THE high incidence of infectious

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1 The Pathology of Infectious Sinusitis of Turkeys S. B. HlTCHNER Section of Animal Pathology, Virginia Agricultural Experiment Station, Blacksburg, Virginia THE high incidence of infectious sinusitis in turkey flocks throughout the United States and certain parts of the world clearly demonstrates the need for more research in the etiology, epizootiology and pathology of this disease in order to bring it under control. Turkey growers in the State of Virginia consider this to be the most important infectious disease of turkeys. Hungerford (1939), in Australia, states that with the exception of blackhead it is probably the most important disease affecting turkeys in the State of New South Wales. Comparatively few studies have been made of infectious sinusitis and the phases most extensively investigated have dealt with treatments. A thorough study of this infection is now being made in an attempt to gain information which may lead to effective control by preventive measures. REVIEW OF LITERATURE Distribution and synonyms. The disease of turkeys usually referred to as infectious sinusitis has been reported in England by Dodd (1905) and Graham-Smith (1907), in Australia by Hungerford (1939) and Hart (1940), and by various investigators in many parts of the United States. The infection has been described under several names, being referred to as sinusitis (Hart 1940, Hungerford 1939, Madsen 1938, and Tyzzer 1926), "swelled head" (Dodd 1905, Hungerford 1939), "swell head" (Dickinson and Hinshaw, 1939) "swollen head" (Dodd 1905, and (Received for publication July 16,1948) Graham-Smith 1907), epizootic pneumoenteritis (Dodd 1905), and turkey "roup" (Hungerford 1939). Dodd's (1905) description of the outbreak which he reported would indicate that he was dealing with infectious sinusitis complicated with fowl cholera. A bronchopneumonia of turkeys described in a report by Gierke (1934), and a respiratory disease of young turkeys reported by Harr (1943), may be the same disease entity as infectious sinusitis. As yet there is no known confirmation of this relationship and because of this these two references will not be given further consideration in this review. Of the various names given, infectious sinusitis would appear to be the preferable term to use because of its currently wide acceptance and to differentiate this type of inflammation of the sinuses from that caused by avitaminosis, described by Hinshaw and Lloyd (1934). Symptoms and lesions. The disease is characterized by shaking of the head, nasal discharge, frothy lacrimation and distention of one or both infraorbital sinuses by an almost colorless, gelatinous exudate, which, according to several reports may later become caseated (Dickinson and Hinshaw 1938, Graham-Smith 1907, Hart 1940, Hinshaw 1943, Hungerford 1939, Madsen 1938, and Tyzzer 1926). Hinshaw (1943) states that caseation of the exudate is the exception in typical outbreaks, and according to Dickinson and Hinshaw (1938) this occurs in instances of long standing or after improper 106

2 THE PATHOLOGY or INFECTIOUS SINUSITIS OF TURKEYS 107 treatment. Hart (1940) reported caseation subsequently occurring in a large number of cases, and likewise Hungerford (1939) noted some outbreaks in which the exudate became caseous in many birds one to two weeks after exudate appeared. He believed the development of the caseous material probably resulted from the presence of secondary bacterial invaders. From post-mortem examinations he reports about 10 percent of the birds contain cheesy material in the sinuses. Madsen (1938), according to his records, also found the mucus becoming cheesy after a few weeks duration in about 10 percent of the affected turkeys. In addition to the above symptoms connected with the inflammation of the sinuses, another form of the disease has been observed involving the lower respiratory tract (Delaplane 1944, Dodd 1905, Hinshaw 1943, Hinshaw and McNeil 1946, and McNeil and Hinshaw 1946). This type of infection may or may not occur in conjunction with the sinus infection, and is evidenced by labored breathing (Hinshaw 1943), coughing, and caseous exudate on the air sacs, most pronounced on the posterior thoracic and abdominal sacs (McNeil and Hinshaw 1946), pneumonia and pleuritis (Dodd 1905, Hinshaw 1943, McNeil and Hinshaw 1946). McNeil and Hinshaw (1946) mentioned a marked congestion of the duodenum and pancreas as frequently occurring in this disease. Histopathology. Very little has been published on the microscopic pathology of infectious sinusitis. Graham-Smith (1907) sectioned fragments of material discharged from living turkeys and observed that they were largely composed of cells in various stages of degeneration, and numerous bacteria. Sections of the nasal mucous membrane showed some dilatation of the vessels and many inflammatory cells. The lining membrane of the infraorbital sinus revealed that, with the exception of some of the recesses, the epithelium had disappeared and was replaced by granulation tissue. The submucosa contained proliferated fibrous tissue infiltrated with inflammatory