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Transmission Studies of Avian Infectious Synovitis by Selected Arthropod Vectors1
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G. F . G E E AND P . C. H A R R I S
concentrations into the egg before incubation resulted in sex reversal of genetic males and alteration of eminence types of both sexes. ACKNOWLEDGMENTS The authors are indebted to Wirthmore Feeds, Inc. for financial support and to Mr. Charles Green, Winterport, Maine, for his sincere interest and willing cooperation in classification of genital eminence types. REFERENCES Canfield, T. H., 1941. Sex determination of day-old chicks. 2. Type variations. Poultry Sci. 20: 327328. Dove, W. F., 1928. Sex sterility and the diminutive copulatory organ in the domestic fowl. Science, 68: 327-328. Hammond, J. C , and W. H. Burrows, 1937. The female genital eminence is not a measure of future egg production. Poultry Sci. 16: 285-286. van Tienhoven, A., 1957. A method of "controlling sex" by dipping of eggs in hormone solutions. Poultry Sci. 36: 628-632.
Transmission Studies of Avian Infectious Synovitis by Selected Arthropod Vectors1 E. C. T U R N E R , J R . , N A N C Y L. W E H R H E I M 2 AND D. H.
MESSERSMITH3
Department of Entomology AND J. W.
DAVIS
Department of Veterinary Science, Virginia Polytechnic Institute, Blacksburg, Va. (Received for publication May 2, 1963)
T
H E disease infectious synovitis was first reported by Wills (1954a, b) and Olson el al. (1954). Since t h a t time ex-
1 The research reported herein was supported by the National Institutes of Health as Project No. E2717. Presented at the 34th Annual Meeting, Eastern Branch, Entomological Society of America, Philadelphia, Pa. 1962. 2 Present Address, Department of Pathology, University of North Carolina, Chapel Hill, North Carolina.
tensive research has been done by numerous workers on the etiology, therapy, and control of this disease, however, the exact nature of how this disease was transmitted from bird to bird has not been proven. Wills and Delaplane (1955) demonstrated transmission of infectious synovitis b y eggs. This was later con3 Present Address, Department of Biology, Radford College, Radford, Virginia.
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show any relationships with genital eminence type: hatch, age of egg before incubation, size of egg, length of incubation period, body weight at day-old and at four and eight weeks of age, tail feather length, age a t first egg, and rate of lay. Chicks from an auto-sexing cross were utilized to investigate the possible relationships between endocrine gland weights and genital eminence type. This mating provided eminence type ratios for 503 males of 7 5 % type A, 1 6 % type B, and 9 % type C; and for 514 females of 8 5 % type A, 10% type B, and 4 % type C. T h e weights of the hypophysis, thyroids, gonads, adrenals, and bursa of Fabricius were not related to eminence type. No differences in oviduct weights were noted between the A and C eminence type female chicks in response to 0, 0.1, and 0.25 mg. of D.E.S. administered subcutaneously on alternate days over a 12-day period. Injection of D.E.S. in varying
I N F E C T I O U S SYNOVITIS
Insect surveys were initiated in poultry houses in Virginia. Particular attention was given to houses with a history of infectious synovitis. These surveys, using standard " N e w J e r s e y " light traps revealed t h a t species of the genus Culicoides (Order: Diptera; Family: Ceratopogonidae) were by far the most a b u n d a n t group of blood-sucking insects associated with poultry. I t is known t h a t several species of Culicoides are avian feeders, as shown by Judd (1957). Fallis and Wood (1957) and Fallis and Bennett (1960, 1961) reported t h a t Culicoides were intermediate hosts for Haemoproteus, a parasitic protozoan found in the blood of birds. A number of species of Culicoides were collected in poultry houses by Snow et al. (1957), Wirth and Bottimer (1956) and others. M a n y of these were engorged females. Culicoides are known vectors of certain tropical filaria as reported by Sharp (1927, 1928) and most recently by Hopkins and Nicholas (1952). Of even greater
significance is the fact t h a t they have been shown by D u T o i t (1944) and later by Price and H a r d y (1954) to transmit blue-tongue disease in sheep. Also of interest is the fact t h a t Tokunaga (1937) showed Culicoides to be a vector of fowlpox. Blood-sucking mites were also suspected as possible vectors of synovitis since they are known vectors of other diseases. Smith et al. (1944) demonstrated the relationship between Dermanyssus gallinae and St. Louis encephalitis. Smith et al. (1947) later proved transmission of the virus to normal chickens by the bite of infected mites. They demonstrated t h a t both experimentally infected and naturally infected mites were capable of the transfer. Benton (1959) reported t h a t cage-mate transmission of infectious synovitis occurred in correlation with northern fowl mite infestations. Peterson (1962) was unable to demonstrate cagem a t e transmission in the absence of mites. Hinshaw and McNeil (1952) noticed a correlation between the incidence of infection and the presence of mosquitoes while working with staphylococcus in turkeys. T h e y reported t h a t a decrease in the incidence of the disease was observed in one area following mosquito control operations. T h e y also suggested the possibility of transmission through open wounds. However Fahey (1954) found no mosquito correlation in his work with staphylococcal arthritis in turkey poults. In view of the work of these and other authors it was felt t h a t an investigation of the possibilities of arthropod transmission of infectious synovitis was justified. Certain arthropods t h a t could be reared in the laboratory were selected for this study. PROCEDURE Rearing Techniques: The source of the infective agent was obtained from a field
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firmed by Snoeyenbos and Basch (1958). I n these reported cases positive transmission via the egg occurred only in a very small number of times. Field outbreaks of the disease occurred in a much larger percentage of cases, and it was suspected t h a t there was probably other means of transmitting the disease within a flock of chickens. I n an early report, Wills (1954a) suggested the possibility of insect transmission. Olson et al. (1957) reported t h a t the causative agent was found in the blood of affected birds. Later, Benton and Cover (1959) found t h a t the agent was present in infectious levels in the blood by the eighth hour after intravenous inoculation and persisted until at least the fifteenth day. These investigations further suggested the possibility of transmission by a blood-sucking arthropod.
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E. C. T U R N E R , J R . , N . L. W E H R H E I M , D . H. M E S S E R S M I T H AND J. W.
