives from the lethargic condition, convalesence being followed by a favorable termination. Often, however, relief is but temporary and is followed by one or more relapses. In this course of the disease convalesence is prolonged if recoverj' takes place, but frequently the bird dies in an emaciated condition.
ETIOLOGY
OF ROUP
J3
ISOLATION OF ORGANISM
Because of the constancy in which the bipolar organism alread}^ mentioned had been found in roup lesions, attempts Avere made to isolate this bacillus. The first attempts were made with material from open lesions but the media Avas over-grown by a great number of different saprophytes. At last a lesion from a case of pneumonic roup Avas cultured, and from this we procured our first strain of the bipolar bacillus. This strain was injected into an adult fowl (407) intraperitoneally but produced no ill effects except a transient sickness. A small quantity of caseous material rich in bipolar organisms was taken from an orbital lesion and injected into the peritoneal cavity of a rabbit. A rapid infection folloAved and death took place in forty-eight hours. A pure culture of the bipolar organism was isolated from peritoneal fluid and blood of this rabbit. Third generation of this culture was found pathogenic for a pigeon and fowls (591, 627 and 305). The organism was recovered direct from lesion on breast of pigeon by cultural methods and from orbital cavity of fowls through rabbit inoculation. A rabbit (222) was infected with material from a natural roup case. It died in eighteen hours and a pure culture was isolated from peritoneal fluid. A small quantity of agar culture from the third generation
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the disease is most prevalent. TJie summer sees an abatement with almost if not total disappearance of visible or clinical cases. We may, therefoi'e, say that tlie prevalence of roup is associated with climatic conditions. Bacterial Flora of Lesions In the majority of instances specimens taken from the lesions show a varied bacterial flora. A gram negative bipolar bacillus is always in evidence, and this is accompanied by a multitude of different organisms such as colon bacilli, staphlococci, molds, etc. But in the lesions which we more seldom encounter, that is those protected from outside influence, and therefore, not contaipinated by various organisms, we find but one type of microorganism—the gram negative bipolar bacillus.
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FOUhTRY
SCIKKCE
was inoculated into the s\ibeoiijuiictiva of a fowl ((,' l o i — F i g . 1 ) producing symijtoms identical with those of rou]). On the tliird dav after infection cultures were made direct from the e\-e.
The organism was found to be in pure culture, and was grown on agar for two genei'atioiis, then iiiocidated into fowl (5203). This bird became infected with roup. REPRODUCTION OP TiiK DISEASE The inoculation of adult fowls with p u r e cultures of a bacterial organism isolated from n a t u r a l l y infected roup cases, had been productiA'c in the inoculated birds of a disease M'ith general systemic disturbances and local lesions indistinguishable from those of roup. A culture of the organism when f(Hl to young fowl (chicks) has piroduced a like result. FIRST ATTEMPT RESTM/rED IN A FAILURE Experiment N^o. 1 On J a n . 25th, 192;}, a cockerel (No. 407) was inoculated iutraperitoneally Avith .5 cc. of a broth culture isolated from a case of pneumonic r o u p . F o u r da.ys later the bird showed symptoms of ])eritonitis Avhich gradua]l.y subsided. Feb. 8tli the bird Avhile appearing normal was killed and aii1oi)sied, all organs being found in a normal state. Experiment No. 2 A series of six intra])eritoneal inoculations were then made
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Fio. 1
Specimen numbei· C151. Inoculated December 14, 1928. Culture, bacillary saline-emulsion. Died January 2, 1924. Photographed December 27, 192 3. Organism recovered direct fronv lesion involving the left orbital region—the point ot inoculation.