cells. He observed that the bacteria had not penetrated deeply into the tissue. Several microscopic studies were also made of the internal organs of infected turkeys but in every instance findings were negative. Species infected. Infectious sinusitis appears to be specific for the turkey. Ducks (Graham-Smith 1907, and Hart 1940) and chickens (Dickinson and Hinshaw 1938, Dodd 1905, Graham-Smith 1907, Hart 1940, and Hungerford 1939) in close contact with infected turkey flocks have shown no evidence of contracting the disease. Turkeys have manifested typical swellings of the infraorbital sinuses resembling the natural infection when inoculated with exudate from coryza infected chickens (Beach and Schalm 1936), or with saline washings or exudate from chicken sinuses which have previously been inoculated with sinus exudate from turkeys (Delaplane 1944, Hart 1940, and Hinshaw 1943). Hart (1940) by means of sinus exudate was able to infect one of two fowls which showed nasal discharge and the presence of mucus in the sinus on postmortem examination. These results would indicate that the chicken may serve as a carrier for the infection. Madsen (1938) mentions the presence of sinusitis in two turkey -flocks in which there was a history of coryza in the chickens on the same farms. Susceptibility and mortality. Poults may first show evidence of infection as early as three (Hungerford 1939) to four weeks (McNeil and Hinshaw 1946) of age, and may be infected at any age thereafter. According to Hart (1940) and Hungerford

3 108 S. B. HlTCHNER (1939) the disease is most prevalent and occurs in its severest form from three to five months of age. It appears that the Australian type of infectious sinusitis is more virulent than that found in the United States, resulting in heavy mortality (Hart 1940, Hungerford 1939). Hart (1940) reports one flock owner losing the entire flock of 800 birds, and Hungerford (1939) cites two cases, one in which there was 100 per cent mortality in a flock of 500 poults, and 90 per cent mortality in another flock of 800. In England (Graham Smith 1907) considerable mortality is sometimes encountered. Dodd (1905) reported 41 per cent mortality in a flock of 120 turkeys, but undoubtedly much of this was due to fowl cholera. Madsen (1938), and Dickinson and Hinshaw (1938) state that mortality is usually low, but according to the latter morbidity may range from 10 to 90 per cent. McNeil and Hinshaw (1946) report instances of high mortality in flocks affected with the lower respiratory infection. The greatest losses from the disease are those resulting from loss in growth and weight gains (Hinshaw 1943, Hungerford 1939, Madsen 1938), and egg production and fertility in the case of breeding stock (McNeil and Hinshaw 1946). Etiology. The etiology of infectious sinusitis has not been established. Dodd (1905) isolated an ovoid bacillus from the sinus exudate, lungs and heart blood which was indistinguishable from the fowl cholera organism. Due to lack of birds he did not attempt experimental transmission to turkeys. Experimental inoculations into other laboratory animals indicate this was Pasteurella avicida. Graham-Smith (1907) cultured the exudate from several infected turkeys and described the morphology and cultural characteristics of the four predominating types of organisms isolated, designating them as Bacilli types A, B, C, and D. Pure cultures of these organisms injected into the sinuses of susceptible turkeys failed to produce the infection. He suggested that the disease might be due to ultramicroscopic organisms. Delaplane (1944) reported a Pasteurella or Pasteurella-like organism as the cause of infectious sinusitis in turkeys. However, he cited no instances in which direct inoculation of the organism into the sinuses of turkeys produced the disease. In every case of transmission reported the organism was inoculated into chickens and the exudate from the chickens used to produce sinusitis in the turkeys. This raises the question as to whether or not the chicken might have played a passive role in carrying the true infective agent of infectious simusitis. Hinshaw (1943) reported consistent isolation of a pleomorphic Gram-negative rod in pure culture from infected sinuses. With one exception he was unable to reproduce the disease by direct inoculation of this organism. In this instance a first generation transfer was used, and the author suggested the possibility that infection was due to the carry-over of a filterable factor. Hart (1940) in his attempts to establish the etiology of the disease made smears of sinus exudate and of scrapings of the mucous membranes. At the same time attempts to recover organisms were made on several different media. The cultures remained sterile except in a few instances where mixed organisms were also seen on the smears. Several workers have suspected a filterable agent as the cause of the disease but to date its filtrability has not been demonstrated. Unsuccessful attempts at filtration were made by Hinshaw (1943) with Chamberlain (L-5) and sintered glass filters, by Hart (1940) with Elford collodion membranes, and by Madsen (1938) with a Berkefeld filter.