Pure cultures of Culicoides variipennis (Coquillett) larvae were collected in the mud of the salt marshes at Saltville, Virginia, and placed in gallon-size icecream cartons. In the laboratory a 2\ inch hole was cut in the top of these cartons and emergence cages were placed over them. These emergence cages consisted of pint size ice-cream cartons in which a 2\ inch hole was cut in the bottom. The tops of these emergence cages were replaced with clear polyethylene plastic on which two f-inch holes were cut by means of a cork borer. One of the holes was used for ventilation and was covered with a small piece of 80-mesh silk cloth. A cork stopper was placed in the other hole. The emerging adult insects were removed daily by means of a hand aspirator and were placed in similarly constructed gallon icecream cartons. Food in the form of dried raisins and water was provided and the cages were stored in a B.O.D. Incubator at a constant temperature of 25°C. Northern fowl mites were reared in the laboratory on PPLO-free culture chickens. Mites were transferred by being brushed from a culture bird by means of a oneinch nylon brush onto a large white enamel pan. Adult mites were then collected with a small camels hair brush or a hand aspirator and placed on test birds. After
feeding on these birds the mites were removed by means of a hand aspirator and used in either the maceration tests or refeeding tests. Pure cultures of Culex pipiens group and Aedes sollicitans (Walker) mosquitoes were collected in the field and allowed to emerge as adults. These adults were stored and handled in the same manner as described for Culicoides variipennis. Transmission Techniques: For transmission studies, 12 to 14 C. variipennis adult females 5 to 7 days old were removed from the holding cage and placed in another similarly constructed cage. T h e cage was placed on its side and the cork stopper from the plastic top was removed. Immediately, either a healthy or diseased chicken was immobilized, and its head was pushed through the plastic hole and the insects were allowed to feed on or about the head. Although actual feeding by a single female took only about five minutes, it usually took about one or two hours for all of the insects to land on the chicken head and engorge fully. T h e engorged insects were then removed from this cage and macerated by means of a glass tissue grinder in 2 ml. of tryptose broth. The tryptose suspension was then inoculated intramuscularly (1 ml.) or intravenously (.4 ml.) into a 3 to 5-week old healthy bird. In other experiments the engorged insects were used in refeeding experiments. The insects were allowed to feed on the healthy birds in the same manner as they had fed on the diseased birds. All exposed birds were then placed in Modified Horsefall-Bauer Units for observation. The northern fowl mites were removed from culture chickens in the manner previously described. Since the mites do not readily leave their hosts, no special cage was needed for the transmission experiments,
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case of infectious synovitis. Material from the spleen was suspended in tryptose and held at — 50°C. until used in the experiments. All birds used were from a P P L O free flock of White Leghorn chickens t h a t were about 3 to 5 weeks old. Modified Horsefall-Bauer Units were used to house and isolate the birds exposed to the disease. Birds used in the transmission experiments were observed daily for external symptoms of infectious synovitis. On death of a bird or at the termination of an experiment, an autopsy was performed to confirm the preliminary diagnosis.
DAVIS
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INFECTIOUS SYNOVITIS
RESULTS AND DISCUSSION
TABLE 1.—Transmission of infectious synovitis using macerated engorged Culicoides variipennis immediately after feeding on infected chickens No. days from initial infection to insect No. of positive transmissions/ No. of birds inoculated feeding 2 4 7 8 a
iv = intravenous.
iv 1/2 2/2 2/2 2/2
ima 0/1 1/1 1/1 1/1
Experiment 2: The purpose of this test was to determine if the infective agent would remain active enough to be transmitted in or on an infected insect (C. variipennis) and how long this agent would remain active. T h e insects were collected after feeding on diseased birds and held for certain periods of time before maceration and inoculation. The results are shown in Table 2. Transmission was demonstrated in infected insects t h a t were held u p to 24 hours but not at 72 hours. I t is interesting to note t h a t the method of inoculating the macerated material appeared to be important. At the 24-hour interval, transmission was demonstrated only when the material was inouclated intravenously into healthy birds. These results would indicate t h a t the infective agent does not remain active in or on the insect vector for a particularly long period of time. Therefore, transmission would be attained only when the insect would feed on infected birds and then on uninfected birds within a relatively short time such as would occur when the insect was interrupted in its feeding. Experiment 3: In preliminary refeeding experiments, both diseased and healthy chickens were placed in adjoining pens in a manner which would prevent direct contact with each other b u t would allow equal exposure to C. variipennis. Large numbers of 5 to 7-day old adult insects TABLE 2.—Transmission of infectious synovitis using engorged Culicoides variipennis and held for various time intervals before maceration and inoculation No. Hours insects held
No. of positive transmission/ No. of birds inoculated iv 2/2
3 6 24 72
—
4/4 0/2
im = intramuscular.
\
im 2/2 2/2 0/4 0/1
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Experiment 1: An a t t e m p t was made to demonstrate the presence of the infectious synovitis agent in or on the body of C. variipennis after the insect had been allowed to feed on birds inoculated with the disease. An a t t e m p t was made to determine how long the infected agent would remain in the blood of infected birds and hence available to blood-sucking insects. At certain time intervals after initial inoculation of the test birds with spleen suspension of the infective agent, the insects were allowed to engorge on these birds. T h e y were then immediately macerated and inoculated into healthy chickens as previously described. In Table 1, positive transmission was demonstrated in almost all cases. This indicates t h a t the infective agent was present in the blood spleen inoculated birds for at least 2 to 8 days, thus partially confirming the results obtained by Benton and Cover (1959) on the infectivity of the blood of birds infected with infectious synovitis. The agent was also demonstrated to be present in or on the engorged insects. Since it has been shown previously t h a t Culicoides are capable of transmitting certain animal diseases, it was felt t h a t further tests using this insect should be carried out in order to determine definitely if infectious synovitis can be carried from bird to bird by this method.
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E. C. T U R N E R , J R . , N . L. W E H R H E I M , D . H . M E S S E R S M I T H AND J. W.
This experiment was set up in such a manner t h a t feeding and refeeding by individual insects was definitely observed. Partially engorged females were removed from diseased birds and placed on healthy birds where these insects continued to feed until they were fully engorged, then they were removed and macerated in tryptose broth. The material thus obtained was inoculated into other healthy birds. Table 3 shows the results of this test. Transmission was not demonstrated by interrupted feeding of C. variipennis although the infective agent was present in or on the insect. This was demonstrated by the positive transmission results obtained when the insect used in the refeeding tests were macerated and inoculated into other healthy birds. I t thus TABLE 3.—Direct transmission ofinfectious synovitis by interrupted feeding of Culicoides variipennis
Type of transmission
No. of positive transmissions/ No. birds tested
Refeeding Maceration1
0/2 2/2
a After the insects had completed feeding on healthy chickens, the engorged insects were then macerated in tryptose and inoculated (im) into other healthy chickens.