ETIOLOdY OF h'OlP
15
A culture was isolated from material taken from a roup case of n a t u r a l infection, through rabbit inoculation. A pigeon (No. 15) was then inoculated, the resulting peritoneal exudate being used as an inoculum for 9 chicks; all succumbed while controls, 10 in muiiber. remained in u'ood health. The same
Fio. 2
Subject number 616. Inoculated on May 9, 1923, with peritoneal exudate from an experimentally infected pigeon. Died May 16, 1923. Photographed May 14, 1923. See also Fig. 3. exudate was inoculated into a pigeon ( x ) , the p r o d u c t from the lesions of Avhich, and a subsequent transfer from the original culture, (bacillary saline emulsion), were inoculated into 5
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upon adult fowl with roupy products. Almost or completely negative results followed, Avith one exception. No. 70, a cock bird was given 3 ec. of a heavy suspension of tracheal exudate on March 12th, 1923, a n d succumbed on March 23rd, 1923. Period of incubation in this instance was five days (appeai'ance of first symptoms). The following autopsy was recorded: Adhesions and lieavy caseous membrane on peritoneum a t point of inoculation. Peritonitis of a general distribution. A b u n d a n t exudate at point of inoculation. Air sac on inoculated side filled with caseous material. Eight lung patchy consolidated areas ; left coinpetelj' solidified Avith plugging of bronchus, extending u p into trachea. 10 cc. of straw colored fluid in pericardial sac. L a r y n x , mouth anti other head p a r t s nornud. Experiment No. 3
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POULTRY
HGIENCE
yearling hens. Three of the birds, (Nos. 591, 627 and 305), receiA'ed the culture, the remaining two, (Nos. 616 and 301), being inoculated with the pigeon peritoneal exudate. All five suffered Λ\'ith roup typical in appearance of natural cases. One bird. No. 616, succumbed seven days following inoculation. See Fig. 2.
Inoculations were made on the evening of May 9th, 1923, and the two folloAving mornings imperfectly formed eggs Λvere found on the dropping boards. Except for these production ceased entirely and was not resumed for three weeks. One bird Avas not laying previous to or during the experiment. The period of incubation was the same in both lots A and B, viz. 12 hours. The second day after inoculation the following observations were made on the location and extent of the lesions, and the general condition of the birds. lîilMi's Nuililier
l.osion e.vteut and IjOcation
(Ifiioi-nl Conditiuii
591 627 305 616 301
** Eye *** Bye * Mouth *** E y e * Mouth
Fair Very weak Huddled, abdomen tense Prostrated Fair
Diarrhoea Avas present in all eases, the excrement being of a greenish-yellow color. A culture of the organism Avas recovered from the eye lesion of No. 627, and was isolated through a rabbit. The disease was transmitted from No. 627 to tliree other birds (Nos. 2812, 2898 and 2591), by swabbing the aff'eeted eye and smearing an eye of each of the other three. In all cases the disease developed in the eye that was swabbed. Two of the birds recovered while the third, No. 2812, succumbed to the disease. Lesions developed in 14 days. No. 2898 suft'ered a mild attack. Controls Nos. 1297, 2099 and 2768 remained healthy.
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lNO
MTIOLOdY OF ROVP
\-J
Experiment No. 4 A new culture was isolated from roup exudate by rabbit inoculation and on Sept. 11th, 1923, three adnlt birds were inoculated Avith a broth culture as follows : Aiiiminl; of C\lltni*e .25 cc .25 cc .50 cc
P o i n t of InoiMilatioii
Mucosa of eye lid Wing vein Roof of mouth—submucosa I n all tliree birds the results were negative, due to death of the organisms in the culture. This Avas proved by transfers from tlic l)roth culture to new media, tlie tubes remaining f»'ee
FJ(!. 3 Specimen number 7 9 6. Inoculated with culture September 14, 1923. Died October 23, 1923. Photographed October 16, 192 3. The left eye is destroyed, the cavity being filled with a caseous exudate. Subject emaciated and very weak. Note smearing of hackle.
of growth, and by rabbit inoculation. Rabbit No. 414 was given .25 cc. intravenously and suffered no ill effects. A bacillarj' saline emulsion was t h e n made from the a g a r c u l t u r e from Avliich the broth culture h a d been made and .25 cc. was introduced into the conjunctiva of an adult foAvl No.
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HirtVs Nuiubor 790 792 794
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POULTRY
SCIENCE.
Tlie disease became chronic in its course and the subject suc cumbed on Oct. 23rd, 1923. A u t o p s y — E x t r e m e emaciation, organs iioruial in appearance except for the effects of the general wasted condition. Left eye destroyed and orbital cavity filled Λvith caseous material. Body weight 625 grams. See F i g . 3. Experiment
No. 5
This experiment Avas conducted to determine the effects of the organism Avhen fed to young chicks. One h u n d r e d and eighteen chicks of one Aveek of age were divided into ΙΛΥΟ equal batelies, a n d Avere maintained u n d e r identical conditions as to housing a n d feeding. Outside r u n s were available p a r t of the time and d u r i n g the intervening intervals lawn clippings, with a high clover leaf content, M'ere used for green feed. Symptoms or autopsj' findings attributable to avitainosis Avere never in evidence throughout the experiment. These are designated pen " Λ " and pen " ' B " ' culture feedingexperiment. P e n A were given once only on Oct. 2, 1923, a broth culture p a r t of which Avas incorporated in a moist mash, tlie remainder being added to the drinking water. ΤΛΥΟ days following, the chicks appeared less active, and took their food sparingly. This condition persisted and as a result groAvth slackened in some and ceased in others, tlie lot assuming a general u n t h r i f t y appearance. On Oct. 11th the first death occurred, from u n k n o w n cause, and two days later definite symptoms of r o u p appeared a n d fatalities occurred in r a p i d succession. The course of the disease reached its peak on the 16th a n d 17th, gradually subsided and cleared u p by the end of the month. Mortality 33.9%.