4 THE PATHOLOGY or INFECTIOUS SINUSITIS OF TURKEYS 109 Viability and infectivity of exudate. Hart (1940) in testing the infectivity of exudate was able to transmit the disease with material taken from a bird which had been infected three months previously. He was also able to infect two turkeys with exudate taken from a turkey 11 months after infection. Survival tests of the infective agent in vitro demonstrated that when diluted one part to seven parts of 50 per cent glycerine in normal saline it withstood refrigeration at 36 F. for six days. In another experiment in which the exudate was diluted 1 to 20 in 50 per cent glycerine in normal saline and held under the same refrigeration, the material was still infective after 14 days but failed to produce the disease after holding for two months. A third experiment, in which exudate originating from the same turkey as in the previous experiment was diluted 1 to 20 in sterile normal saline, failed to show infectivity after 2 weeks duration in the refrigerator. In determining the minimal infective dose Hart (1940) found that 1 ml. of exudate injected into each sinus produced infection when diluted 1 to 10,000 in normal saline, the highest dilution tested. Transmission. Field observations, according to Hungerford (1939), indicate that the disease is of a highly infectious nature. Madsen (1938), on the other hand, is of the opinion that it is not highly contagious. Tyzzer (1926) attributed more significance to environmental factors in the transmission of the disease, such as chilling and drafts, than to the presence of an infectious agent. Hinshaw (1943) also mentions drafts, windstorms, sandstorms and vitamin A deficiency as factors in the spread of the disease. Madsen (1938) concurs in the opinion that vitamin A deficiency is a predisposing cause. Graham-Smith (1907), Hart (1940), Hinshaw (1943), Hungerford (1939), Madsen (1938), and Tyzzer (1926) have successfully transmitted the disease experimentally by direct inoculation of the sinus exudate into the sinuses of susceptible turkeys. Incubation periods varied from 1 to 12 days. Hart (1940), Hinshaw (1943), and Madsen (1938) were unable to effect transmission by close contact in cages, whereas Graham-Smith (1907) reported contact transmission in two of four trials. Hart (1940), and Hinshaw (1943), obtained negative results by intranasal and intraocular instillation. Graham-Smith (1907) records transmission once out of three trials by the intranasal route. Transmission by atomization was unsuccessful by Hart (1940). Contamination of the food with exudate gave transmission in one trial by Graham- Smith (1907). Immunity. Hungerford (1939) is the only author to mention that certain strains of turkeys are more resistant than others, based upon field evidence. Natural recovery of some of the infected birds has been observed by Dickinson and Hinshaw (1938), Graham-Smith (1907), Hart (1940) and Madsen (1938). Immunity is established in recovered birds according to flock owner's reports cited by Hart (1940), and Hungerford (1939). Hart (1940) challenged two recovered birds with known virulent exudate and was unable to produce infection. He was unsuccessful in inducing artificial immunity in two birds by subcutaneous injection of exudate, and likewise failed to produce immunity in two other turkeys by applying the exudate to the cloacal mucous membrane. Madsen (1938) tried vaccinating two flocks with "roup" bacterin but results were of doubtful significance. Treatment. Tyzzer (1926) was first to report successful treatment of cases of sinusitis with silver preparations. He

5 110 S. B. HlTCHNER used 1 ml. of 15 per cent argyrol injected into each sinus after expressing the exudate by gentle pressure. Second and third administrations of the drug were needed in a few birds to bring about complete recovery. Madsen (1938) conducted a series of tests using the following medicaments in infected sinuses: 8, 4, and 2 per cent aqueous solutions of silver nitrate, calomel, 20 per cent argyrol, tincture of metaphen, and equal parts of tincture of iodine and glycerine. He concluded that 4 per cent- silver nitrate gave the highest percentage of recoveries, and that the technique of using a syringe and needle to aspirate the exudate and apply the silver nitrate was superior to lancing the sinus with a knife and then administering the drug. Dickinson and Hinshaw (1938) report 4 per cent silver nitrate slightly more effective than 15 per cent argyrol. Hart (1940) obtained good results with 2 to 5 per cent silver nitrate. Hungerford (1939) reported on the efficacy of 1 and 5 per cent silver nitrate, 1 per cent copper sulphate and 1 per cent zinc sulphate, best results being obtained with5 per cent silver nitrate. As an alternative method of treatment he suggests lancing the sinus followed by frequent