A
TABLE 4.—Transmission of infectious synovitis using macerated Ornithonyssus sylviarum immediately after feeding on infected birds Time mites allowed on infected birds 3 10 12 23
hours days days days
No. of positive transmission/ No. of birds inoculated iv 2/3 1/2 4/4 0/2
im 0/1
— —
appears that, under the conditions outlined in this experiment, the infectious synovitis agent cannot be transmitted by the bite of C. variipennis even though the agent is present in or on the vector. Experiment 4: This experiment was set up to determine whether the infectious synovitis agent would be found present in or on blood-sucking mites t h a t had fed on diseased birds. Twelve to fifteen adult northern fowl mites were removed from culture birds and placed on previously inoculated chickens. These mites were allowed to feed on the infected birds for various lengths of time as shown on Table 4. I t was noted t h a t only about half of the mites were engorged when they were removed from the infected birds. The mites were then macerated in 2 ml. of tryptose broth and inoculated into healthy chickens as previously described. The results show t h a t the agent is present in or on the mites although 100% transmission was not demonstrated. Experiment 5: Since it was indicated in experiment 4 t h a t the agent was present in or on blood-sucking mites, a refeeding test was initiated to demonstrate natural transmission. The mites were handled as previously described and allowed to feed on infected birds as shown in Table 5. At certain time intervals the infected mites were removed and placed on healthy birds where they were allowed to remain for several days. The results show t h a t no transmission was demon-
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were released in these pens, however, it was difficult to determine if the insects fed at all or if they moved from sick birds to healthy birds. The results from these preliminary refeeding experiments were thus inconclusive. Messersmith (1961) live collected several species of Culicoides from poultry houses in which some birds were infected with infectious synovitis. These insects were macerated and injected into healthy birds b u t negative results were obtained. Unfortunately the birds t h a t were inoculated had been receiving aureomycin in their feed. Since the material is used as a control for infectious synovitis, the results of the experiment must be regarded as inconclusive.
DAVIS
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INFECTIOUS SYNOVITIS TABLE 5.—Refeeding or natural transmission of infectious synovitis by Ornithonyssus sylviarum Time mites allowed on infected birds 2 hours 3 hours 2 days 3 days 7 days 10 days 23 days
No. positive transmissions/ No. birds tested 0/2 0/2 0/4 0/4 0/7 0/2 0/2
Time mites allowed on infected birds
No. of positive transmissions/ No. birds tested
3 hours 23 days
0/4 0/2
to feed on infected birds and immediately macerated in a tissue grinder as described previously. The macerated material was then inoculated into healthy birds as shown in Table 7. Positive transmission of the disease was obtained in every case using either species of mosquitoes, indicating that the agent of infectious synovitis can therefore be carried in or on the bodies of these mosquitoes. Benton (1959) and Peterson (1962) were unable to demonstrate positive transmission of infectious synovitis using macerated mosquitoes. Refeeding tests by Peterson with Aedes aegypti were also negative. It is concluded that under the conditions of the experiment described herein that direct mechanical transmission of the infectious synovitis agent by C. variipennis (Coquillett) and the northern fowl mite probably does not occur. Negative transmission was obtained with C. variipennis even when the agent producing the disease was shown to be present in or on the body of this insect. Refeeding tests using the northern fowl mite also resulted in negative transmission. The method of inoculation of the disease in the bird may be quite important in transmission studies. As shown by Skamser and Seeger (1960), sinus inoculated birds were able to infect a greater percent of cage mates than footpad-inoculated birds. They stated that the increased transmission of synovitis in the sinus infected groups could occur by contact or by contaminated feed and water. Experiments to investigate the possibility of biological
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strated under the conditions of this experiment. Similar results were obtained by Chamberlain and Sikes (1955) and Chamberlain et al. (1957) working with the transmission of eastern and western equine encephalitis. In extensive experiments they were unable to demonstrate transmission of these viruses by several species of mites including, Ornithonyssus sylviarum. Experiment 6: Chamberlain and Sikes (1955) and Chamberlain et al. (1957) also attempted to demonstrate the transmission of eastern and western equine encephalitis by crushing infected mites into skin break of birds. Results were again negative. In this experiment the mites were allowed to feed on infected birds. After feeding, the engorged mites were removed by sterile forceps and crushed into wounds made on the backs of healthy birds, such as might be encountered in peck wounds. Table 6 shows the results of this test. Again no transmission of infectious synovitis was demonstrated. Thus, it appears that the disease is not transmitted in this manner by the northern fowl mite. Experiment 7: Since mosquitoes are known vectors of many virus diseases as well as other types of diseases it was felt that preliminary maceration tests should be carried out. Using pure cultures of Culex pipiens group and Aedes sollicitans (Walker), the adult females were allowed
TABLE 6.—Transmission of infectious synovitis by crushing infected Ornithonyssus sylviarum into skin breaks of susceptible birds
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E. C. T U R N E R , J R . , N . L. W E H R H E I M , D . H. M E S S E R S M I T H AND J. W.
TABLE 7.—Transmission of infectious synovitis using macerated engorged mosquitoes No. of positive transmissions/ No. of birds inoculated
Species
Culex pipiens Gp. Aedes sollicitans
iv 2/2 4/4
im —• 2/2
SUMMARY Preliminary studies on the infectivity of blood of synovitis infected birds, have indicated t h a t certain blood-sucking arthropods known to feed on poultry could possibly transmit the agent causing this disease. The purpose of these experiments was therefore to investigate possible vectors of infectious synovitis among certain arthropods known to transmit other diseases. Culicoides variipennis (Coquillett) and the northern fowl mite, Ornilhonyssus sylviarum (C. & F.) were investigated as possible vectors of this disease. Immediately after being allowed to engorge on .diseased chickens, C. variipennis adults were macerated and inoculated into healthy birds. This resulted in positive transmission of the disease. Tests using infected engorged C. variipennis which were held for various time intervals up to 24 hours before maceration and inoculations into healthy chickens also resulted in positive transmission. However, in interrupted feeding tests using infected C. variipennis, no transmission occurred.
