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Stahler, N. & L. A. Terzian. 1953. Studies in the laboratory the caloric reserves of mosquitoes. Mosquito News 32: mating habits of Anopheles quadrimaculatus Say. J. Exp. Zool. 589-91. 124: 317-28. Volozina, N. V. 1967. The effects of the amount of blood Van Handel, E. 1965a. The obese mosquito. J. Physiol. 181: taken and additional carbohydrate nutrition on oogenesis in females of blood-sucking mosquitoes of the genus Aedes 478-S6. (Diptera, Culicidae) of various weights and ages. Ent. Rev. 1965b. Microseparation of glycogen, sugars and lipids. USSR 46: 27-32. J. Anal. Biochem. 11: 266-71. 1972. Simple biological and chemical methods to determine

J. Med. Ent. Vol. 12, no. 2: 225-227

30 June 1975

By W. M. Rogoff2, E. G. Carbrey3, R. A. Bram4, Truman B. Clark2 and Gordon H. Gretz5 Abstract: Field collections of insects in the vicinity of poultry flocks infected with the virus of exotic viscerotropic, velogenic Newcastle disease ( W N D ) were made in Riverside and San Bernardino Counties, southern California. Virus was isolated from 3 pools ofFannia canicularis (L.) and 1 ofF.femoralis (Stein). Another pool of F. canicularis contained a mesogenic ND virus, and 1 pool of Musca domestica L. larvae had W N D virus. Newcastle disease virus was not isolated from 117 pools of M. domestica adults nor from 19 pools of Muscina stabulatis (Fallen) tested. None of the other insect species collected showed evidence of the presence of Newcastle disease virus.

Newcastle disease (ND) is a well known, essentially cosmopolitan viral affliction of domestic poultry. Different isolates of the virus vary in virulence and in the primary location of the lesions in the bird's organ systems (e.g., "pneumotropic" or "viscerotropic"). ND strains are classified as to relative virulence by the length of time it takes the virus to kill chick embryos. A velogenic strain (highly virulent) has a mean death time of less than 60 hr. A detailed description of the disease is given by Hanson (1972, 1973) and by Hanson et al. (1973). During late 1971 and 1972, a velogenic, vis1 Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA. 'Research Entomologist, Western Insects Affecting Man and Animals Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Fresno, Calif. 93727, U.S.A. 3 Chief, Diagnostic Virology, National Animal Disease Laboratory, Animal and Plant Health Inspection Service, U. S. Department of Agriculture, Ames, Iowa 50010, U.S.A. 4 Principal Staff Officer, Vector Biology, Surveillance, and Control, Emergency Programs, Animal and Plant Health Inspection Service, U. S. Department of Agriculture, Hyattsville, Md. 20782, U.S.A. Agricultural Research Technician, Western Insects Affecting Man and Animals Laboratory. Agricultural Research Service, U. S. Department of Agriculture, Fresno, Calif. 93727, U.S.A.

cerotropic strain (WND, exotic ND, or Asiatic ND) was introduced into southern California, spread rapidly to the dense population of poultry in the area (Omohundro 1972) and was causing major problems among egg producers in southern California. Therefore, in March 1972, a cooperative effort to eradicate the disease was initiated by the California State Department of Agriculture and the USDA (Sharman & Walker 1973). A complete review of the epizootiology of this outbreak was published by Walker et al. (1973) and by Utterback & Schwartz (1973). Transmission of ND was known to occur by many routes, such as contaminated objects, poultry moved from one farm to another, air-borne particles, etc. The possibility of insect transmission had previously been ignored but was considered a possible threat to a successful eradication of the southern California epizootic. The present paper describes efforts to determine whether virus was present in or on insects found in association with flocks infected with the W N D virus. MATERIALS AND METHODS

Infected poultry flocks were identified by postmortem examination and virus isolation and characterization by personnel of the Newcastle Disease Task Force (McDaniel & Orsborn 1973). We made collections on such contaminated premises before, during, and/or after "depopulation" (destruction and removal of birds). Sampling was conducted on 29 premises in Riverside and San Bernardino Counties, primarily within 40 km of the City of Riverside. Flocks ranged up to 114,000

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TRANSMISSION OF NEWCASTLE DISEASE VIRUS BY INSECTS: DETECTION IN WILD FANNIA SPP. (DIPTERA: MUSCIDAE)i

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RESULTS

The first collections of insects were made during the week of 8 May 1972. Of 68 pools (from 1 infected and 2 exposed premises) representing collections of 14 species of insects, 2 pools of Fannia canicularis (L.) were found positive for VVND virus. These 2 positive pools were collected on successive days from the same infected premises (on the day of depopulation and on the day afterward). The remaining 66 pools were negative for the virus. The findings for later collections (between 25 July 1972, and 26 April 1973) are summarized in TABLE 1. VVND virus was detected in 1 pool each of adult F. canicularis (L.) and adult F. femoralis (Stein), both collected on 25 July, and in 1 pool of larval Musca domestica L. collected on 7 September. Mesogenic ND virus was detected in 1 pool of adult F. canicularis collected on 14 August.