flushing with 5 per cent silver nitrate or a strong solution of potassium permanganate. McNeil and Hinshaw (1946) compared the efficacy of 4 percent silver nitrate, Protargol (Winthrop), 15 per cent argyrol, Novoxil (Squibb) and two ephedrine sulphonamide preparations. From these tests they concluded freshly prepared solutions of 4 per cent aqueous silver nitrate gave the best results. There appears to be agreement among the various investigators that silver nitrate in freshly prepared aqueous solution is the most effective treatment for the involved sinuses, giving between 80 to 90 per cent recoveries. With the development of the sulfonamide drugs several of these have been investigated in the anticipation that they might prove an effective treatment for infectious sinusitis, particularly for the lower respiratory form of the disease. Delaplane (1944) reported field treatment of an adult flock showing sinus infection with 0.75 to 1.0 gram of sulfathiazole twice daily. Recovery resulted in those which showed no caseation of the exudate. Favorable response was also noted in birds showing lower respiratory involvement without sinus infection. Hinshaw and McNeil (1946) conducted several field experiments using 0.5 per cent sulfathiazole in the mash, 1 to 2,000 dilution in the drinking water, and individual treatment of 0.5 gram twice daily, for periods of 5 days each. They concluded that sulfathiazole was too unpalatable for turkeys to obtain effective blood levels of the drug. Improvement, when noted, appeared to be only temporary. McNeil and Hinshaw (1946) conducted eight trials in the use of sulfamerazine on turkeys affected with the respiratory form of the disease. By feeding the drug in the mash at 0.25 and 0.5 per cent levels beneficial results were noted in at least three of the experiments. However, symptoms in the flock reappeared when tretament was discontinued. EXPERIMENTAL PROCEDURE AND RESULTS The first objective in this study was to attempt to determine the cause. To do this it was considered of primary importance to learn if the causative agent could be propagated by chick-embryo culture techniques and, if so, attempt to reproduce the disease in uncomplicated form. Isolation and propagation of infective agent. The original material for this study was obtained from a 5 weeks-old turkey

6 THE PATHOLOGY OF INFECTIOUS SINUSITIS OF TURKEYS 111 i T347 I T371 Air sac material from naturally infected turkey originating in a nock which was experiencing an acute outbreak of infectious sinusitis. Caseous air sac material from this turkey was collected aseptically, ground in a mortar with nutrient broth and 0.5 ml. of this material was injected into the right infraorbital sinus of a 20- day-old poult T388. A sterility check on nutrient agar disclosed two bacterial colonies. Nine days after injection the right sinus showed evidence of slight distension and on the 13th day the left sinus also became distended. Exudate was immediately aspirated from the left sinus, suspended in 2 parts of sterile saline, and 0.2 ml. was injected into the right sinus of each of two poults, T371 and T335. Some of the same material streaked on blood agar was found to be free from bacterial growth during several days incubation at 37 C. Both of these poults showed evidence of distension of the right sinus on the 4th day after injection. A third transfer of exudate from these i 4- T388- I T335 T321 T334- two birds was made to three other poults as is shown in Figure 1. The exudate used for injection was also sterile when checked on blood agar. Distension of the sinuses was visible at 5, 6, and 7 days respectively. From the results of these serial transfers it was apparent that the type of sinusitis being dealt with was of an infectious nature. It was also evident from the results of the sterility tests on blood agar that either bacteria played no part in Sl 3rd Gen. S2 3rd Gen. S3 ->T384 T354 the etiology of the disease, or, if present, they possessed fastidious growth requirements. The absence of any visible microorganisms in smears of the exudate stained by methylene blue, Gram's, and acidfast stains favored the former view. Since the etiology of infectious sinusitis now appeared to be an ultramicroscopic agent, the next procedure was to try propagating it on embryonating chicken eggs. Some of the same saline suspension of exudate from T388 used to infect T371 and T33S was inoculated on the chorioallantoic membrane and into the S4 2nd Gen. 1st Gen. 5th Gen. 9th Gen. 10th Gen. 11th Gen. 17th Gen. T330 -T320 -+T348 -> T366 ->T389 ->T324 ->T313 FIG. 1. Origin of strains of infectious sinusitis. Vertical arrows show serial passages of field strain made in turkeys. Horizontal arrows show origin of exudate used in recovery of infective agent on embryonating eggs and the generation each was tested.

