AN ABSTRACT OF THE THESISOF William Edward Noonanfor the Doctor of Philosophy (Name) (Degree) in Zoology presented on July 7, 1972 (Major) (Date) Title: NEORICKETTSIAHELMINTHOEC.6.-IficzQ.--K,LL CULTURE Redacted for Privacy Abstract approved: Dr. Ivan Pratt The etiologic agent of salmonpoisoning disease was found to be Neorickettsia helminthoeca, although asecond organism, the Eloko- min fluke fever agent, mayalso be involved in other areas.Primary cultures of dog leucocytes werefound to support the in vitro cultiva- tion of Neorickettsiahelminthoeca as were canine sarcoma503 cells and mouse lymphoblasts MBIII.Quantitative methods were not applied to in vitro studies ofmultiplication of the rickettsiabecause it failed to grow sufficientlyin laboratory animals or inchicken embryo yolk sacs to do so. By ultrastructural analysisit was learned thatNeorickettsia helminthoeca was structurallysimilar to other rickettsiae.Rickett- sial cells were seen ascircular profiles or rod-shapedcells 0. 5µ wide and up to 0.7p. long.They were bounded by a cellwall and an underlying cytoplasmic membrane.Each of the membranes was a trilayered structure andshowed the unit membrane structure. Koch's postulates werefulfilled as completely as possiblefor an organism that is an obligateintracellular parasite.Neorickettsia helminthoeca was found in all casesof salmon poisoning disease. The rickettsia was isolatedand grown in culture.The isolated culture was found to reproducethe disease when inoculated into susceptible dogs.The rickettsia was observed inand recovered from the inoculated dog. Neorickettsia helminthoeca incell systems can be used as a model to further investigate thehost-parasite relationship. Neorickettsia helminthoeca inCell Culture by William Edward Noonan A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1973 APPROVED: Redacted for Privacy Professor of Zoology in charge of major Redacted for Privacy Chairman ofDepartiment of Zoology Redacted for Privacy Dean of Graduate School Date thesis is presented July 7, 1972 Typed by Cheryl E. Curbfor William Edward Noonan Parasitism may be regarded, not as a pathological manifestation but as anormal condition having its roots inthe inter- dependence of all living organisms. The obald Smith ACKNOWLEDGMENTS I gratefully acknowledgethe guidance and adviceof Dr. Ivan Pratt under whosedirection this study wasmade.I wish particularly to thank Dr. A.Owczarzak without whoseessential advice and facili- ties for tissue culturethe project could not havebeen carried out.I individuals: Mr. Lyle also acknowledge theassistance of the following Bogenhagen, Dr. Stuart Knapp,Dr. Donald Mattson andDr. Paul Presidente of the Departmentof Veterinary Medicine,Oregon State University; Dr. K. Farrell,Department of VeterinaryMedicine, Washington State University;Dr. James Akiyama,Department of College; Mr. Kay Biological Sciences andMicrobiology, San Jose State Fernald, Dr. H.K. Phinneyand Mr. Al Soeldner,Department of Bo- Department of Micro- tany, Oregon StateUniversity; Dr. S. Pilcher, biology, Oregon StateUniversity; Mr. DonaldMorrison, Department of Zoology, OregonState University; and Mr.Paul Vroman, Oregon State Game Commission. TABLE OF CONTENTS Page 1 INTRODUCTION 20 METHODS AND MATERIALS Metacercarial Cysts 20 Rickettsial Strains 20 Experimental Canid Hosts 23 23 Canine Leucocytes Transmission ElectronMicroscopy 26 Time-Lapse Cinemicrography 26 Preservation of Tissues inLiquid Nitrogen 27 Primary Tissue Cultures 27 Isolation of the Rickettsiafrom Experimentally Infected Tissue 28 Rickettsial Agents in Mice, Isolation Procedure for 28 Rats and Guinea Pigs 29 Immunosuppressants Rickettsiae in Yolk Sacs 29 Isolation Procedure for 31 Mycoplasma Isolation 31 Tissue Culture Methods 32 Chicken Fibroblasts 32 Salmonid Cells 33 Canine Kidney Cells 34 Canine Sarcoma Cells 34 Mouse Lymphoblasts 35 Lepidopte ran Hemocyte s Preparation and Infection ofCells 36 Fate of InactivatedRickettsiae 37 38 RESULTS Observations of ExperimentalInfections 38 Microscopy 41 Transmission Electron 50 Time Lapse Cinemicrography Preservation of Tissues inLiquid Nitrogen 51 Infected Tissue 52 Isolation from Experimentally 53 Immunosuppressants in Yolk Sacs 53 Isolation of Rickettsiae 53 Mycoplasma Isolation 54 Tissue Cultures Rickettsiae 55 Fate of Inactivated 56 Inoculation of Dogs withCultures TABLE OF CONTENTS(Cont. ) Page 57' DISCUSSION 64 BIBLIOGRAPHY LIST OF FIGURES Figure Page 1 Schema of experiments used todetermine the relationship between Neorickettsiahelminthoeca and the Elokomin fluke feveragent, and the pro- cedures used in the isolationcultivation and characterization of Neorickettsiahelminthoeca, 22 2 Comparative body temperaturesand weights of principal (1,000 metacercarialcysts) and non-infected control dogs. 39 3 Comparative body temperaturesof principals (infected with the Elokomin flukefever agent) and non-infected control dogs. 39 4 Comparative body temperaturesof principals (Dog D-3 was given 1,000metacercarial cysts after recovery from Elokominfluke fever; Dog D-4 challenged with theElokomin fluke fever agent after recoveryfrom Elokomin fluke fever) and non-infected controldogs. 