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Epidemiology of a Maltose Fermenting Variant of Salmonella Pullorum
Epidemiology of a Maltose Fermenting Variant of Salmonella Pullorum W. R. HINSHAW, E. MCNEIL, AND T. J. TAYLOR
Division of Veterinary Science, University of California, Davis (Received for publication August 18, 1943)
HE ability of Salmonella pullorum to ferment maltose is discussed by Hinshaw, Browne, and Taylor, (1943). In that paper the authors review the literature and present a study on the ability of certain strains to segregate into maltose fermenting and maltose non-fermenting substrains. In this paper is given epidemiological observations on a group of 146 nonmotile Salmonella cultures, antigenically indistinguishable from, 5. pullorum, which, on original isolation, fermented maltose with gas production in 24 to 72 hours, and which have continued to be rapid fermenters since isolation. SOURCE OF CULTURES
In 1935, one of the writers (W. R. H.), isolated a culture of Salmonella from the ovary of a turkey hen. This proved to have all the characteristics of S. pullorum except that it produced acid and gas in maltose. The following spring two identical cultures were received from the laboratories of the California State Department of Agriculture with statements that each had been isolated from an outbreak of paratyphoid; one in chicks and one in poults. The chick strain came from southern California and the poult strain from the northern part of the state. For the next few years the various diagnostic laboratories of the State Department of Agriculture supplied us with all the similar cultures isolated by them. In 1937, four strains from three outbreaks were received, and, in 1938, two strains from two outbreaks. In 1939, this variant began to be isolated frequently from acute
outbreaks in both chicks and poults. Since the isolation of the first strain in 1935, 146 cultures from 52 outbreaks have been either isolated by us, or submitted to us for study. All of the cultures have been rapid fermenters of maltose (acid and gas in 1 percent maltose broth within 72 hours incubation at 37°C), but otherwise similar to S. pullorum. Of these 52 outbreaks, 12 occurred in chickens and 40 in turkeys. The chicken outbreaks occurred in five counties of California and in one other state. The turkey outbreaks were' found in nine counties of California and three other states. Eighteen of the 40 turkey outbreaks occurred in two adjacent counties of southern California which have had the greatest intertraffic of hatching eggs over a period of years. The 10 remaining California outbreaks were distributed as follows: northern California, 1; central California, 6; southern California, 3. The 12 out-of-state cultures from turkeys were isolated from poults hatched from eggs purchased from three different California hatching-egg-co6peratives. The source of one out-of-state culture was traced to an individual flock which was found to have carriers in it. The others were traced to pooled eggs supplied by two different cooperatives, both of which had flocks known to be infected with the variant. The outof-state culture from a chick could not be traced to a California source.
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EPIDEMIOLOGY
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fornia. The probable reason for this is mens were submitted for autopsy. More the interstate sale of hatching eggs, poults, complete epidemiological data are, availchicks, and breeding turkeys and chickens able from the area where groups C and D from infected areas to non-infected areas. are located than from the other sections. Many examples of such distribution have Certain observations made in this comcome to light in our investigations which munity are given below. include the out-of-state transmissions menGroup C. As stated above, the maltose tioned above. Examples of these are given fermenter was introduced into group O b y below. a new member who transferred from group Groups A, B, C, and D are four turkey A in the spring of 1939. There were at that egg handling organizations within the two time 80 members in group C. Forty of counties mentioned above. They have their ranches (50 percent) were found handled the majority of hatching eggs pro- to be infected when tested for pullorum duced in these counties. One of the first disease carriers in the 1939-1940 season. known outbreaks caused by this variant Bacteriological data are not available on occurred in 1937 on the key ranch of group all the ranches, but a survey of 23 showed A. In 1938, another outbreak occurred that 3 were infected with the variant and in group A, and also one in group B. Both 20 with the usual type of S. pullorum. A and B are located in the same county A cooperative project on eradication of and have frequently bought eggs from each pullorum disease was in effect at the time other to control shortages and surpluses. between the writers and group C, and every In 1939, there was one outbreak in each effort was made to eliminate all infected of groups A and B, and three in group C. flocks as sources of hatching eggs for the In the spring of 1939, one of the group 1940-1941 replacements. In 1940-1941, there were 58 growers in A members resigned and joined group C. This rancher supplied a large number of group C, and 11 of these had infection early hatching eggs for use as replacements on their premises. Two of the 11 cases were for group C. The three outbreaks in this found by our studies to be due to the malgroup could all be traced to eggs supplied tose fermenter. Two of the three ranches by the new member, and one of them which had had the variant on them in occurred on his ranch. In 1940, there were 1939-1940 were free from pullorum disease two outbreaks in group C, and none were in 1940-1941, and the third had one lot reported from the others. In 1941, one of poults infected with the usual type of outbreak was reported by groups B and C S. pullorum as a result of buying poults and at least four, in group D. It is known from another infected source. that group D had purchased eggs from the There is evidence that one of the two other groups, but, previous to 1941, very 1940-1941 infected ranches had the variant few laboratory records were available from on it in 1939-1940, as it had a reacting its members. flock. No reactors were examined bacteriThese data show that the intertraffic of ologically. Detailed studies on the results hatching eggs between the four groups of studies on this ranch are given below. has caused the spread of this variant from In February, 1939, a diagnosis of pullorum the original focus of infection in group A. disease was made in poults from this ranch. Doubtless other outbreaks than those The culture isolated from the outbreak mentioned occurred in the various groups, was a slow maltose fermenter (18 days). but information is lacking because no speci- When 400 survivors of the outbreak were
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all available eggs laid by it were recalled to prevent further outbreaks. By the time arrangements were completed to retest the flock, all but 472 had been sold, and these were ready to be hauled to the killing plant. Blood samples were taken as the birds were loaded on the buyers' trucks. To our surprise, 373 (79.0 percent) gave complete reactions at 1-25 dilution. The blood serums from these reactors were pooled into five lots and reset to titer. All five pooled samples reacted to a titer of 1-640, showing that the average titer was at least this dilution. Attempts to secure reactors for autopsy were futile, because the dealer already had disposed of them. Fifty of the positive blood samples were pooled and put into 100 ml. of tetrathionate broth (Difco) and after 20 hours' incubation at 37°C. were plated on several media. Salmonella species were not isolated.