Maceration tests using engorged infected northern fowl mites resulted in positive transmission, b u t direct refeeding experiments resulted in no transmission of the disease. Also, tests on the transmission of the disease by crushing infected mites into skin-breaks of susceptible birds were negative. Preliminary maceration tests using infected Culex pipiens group and Aedes sollicitans (Walker) mosquitoes also resulted in positive transmission of infectious synovitis in chickens.
REFERENCES Benton, W. J., 1959. Studies on the transmission of infectious synovitis of chickens. M. S. Thesis, University of Del., Newark, Del. Benton, W. J., and M. S. Cover, 1959. The infectivity of blood and tissue from chickens with infectious synovitis. Avian Dis. 3: 361-370. Chamberlain, R. W., and R. K. Sikes, 1955. Laboratory investigations on the role of bird mites in the transmission of eastern and western equine encephalitis. Am. J. Trop. Med. Hyg. 4: 106-108. Chamberlain, R. W., R. K. Sikes and W. D. Sudia, 1957. Attempted laboratory infection of bird mites with the virus of St. Louis encephalitis. Am. J. Trop. Med. Hyg. 6: 1047-1053. DuToit, R. M., 1944. The transmission of bluetongue and horse sickness by Culicoides. Onderstepoort. J. Vet. Sci. Animal Indus. 19: 7-16. Fahey, J. E., 1954. An outbreak of staphylococcal arthritis in turkey poults Poultry Sci. 33-661664. Fallis, A. M., and D. M. Wood, 1957. Biting midges (Diptera: Ceratopogonidae) as intermediate hosts for Haemoproteus of ducks. Can. J. Zool. 35: 425-435. Fallis, A. M., and G. F. Bennett, 1960 Description of Haemoproteus canachites n. sp. (Sporozoa: Haemoproteidae) and sporogony in Culicoides (Diptera: Ceratopogonidae). Can. J. Zool. 38: 455-464. Fallis, A. M., and G. F. Bennett, 1961. Ceratopogonidae as intermediate hosts for Haemoproteus and other parasites. Mosquito News, 21: 21-28. Hinshaw, W. R., and E. McNeil, 1952. Staphylococcus (synovitis) in turkeys. Poultry Sci. 31: 320327. Hopkins, C. A., and W. L. Nicholas, 1952. Culicoides austeni, the vector of Acanthocheilonema perstans.
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or propagative transmission were not carried out. Several other species of the genus Culicoides known to feed on poultry as well as other external parasites of poultry need to be investigated. I t is also possible t h a t the potential vector may have to be subjected to certain environmental factors before transmission can occur.
DAVIS
INFECTIOUS SYNOVITIS
perimental synovitis in chickens. Avian Dis. 4: 42-48. Smith, M. F., R. J. Blattner and F. M. Heys, 1944. The isolation of the St. Louis Encephalitis virus from chicken mites {Dermanyssus gallinae) in nature. Science, 100: 362-363. Smith, M. F., R. J. Blattner and F. M. Heys, 1947. St. Louis Encephalitis-Transmission of virus to chickens by infected mites Dermanyssus gallinae and resulting viremia as a source of virus for infection of mites. J. Exp. Med. 86: 229-237. Snoeyenbos, G. H., and H. I. Basch, 1958. A further indication of egg transmission of infectious synovitis. Avian Dis. 2: 494-498. Snow, W. E., E. Pickard and J. B. Moore, 1957. The Heleidae of the Tennessee River Basin. Tennessee Acad. Sci. J. 32: 18-36. Tokunaga, M., 1937. Sand-flies (Ceratopogonidae, Diptera) from Japan. Tenthrede, 1: 233-337. Wills, F. K., 1954a. Preliminary report on transmission of an agent producing an arthritis in chickens. Progress Report 1674. Texas Agric. Exp. Sta., College Station, Texas. April 26, 1954. Wills, F. K., 1954b. Observations on an agent producing arthritis in chickens. Progress Report 1723. Texas Agric. Exp. Sta., College Station, Texas, October 25, 1954. Wills, F. K., and J. P. Delaplane, 1955. Transmission and therapy studies on an agent which produces arthritis in chickens. (Pro.) 92nd Annual Meeting. Vet. Med. Assoc. 350-356. Wirth, W. W., and L. J. Bottimer, 1956. A population study of the Culicoides midges of the Edwards plateau region of Texas. Mosquito News, 16: 256-266.
The Erfect of CC, Cc and cc Plumage Color Genotypes on Body Weight in the Fowl J. ROBERT SMYTH, JR. AND T. W. FOX Poultry Science Department, University of Massachusetts, A mherst (Received for publication May 2, 1963)
A
RELATIONSHIP between the recessive white alleles, C and c, and body weight at 8 and 9 weeks of age has been suggested by the results of Jerome and Contribution from Massachusetts Agricultural Experiment Station.
Huntsman (1959) and Smyth and Fox (1961). In both studies birds with colored plumage, and heterozygous for the recessive white gene (Cc), were significantly heavier than were those homozygous for the recessive gene (cc) and segregating from the same population. On the other
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Ann. Trop. Med. Parasit. 46: 276-283. Judd, W. W., 1957. Dipteracollectedfrombirds'nests at London, Ontario, Ent. News, 68: 32-34. Messersmith, D. H., 1961. Studies on Culicoid.es (Diptera: Ceratopogonidae) and their relationship to infectious synovitis in poultry in Virginia. Ph.D. Thesis, Virginia Polytechnic Institute, Blacksburg, Va. Olson, N. O., J. K. Bletner, D. C. Shelton, D. A. Monro and G. C. Anderson, 1954. Enlarged joint condition in poultry caused by infectious agent. Poultry Sci. 33: 1075. Olson, N. O., D. C. Shelton, D. A. Monro and R. Bletner, 1957. Preliminary blood studies in chickens with a synovitis caused by the infectious synovitis agent, pleuropneumonia-like organisms and a combination of the two agents. Avian Dis. 1: 8291. Peterson, R. V., 1962. Studies on the transmission of the poultry disease, Infectious synovitis, by using direct contact and insect vector methods. M. S. Thesis, Virginia Polytechnic Institute, Blacksburg, Va. Price, D. A., and W. T. Hardy, 1954. Isolation of the blue tongue virus from Texas sheep-Culicoides shown to be a vector. J. Am. Vet. Med. Assoc. 124:255-258. Sharp, N. A. D., 1927. Development of Microfilaria Persians in Culicoii.es grahami: a preliminary note. Trans. Roy. Soc. Trop. Med. Hyg. 21: 70. Sharp, N. A. D., 1928. Filaria perstans: its development in Culicoides austani Trans. Roy. Soc. Trop. Med. Hyg. 21: 371-396. Skamser, L. M., and K. C. Seeger, 1960. Aureomycin chlortetracycline as a preventative for ex-
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concentrations into the egg before incubation resulted in sex reversal of genetic males and alteration of eminence types of both sexes. ACKNOWLEDGMENTS The authors are indebted to Wirthmore Feeds, Inc. for financial support and to Mr. Charles Green, Winterport, Maine, for his sincere interest and willing cooperation in classification of genital eminence types. REFERENCES Canfield, T. H., 1941. Sex determination of day-old chicks. 2. Type variations. Poultry Sci. 20: 327328. Dove, W. F., 1928. Sex sterility and the diminutive copulatory organ in the domestic fowl. Science, 68: 327-328. Hammond, J. C , and W. H. Burrows, 1937. The female genital eminence is not a measure of future egg production. Poultry Sci. 16: 285-286. van Tienhoven, A., 1957. A method of "controlling sex" by dipping of eggs in hormone solutions. Poultry Sci. 36: 628-632.