TABLE 1. Isolation of Newcastle disease virus from adult insects collected on contaminated premises, 25 July 1972 to 26 April, 1973. POOLS OF 10 OR MORE SPECIMENS* TAKEN PRIOR TO 2 DAYS AFTER DEPOPULATION

Avg.

TAKEN 2 OR MORE DAYS AFTER DEPOPULATION

Avg. per No. of Pools TAXA pool pools pool + + ** + + ** 0 Musca domestica*** 91 47 26 50 0 Fannia canicularis 44 55 65 0 51 2t Fannia femoralis 13 6 50 47 0 Iff Muscina stabulans 13 27 0 6 23 0 Stomoxys calcitrans 0 0 1 10 0 0 0 0 0 3 27 0 Ophyra sp.ftt Phoenicia sp. 0 0 0 1 13 0 Culex pens 0 0 0 2 178 0 1 Culex pipiens quinquefascialus 18 0 0 0 0 *An additional 67 pools of larvae, unidentified insects, or pools of less than 10 specimens were also evaluated (none showed evidence of ND). **Virus isolated and characterized. ***One pool of larvae collected 2 days after depopulation contained VVND virus. fOne pool was mesogenic, 1 pool VVND. ffWND. ff f Ophyra leucostoma (Wiedemann) in 2 pools. No. of pools

per

Pools

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lated via the allantoic route (0.2 ml per egg) into ten 9- to 11-day-old embryonated eggs. The eggs were incubated at 37 °C through 10 days and examined daily. Allantoic fluids were harvested from dead embryos and tested for the presence of a hemagglutinating virus. The presence of ND virus in the fluids was confirmed by hemagglutination inhibition with specific antiserum. The ND isolates were characterized by the determination of mean death time in chick embryos (National Academy of Sciences 1971), viscerotropism in chickens (Hanson et al. 1973, McDaniel & Orsborn 1973), and plaquing properties on chick embryo fibroblasts (Schloer & Hanson 1968).

chickens, though most contained 25,000 or fewer. Collections were made primarily by hand netting, but also by light trap, bait trap, or garden trowel (larvae). During the initial probe, insects were immobilized in the field by chilling, sorted to species, pooled in groups of approximately 25 to 50 in tightly stoppered vials, frozen and packed on dry ice, and immediately sent to the laboratory for virus isolation, identification, and characterization. Subsequently, insects were placed on dry ice as they were taken in the field (after ethyl acetate or chloroform anesthetization) and later sorted by species and pooled, where possible, in groups of 30 to 50 specimens. In those situations in which smaller numbers of specimens were taken, pools were assembled from the number available. In most cases, specimens reached ambient temperature during the sorting and pooling process but were then immediately returned to dry ice or equivalent temperature. Insect pools packed in dry ice were sent by air express to the Veterinary Services Laboratory (USDA) at Ames, Iowa, for virus isolation, identification, and characterization. At Ames the insect pools were maintained frozen (at — 70 °C) until processed. In processing, each pool was triturated with a sterile mortar and pestle, and 10% suspensions were prepared in phosphatebuffered saline solution enriched with 2% fetal calf serum, pH 7.2. The suspensions were centrifuged in a PR6, refrigerated centrifuge (International Equipment Co., Needham Heights, Mass. 02194, U.S.A.) at 2500 rpm for 20 min. (head # 253 was used). The supernate from each pool was inocu-

1975

Rogoff et al.:

Newcastle disease virus detection in Fannia spp.

DISCUSSION

The isolation of W N D virus from free-flying insects taken on premises with infected flocks is not in itself remarkable. Greenberg (1971) listed 59 strains of virus from vertebrates associated with flies of various species. Eleven of these strains were detected in the genus Fannia; 37 were described associated with Musca domestica. Also, Hofstad (1949) isolated NDV from the northern fowl mite, Omithonyssus (—Liponyssus) sylviarum (Canestrini &

227

greater than normal fly dispersion. Thus, if transmission of W N D from bird to fly and from fly to bird can reasonably be postulated, fly-control operations on infected and exposed premises would appear to be a proper tactic in support of an eradication campaign. LITERATURE CITED