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796. Inoculation Avas made Sept. 14, 1923, a t 11:30 a. m. antl when examined the next m o r n i n g at 7 a. m. the following results were n o t e d : t e m p e r a t u r e 43° C. E y e into which culture was given was swollen shut and lids glued together. Mouth, throat and other eye appeared noi'mal, except t h a t the swelling about the affected eye p a r t i a l l y closes the passages from t h a t side. Marked systemic disturbances, depression, and diarrlioea.
ETIOLOGY OF ROUP
I9
The organism was recovered from these chicks by both direct culture methods a n d t h r o u g h animal passage. P e n Β chicks remained free of the disease and made satis factory gains, Avith a moderately normal mortality of 8.4%. Sec F i g u r e 4. Experiment
No. 6
Experiment
No. 7
This experiment was made to test the patliogenicity of the bi])o]ar organism on rabbits. A n agar culture (fourth generation) was washed off with suiïieient normal saline, to give to the resultant suspension, faintly opaque appearance. 0.5 cc. of this suspension Avas given intraperitoneally to each of two rabbits, and 0.5 Avas given subcutaneously to each of two rabbits. All rabbits showed illness Avithin twelve hours. Those inoculated subcutaneouslj" showed considerable local disturbance at point of inoculation. The two rabbits inoculated intraperitoneally died within tAventy-four hours a n d cultures made from heart blood a n d peritoneal fluid showed the organism in p u r e culture. One of the rabbits inoculated subcutaneously died Avithin three days. T h i s rabbit shoAved a p u r e culture in blood, and at the point of inoculation.
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The organism was again isolated from a n a t u r a l roup case and the disease reproduced in an adult foAvl No. C 1.51, from the lesions of which the organism Avas recovered, cultured in p u r e state on artificial media, reinoeulated into a fowl No. 5203 again producing the disease. Inoculation for comparative results Avere made with patho genic strains of Staphlococcus, Bacillus Welchii and an in.iection with spirits of tui'pentine on adult foAvls, numbers Π 946, 6241 and Η 990, respectively. The cultures a n d chemical Avere in jected into the submucosa of an eyelid in the same m a n n e r as the organism isolated from roup lesions and with Avhich the disease was reproduced. Reactions folloAved injections Avhich Avere local in cliaracter, unaccompanied by systemic disturbances and having no simi larity to the disease roup.
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POULTRY
SCIENCE
R i g h t eye
PKX " B " CiiKK N o r m a l d e v e l o p m e n t — B o d y g r o w t h , size of legs, comb a n d w a t tles and clear b r i g h t eye. Pio. 4 Chicks (rom C u l t u r e F e e d i n g E.xperiment.
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Ρκ.Ν " A " Ciuc'K Underdeveloped a n d e m a c i a t e d , b a r e l y able to s t a n d . closed a n d filled "with caseous e x u d a t e .
E'VIOLOGY
OF ROUP
21
The fourth rabbit after a rather stormy illness of three weeks duration recovered. T H E ORGANISM
BIOLOGICAL CHAEACTEES
It is a bacillus measuring approximately .5 microns in length by .25 microns in Avidth, having a tendeiiej!- to bipolar staining especially in the tissues. In culture it is exceedingly pleomorI)hic and frequently assumes oval and coccoid forms. It does not form spores. Stains with the ordinary aniline dyes and does not retain the Gram stain. It produces a slight amount of Indol (Salkonski-Kilasato method). Nitrates are not reduced. Aerobic and facultative anaerobic. Optimum temperature 37° C. Litmus milk is not altered. Agar : Growth comes up as dew-drop like colonies which are at first discreet and show little tendency to spread. In older generations cultures take on a white color and lose to some extent their transparency, but there always remains a wax-like look whether recently isolated or in older cultures. Tlie growth is exceedingly sticky and when the inoculation loop is touched to a colony it will string out. Broth: Growth on broth is rather characteristic. If the media is inoculated and not disturbed the organism floats in small islands on the surface, and through the media will be noticed groups of bacteria which give to it the appearance of a positiA^e agglutination reaction. After two or three days the (Continued on page 24)
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As pointed out earlier in this paper it is imiDossible to isolate tliis organism from an open lesion by ordinary ctiltural methods. This is what one Avould expect, when the number of contaminating organisms that are scratched into the eye by a diseased bird is considered. On the other hand if a bird is secured in the early stages of the disease, Avhile the lids are still adhered together and consequently protected against outside influence, the securing of pure cultures is a comparatively simple matter. Experience has taught us that the simplest method for the obtaining of pure cultures is to inoculate a rabbit intraperitoneally with a small quantity of the caseous material taken from tlie eye of an affected bird. The rabbit rapidly dcA'clops an acute infection, and dies usually within twentj'-four hours. Dilute agar plates planted from the blood and peritoneal fluid of the rabbit usually give pure cultures of the organism.