A total of 381 eggs were laid by these breeders during the two days previous to being sold to market. These eggs were donated by the owner for hatching. A very poor hatch was secured due to incubator With the exception of one pen of 79 difficulties, pullorum disease, and poor birds, all the other lots were free-on- fertility. From the 247 fertile eggs, only first test, and were not retested. The 79 80 poults were put in the brooder. A total birds had two reactors when tested in Sep- of 134 infertile eggs were cultured and 22 tember, and the 28 kept for breeders had (16.4 percent) yielded the maltose fera single reactor among them in October. menting variant. A total of 24 (16.4 perTwo weeks later, the remaining 27 were cent) of 146 dead embryos yielded it. Thus, negative to the test. At the end of the 46 (16.4 percent) of 280 eggs were intesting season all the breeders were put in fected. No other Salmonellas were isolated. one pen. The flock was considered safe for During the first three weeks, 29 of the 80 egg production until April, 1941, when an poults (36.3 percent) died, and the maltose outbreak of disease caused by the maltose fermenting variant was isolated from 24 of fermenter occurred in an eastern state, and these. The symptoms and pathology in this was traced to eggs laid by this flock. Cul- outbreak were typical of pullorum disease. tures from this outbreak were submitted The surviving poults were artificially into us and they proved to be identical to the fected with Hexamita meleagridis at the age one isolated from the reactor mentioned of 15 days. Losses from hexamitiasis started above. As soon as this outbreak was re- in about three weeks and in the fourth week ported, , and the source determined, the 31 poults died. An additional 15 strains of grower was ordered to sell his flock, and the variant were isolated from these. From
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tested at seven months of age, there were 35 reactors (8.8 percent). A month later the non-reactors were again tested and one more was detected. This was removed, but the owner failed to retest again that season. During the 1940 testing season, a total of 836 turkey breeders from this ranch were tested by the tube agglutination method (1-25 dilution). In one lot of 464 fivemonth-old birds tested in July, 14 reactors were detected (3.0 percent). One month later 13 more reactors (2.9 percent) were found in the remaining 450 birds and on the third test, made in September, four (0.9 percent) were detected. At this time, two reactors were submitted for autopsy and the maltose fermenting variant was isolated from the unabsorbed yolk of one of them. The second was negative for Salmonella species. In October, a fourth test was made, and a single reactor (0.2 percent) was found in the 440 birds. Two weeks after removal of the reactor, the 162 birds in the same pen were retested and all were negative to the test.
EPIDEMIOLOGY OF A MALTOSE FERMENTING VARIANT OF S. PULLORUM
The source of the infection of this flock after it was thought to be free from the disease (negative to the test) is not known. Possibilities are: failure of an infected bird to react to a dilution of 1-25 on the last test; transmission from untested birds in an isolated pen being held till ready for market; some animal source on the ranch; and, from an unidentified bird found in the pen at the end of the season. It has also been impossible to determine when the reinfection occurred. The owner did not notice any indication of illness in
the flock during the spring, nor was there any drop in egg production that would suggest even a sub-acute outbreak. The number of reactors, and the high incidence of the organism in the eggs laid by the flock the last two days before killing, shows a very high morbidity. The egg incidence is higher than any previously recorded for pullorum disease carriers. The fact that the flock was a good laying one, and eggs from it were in many shipments of pooled eggs without any reported cases of pullorum disease previous to the one in March, 1941, would suggest that the reinfection did not take place till late in the season. The high titers (average 640) of the reactors in the April test would also suggest that the birds were then in an active stage of the disease. Group D. The history of the flocks of group D is not as well known as those of group C, because the growers .did not cooperate in taking poults to the laboratory. Testing records are, however, available for the seasons of 1938-1939 to 1941-1942. One of the growers who has been with this group since its beginning, had had reactors in his flock each season, and had never retested until he received a negative report. Many of his reactors had very low titers. This flock was one of the largest and earliest laying ones, therefore, many of the eggs were used for local replacements. This was particularly true in the hatching season of 1941 when eggs from his flock were in every early hatch. The writers tested the flocks for this group during the 1941-1942 season, and attempted to obtain histories of the various Tanches. It was found that 20 (80 percent) of the 25 flocks had reactors to the test for pullorum disease. Reactors from four of the infected ranches (including the one mentioned above) were procured for autopsy, and the maltose fermenting variant was isolated from all of them. It was
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the infertile eggs, dead embryos, and poults, a total of 85 cultures were isolated. All of them fermented maltose within 48 hours, and were culturally identical to the strain isolated from the reactor in this flock in the fall of 1940. Representative strains were tested and found antigentically identical to S. pullorum. The 20 survivors were kept until they were 26 weeks of age; none died after the seventh week. When they were 18 weeks old, they were tested with S. pullorum antigen. A total of four reactors (20 percent) were detected. These were left in the flock with the non-reactors and all were tested again at 24 weeks of age. This time only the same four birds reacted. Two weeks later the four reactors (all males) were rebled and then killed for autopsy. With the exception of unabsorbed umbilical yolks in three, and small caseated masses of exudate in the abdominal cavity of one, the reactors were normal. The maltose variant was isolated from three of the four birds, but only from the unbilical yolk masses of two, and the abdominal lesions of the third. Spleens, livers, testes, and intestinal contents were negative in all of them. The agglutination titers were low in all four reactors. Two of the ones which yielded the variant reacted only to a dilution of 1-25 and the other to a dilution of 1-40. The reactor which was negative on autopsy had a titer of 40.