Transmission Studies of Avian Infectious Synovitis by Selected Arthropod Vectors1 E. C. T U R N E R , J R . , N A N C Y L. W E H R H E I M 2 AND D. H.
MESSERSMITH3
Department of Entomology AND J. W.
DAVIS
Department of Veterinary Science, Virginia Polytechnic Institute, Blacksburg, Va. (Received for publication May 2, 1963)
T
H E disease infectious synovitis was first reported by Wills (1954a, b) and Olson el al. (1954). Since t h a t time ex-
1 The research reported herein was supported by the National Institutes of Health as Project No. E2717. Presented at the 34th Annual Meeting, Eastern Branch, Entomological Society of America, Philadelphia, Pa. 1962. 2 Present Address, Department of Pathology, University of North Carolina, Chapel Hill, North Carolina.
tensive research has been done by numerous workers on the etiology, therapy, and control of this disease, however, the exact nature of how this disease was transmitted from bird to bird has not been proven. Wills and Delaplane (1955) demonstrated transmission of infectious synovitis b y eggs. This was later con3 Present Address, Department of Biology, Radford College, Radford, Virginia.
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show any relationships with genital eminence type: hatch, age of egg before incubation, size of egg, length of incubation period, body weight at day-old and at four and eight weeks of age, tail feather length, age a t first egg, and rate of lay. Chicks from an auto-sexing cross were utilized to investigate the possible relationships between endocrine gland weights and genital eminence type. This mating provided eminence type ratios for 503 males of 7 5 % type A, 1 6 % type B, and 9 % type C; and for 514 females of 8 5 % type A, 10% type B, and 4 % type C. T h e weights of the hypophysis, thyroids, gonads, adrenals, and bursa of Fabricius were not related to eminence type. No differences in oviduct weights were noted between the A and C eminence type female chicks in response to 0, 0.1, and 0.25 mg. of D.E.S. administered subcutaneously on alternate days over a 12-day period. Injection of D.E.S. in varying
I N F E C T I O U S SYNOVITIS
Insect surveys were initiated in poultry houses in Virginia. Particular attention was given to houses with a history of infectious synovitis. These surveys, using standard " N e w J e r s e y " light traps revealed t h a t species of the genus Culicoides (Order: Diptera; Family: Ceratopogonidae) were by far the most a b u n d a n t group of blood-sucking insects associated with poultry. I t is known t h a t several species of Culicoides are avian feeders, as shown by Judd (1957). Fallis and Wood (1957) and Fallis and Bennett (1960, 1961) reported t h a t Culicoides were intermediate hosts for Haemoproteus, a parasitic protozoan found in the blood of birds. A number of species of Culicoides were collected in poultry houses by Snow et al. (1957), Wirth and Bottimer (1956) and others. M a n y of these were engorged females. Culicoides are known vectors of certain tropical filaria as reported by Sharp (1927, 1928) and most recently by Hopkins and Nicholas (1952). Of even greater
significance is the fact t h a t they have been shown by D u T o i t (1944) and later by Price and H a r d y (1954) to transmit blue-tongue disease in sheep. Also of interest is the fact t h a t Tokunaga (1937) showed Culicoides to be a vector of fowlpox. Blood-sucking mites were also suspected as possible vectors of synovitis since they are known vectors of other diseases. Smith et al. (1944) demonstrated the relationship between Dermanyssus gallinae and St. Louis encephalitis. Smith et al. (1947) later proved transmission of the virus to normal chickens by the bite of infected mites. They demonstrated t h a t both experimentally infected and naturally infected mites were capable of the transfer. Benton (1959) reported t h a t cage-mate transmission of infectious synovitis occurred in correlation with northern fowl mite infestations. Peterson (1962) was unable to demonstrate cagem a t e transmission in the absence of mites. Hinshaw and McNeil (1952) noticed a correlation between the incidence of infection and the presence of mosquitoes while working with staphylococcus in turkeys. T h e y reported t h a t a decrease in the incidence of the disease was observed in one area following mosquito control operations. T h e y also suggested the possibility of transmission through open wounds. However Fahey (1954) found no mosquito correlation in his work with staphylococcal arthritis in turkey poults. In view of the work of these and other authors it was felt t h a t an investigation of the possibilities of arthropod transmission of infectious synovitis was justified. Certain arthropods t h a t could be reared in the laboratory were selected for this study. PROCEDURE Rearing Techniques: The source of the infective agent was obtained from a field
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firmed by Snoeyenbos and Basch (1958). I n these reported cases positive transmission via the egg occurred only in a very small number of times. Field outbreaks of the disease occurred in a much larger percentage of cases, and it was suspected t h a t there was probably other means of transmitting the disease within a flock of chickens. I n an early report, Wills (1954a) suggested the possibility of insect transmission. Olson et al. (1957) reported t h a t the causative agent was found in the blood of affected birds. Later, Benton and Cover (1959) found t h a t the agent was present in infectious levels in the blood by the eighth hour after intravenous inoculation and persisted until at least the fifteenth day. These investigations further suggested the possibility of transmission by a blood-sucking arthropod.