7 112 S. B. HlTCHNEE allantoic sac of three 10-day-old embryonating eggs. After four days of incubation the chorioallantoic membranes were harvested and stored in a frozen condition for subsequent inoculations. Egg material streaked on blood agar proved to be sterile. After two more similar passages in eggs two membranes of the 3rd generation were pooled, ground with sand and suspended in approximately 1 to 3 parts of broth. Sterile technique was used throughout the procedure. The left sinus of T384 was injected with 0.75 ml. of this suspension. Distension of the sinus appeared on the 11th day following the injection. This egg-propagated material was designated as SI. Figure 1 shows the origin of three other strains of the infectious agent recovered similarly from sinus exudate on embryonating eggs. These other recoveries were designated as S2, S3, and S4. The horizontal arrows designate the strain and the egg generation in which each was tested. To the right of the arrow is the number of the turkey injected with each generation. The S4 strain was recovered from T354 which had been infected by material of the S2 strain. This procedure, therefore, satisfied Kock's postulate for the isolation of a pure culture. The S3 strain was recovered from sterile exudate aspirated from T334 fifty-one days after being infected. This was the only strain which was serially passed in embryonating eggs. At the present time it is in the 28th passage and is still infective when inoculated into susceptible turkeys. Although the original recovery of the infectious agent was made on 10-day-old embryonating eggs, almost all subsequent transfers were made on 11-day-old embryos. Inoculation was tried on the chorioallantoic membrane, and in the allantoic and amnionic sacs. The route of inoculation made no noticeable difference in the lesions produced, and consequently the allantoic sac was the route of choice because of lower embryo mortality and ease of administration. The chorioallantoic membranes were harvested and preserved by freezing. Original harvests were made on the 4th and 5th days after inoculation. Later they were allowed to incubate for 6 days, and since death did not occur in the embryos many harvests were made on the 8th day following inoculation. Subsequent transfers were made by grinding the membrane in a mortar with sand and adding sufficient nutrient broth to make approximately a 1 to 3 suspension. A quantity of 0.1 ml. was used for embryo inoculation. The fact that the infective agent was ultramicroscopic and could be propagated on- embryonating eggs indicated that a virus might be the causative agent of the disease. However, direct inoculation into the sinus of filtrates of infected egg membranes from a Seitz, Berkefeld N and V filters were negative. Gross lesions in embryos. Gross lesions were not consistently observed in infected embryos, and consequently it was necessary to resort to many blind passages. Frequent inoculation of broth suspensions of the membranes into the sinuses of susceptible turkeys was used as a means of verifying the presence of the infective agent in eggs. When inoculated on the chorioallantoic membrane, embryos at the time of harvest frequently showed a small, moist, raised nodule about 2 mm. in diameter at the point of inoculation. This nodule protruded through the shell membrane so that it was readily visible following removal of the egg-shell cap. The chorioallantoic membrane appeared to be thickened and somewhat edematous in some cases, but often these changes were so slight that they could not be relied upon as a measure of growth. Another lesion occasionally observed was

8 THE PATHOLOGY or INFECTIOUS SINUSITIS OF TURKEYS 113 the presence of a dark green-colored liver, undoubtedly due to the presence of bile pigment. The only consistent gross lesion in infected embryos was the presence of granular urates in the allantoic sac, which, when observed under the microscope were composed of long, needle-like crystals. Urates of control non-infected embryos were amorphous in character and did not show the crystalline structure when observed under the microscope. Although no significant mortality occurred in inoculated embryos up to the time of harvest at 19 days of age, some mortality did occur after this and many of the eggs which were allowed to hatch would pip the shell but seldom emerged from it. Of 57 eggs which were allowed to hatch 36 pipped the shell but failed to emerge, 15 died on the 19th and 20th day of incubation, and only 6 hatched. Five of these 6 showed gasping symptoms a few days later and either succumbed to the infection or were destroyed for postmortem examination when in a weakened condition. Post-mortem examination revealed a dry caseous exudate in the lower part of the trachea, prominent caseation of the small abdominal air sacs and varying degrees of consolidation of the lungs. The embryos which pipped the shell but failed to hatch showed complete or nearly complete consolidation of both lungs. Some of the lungs seemed friable and when ruptured revealed what appeared to be branched caseous casts of the bronchi. In nearly every instance the small abdominal air sacs were readily visible due to thick, yellow deposits on the membrane, frequently forming a caseous cast of the sac. Symptoms and lesions of experimentally produced sinusitis. The pathological study of infectious sinusitis contained in this report was made from thirteen experimentally infected turkeys infected between 2 to 7 months of age by egg propagated material. Eleven of the 13 cases herein cited were infected by direct inoculation into the sinuses with broth suspensions of infected egg membranes. The other two turkeys were infected following vent and feather follicle inoculations respectively. The disease