1 MooKE, VEKANUS A. A Preliminary Investigation of Diphtheria in Fowls. Bulletin No. 8, U. S. Dept. of Agri., B. A. I. 1895. 2 HABBISON AND STUKIT. Roup. Bulletin 125, Ont. Agri. College 1902. 9
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Moore^ in 1895 investigated Avian d i p h t h e r i a or so called Roup for the United States B u r e a u of A n i m a l I n d u s t r y , and gave a comprehensive p a p e r on the disease as a p p e a r i n g on this continent. I t was shown t h a t r o u p was then widespread, of considerable economic importance, and, while seemingly com municable in n a t u r e was, nevertheless influenced by the en vironment to which the birds were subjected. A n organism, con stantly associated with the disease process, was isolated and found to be pathogenic for laboratory animals, but the disease was not reproduced in fowls although inoculations were made. F r o m this investigation Moore concluded in p a r t as follows :— T h a t " R o u p , diphtheria and influenza resemble each other so closely in their manifestations t h a t they may be considered as belonging to one a n d t h e same disease. The same species of pathogenic bacillus is associated with a p p a r e n t l y different forms or lesions. This bacillus simulates very closely the fowl cholera organism a n d the bacillus of swine plague. The lesions are usually diphtheritic in n a t u r e , but in the beginning are serous or muco-purulent ; the chronic n a t u r e of the disease p e r m i t t i n g more or less modification of the lesions due to introduction of secondary o r g a n i s m s . " I n 1902 H a r r i s o n & Streit- reported on a series of experiments covering four y e a r s ' work in which a bacillus was isolated with M'hich the disease, with all its varied symptoms, Avas reproduced in fowls. The same article states of the disease having been produced by inoculation of other organisms, a n d conclusions are d r a w n to the effect t h a t roup, fowl diphtheria and canker is a complex of s u p p u r a t i v e processes t a k i n g place especially in the head of
10
POULTRY
SCIENCE
ROUP .\S IT OCCURS I N CAN.\U.V
Description of the Disease Roup, in t h e common acceptance of the term, is a disease be ginning as a c a t a r r h a l condition, often associated \vith coughing, 3 SwEKT, CLIFFOBO D. A Study o£ Epithelioma Contagiosum of the Common Fowl. U. of California publication in Zoology, Vol. II, No, 3, Jan. 30, 1913. *WARD AND GALLAGIIEK. Diseases of Domesticated Birds. 5 HAHISG AND KOFOID. Observations Concerning the Pathology of Roup and Chicken-pox. Am. Vet. Rev. Vol. XL, No. 2, 1911-12. 0 HADLEY AND BEACH. Controlling Chicken Pox, Sorehead or Con tagious Epithelioma by Vaccination. Am. Vet. Rev. Vol. XLIV, No. 2, 1913-14.
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fowls. This s u p p u r a t i o n may be caused by different species of bacteria. While investigating epithelioma contagiosum in 1913 Sweet" also a r r a n g e d his studies to determine whether or not a relation ship existed between the disease and roup, a n d he concluded therefrom t h a t the two diseases Λyere entirely independent from each other. W a r d a n d Gallagher^ in their text-book t r e a t m e n t of avian diphtheria and bird pox, characterize the two as internal and external respectively manifested forms of the one disease. Both Avere considered as being due to the filterable virus of cliickenpox. A few p a r a g r a p h s are devoted to the bacteriology of avian diphtheria. H e r e it is pointed out t h a t many bacteriological in vestigations were m a d e previous to the recognition of the role of pox virus in causing diphtheritic lesions. They go on to say t h a t reports of investigations conducted more recently do not contain evidence that pox virus was not concerned in the causa tion of the lesions. Mention is made, however, of the finding of a member of the hemorrhagic septicemia group in diphtheritic lesions in fowls, which observation has been confirmed by a large number of investigators. H a r i n g and Kofoid" r e p o r t i n g upon their observations concern ing the pathology of r o u p a n d chicken pox state t h a t i m m u n i t y to pox does not confer immunity to r o u p and vice versa. Hadley a n d Beach" conclude t h a t it is logical to presume t h a t chicken jwx and roup are the same disease.