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Pure cultures of Culicoides variipennis (Coquillett) larvae were collected in the mud of the salt marshes at Saltville, Virginia, and placed in gallon-size icecream cartons. In the laboratory a 2\ inch hole was cut in the top of these cartons and emergence cages were placed over them. These emergence cages consisted of pint size ice-cream cartons in which a 2\ inch hole was cut in the bottom. The tops of these emergence cages were replaced with clear polyethylene plastic on which two f-inch holes were cut by means of a cork borer. One of the holes was used for ventilation and was covered with a small piece of 80-mesh silk cloth. A cork stopper was placed in the other hole. The emerging adult insects were removed daily by means of a hand aspirator and were placed in similarly constructed gallon icecream cartons. Food in the form of dried raisins and water was provided and the cages were stored in a B.O.D. Incubator at a constant temperature of 25°C. Northern fowl mites were reared in the laboratory on PPLO-free culture chickens. Mites were transferred by being brushed from a culture bird by means of a oneinch nylon brush onto a large white enamel pan. Adult mites were then collected with a small camels hair brush or a hand aspirator and placed on test birds. After
feeding on these birds the mites were removed by means of a hand aspirator and used in either the maceration tests or refeeding tests. Pure cultures of Culex pipiens group and Aedes sollicitans (Walker) mosquitoes were collected in the field and allowed to emerge as adults. These adults were stored and handled in the same manner as described for Culicoides variipennis. Transmission Techniques: For transmission studies, 12 to 14 C. variipennis adult females 5 to 7 days old were removed from the holding cage and placed in another similarly constructed cage. T h e cage was placed on its side and the cork stopper from the plastic top was removed. Immediately, either a healthy or diseased chicken was immobilized, and its head was pushed through the plastic hole and the insects were allowed to feed on or about the head. Although actual feeding by a single female took only about five minutes, it usually took about one or two hours for all of the insects to land on the chicken head and engorge fully. T h e engorged insects were then removed from this cage and macerated by means of a glass tissue grinder in 2 ml. of tryptose broth. The tryptose suspension was then inoculated intramuscularly (1 ml.) or intravenously (.4 ml.) into a 3 to 5-week old healthy bird. In other experiments the engorged insects were used in refeeding experiments. The insects were allowed to feed on the healthy birds in the same manner as they had fed on the diseased birds. All exposed birds were then placed in Modified Horsefall-Bauer Units for observation. The northern fowl mites were removed from culture chickens in the manner previously described. Since the mites do not readily leave their hosts, no special cage was needed for the transmission experiments,
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case of infectious synovitis. Material from the spleen was suspended in tryptose and held at — 50°C. until used in the experiments. All birds used were from a P P L O free flock of White Leghorn chickens t h a t were about 3 to 5 weeks old. Modified Horsefall-Bauer Units were used to house and isolate the birds exposed to the disease. Birds used in the transmission experiments were observed daily for external symptoms of infectious synovitis. On death of a bird or at the termination of an experiment, an autopsy was performed to confirm the preliminary diagnosis.
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INFECTIOUS SYNOVITIS
RESULTS AND DISCUSSION
TABLE 1.—Transmission of infectious synovitis using macerated engorged Culicoides variipennis immediately after feeding on infected chickens No. days from initial infection to insect No. of positive transmissions/ No. of birds inoculated feeding 2 4 7 8 a
iv = intravenous.
iv 1/2 2/2 2/2 2/2
ima 0/1 1/1 1/1 1/1
Experiment 2: The purpose of this test was to determine if the infective agent would remain active enough to be transmitted in or on an infected insect (C. variipennis) and how long this agent would remain active. T h e insects were collected after feeding on diseased birds and held for certain periods of time before maceration and inoculation. The results are shown in Table 2. Transmission was demonstrated in infected insects t h a t were held u p to 24 hours but not at 72 hours. I t is interesting to note t h a t the method of inoculating the macerated material appeared to be important. At the 24-hour interval, transmission was demonstrated only when the material was inouclated intravenously into healthy birds. These results would indicate t h a t the infective agent does not remain active in or on the insect vector for a particularly long period of time. Therefore, transmission would be attained only when the insect would feed on infected birds and then on uninfected birds within a relatively short time such as would occur when the insect was interrupted in its feeding. Experiment 3: In preliminary refeeding experiments, both diseased and healthy chickens were placed in adjoining pens in a manner which would prevent direct contact with each other b u t would allow equal exposure to C. variipennis. Large numbers of 5 to 7-day old adult insects TABLE 2.—Transmission of infectious synovitis using engorged Culicoides variipennis and held for various time intervals before maceration and inoculation No. Hours insects held
No. of positive transmission/ No. of birds inoculated iv 2/2
3 6 24 72
—
4/4 0/2
im = intramuscular.
\
im 2/2 2/2 0/4 0/1
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Experiment 1: An a t t e m p t was made to demonstrate the presence of the infectious synovitis agent in or on the body of C. variipennis after the insect had been allowed to feed on birds inoculated with the disease. An a t t e m p t was made to determine how long the infected agent would remain in the blood of infected birds and hence available to blood-sucking insects. At certain time intervals after initial inoculation of the test birds with spleen suspension of the infective agent, the insects were allowed to engorge on these birds. T h e y were then immediately macerated and inoculated into healthy chickens as previously described. In Table 1, positive transmission was demonstrated in almost all cases. This indicates t h a t the infective agent was present in the blood spleen inoculated birds for at least 2 to 8 days, thus partially confirming the results obtained by Benton and Cover (1959) on the infectivity of the blood of birds infected with infectious synovitis. The agent was also demonstrated to be present in or on the engorged insects. Since it has been shown previously t h a t Culicoides are capable of transmitting certain animal diseases, it was felt t h a t further tests using this insect should be carried out in order to determine definitely if infectious synovitis can be carried from bird to bird by this method.
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This experiment was set up in such a manner t h a t feeding and refeeding by individual insects was definitely observed. Partially engorged females were removed from diseased birds and placed on healthy birds where these insects continued to feed until they were fully engorged, then they were removed and macerated in tryptose broth. The material thus obtained was inoculated into other healthy birds. Table 3 shows the results of this test. Transmission was not demonstrated by interrupted feeding of C. variipennis although the infective agent was present in or on the insect. This was demonstrated by the positive transmission results obtained when the insect used in the refeeding tests were macerated and inoculated into other healthy birds. I t thus TABLE 3.—Direct transmission ofinfectious synovitis by interrupted feeding of Culicoides variipennis
Type of transmission
No. of positive transmissions/ No. birds tested
Refeeding Maceration1
0/2 2/2
a After the insects had completed feeding on healthy chickens, the engorged insects were then macerated in tryptose and inoculated (im) into other healthy chickens.