produced showed the characteristic symptoms previously described by other workers, namely, shaking of the head, distension of the infraorbital sinuses, nasal discharge, frothy lacrimation, and occasionally listlessness and labored breathing. Daily temperatures taken on three infected birds showed only a low grade fever never exceeding 1 F. above normal temperature. Swelling of the sinuses appeared in 2 to 12 days following direct inoculation with 0.25 to 0.75 ml. amounts per sinus. The S3 strain of material was tested in turkeys from egg generations 1, 5, 9, 10, 11, and 17, and in every instance produced distension of the infraorbital sinuses after an incubation period of 12, 4, 3, 3, 2, 2, days, respectively. Since all turkeys used for these inoculations came from the same lot, it was not known whether the decrease in incubation period was due to an increase in virulence of the egg propagated material or whether the birds became more susceptible as they became progressively older. Following the appearance of the infraorbital swellings the exudate was aspirated aseptically every few days in order to study its character and to examine it for bacterial sterility. In 10 of the 11 birds which developed swelling the exudate was yellow or amber colored. This coloration persisted from 7 days (3 birds) to 21 days (2 birds), at which time it became almost colorless or slightly turbid and more viscous. This color of the exudate of turkeys infected with egg propagated material distinguishes it from the

9 114 S. B. HlTCHNER exudate in naturally infected or exudate-infected turkeys, which begins as a slightly turbid, almost colorless viscous exudate. Several previous investigators observed that in some instances the exudate became hard and caseous after several weeks' duration of the swellings. FIG. 2. Experimentally infected turkey 12 days after injection showing distended sinus and enlargement of Harder's gland. T324. In one of the experimentally infected birds the exudate was too caseous to be aspirated on the 5th day and another was in a similar condition on the 9th day after infection, both of which were sterile when streaked on blood agar. The sterility of the aspirated fluid was checked each time by streaking some of the exudate on blood agar. Small hemolytic areas were frequently observed on the plate along the streaked lines but stained smears from these areas failed to reveal any organisms. The elapse of time before bacterial contamination appeared in the exudate was variable. The two longest periods in which the sinus exudate remained sterile were 60 and 66 days, respectively. A heretofore unreported symptom of infectious sinsusitis was observed in 5 of the 11 turkeys showing involvement of the sinuses. This was manifested by a marked enlargement and protrusion of the Harder's gland from the inner canthus of the eye. This lymphoid gland is found in the nictitans membrane and is not noticeable in the normal turkey. Its appearance was visible shortly after exudation was evident in the sinuses, and in most instances was so enlarged as to cover one-half of the eyeball (Figure 2). Recession of the gland was observed in 2 to 3 weeks. Post-mortem examination of the involved sinuses revealed an accumulation of gelatinous exudate, sometimes interspersed with a white, flaky material. The lining of the cavity was thickened and frequently the mucosal surface was studded with petechial hemorrhages. In several turkeys hyperemia of the lacrimal gland in the ventral palpebral conjunctiva was apparent. Exposure of the viscera revealed clouding of, and caseous deposits on the peritoneum, the abdominal air sacs, and the thoraco-abdominal and pulmonary diaphragms. Among the air sacs involved the small abdominal sac appeared more acutely affected than the others. In long standing cases the sac lining became greatly thickened, giving the appearance of a fibrous cord being attached to the caudal extremity of the lung. Eleven of the 13 birds reported had involvement of the airs sacs. In a few cases a foamy, cream-colored serous fluid was present around the abdominal organs, and large caseous masses were sometimes found unattached in this fluid. Eleven of the 13 turkeys examined had gross lessions of pneumonia, and one of the two which did not revealed previous infection on histopathological examination. Areas of consolidation were evident in the caudal extremities and lateral borders of the lungs. In some instances consolidation appeared to be limited to the peripheral surface of the ventral or

10 THE PATHOLOGY OP INFECTIOUS SINUSITIS OF TURKEYS 115 dorsal borders. In other cases consolidation was more extensive involving practically all of the lung tissue in the caudal extremity. The cross sections of the latter cases disclosed small grey foci from which a white mucoid exudate could readily be expressed when subjected to slight pressure. Histopathology. The histopathological changes in the affected organs were those of a chronic inflammatory process characterized by proliferation of the connective tissue, exudation, cellular infiltration with granulocytes and an increased number of lymphoid nodules. The lining of the infraorbital sinuses of affected turkeys was characterized by catarrhal inflammation (Figure 3). Degenerative changes in the cells of the mucous glands occurred early in the infection followed by degeneration and desquamation of the epithelium. The mucus exudate contained epithelial cells and leucocytes, the predominating type of the latter being granulocytes. A marked cellular infiltration was present in the lamina propria accompanied by vascular congestion. As the disease progressed the tissue of the submucosa became proliferated, somewhat edematous and infiltrated with granulocytes. A moderate to marked increase in the