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RELATION TO THE AGGLUTINATION TEST
There is evidence that this maltose fermenting variant does not always maintain sufficient agglutinins in carriers to cause reactors of high titer. This becomes of importance in the interpretation of results in testing programs. In a group of SO reactors from flocks which yielded the variant by autopsy 34 (68 percent) reacted only at a dilution of 1-25; 7 (14 percent) at 1-40; 4 (8 percent) at 1-80; 4 (8 percent) at 1-160, and 1 (2 percent) at 1-320. The large number of low reactors (68 percent at 1-25) in this group is in contrast to 23.6 percent in a group of 87 reactors yielding S. pullorum reported by Hinshaw, McNeil, and Taylor (1942). It has also been observed that the variant has a tendency to cause "many incomplete reactions even at 1-25 dilution. If there is a history of this organism being in a flock, greater care must, therefore, be exercised in interpreting the reactions obtained. It is, however, capable of producing subacute outbreaks in adult turkeys, and at such times the titers of the infected birds
go much higher. Such a case is described above under group C, in which the average titers of five pooled samples of blood from 373 reactors was 1-640. DISCUSSION
The origin of these maltose fermenting variants of S. pullorum can only be hypothesized, but certain evidence indicates that they are segregated from strains that contained both maltose fermenting and maltose non-fermenting types (Hinshaw, Browne, and Taylor, 1943). An example from our field experience also suggests this possibility. In 1939 two different outbreaks of pullorum disease originating from the same hatch were caused by strains which fermented maltose in 24 hours on original isolation. A strain from one of these outbreaks soon became a very slow fermenter (three to four weeks). A strain from the second outbreak remained a rapid fermenter, but survivors of this outbreak yielded a strain which failed to ferment maltose for nearly three weeks, and then produced both acid and gas. These strains are undoubtedly true variants of S. pullorum. Whether or not some others are, or whether they are a separate species that has the same antigenic structure, is impossible to say at the present time. Slight variations in biochemical reactions suggest that they may be different in their cultural characteristics, though not as distinct as S. gallinarum. Further biochemical tests may reveal other differences, but at present it can be said that this variant differs from the usual type of S. pullorum in the following ways: 1. It ferments maltose regularly and rapidly with gas production. 2. In the carrier state it produces more "low titer" reactors. 3. It has a greater tendency to cause subacute outbreaks in adult birds. 4. It appears to be more easily carried
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discovered that the key ranch, referred to above, always had a few chickens on the premises. Part of these were bantams which had a habit of roosting with the turkeys. The 117 chickens were tested and three reactors were found (one of them a bantam) . These were autopsied and the maltose fermenting variant isolated from two of them. Group D did not consent to retest until its flocks were free, and further bacteriological records are not available locally. However, numerous outbreaks in other areas, caused by the variant, have been traced to poults purchased from this group during the past two seasons, and cultures isolated from these have all been the maltose fermenting variant.
EPIDEMIOLOGY OF A MALTOSE FERMENTING VARIANT or S. PULLORUM
Many diagnostic laboratories pay little or no attention to motility and base their diagnosis of pullorum disease on the failure of cultures to ferment maltose, lactose, and sucrose, and on the ability to ferment glucose. We recommend the routine use of glucose, lactose, maltose, sucrose, and dulcitol for fermentation tests; the use of Tittsler and Sandholzer's (1935) semi-solid agar for motility; and Jordan and Harmon's (1928) d-tartrate agar for preliminary identification of cultures isolated from avian specimens. Cultures that are nonmotile, produce acid and gas in glucose (or glucose and maltose), but not in the other media, may be safely considered tp be S. pullorum. S. pullorum does not ferment Jordan and Harmon's d-tartrate medium while most of the other Salmonellas produce an acid reaction when grown in it. Dulcitol is also fermented by most of the Salmonellas and aids in identifying the maltose fermenting variant from an occasional nonmotile Salmonella variant. Antigenic tests are recommended whenever there is a doubt TO to the proper classification.