A
TABLE 4.—Transmission of infectious synovitis using macerated Ornithonyssus sylviarum immediately after feeding on infected birds Time mites allowed on infected birds 3 10 12 23
hours days days days
No. of positive transmission/ No. of birds inoculated iv 2/3 1/2 4/4 0/2
im 0/1
— —
appears that, under the conditions outlined in this experiment, the infectious synovitis agent cannot be transmitted by the bite of C. variipennis even though the agent is present in or on the vector. Experiment 4: This experiment was set up to determine whether the infectious synovitis agent would be found present in or on blood-sucking mites t h a t had fed on diseased birds. Twelve to fifteen adult northern fowl mites were removed from culture birds and placed on previously inoculated chickens. These mites were allowed to feed on the infected birds for various lengths of time as shown on Table 4. I t was noted t h a t only about half of the mites were engorged when they were removed from the infected birds. The mites were then macerated in 2 ml. of tryptose broth and inoculated into healthy chickens as previously described. The results show t h a t the agent is present in or on the mites although 100% transmission was not demonstrated. Experiment 5: Since it was indicated in experiment 4 t h a t the agent was present in or on blood-sucking mites, a refeeding test was initiated to demonstrate natural transmission. The mites were handled as previously described and allowed to feed on infected birds as shown in Table 5. At certain time intervals the infected mites were removed and placed on healthy birds where they were allowed to remain for several days. The results show t h a t no transmission was demon-
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were released in these pens, however, it was difficult to determine if the insects fed at all or if they moved from sick birds to healthy birds. The results from these preliminary refeeding experiments were thus inconclusive. Messersmith (1961) live collected several species of Culicoides from poultry houses in which some birds were infected with infectious synovitis. These insects were macerated and injected into healthy birds b u t negative results were obtained. Unfortunately the birds t h a t were inoculated had been receiving aureomycin in their feed. Since the material is used as a control for infectious synovitis, the results of the experiment must be regarded as inconclusive.
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INFECTIOUS SYNOVITIS TABLE 5.—Refeeding or natural transmission of infectious synovitis by Ornithonyssus sylviarum Time mites allowed on infected birds 2 hours 3 hours 2 days 3 days 7 days 10 days 23 days
No. positive transmissions/ No. birds tested 0/2 0/2 0/4 0/4 0/7 0/2 0/2
Time mites allowed on infected birds
No. of positive transmissions/ No. birds tested
3 hours 23 days
0/4 0/2
to feed on infected birds and immediately macerated in a tissue grinder as described previously. The macerated material was then inoculated into healthy birds as shown in Table 7. Positive transmission of the disease was obtained in every case using either species of mosquitoes, indicating that the agent of infectious synovitis can therefore be carried in or on the bodies of these mosquitoes. Benton (1959) and Peterson (1962) were unable to demonstrate positive transmission of infectious synovitis using macerated mosquitoes. Refeeding tests by Peterson with Aedes aegypti were also negative. It is concluded that under the conditions of the experiment described herein that direct mechanical transmission of the infectious synovitis agent by C. variipennis (Coquillett) and the northern fowl mite probably does not occur. Negative transmission was obtained with C. variipennis even when the agent producing the disease was shown to be present in or on the body of this insect. Refeeding tests using the northern fowl mite also resulted in negative transmission. The method of inoculation of the disease in the bird may be quite important in transmission studies. As shown by Skamser and Seeger (1960), sinus inoculated birds were able to infect a greater percent of cage mates than footpad-inoculated birds. They stated that the increased transmission of synovitis in the sinus infected groups could occur by contact or by contaminated feed and water. Experiments to investigate the possibility of biological
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strated under the conditions of this experiment. Similar results were obtained by Chamberlain and Sikes (1955) and Chamberlain et al. (1957) working with the transmission of eastern and western equine encephalitis. In extensive experiments they were unable to demonstrate transmission of these viruses by several species of mites including, Ornithonyssus sylviarum. Experiment 6: Chamberlain and Sikes (1955) and Chamberlain et al. (1957) also attempted to demonstrate the transmission of eastern and western equine encephalitis by crushing infected mites into skin break of birds. Results were again negative. In this experiment the mites were allowed to feed on infected birds. After feeding, the engorged mites were removed by sterile forceps and crushed into wounds made on the backs of healthy birds, such as might be encountered in peck wounds. Table 6 shows the results of this test. Again no transmission of infectious synovitis was demonstrated. Thus, it appears that the disease is not transmitted in this manner by the northern fowl mite. Experiment 7: Since mosquitoes are known vectors of many virus diseases as well as other types of diseases it was felt that preliminary maceration tests should be carried out. Using pure cultures of Culex pipiens group and Aedes sollicitans (Walker), the adult females were allowed
TABLE 6.—Transmission of infectious synovitis by crushing infected Ornithonyssus sylviarum into skin breaks of susceptible birds
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E. C. T U R N E R , J R . , N . L. W E H R H E I M , D . H. M E S S E R S M I T H AND J. W.
TABLE 7.—Transmission of infectious synovitis using macerated engorged mosquitoes No. of positive transmissions/ No. of birds inoculated
Species
Culex pipiens Gp. Aedes sollicitans
iv 2/2 4/4
im —• 2/2
SUMMARY Preliminary studies on the infectivity of blood of synovitis infected birds, have indicated t h a t certain blood-sucking arthropods known to feed on poultry could possibly transmit the agent causing this disease. The purpose of these experiments was therefore to investigate possible vectors of infectious synovitis among certain arthropods known to transmit other diseases. Culicoides variipennis (Coquillett) and the northern fowl mite, Ornilhonyssus sylviarum (C. & F.) were investigated as possible vectors of this disease. Immediately after being allowed to engorge on .diseased chickens, C. variipennis adults were macerated and inoculated into healthy birds. This resulted in positive transmission of the disease. Tests using infected engorged C. variipennis which were held for various time intervals up to 24 hours before maceration and inoculations into healthy chickens also resulted in positive transmission. However, in interrupted feeding tests using infected C. variipennis, no transmission occurred.
Maceration tests using engorged infected northern fowl mites resulted in positive transmission, b u t direct refeeding experiments resulted in no transmission of the disease. Also, tests on the transmission of the disease by crushing infected mites into skin-breaks of susceptible birds were negative. Preliminary maceration tests using infected Culex pipiens group and Aedes sollicitans (Walker) mosquitoes also resulted in positive transmission of infectious synovitis in chickens.