number of lymphoid nodules was observed in the submucosa. Changes in the Harder's gland were characterized by marked proliferation of connective tissue and lymphoid nodules, the latter being present in all stages of growth and rarefaction (Figure 4). Also prominent was the increased vascularization of the connective and lymphoid tissues. Capillaries, as well as large sinuses filled with erythrocytes, were observed in these nodules. The lung involvement was typical of bronchopneumonia. Active hyperemia of the vessels and capillaries was present in the affected areas. The bronchi were filled with an inflammatory exudate, highly cellular in character with granulocytes predominating. The tertiary bronchus with its network of air and blood capillaries was consolidated as a unit, comparable to the consolidation of the lobules in the mammalian lung in bronchopneumonia. Some of the air capillaries of adjacent bronchi showed no consolidation, but only congestion of the vascular capillaries. Cases in which consolidation persisted for some time exhibited an interesting lymphoid reaction to the presence of the disease. A marked increase in number of lymphoid nodules was observed scattered throughout the consolidated tissue (Figure 5). In more advanced cases in which resolution had taken place the lymphoid nodules were found to be more numerous and were observed to increase in the wall of the bronchus to a point where the lumen of the bronchus was almost occluded. In the avian species a fixed glandular system does not exist and the presence of a few lymphoid nodules occurs in most organs. The marked increase in the number of these nodules in the affected organs was undoubtedly a compensatory response to the demand for granulocytes created by the presence of this infection. The air sac membranes showed hyperplasia and edema of the connective tissue with marked cellular infiltration with granulocytes, disruption of the continuity of the respiratory epithelium, and degeneration and desquamation of the epithelial cells (Figure 6). Several suppurative foci were visible. Although lymphoid nodules were present their numbers were not as significant as in the lung tissue. The affected peritoneum was swollen and edematous with cellular infiltration and necrotic foci similar to that seen in the air sacs.

11 r, ' * '' ^i^-'-" ; / '.... -' - * ''^ -n ~ " 1 * S. B. HlTCHNER 116

12 THE PATHOLOGY OF INFECTIOUS SINUSITIS OF TURKEYS 117 DISCUSSION The fact that the causative agent of infectious sinusitis can be propagated in embryonating eggs supports the supposition proposed by many workers that the disease is caused an an ultramicroscopic organism, probably a virus. The marked increase in the lymphoid nodules of affected organs and tissues as seen in the disease has been observed in avian virus infections and would lend support to this view. However, other supportive evidence, such as cellular inclusion bodies, has not been observed in tissue sections. Unsuccessful filtration by the author and previous investigators would tend to rule out a filterable virus as being the causative agent. There is a probability that the infection may be due to a rickettsia, and further investigation of this possibility seems to be warranted. Topley and Wilson (1946) in their discussion of the growth requirements of the rickettsiae point out that these organisms prefer tissue cells which have ceased active growth, in contrast to the filterable viruses which prefer actively growing tissue. They also grow best in regions where there is an abundant supply of oxygen. The cultural behavior of the eggpropagated agent described in this study fulfills these requirements, in that death and marked lesions do not occur until the later stages of embryo development. Development of the embryo in many instances was not retarded until after the shell was pipped at which time an abundant supply of oxygen became available, and from the appearance of the air sacs and lungs undoubtedly favored rapid growth of the agent. Another factor which would tend to support the rickettsial theory is the inhibition of the infective agent by a mixture of penicillin and streptomycin.* Only recently smears of sinus exudate and yolk sac material from infected eggs stained with Giemsa's Stain have revealed minute bodies which may play some part in the infection. It is understood that the evidence to date is not conclusive and that further attempts at filtration of the agent and additional studies of its morphological and cultural characteristics are indicated. SUMMARY Transmission of sinusitis was accomplished by a bacteria-free egg-propagated agent passed through 28 generations in 11-day-old embryonating chicken eggs. Significant lesions were not consistently observed in embryos when inoculated by the chorioallantoic, amnionic or allantoic routes. Mortality of the embryos was not observed until the 19th to the 21st day of incubation and the majority of the mortality at this time occurred after the shells were pipped. Attempts at filtering the infectious agent were negative with Seitz and with Berkefeld N and V filters. Direct inoculations of broth suspensions of infected egg membranes into the infraorbital sinuses of susceptible turkeys produced infection after an incubation period of 2 to 12 days, with symptoms typical of the disease. In addition to the symptoms observed in natural infection, the egg-propagated agent produced an * Unpublished data. FIG. 3. Mucous membrane of infraorbital sinus 29 days after evidence of distension of sinus. X80. T3S4 FIG. 4. Harder's gland 14 days after injection of the sinus. X80. T324. FIG. 5. Consolidated portion of lung showing lymphoid nodules. X100. T3S4. FIG. 6. Membrane of small abdominal air sac from turkey destroyed 56 days after infection. X80. T3S4.