The question of the possibility of a new species always arises when one finds such a variant as is described herein. That there may be several variants that could be called sub-species is suggested by our observations and those of Pacheco and Rodrigues (1935, 1935A), Nobrega (1935), Barboni (1937), and Delpy and Rastegar (1938). These authors have all reported on S. pullorum-like strains which rapidly ferment maltose. They differ from the strain reported herein in that they ferment dulcitol and fail to ferment xylose. A more complete discussion of these variants which Delpy and Rastegar suggest be called S. intermedium will be found in Hinshaw (1941). Until more differences than are now apparent are discovered, we question the advisability of considering this variant a new species. SUMMARY
Epidemiological studies are reported on a group of 146 non-motile, maltose fermenting Salmonella cultures which are antigenically identical to Salmonella pullorum. These cultures fermented maltose with gas production in 24 to 72 hours on original isolation and have continued to be rapid fermenters since isolation. Of the 52 outbreaks from which the 146 cultures came, 12 occurred in chickens and 40 in turkeys. Eighteen, of the outbreaks in turkeys occurred in two counties and on ranches controlled by four turkey hatching egg cooperatives (A, B, C, and D). In addition, 13 of the outbreaks could be traced to eggs or poults furnished by these cooperatives, and 10 of these 13 were traceable to a single cooperative hatchery. In these groups the variant was first isolated from a flock belonging to group A. By interchange of members and stock, it was transmitted to groups B, C, and D. Detailed results of studies made on outbreaks in groups C and D are reported.
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by other birds or mammals and may thus be more difficult to eradicate from,a community. Morphologically, it cannot be distinguished from S. pullorum. Growth on solid media is identical and most biochemical reactions are the same. Antigenically, it appears to be identical. Representative strains sent to Dr. P. R. Edwards, University of Kentucky, were found by him to be indistinguishable from S. pullorum. Further evidence of the antigenic likeness was obtained by absorption of the pooled sera from redactors in the subacute outbreak referred to above, with an antigen prepared from a standard strain of S. pullorum. All the agglutinins for S. pullorum and for the variant were absorbed by this procedure. Likewise, the same sera absorbed by the variant contained no agglutinins for S. pullorum.
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ACKNOWLEDGMENTS
Acknowledgment is made to H. R. Baker, E. M. Dickinson, C. B. Hudson, D. E. Madsen, and the California State Department of Agriculture for furnishing cultures and epidemiological data; to P. R. Edwards for antigenic typing of representative strains; and to the Los Angeles County Livestock Department for use of facilities at its Pomona Poultry Demonstration Plant where part of the transmission studies were conducted.
REFERENCES
Barboni, E., 1937, Contributo alia classiflcazione delle Salmonelle "Gallinarum-Pullorum." Clin. Vet. 60:741-757. Delpy, L., and R. Rastegar, 1938. Etude de souches americanis, asiatiques et europeennes de microbes du groupe pullorum-gallinarum. Ann. Inst. Pasteur 61:536-564. Hinshaw, W. R., 1941. Cysteine and related compounds for differentiating members of the genus Salmonella. Hilgardia (U. of Calif.) 13:583-621. Hinshaw, W. R., A. S. Browne, and T. J. Taylor, 1943. Fermentation of maltose by Salmonella pullorum. Jour. Infect. Dis. 72:197-201. Hinshaw, W. R., E. McNeil, and T. J. Taylor, 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meet. U. S. Livestock Sanitary Assoc, pp. 224-237. Jordan, E. 0., and P. H . Harmon, 1928. A new differential medium for the paratyphoid group. Jour. Infect. Dis. 42:238-241. Nobrega, P., 1935. Differenciacao entre "S. Pullorum" e "S. Gallinarum." Papel importante do bacteriophage Archives do Instituto Biologico. 6:71-84. Pacheco, G., and C. Rodrigues, 1935. Nouveau representent des bacteries du groupe pullorumgallinarum. Morphologie des colonies du groupe. C. R. Soc. Biol. 118:905-907. , 1935A. Biologie des bacteries du groupe pullorum-gallinarum. Action sur les mileaux au lait et sur le rouge neutre. C. R. Soc. Biol. 118:1019-1022. Tittsler, R. P., and L. A. Sandholzer, 1935. The use of semi-solid agar for the detection of bacterial motility. Jour. Bact. 29:15.
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The epidemiological picture presented by the variant is similar to that of pullorum disease. The organism differs from S. pullorum mainly in its consistent ability to ferment maltose within 72 hours. In adult carriers more low titer reactors have been encountered, but there appears to be a greater tendency to produce sub-acute outbreaks in adults than in case of pullorum disease. Isolations from eggs laid by reactors, and the production of an acute disease in poults hatched from eggs laid by reactors, show that it has an identical cycle of infection to 5. pullorum. The present studies indicate that the organism is closely related to S. pullorum and that there is little justification for considering it a new species unless more differences are found by future investigation.