ETIOLOGY
Of ROUP
H
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sneezing and noisy breathing. A yellowisli colored caseous mass forms in the nostrils, orbital cavity, sinuses of the head and larynx, and a similar appearing- membrane may appear on the floor of the mouth, or tongue, which Avhen removed leaves a raw bleeding surface. Canker, diphtheria, diplitheritic roup or roup are various terms given to the disease according- as some particular symptom is more prominent. It is more properly characterized as an infectious disease the lesions of which first appear on the mucosa of the nasal passages, the eyes, the mouth, the pharynx and larynx, and may extend to the trachea, bronchi, the airsacs, the intestines and possibly to other abdominal organs. The serous discharge, earlj' associated Avitli tlie inflamed membranes, soon becomes flecked with mucous particles, which gradually increase until the substance becomes entirely mucous in character. This is followed by the formation of caseous yellowish colored masses or membranes according to the location of the disease process. Although the disease commences with a marked suddenness of onset, it, nevertheless, is usually chronic in its course. Average cases under normal conditions will occupy approximately a month's time to recover, about one week of which is required for convalesence. Deviations from the normal course are frequent, both among- individuals comprising a flock unit, and between separate flocks. Extremes are found on the one hand wliere birds appearing- normal at night arc found dead in the morning, the larjmx being filled with exudate causing- death bj- suffocation, and on the other where local lesions make very little if any change for the better, the case dragging on for weeks or even months. A pneumonic form is seen from time to time. With it tiie course is more acute and accompanied by high mortality. As the outbreak begins to wane the lung lesions become less in evidence and the disease settles down to cases with lesions of the usual form. Constitutional disturbances are quite pronounced. Digestive derangement is the rule. The evacuations become more fluid in consistency and take on a greenish color. Weakness, prostra-
J2
POULTRY
SCIElfCE
Economic Importance The main financial loss incurred from this disease is not due so much to that of wasted flesh through deaths, great as that may be at times, but is the result of shrinkage in production. An indication of this loss is afforded by the following examples : A pen of Single Comb White Leghorn pullets of a given strain, was divided into two pens on November 22, and are designated pens A and B. By the end of the month roup made its appear ance in pen A and increased in virulence throughout the month of December. I t remained at its height during the following two months, and subsided again in March. Pen Β remained free of the disease. There is a marked difference in the production Λ'3ΐυβ8 of these two pens accountable for only by the presence of roup in the one pen and absence of the disease in the other. Egg values for pen A per bird for the three months of December, January and February equal 70 cents. Pen Β egg values per bird for the corresponding period total $2.50 or a difference in favor of the latter of $1.80. For the six Λvinter months the production values are $3.12 and $5.38 respectively or a difference of $2.26 per bird in favor of pen B. Prevalence In geographical distribution roup has become quite general, but it appears more in evidence in sections Avhere poultry farm ing occupies a relativelj^ large part of the agricultural activities. Thus in sections adjacent to the larger urban centers or where henneries thrive for other reasons one may expect to find the disease most prevalent. During the winter season, when weather is changeable or broken, particularly at the beginning or ending of the season,
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tion or a comatose state are symptoms which appear early in the course of the disease. When permanent relief comes early the subject soon revives from the lethargic condition, convalesence being followed by a favorable termination. Often, however, relief is but temporary and is followed by one or more relapses. In this course of the disease convalesence is prolonged if recoverj' takes place, but frequently the bird dies in an emaciated condition.