13 118 S. B. HlTCHNER amber color in the initial sinus exudate and enlargement and protrusion of the Harder's gland. The post-mortem findings were thickening of and petechial hemorrhages in the sinus membranes, areas of consolidation in the caudal extremities and lateral borders of the lungs, thickening and deposition of caseous material on the air sacs. Microscopically, the lung lesions were typical of bronchopneumonia in which an increased number of lymphoid nodules appeared. Affected sinuses and air sacs were characterized by a proliferation of connective and epithelial tissues with varying degrees of degenerative changes. Smears of sinus exudate and yolk sac material stained with Giemsa's stain revealed minute bodies suggestive of an infectious agent. The possibility of the etiological agent being a rickettsial organism was discussed. ACKNOWLEDGMENT Grateful acknowledgment is extended to Dr. E. P. Johnson for helpful suggestions and criticisms, to Dr. W. L. Threlkeld and W. Schaal of the Virginia Agricultural Experiment Station for photographic assistance and to Mrs. K. A. King for technical assistance. ADDENDUM Since completion of this report the author's attention has been called to recent work in which an agent causing infectious sinusitis has been propagated on chicken embryos 1 and also to a preliminary report on the pathology of infectious sinusitis. 2 1 Groupg, V., J. D. Winn, and E. Jungherr, Isolation of an agent in chicken embryos causing infectious sinusitis of turkeys. Proc. Soc. Exp. Biol, and Med. 67: Jungherr, E., The pathology of turkey REFERENCES Beach, J. R., and C. W. Schalm, Studies on the clinical manifestations and transmissibility of infectious coryza of chickens. Poultry Sci. 15: Delaplane, J. P., A Pasteurella or Pasteurellalike organism as the cause of an infectious sinusitis of turkeys. Poultry Sci. 23: Dickinson, E. M., and W. R. Hinshaw, Treatment of infectious sinusitis of turkeys with argyrol and silver nitrate. J.A.V.M.A. 93: Dodd, S., Epizootic pneumo-enteritis of the turkey. Jour, of Comp. Path, and Ther. 18: Gierke, A. G., A report on miscellaneous diseases. Cal. Dept. Agr. Bui. 23: 364. Graham-Smith, G. S., Some observations on "swollen head" in turkeys. Jour. Agr. Sci. 2: Harr, J. F., A respiratory disease of young turkeys. Cal. Dept. Agr. Bui. 32: Hart, L., Sinusitis in turkeys. Australian Vet. 16: , Infectious sinusitis, a common disease in turkeys in New South Wales. Agr. Gaz. of N.S.W. 58: Hinshaw, W. R., (revised). Diseases of turkeys. Cal. Agr. Exp. Sta. Bui. 613: , and W. E. Lloyd, Vitamin A deficiency in turkeys. Hilgardia. 8: , and E. McNeil, Experiments in the use of sulfathiazole for turkeys. Cornell Vet. 36: Hungerford, T. G., Sinusitis common disease of turkeys in N.S.W. Agr. Gaz. of N.S.W. 50: , Madsen, D. E., Sinusitis of turkeys and its treatment. Utah Agr. Exp. Sta. Bui. 280:1-12. McNeil, E., and W. R. Hinshaw, Recent studies on the treatment of infectious sinusitis in turkeys. J.A.V.M.A. 108: , Field trials on the use of sulfamerazine for a respiratory disease of turkeys. Poultry Sci. 25: Topley, W. W. C, G. S. Wilson, and A. A. Miles, Topley and Wilson's principles of bacteriology and immunity. 3rd Ed., Vol. 1, Williams and Wilkins Co., Baltimore, Md. Tyzzer, E. E., The injection of argyol for the treatment of sinusitis in turkeys. Cornell Vet. 16: sinusitis. Proc. of the Twentieth Annual Conf. of Lab. Workers in Pullorum Dis. Control.