Division of Veterinary Science, University of California, Davis (Received for publication August 18, 1943)
HE ability of Salmonella pullorum to ferment maltose is discussed by Hinshaw, Browne, and Taylor, (1943). In that paper the authors review the literature and present a study on the ability of certain strains to segregate into maltose fermenting and maltose non-fermenting substrains. In this paper is given epidemiological observations on a group of 146 nonmotile Salmonella cultures, antigenically indistinguishable from, 5. pullorum, which, on original isolation, fermented maltose with gas production in 24 to 72 hours, and which have continued to be rapid fermenters since isolation. SOURCE OF CULTURES
In 1935, one of the writers (W. R. H.), isolated a culture of Salmonella from the ovary of a turkey hen. This proved to have all the characteristics of S. pullorum except that it produced acid and gas in maltose. The following spring two identical cultures were received from the laboratories of the California State Department of Agriculture with statements that each had been isolated from an outbreak of paratyphoid; one in chicks and one in poults. The chick strain came from southern California and the poult strain from the northern part of the state. For the next few years the various diagnostic laboratories of the State Department of Agriculture supplied us with all the similar cultures isolated by them. In 1937, four strains from three outbreaks were received, and, in 1938, two strains from two outbreaks. In 1939, this variant began to be isolated frequently from acute
outbreaks in both chicks and poults. Since the isolation of the first strain in 1935, 146 cultures from 52 outbreaks have been either isolated by us, or submitted to us for study. All of the cultures have been rapid fermenters of maltose (acid and gas in 1 percent maltose broth within 72 hours incubation at 37°C), but otherwise similar to S. pullorum. Of these 52 outbreaks, 12 occurred in chickens and 40 in turkeys. The chicken outbreaks occurred in five counties of California and in one other state. The turkey outbreaks were' found in nine counties of California and three other states. Eighteen of the 40 turkey outbreaks occurred in two adjacent counties of southern California which have had the greatest intertraffic of hatching eggs over a period of years. The 10 remaining California outbreaks were distributed as follows: northern California, 1; central California, 6; southern California, 3. The 12 out-of-state cultures from turkeys were isolated from poults hatched from eggs purchased from three different California hatching-egg-co6peratives. The source of one out-of-state culture was traced to an individual flock which was found to have carriers in it. The others were traced to pooled eggs supplied by two different cooperatives, both of which had flocks known to be infected with the variant. The outof-state culture from a chick could not be traced to a California source.
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EPIDEMIOLOGY
As mentioned above, this variant has now become distributed throughout Cali-
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fornia. The probable reason for this is mens were submitted for autopsy. More the interstate sale of hatching eggs, poults, complete epidemiological data are, availchicks, and breeding turkeys and chickens able from the area where groups C and D from infected areas to non-infected areas. are located than from the other sections. Many examples of such distribution have Certain observations made in this comcome to light in our investigations which munity are given below. include the out-of-state transmissions menGroup C. As stated above, the maltose tioned above. Examples of these are given fermenter was introduced into group O b y below. a new member who transferred from group Groups A, B, C, and D are four turkey A in the spring of 1939. There were at that egg handling organizations within the two time 80 members in group C. Forty of counties mentioned above. They have their ranches (50 percent) were found handled the majority of hatching eggs pro- to be infected when tested for pullorum duced in these counties. One of the first disease carriers in the 1939-1940 season. known outbreaks caused by this variant Bacteriological data are not available on occurred in 1937 on the key ranch of group all the ranches, but a survey of 23 showed A. In 1938, another outbreak occurred that 3 were infected with the variant and in group A, and also one in group B. Both 20 with the usual type of S. pullorum. A and B are located in the same county A cooperative project on eradication of and have frequently bought eggs from each pullorum disease was in effect at the time other to control shortages and surpluses. between the writers and group C, and every In 1939, there was one outbreak in each effort was made to eliminate all infected of groups A and B, and three in group C. flocks as sources of hatching eggs for the In the spring of 1939, one of the group 1940-1941 replacements. In 1940-1941, there were 58 growers in A members resigned and joined group C. This rancher supplied a large number of group C, and 11 of these had infection early hatching eggs for use as replacements on their premises. Two of the 11 cases were for group C. The three outbreaks in this found by our studies to be due to the malgroup could all be traced to eggs supplied tose fermenter. Two of the three ranches by the new member, and one of them which had had the variant on them in occurred on his ranch. In 1940, there were 1939-1940 were free from pullorum disease two outbreaks in group C, and none were in 1940-1941, and the third had one lot reported from the others. In 1941, one of poults infected with the usual type of outbreak was reported by groups B and C S. pullorum as a result of buying poults and at least four, in group D. It is known from another infected source. that group D had purchased eggs from the There is evidence that one of the two other groups, but, previous to 1941, very 1940-1941 infected ranches had the variant few laboratory records were available from on it in 1939-1940, as it had a reacting its members. flock. No reactors were examined bacteriThese data show that the intertraffic of ologically. Detailed studies on the results hatching eggs between the four groups of studies on this ranch are given below. has caused the spread of this variant from In February, 1939, a diagnosis of pullorum the original focus of infection in group A. disease was made in poults from this ranch. Doubtless other outbreaks than those The culture isolated from the outbreak mentioned occurred in the various groups, was a slow maltose fermenter (18 days). but information is lacking because no speci- When 400 survivors of the outbreak were
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all available eggs laid by it were recalled to prevent further outbreaks. By the time arrangements were completed to retest the flock, all but 472 had been sold, and these were ready to be hauled to the killing plant. Blood samples were taken as the birds were loaded on the buyers' trucks. To our surprise, 373 (79.0 percent) gave complete reactions at 1-25 dilution. The blood serums from these reactors were pooled into five lots and reset to titer. All five pooled samples reacted to a titer of 1-640, showing that the average titer was at least this dilution. Attempts to secure reactors for autopsy were futile, because the dealer already had disposed of them. Fifty of the positive blood samples were pooled and put into 100 ml. of tetrathionate broth (Difco) and after 20 hours' incubation at 37°C. were plated on several media. Salmonella species were not isolated.