ETIOLOGY
OF ROUP
J3
ISOLATION OF ORGANISM
Because of the constancy in which the bipolar organism alread}^ mentioned had been found in roup lesions, attempts Avere made to isolate this bacillus. The first attempts were made with material from open lesions but the media Avas over-grown by a great number of different saprophytes. At last a lesion from a case of pneumonic roup Avas cultured, and from this we procured our first strain of the bipolar bacillus. This strain was injected into an adult fowl (407) intraperitoneally but produced no ill effects except a transient sickness. A small quantity of caseous material rich in bipolar organisms was taken from an orbital lesion and injected into the peritoneal cavity of a rabbit. A rapid infection folloAved and death took place in forty-eight hours. A pure culture of the bipolar organism was isolated from peritoneal fluid and blood of this rabbit. Third generation of this culture was found pathogenic for a pigeon and fowls (591, 627 and 305). The organism was recovered direct from lesion on breast of pigeon by cultural methods and from orbital cavity of fowls through rabbit inoculation. A rabbit (222) was infected with material from a natural roup case. It died in eighteen hours and a pure culture was isolated from peritoneal fluid. A small quantity of agar culture from the third generation
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the disease is most prevalent. TJie summer sees an abatement with almost if not total disappearance of visible or clinical cases. We may, therefoi'e, say that tlie prevalence of roup is associated with climatic conditions. Bacterial Flora of Lesions In the majority of instances specimens taken from the lesions show a varied bacterial flora. A gram negative bipolar bacillus is always in evidence, and this is accompanied by a multitude of different organisms such as colon bacilli, staphlococci, molds, etc. But in the lesions which we more seldom encounter, that is those protected from outside influence, and therefore, not contaipinated by various organisms, we find but one type of microorganism—the gram negative bipolar bacillus.
14
FOUhTRY
SCIKKCE
was inoculated into the s\ibeoiijuiictiva of a fowl ((,' l o i — F i g . 1 ) producing symijtoms identical with those of rou]). On the tliird dav after infection cultures were made direct from the e\-e.
The organism was found to be in pure culture, and was grown on agar for two genei'atioiis, then iiiocidated into fowl (5203). This bird became infected with roup. REPRODUCTION OP TiiK DISEASE The inoculation of adult fowls with p u r e cultures of a bacterial organism isolated from n a t u r a l l y infected roup cases, had been productiA'c in the inoculated birds of a disease M'ith general systemic disturbances and local lesions indistinguishable from those of roup. A culture of the organism when f(Hl to young fowl (chicks) has piroduced a like result. FIRST ATTEMPT RESTM/rED IN A FAILURE Experiment N^o. 1 On J a n . 25th, 192;}, a cockerel (No. 407) was inoculated iutraperitoneally Avith .5 cc. of a broth culture isolated from a case of pneumonic r o u p . F o u r da.ys later the bird showed symptoms of ])eritonitis Avhich gradua]l.y subsided. Feb. 8tli the bird Avhile appearing normal was killed and aii1oi)sied, all organs being found in a normal state. Experiment No. 2 A series of six intra])eritoneal inoculations were then made
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Fio. 1
Specimen numbei· C151. Inoculated December 14, 1928. Culture, bacillary saline-emulsion. Died January 2, 1924. Photographed December 27, 192 3. Organism recovered direct fronv lesion involving the left orbital region—the point ot inoculation.
ETIOLOdY OF h'OlP
15
A culture was isolated from material taken from a roup case of n a t u r a l infection, through rabbit inoculation. A pigeon (No. 15) was then inoculated, the resulting peritoneal exudate being used as an inoculum for 9 chicks; all succumbed while controls, 10 in muiiber. remained in u'ood health. The same
Fio. 2
Subject number 616. Inoculated on May 9, 1923, with peritoneal exudate from an experimentally infected pigeon. Died May 16, 1923. Photographed May 14, 1923. See also Fig. 3. exudate was inoculated into a pigeon ( x ) , the p r o d u c t from the lesions of Avhich, and a subsequent transfer from the original culture, (bacillary saline emulsion), were inoculated into 5
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upon adult fowl with roupy products. Almost or completely negative results followed, Avith one exception. No. 70, a cock bird was given 3 ec. of a heavy suspension of tracheal exudate on March 12th, 1923, a n d succumbed on March 23rd, 1923. Period of incubation in this instance was five days (appeai'ance of first symptoms). The following autopsy was recorded: Adhesions and lieavy caseous membrane on peritoneum a t point of inoculation. Peritonitis of a general distribution. A b u n d a n t exudate at point of inoculation. Air sac on inoculated side filled with caseous material. Eight lung patchy consolidated areas ; left coinpetelj' solidified Avith plugging of bronchus, extending u p into trachea. 10 cc. of straw colored fluid in pericardial sac. L a r y n x , mouth anti other head p a r t s nornud. Experiment No. 3
16
POULTRY
HGIENCE
yearling hens. Three of the birds, (Nos. 591, 627 and 305), receiA'ed the culture, the remaining two, (Nos. 616 and 301), being inoculated with the pigeon peritoneal exudate. All five suffered Λ\'ith roup typical in appearance of natural cases. One bird. No. 616, succumbed seven days following inoculation. See Fig. 2.