REFERENCES Benton, W. J., 1959. Studies on the transmission of infectious synovitis of chickens. M. S. Thesis, University of Del., Newark, Del. Benton, W. J., and M. S. Cover, 1959. The infectivity of blood and tissue from chickens with infectious synovitis. Avian Dis. 3: 361-370. Chamberlain, R. W., and R. K. Sikes, 1955. Laboratory investigations on the role of bird mites in the transmission of eastern and western equine encephalitis. Am. J. Trop. Med. Hyg. 4: 106-108. Chamberlain, R. W., R. K. Sikes and W. D. Sudia, 1957. Attempted laboratory infection of bird mites with the virus of St. Louis encephalitis. Am. J. Trop. Med. Hyg. 6: 1047-1053. DuToit, R. M., 1944. The transmission of bluetongue and horse sickness by Culicoides. Onderstepoort. J. Vet. Sci. Animal Indus. 19: 7-16. Fahey, J. E., 1954. An outbreak of staphylococcal arthritis in turkey poults Poultry Sci. 33-661664. Fallis, A. M., and D. M. Wood, 1957. Biting midges (Diptera: Ceratopogonidae) as intermediate hosts for Haemoproteus of ducks. Can. J. Zool. 35: 425-435. Fallis, A. M., and G. F. Bennett, 1960 Description of Haemoproteus canachites n. sp. (Sporozoa: Haemoproteidae) and sporogony in Culicoides (Diptera: Ceratopogonidae). Can. J. Zool. 38: 455-464. Fallis, A. M., and G. F. Bennett, 1961. Ceratopogonidae as intermediate hosts for Haemoproteus and other parasites. Mosquito News, 21: 21-28. Hinshaw, W. R., and E. McNeil, 1952. Staphylococcus (synovitis) in turkeys. Poultry Sci. 31: 320327. Hopkins, C. A., and W. L. Nicholas, 1952. Culicoides austeni, the vector of Acanthocheilonema perstans.
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or propagative transmission were not carried out. Several other species of the genus Culicoides known to feed on poultry as well as other external parasites of poultry need to be investigated. I t is also possible t h a t the potential vector may have to be subjected to certain environmental factors before transmission can occur.
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perimental synovitis in chickens. Avian Dis. 4: 42-48. Smith, M. F., R. J. Blattner and F. M. Heys, 1944. The isolation of the St. Louis Encephalitis virus from chicken mites {Dermanyssus gallinae) in nature. Science, 100: 362-363. Smith, M. F., R. J. Blattner and F. M. Heys, 1947. St. Louis Encephalitis-Transmission of virus to chickens by infected mites Dermanyssus gallinae and resulting viremia as a source of virus for infection of mites. J. Exp. Med. 86: 229-237. Snoeyenbos, G. H., and H. I. Basch, 1958. A further indication of egg transmission of infectious synovitis. Avian Dis. 2: 494-498. Snow, W. E., E. Pickard and J. B. Moore, 1957. The Heleidae of the Tennessee River Basin. Tennessee Acad. Sci. J. 32: 18-36. Tokunaga, M., 1937. Sand-flies (Ceratopogonidae, Diptera) from Japan. Tenthrede, 1: 233-337. Wills, F. K., 1954a. Preliminary report on transmission of an agent producing an arthritis in chickens. Progress Report 1674. Texas Agric. Exp. Sta., College Station, Texas. April 26, 1954. Wills, F. K., 1954b. Observations on an agent producing arthritis in chickens. Progress Report 1723. Texas Agric. Exp. Sta., College Station, Texas, October 25, 1954. Wills, F. K., and J. P. Delaplane, 1955. Transmission and therapy studies on an agent which produces arthritis in chickens. (Pro.) 92nd Annual Meeting. Vet. Med. Assoc. 350-356. Wirth, W. W., and L. J. Bottimer, 1956. A population study of the Culicoides midges of the Edwards plateau region of Texas. Mosquito News, 16: 256-266.
The Erfect of CC, Cc and cc Plumage Color Genotypes on Body Weight in the Fowl J. ROBERT SMYTH, JR. AND T. W. FOX Poultry Science Department, University of Massachusetts, A mherst (Received for publication May 2, 1963)
A
RELATIONSHIP between the recessive white alleles, C and c, and body weight at 8 and 9 weeks of age has been suggested by the results of Jerome and Contribution from Massachusetts Agricultural Experiment Station.
Huntsman (1959) and Smyth and Fox (1961). In both studies birds with colored plumage, and heterozygous for the recessive white gene (Cc), were significantly heavier than were those homozygous for the recessive gene (cc) and segregating from the same population. On the other
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Ann. Trop. Med. Parasit. 46: 276-283. Judd, W. W., 1957. Dipteracollectedfrombirds'nests at London, Ontario, Ent. News, 68: 32-34. Messersmith, D. H., 1961. Studies on Culicoid.es (Diptera: Ceratopogonidae) and their relationship to infectious synovitis in poultry in Virginia. Ph.D. Thesis, Virginia Polytechnic Institute, Blacksburg, Va. Olson, N. O., J. K. Bletner, D. C. Shelton, D. A. Monro and G. C. Anderson, 1954. Enlarged joint condition in poultry caused by infectious agent. Poultry Sci. 33: 1075. Olson, N. O., D. C. Shelton, D. A. Monro and R. Bletner, 1957. Preliminary blood studies in chickens with a synovitis caused by the infectious synovitis agent, pleuropneumonia-like organisms and a combination of the two agents. Avian Dis. 1: 8291. Peterson, R. V., 1962. Studies on the transmission of the poultry disease, Infectious synovitis, by using direct contact and insect vector methods. M. S. Thesis, Virginia Polytechnic Institute, Blacksburg, Va. Price, D. A., and W. T. Hardy, 1954. Isolation of the blue tongue virus from Texas sheep-Culicoides shown to be a vector. J. Am. Vet. Med. Assoc. 124:255-258. Sharp, N. A. D., 1927. Development of Microfilaria Persians in Culicoii.es grahami: a preliminary note. Trans. Roy. Soc. Trop. Med. Hyg. 21: 70. Sharp, N. A. D., 1928. Filaria perstans: its development in Culicoides austani Trans. Roy. Soc. Trop. Med. Hyg. 21: 371-396. Skamser, L. M., and K. C. Seeger, 1960. Aureomycin chlortetracycline as a preventative for ex-
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