A total of 381 eggs were laid by these breeders during the two days previous to being sold to market. These eggs were donated by the owner for hatching. A very poor hatch was secured due to incubator With the exception of one pen of 79 difficulties, pullorum disease, and poor birds, all the other lots were free-on- fertility. From the 247 fertile eggs, only first test, and were not retested. The 79 80 poults were put in the brooder. A total birds had two reactors when tested in Sep- of 134 infertile eggs were cultured and 22 tember, and the 28 kept for breeders had (16.4 percent) yielded the maltose fera single reactor among them in October. menting variant. A total of 24 (16.4 perTwo weeks later, the remaining 27 were cent) of 146 dead embryos yielded it. Thus, negative to the test. At the end of the 46 (16.4 percent) of 280 eggs were intesting season all the breeders were put in fected. No other Salmonellas were isolated. one pen. The flock was considered safe for During the first three weeks, 29 of the 80 egg production until April, 1941, when an poults (36.3 percent) died, and the maltose outbreak of disease caused by the maltose fermenting variant was isolated from 24 of fermenter occurred in an eastern state, and these. The symptoms and pathology in this was traced to eggs laid by this flock. Cul- outbreak were typical of pullorum disease. tures from this outbreak were submitted The surviving poults were artificially into us and they proved to be identical to the fected with Hexamita meleagridis at the age one isolated from the reactor mentioned of 15 days. Losses from hexamitiasis started above. As soon as this outbreak was re- in about three weeks and in the fourth week ported, , and the source determined, the 31 poults died. An additional 15 strains of grower was ordered to sell his flock, and the variant were isolated from these. From
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tested at seven months of age, there were 35 reactors (8.8 percent). A month later the non-reactors were again tested and one more was detected. This was removed, but the owner failed to retest again that season. During the 1940 testing season, a total of 836 turkey breeders from this ranch were tested by the tube agglutination method (1-25 dilution). In one lot of 464 fivemonth-old birds tested in July, 14 reactors were detected (3.0 percent). One month later 13 more reactors (2.9 percent) were found in the remaining 450 birds and on the third test, made in September, four (0.9 percent) were detected. At this time, two reactors were submitted for autopsy and the maltose fermenting variant was isolated from the unabsorbed yolk of one of them. The second was negative for Salmonella species. In October, a fourth test was made, and a single reactor (0.2 percent) was found in the 440 birds. Two weeks after removal of the reactor, the 162 birds in the same pen were retested and all were negative to the test.
EPIDEMIOLOGY OF A MALTOSE FERMENTING VARIANT OF S. PULLORUM
The source of the infection of this flock after it was thought to be free from the disease (negative to the test) is not known. Possibilities are: failure of an infected bird to react to a dilution of 1-25 on the last test; transmission from untested birds in an isolated pen being held till ready for market; some animal source on the ranch; and, from an unidentified bird found in the pen at the end of the season. It has also been impossible to determine when the reinfection occurred. The owner did not notice any indication of illness in
the flock during the spring, nor was there any drop in egg production that would suggest even a sub-acute outbreak. The number of reactors, and the high incidence of the organism in the eggs laid by the flock the last two days before killing, shows a very high morbidity. The egg incidence is higher than any previously recorded for pullorum disease carriers. The fact that the flock was a good laying one, and eggs from it were in many shipments of pooled eggs without any reported cases of pullorum disease previous to the one in March, 1941, would suggest that the reinfection did not take place till late in the season. The high titers (average 640) of the reactors in the April test would also suggest that the birds were then in an active stage of the disease. Group D. The history of the flocks of group D is not as well known as those of group C, because the growers .did not cooperate in taking poults to the laboratory. Testing records are, however, available for the seasons of 1938-1939 to 1941-1942. One of the growers who has been with this group since its beginning, had had reactors in his flock each season, and had never retested until he received a negative report. Many of his reactors had very low titers. This flock was one of the largest and earliest laying ones, therefore, many of the eggs were used for local replacements. This was particularly true in the hatching season of 1941 when eggs from his flock were in every early hatch. The writers tested the flocks for this group during the 1941-1942 season, and attempted to obtain histories of the various Tanches. It was found that 20 (80 percent) of the 25 flocks had reactors to the test for pullorum disease. Reactors from four of the infected ranches (including the one mentioned above) were procured for autopsy, and the maltose fermenting variant was isolated from all of them. It was
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the infertile eggs, dead embryos, and poults, a total of 85 cultures were isolated. All of them fermented maltose within 48 hours, and were culturally identical to the strain isolated from the reactor in this flock in the fall of 1940. Representative strains were tested and found antigentically identical to S. pullorum. The 20 survivors were kept until they were 26 weeks of age; none died after the seventh week. When they were 18 weeks old, they were tested with S. pullorum antigen. A total of four reactors (20 percent) were detected. These were left in the flock with the non-reactors and all were tested again at 24 weeks of age. This time only the same four birds reacted. Two weeks later the four reactors (all males) were rebled and then killed for autopsy. With the exception of unabsorbed umbilical yolks in three, and small caseated masses of exudate in the abdominal cavity of one, the reactors were normal. The maltose variant was isolated from three of the four birds, but only from the unbilical yolk masses of two, and the abdominal lesions of the third. Spleens, livers, testes, and intestinal contents were negative in all of them. The agglutination titers were low in all four reactors. Two of the ones which yielded the variant reacted only to a dilution of 1-25 and the other to a dilution of 1-40. The reactor which was negative on autopsy had a titer of 40.