42 5 Comparative body temperatures ofprincipal (Dog D-7 infected with Neorickettsiahelminthoeca from infected tissue culture)and non-infected control dogs. 42 6 Mesenteric lymph node of anon-infected control dog. 43 7 Mesenteric lymph node of a doginfected with Neorickettsia helminthoeca. 44 8 Mesenteric lymph node of adog infected with Neorickettsia helminthoeca. 45 9 Mesenteric lymph node of a doginfected with Neorickettsia helminthoeca. 46 47 10 Thymus of a non-infectedcontrol dog. LIST OF FIGURES (Cont.) Page Figure 11 Thymus of a dog infectedwith Neorickettsia helminthoeca. 48 12 Thymus of a dog infectedwith Neorickettsia helminthoeca, 49 NEORICKETTSIA HELMINTHOECAIN CELL CULTURE INTRODUCTION Salmon poisoning, a diseaseof the Canidae, was indigenousto southwestern Washington, westernOregon and northwesternCalifornia before this region was settledby the pioneers. Henry (1814) mentioned thatdogs from Astoria were sent upto the Willamette for thewinter to prevent their death, asliving on raw salmon the previous summerand fall had caused manylosses. Thornton (1849) furtherqualified this relationshipby noting that dogs He con- are very fondof raw salmon, but itis usually fatal to them. sidered only fresh salmonto be injurious andrelated that an opinion generally prevailed that apoison was contained inthe blood of the sal- dogs the mon.This poison causedalidull and moping "condition in second day after ingestion.This was followed on thefourth day by rejection of food exceptsalmon which was eatenuntil expiration about the tenth day.Cooper and Suck ley (1859)observed that blooded dogs introduced from the Statesbecame very ill after eating rawsalmon although the native dogs ofOregon subsisted well uponit.Bonebrake (1925), having observed thedisease in 1862, alsoreported this condi- tion which was called"salmon sickness".It was, however, thought by some to be commondog distemper. 2 Nash (1882) explained that salmon returning to spawn were deadly poison to dogs and wolves.He did not have any faith in the remedies such as epsom salts, castor-oil,large doses of mustard, shot in quantities forced down thethroat, calomel, aloes and black- berry-tea that were prescribed as antidotesfor the poison.Since most mature dogs became"salmoned" and died he suggested immunity could be obtained by allowing puppiesfree run of salmon.Survivors having passed the ordeal weresalmon-proof and lived to their full age. Preventive advice of keeping valuable dogstied up was offered to newcomers into the coast country. Pernot (1911) related that the mostfatal disease of dogs in Oregon was commonly known as"salmon poisoning".This disease was commonly explainedby the theory that salmon possess apoison which when eaten by dogs producedsickness and death.He reasoned since all diseases have a specific causeit was necessary to determine "the specific cause". He assumedthat since the disease had an incubation period of nine days after ingestionof salmon that it must be bacterial in nature.Pernot isolated pure cultures ofmicroorganisms from sal- mon, cultured them inbouillon and fed them separately to dogs.None of the cultures, either singly ormixed, gave symptoms of the disease. He reasoned that the diseasemust be caused by the multiplicationof some living parasiteduring the incubation period.This led him to make a systematic study of thesalmon and the blood of an infecteddog. 3 He fed a dog one-quarterof a teaspoonful of thedark bloody substance taken from along the backboneof the inside of the salmon(kidney tis- sue) and produced salmonpoisoning disease.He drew a fraction of a drop of blood from this dogand injected it subcutaneouslyinto a healthy dog.After nine days the dogdeveloped a typical case of sal- mon poisoning.The experiment was repeatedusing a guinea pig which did not show any signsof disease after 15 days.However, the disease was produced byfeeding one inch of the lowerintestine of an infected dog to a susceptibledog.These facts convinced Pernotthat some living parasite wastransferred from a diseaseddog to a healthy one and that theparasite multiplying duringthe incubation period pro- duced the disease andsubsequent death.The disease signs did not occur uponchallenge with infectivesalmonid kidney if the dog had been "salmoned" previously or ifthe salmon had been cooked.Upon close