Inoculations were made on the evening of May 9th, 1923, and the two folloAving mornings imperfectly formed eggs Λvere found on the dropping boards. Except for these production ceased entirely and was not resumed for three weeks. One bird Avas not laying previous to or during the experiment. The period of incubation was the same in both lots A and B, viz. 12 hours. The second day after inoculation the following observations were made on the location and extent of the lesions, and the general condition of the birds. lîilMi's Nuililier
l.osion e.vteut and IjOcation
(Ifiioi-nl Conditiuii
591 627 305 616 301
** Eye *** Bye * Mouth *** E y e * Mouth
Fair Very weak Huddled, abdomen tense Prostrated Fair
Diarrhoea Avas present in all eases, the excrement being of a greenish-yellow color. A culture of the organism Avas recovered from the eye lesion of No. 627, and was isolated through a rabbit. The disease was transmitted from No. 627 to tliree other birds (Nos. 2812, 2898 and 2591), by swabbing the aff'eeted eye and smearing an eye of each of the other three. In all cases the disease developed in the eye that was swabbed. Two of the birds recovered while the third, No. 2812, succumbed to the disease. Lesions developed in 14 days. No. 2898 suft'ered a mild attack. Controls Nos. 1297, 2099 and 2768 remained healthy.
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lNO
MTIOLOdY OF ROVP
\-J
Experiment No. 4 A new culture was isolated from roup exudate by rabbit inoculation and on Sept. 11th, 1923, three adnlt birds were inoculated Avith a broth culture as follows : Aiiiminl; of C\lltni*e .25 cc .25 cc .50 cc
P o i n t of InoiMilatioii
Mucosa of eye lid Wing vein Roof of mouth—submucosa I n all tliree birds the results were negative, due to death of the organisms in the culture. This Avas proved by transfers from tlic l)roth culture to new media, tlie tubes remaining f»'ee
FJ(!. 3 Specimen number 7 9 6. Inoculated with culture September 14, 1923. Died October 23, 1923. Photographed October 16, 192 3. The left eye is destroyed, the cavity being filled with a caseous exudate. Subject emaciated and very weak. Note smearing of hackle.
of growth, and by rabbit inoculation. Rabbit No. 414 was given .25 cc. intravenously and suffered no ill effects. A bacillarj' saline emulsion was t h e n made from the a g a r c u l t u r e from Avliich the broth culture h a d been made and .25 cc. was introduced into the conjunctiva of an adult foAvl No.
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HirtVs Nuiubor 790 792 794
38
POULTRY
SCIENCE.
Tlie disease became chronic in its course and the subject suc cumbed on Oct. 23rd, 1923. A u t o p s y — E x t r e m e emaciation, organs iioruial in appearance except for the effects of the general wasted condition. Left eye destroyed and orbital cavity filled Λvith caseous material. Body weight 625 grams. See F i g . 3. Experiment
No. 5
This experiment Avas conducted to determine the effects of the organism Avhen fed to young chicks. One h u n d r e d and eighteen chicks of one Aveek of age were divided into ΙΛΥΟ equal batelies, a n d Avere maintained u n d e r identical conditions as to housing a n d feeding. Outside r u n s were available p a r t of the time and d u r i n g the intervening intervals lawn clippings, with a high clover leaf content, M'ere used for green feed. Symptoms or autopsj' findings attributable to avitainosis Avere never in evidence throughout the experiment. These are designated pen " Λ " and pen " ' B " ' culture feedingexperiment. P e n A were given once only on Oct. 2, 1923, a broth culture p a r t of which Avas incorporated in a moist mash, tlie remainder being added to the drinking water. ΤΛΥΟ days following, the chicks appeared less active, and took their food sparingly. This condition persisted and as a result groAvth slackened in some and ceased in others, tlie lot assuming a general u n t h r i f t y appearance. On Oct. 11th the first death occurred, from u n k n o w n cause, and two days later definite symptoms of r o u p appeared a n d fatalities occurred in r a p i d succession. The course of the disease reached its peak on the 16th a n d 17th, gradually subsided and cleared u p by the end of the month. Mortality 33.9%.
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796. Inoculation Avas made Sept. 14, 1923, a t 11:30 a. m. antl when examined the next m o r n i n g at 7 a. m. the following results were n o t e d : t e m p e r a t u r e 43° C. E y e into which culture was given was swollen shut and lids glued together. Mouth, throat and other eye appeared noi'mal, except t h a t the swelling about the affected eye p a r t i a l l y closes the passages from t h a t side. Marked systemic disturbances, depression, and diarrlioea.