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RELATION TO THE AGGLUTINATION TEST
There is evidence that this maltose fermenting variant does not always maintain sufficient agglutinins in carriers to cause reactors of high titer. This becomes of importance in the interpretation of results in testing programs. In a group of SO reactors from flocks which yielded the variant by autopsy 34 (68 percent) reacted only at a dilution of 1-25; 7 (14 percent) at 1-40; 4 (8 percent) at 1-80; 4 (8 percent) at 1-160, and 1 (2 percent) at 1-320. The large number of low reactors (68 percent at 1-25) in this group is in contrast to 23.6 percent in a group of 87 reactors yielding S. pullorum reported by Hinshaw, McNeil, and Taylor (1942). It has also been observed that the variant has a tendency to cause "many incomplete reactions even at 1-25 dilution. If there is a history of this organism being in a flock, greater care must, therefore, be exercised in interpreting the reactions obtained. It is, however, capable of producing subacute outbreaks in adult turkeys, and at such times the titers of the infected birds
go much higher. Such a case is described above under group C, in which the average titers of five pooled samples of blood from 373 reactors was 1-640. DISCUSSION
The origin of these maltose fermenting variants of S. pullorum can only be hypothesized, but certain evidence indicates that they are segregated from strains that contained both maltose fermenting and maltose non-fermenting types (Hinshaw, Browne, and Taylor, 1943). An example from our field experience also suggests this possibility. In 1939 two different outbreaks of pullorum disease originating from the same hatch were caused by strains which fermented maltose in 24 hours on original isolation. A strain from one of these outbreaks soon became a very slow fermenter (three to four weeks). A strain from the second outbreak remained a rapid fermenter, but survivors of this outbreak yielded a strain which failed to ferment maltose for nearly three weeks, and then produced both acid and gas. These strains are undoubtedly true variants of S. pullorum. Whether or not some others are, or whether they are a separate species that has the same antigenic structure, is impossible to say at the present time. Slight variations in biochemical reactions suggest that they may be different in their cultural characteristics, though not as distinct as S. gallinarum. Further biochemical tests may reveal other differences, but at present it can be said that this variant differs from the usual type of S. pullorum in the following ways: 1. It ferments maltose regularly and rapidly with gas production. 2. In the carrier state it produces more "low titer" reactors. 3. It has a greater tendency to cause subacute outbreaks in adult birds. 4. It appears to be more easily carried
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discovered that the key ranch, referred to above, always had a few chickens on the premises. Part of these were bantams which had a habit of roosting with the turkeys. The 117 chickens were tested and three reactors were found (one of them a bantam) . These were autopsied and the maltose fermenting variant isolated from two of them. Group D did not consent to retest until its flocks were free, and further bacteriological records are not available locally. However, numerous outbreaks in other areas, caused by the variant, have been traced to poults purchased from this group during the past two seasons, and cultures isolated from these have all been the maltose fermenting variant.
EPIDEMIOLOGY OF A MALTOSE FERMENTING VARIANT or S. PULLORUM
Many diagnostic laboratories pay little or no attention to motility and base their diagnosis of pullorum disease on the failure of cultures to ferment maltose, lactose, and sucrose, and on the ability to ferment glucose. We recommend the routine use of glucose, lactose, maltose, sucrose, and dulcitol for fermentation tests; the use of Tittsler and Sandholzer's (1935) semi-solid agar for motility; and Jordan and Harmon's (1928) d-tartrate agar for preliminary identification of cultures isolated from avian specimens. Cultures that are nonmotile, produce acid and gas in glucose (or glucose and maltose), but not in the other media, may be safely considered tp be S. pullorum. S. pullorum does not ferment Jordan and Harmon's d-tartrate medium while most of the other Salmonellas produce an acid reaction when grown in it. Dulcitol is also fermented by most of the Salmonellas and aids in identifying the maltose fermenting variant from an occasional nonmotile Salmonella variant. Antigenic tests are recommended whenever there is a doubt TO to the proper classification.