Fanzago). However, this report is the first of which we are aware that documents an association between a Newcastle disease virus and a free-flying insect. Because this association was established during a major campaign to eradicate the W N D strain involved, the possibility of transmission by flies (whatever the degree of probability) suggested that fly control was a desirable component of the total Hanson, R. P., J. Spalatin & G. S. Jacobson. 1973. The viscerotropic pathotype of Newcastle disease virus. Avian eradicative effort. In fact, when the relationship Dis. 17: 354-61. between Fannia spp. and W N D became established, Hofstad, M. S. 1949. Recovery of Newcastle disease (pneua routine fly-control program was instituted on moencephalitis) virus from mites, Liponyssus sylviarum, after feeding upon Newcastle-infected chickens. Amer. J. Vet. premises that were being depopulated to reduce the Res. 10: 370-71. potential of spread by flies (Bram et al. 1974). Lempke, B. J. 1962. Insecten gevangen op het lichtschip The observation that all 5 fly pools found with "Noord Hinder." Ent. Ber. 22: 100-01. ND virus were associated with the genus Fannia is McDaniel, H. A. & J. S. Orsborn, Jr. 1973. Diagnosis of velogenic viscerotropic Newcastle disease. J. Amer. Vet. of considerable interest. Despite a similar abunMed. Assoc. 163: 1075-79. dance of M. domestica in our collections, no evidence National Academy of Sciences. 1971. Methods for exof involvement of this species was found, except in amining poultry biologies and for identifying and quantifying avian pathogens. Washington, D. C. 326 p. the 1 pool of larvae. Little significance is attached to the isolation of the virus from larvae, since these Ogata, K. & T. Suzuki. 1960. Release studies on the dispersion of the lesser house fly, Fannia canicularis, in the were taken in manure that may have been incomresidential area of Bibai, Hokkaido. Botyu-Kagaku 25: 51-57. pletely removed from the specimens. Omohundro, R. E. 1972. Exotic Newcastle disease eradication. Proc. U.S. Anim. Hlth Assoc. 76: 264-68. We have no evidence to indicate whether the association of ND virus with Fannia is external or Schloer, G. & R. P. Hanson. 1968. Plaque morphology of Newcastle disease virus as influenced by all types and internal, mechanical or biological, although these environmental factors. Amer. J. Vet. Res. 29: 883-95. are questions of considerable interest. Sharman, E. C. & J. W. Walker. 1973. Regulatory aspects of velogenic viscerotropic Newcastle disease. J. Amer. Vet. The flight range of F. canicularis is not regarded Med. Assoc. 163: 1089-93. as particularly great. Some studies (e.g., Ogata & Shura-Bura, B. L., A. D. Shaikov, E. V. Ivanova, A. I. Suzuki 1960, Williams 1973) indicate that at least Glazunova, M. S. Mitriu Kora & K. G. Fedorova. 1958. The character of dispersion from the point of a low order of dispersion does occur. On the other release in certain species of flies of medical importance. hand, Lempke (1962) captured 2 specimens on a Ent. Rev. 37': 282-90. lightship about 40 km from the nearest coastline, Utterback, W. W. & J. H. Schwartz. 1973. Epizootiology and Shura-Bura et al. (1958) captured radioof velogenic viscerotropic Newcastle disease in southern labeled specimens 4.5 km from the point of release California, 1971-1973. J. Amer. Vet. Med. Assoc. 163: 1080-88. in sparsely populated areas near Leningrad. The Walker, J. W., B. R. Heron & M. A. Mixson. 1973. Exotic situation during the W N D eradication campaign, Newcastle disease eradication program in the United however, provided an abnormal factor that increased States. Avian Dis. 17: 486-503. the likelihood of fly dispersion: depopulation of Williams, J. R. P. 1973. Dispersal of 32P-labelled adult Fannia canicularis. Int. Pest Control 15: 20-21. chickens accompanied by removal of manure and cleaning and disinfection of the premises causes

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Bram, R. A., S. W. Wilson & J. B. Sardesai. 1974. Fly control in support of the exotic Newcastle disease eradication program in southern California. Bull. Ent. Soc. Amer. 20: 228-30. Greenberg, B. 1971. Flies and disease. Vol. 1. Princeton Univ. Press, Princeton. 856 p. Hanson, R. P. 1972. Newcastle disease, p. 619-56. In: M. S. Hofstad et al. [eds.] Diseases of poultry. 6th ed. Iowa State Univ. Press, Ames. 1973. Research support for eradication of velogenic, viscerotropic Newcastle disease and other diseases. J. Amer. Vet. Med. Assoc. 163: 1094-96.

Transmission of Newcastle disease virus by insects: detection in wild Fannia spp. (Diptera: Muscidae).

1975 Nayar & Sauerman: 225 Mosquito utilization of blood and sugar for fecundity Stahler, N. & L. A. Terzian. 1953. Studies in the laboratory the...
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