ETIOLOGY OF ROUP
I9
The organism was recovered from these chicks by both direct culture methods a n d t h r o u g h animal passage. P e n Β chicks remained free of the disease and made satis factory gains, Avith a moderately normal mortality of 8.4%. Sec F i g u r e 4. Experiment
No. 6
Experiment
No. 7
This experiment was made to test the patliogenicity of the bi])o]ar organism on rabbits. A n agar culture (fourth generation) was washed off with suiïieient normal saline, to give to the resultant suspension, faintly opaque appearance. 0.5 cc. of this suspension Avas given intraperitoneally to each of two rabbits, and 0.5 Avas given subcutaneously to each of two rabbits. All rabbits showed illness Avithin twelve hours. Those inoculated subcutaneouslj" showed considerable local disturbance at point of inoculation. The two rabbits inoculated intraperitoneally died within tAventy-four hours a n d cultures made from heart blood a n d peritoneal fluid showed the organism in p u r e culture. One of the rabbits inoculated subcutaneously died Avithin three days. T h i s rabbit shoAved a p u r e culture in blood, and at the point of inoculation.
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The organism was again isolated from a n a t u r a l roup case and the disease reproduced in an adult foAvl No. C 1.51, from the lesions of which the organism Avas recovered, cultured in p u r e state on artificial media, reinoeulated into a fowl No. 5203 again producing the disease. Inoculation for comparative results Avere made with patho genic strains of Staphlococcus, Bacillus Welchii and an in.iection with spirits of tui'pentine on adult foAvls, numbers Π 946, 6241 and Η 990, respectively. The cultures a n d chemical Avere in jected into the submucosa of an eyelid in the same m a n n e r as the organism isolated from roup lesions and with Avhich the disease was reproduced. Reactions folloAved injections Avhich Avere local in cliaracter, unaccompanied by systemic disturbances and having no simi larity to the disease roup.
20
POULTRY
SCIENCE
R i g h t eye
PKX " B " CiiKK N o r m a l d e v e l o p m e n t — B o d y g r o w t h , size of legs, comb a n d w a t tles and clear b r i g h t eye. Pio. 4 Chicks (rom C u l t u r e F e e d i n g E.xperiment.
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Ρκ.Ν " A " Ciuc'K Underdeveloped a n d e m a c i a t e d , b a r e l y able to s t a n d . closed a n d filled "with caseous e x u d a t e .
E'VIOLOGY
OF ROUP
21
The fourth rabbit after a rather stormy illness of three weeks duration recovered. T H E ORGANISM
BIOLOGICAL CHAEACTEES
It is a bacillus measuring approximately .5 microns in length by .25 microns in Avidth, having a tendeiiej!- to bipolar staining especially in the tissues. In culture it is exceedingly pleomorI)hic and frequently assumes oval and coccoid forms. It does not form spores. Stains with the ordinary aniline dyes and does not retain the Gram stain. It produces a slight amount of Indol (Salkonski-Kilasato method). Nitrates are not reduced. Aerobic and facultative anaerobic. Optimum temperature 37° C. Litmus milk is not altered. Agar : Growth comes up as dew-drop like colonies which are at first discreet and show little tendency to spread. In older generations cultures take on a white color and lose to some extent their transparency, but there always remains a wax-like look whether recently isolated or in older cultures. Tlie growth is exceedingly sticky and when the inoculation loop is touched to a colony it will string out. Broth: Growth on broth is rather characteristic. If the media is inoculated and not disturbed the organism floats in small islands on the surface, and through the media will be noticed groups of bacteria which give to it the appearance of a positiA^e agglutination reaction. After two or three days the (Continued on page 24)
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As pointed out earlier in this paper it is imiDossible to isolate tliis organism from an open lesion by ordinary ctiltural methods. This is what one Avould expect, when the number of contaminating organisms that are scratched into the eye by a diseased bird is considered. On the other hand if a bird is secured in the early stages of the disease, Avhile the lids are still adhered together and consequently protected against outside influence, the securing of pure cultures is a comparatively simple matter. Experience has taught us that the simplest method for the obtaining of pure cultures is to inoculate a rabbit intraperitoneally with a small quantity of the caseous material taken from tlie eye of an affected bird. The rabbit rapidly dcA'clops an acute infection, and dies usually within twentj'-four hours. Dilute agar plates planted from the blood and peritoneal fluid of the rabbit usually give pure cultures of the organism.