The question of the possibility of a new species always arises when one finds such a variant as is described herein. That there may be several variants that could be called sub-species is suggested by our observations and those of Pacheco and Rodrigues (1935, 1935A), Nobrega (1935), Barboni (1937), and Delpy and Rastegar (1938). These authors have all reported on S. pullorum-like strains which rapidly ferment maltose. They differ from the strain reported herein in that they ferment dulcitol and fail to ferment xylose. A more complete discussion of these variants which Delpy and Rastegar suggest be called S. intermedium will be found in Hinshaw (1941). Until more differences than are now apparent are discovered, we question the advisability of considering this variant a new species. SUMMARY
Epidemiological studies are reported on a group of 146 non-motile, maltose fermenting Salmonella cultures which are antigenically identical to Salmonella pullorum. These cultures fermented maltose with gas production in 24 to 72 hours on original isolation and have continued to be rapid fermenters since isolation. Of the 52 outbreaks from which the 146 cultures came, 12 occurred in chickens and 40 in turkeys. Eighteen, of the outbreaks in turkeys occurred in two counties and on ranches controlled by four turkey hatching egg cooperatives (A, B, C, and D). In addition, 13 of the outbreaks could be traced to eggs or poults furnished by these cooperatives, and 10 of these 13 were traceable to a single cooperative hatchery. In these groups the variant was first isolated from a flock belonging to group A. By interchange of members and stock, it was transmitted to groups B, C, and D. Detailed results of studies made on outbreaks in groups C and D are reported.
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by other birds or mammals and may thus be more difficult to eradicate from,a community. Morphologically, it cannot be distinguished from S. pullorum. Growth on solid media is identical and most biochemical reactions are the same. Antigenically, it appears to be identical. Representative strains sent to Dr. P. R. Edwards, University of Kentucky, were found by him to be indistinguishable from S. pullorum. Further evidence of the antigenic likeness was obtained by absorption of the pooled sera from redactors in the subacute outbreak referred to above, with an antigen prepared from a standard strain of S. pullorum. All the agglutinins for S. pullorum and for the variant were absorbed by this procedure. Likewise, the same sera absorbed by the variant contained no agglutinins for S. pullorum.
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ACKNOWLEDGMENTS
Acknowledgment is made to H. R. Baker, E. M. Dickinson, C. B. Hudson, D. E. Madsen, and the California State Department of Agriculture for furnishing cultures and epidemiological data; to P. R. Edwards for antigenic typing of representative strains; and to the Los Angeles County Livestock Department for use of facilities at its Pomona Poultry Demonstration Plant where part of the transmission studies were conducted.
REFERENCES
Barboni, E., 1937, Contributo alia classiflcazione delle Salmonelle "Gallinarum-Pullorum." Clin. Vet. 60:741-757. Delpy, L., and R. Rastegar, 1938. Etude de souches americanis, asiatiques et europeennes de microbes du groupe pullorum-gallinarum. Ann. Inst. Pasteur 61:536-564. Hinshaw, W. R., 1941. Cysteine and related compounds for differentiating members of the genus Salmonella. Hilgardia (U. of Calif.) 13:583-621. Hinshaw, W. R., A. S. Browne, and T. J. Taylor, 1943. Fermentation of maltose by Salmonella pullorum. Jour. Infect. Dis. 72:197-201. Hinshaw, W. R., E. McNeil, and T. J. Taylor, 1942. Four years progress in eradication of pullorum disease from turkey flocks. Proc. 46th Ann. Meet. U. S. Livestock Sanitary Assoc, pp. 224-237. Jordan, E. 0., and P. H . Harmon, 1928. A new differential medium for the paratyphoid group. Jour. Infect. Dis. 42:238-241. Nobrega, P., 1935. Differenciacao entre "S. Pullorum" e "S. Gallinarum." Papel importante do bacteriophage Archives do Instituto Biologico. 6:71-84. Pacheco, G., and C. Rodrigues, 1935. Nouveau representent des bacteries du groupe pullorumgallinarum. Morphologie des colonies du groupe. C. R. Soc. Biol. 118:905-907. , 1935A. Biologie des bacteries du groupe pullorum-gallinarum. Action sur les mileaux au lait et sur le rouge neutre. C. R. Soc. Biol. 118:1019-1022. Tittsler, R. P., and L. A. Sandholzer, 1935. The use of semi-solid agar for the detection of bacterial motility. Jour. Bact. 29:15.
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The epidemiological picture presented by the variant is similar to that of pullorum disease. The organism differs from S. pullorum mainly in its consistent ability to ferment maltose within 72 hours. In adult carriers more low titer reactors have been encountered, but there appears to be a greater tendency to produce sub-acute outbreaks in adults than in case of pullorum disease. Isolations from eggs laid by reactors, and the production of an acute disease in poults hatched from eggs laid by reactors, show that it has an identical cycle of infection to 5. pullorum. The present studies indicate that the organism is closely related to S. pullorum and that there is little justification for considering it a new species unless more differences are found by future investigation.