Author Institution: Division of Laboratory Animal Medicine, Medical College of OhioSerological evidence was obtained that demonstrated previous infection with Pasteurella tularensis in 2 of 9 sheep raised in Monroe County, MI. Although no ectoparasites were found on the sheep in April and May when the testing was done, it was assumed that they were infected by ticks. Titers for Brucella were much less than those for P. tularensis and were not considered meaningful. Eight New Zealand white rabbits from nearby Risingsun, OH were serologically negative for tularemia and brucellosis. These animals had been confined in a commercial rabbitry and were free of ectoparasites. One of 8 dogs from Fulton County, OH, had a positive titer for P. tularensis, but not Brucella. No ectoparasites were observed on the dogs, but they had been recently dipped. The possibility that man as well as other animals might become infected with Pasteurella tularensis in the Toledo, OH area exists

Clifford, Donald H.

Long, Matthew M.

The Ohio Journal of Science

KnowledgeBank at OSU

A SEROLOGICAL STUDY OF TULAREMIA IN DOMESTICANIMALS AND THE POTENTIAL THREAT TO HUMANS1MATTHEW M. LONG and DONALD H. CLIFFORD, Division of Laboratory Animal Medicine,Medical College of Ohio, Toledo, OH 43699Abstract. Serological evidence was obtained that demonstrated previous infection withPasteurella tularensis in 2 of 9 sheep raised in Monroe County, MI. Although no ecto-parasites were found on the sheep in April and May when the testing was done, itwas assumed that they were infected by ticks. Titers for Brucella were much lessthan those for P. tularensis and were not considered meaningful. Eight New Zealandwhite rabbits from nearby Risingsun, OH were serologically negative for tularemiaand brucellosis. These animals had been confined in a commercial rabbitry andwere free of ectoparasites. One of 8 dogs from Fulton County, OH, had a positivetiter for P. tularensis, but not Brucella. No ectoparasites were observed on the dogs,but they had been recently dipped. The possibility that man as well as other animalsmight become infected with Pasteurella tularensis in the Toledo, OH area exists.OHIO J. SCI. 78(2): 92, 1978This study was undertaken to deter-mine if tularemia was a potential threatto the human population in the Toledoarea. Initially, we tried to capture wildrabbits but after 2 weeks of observing 7"live traps" in the Lucas County areawithout results, we decided that othermethods should be used for the study. Ithas been reported in the American, Can-adian, and Russian literature (Olsofieuand Rodneva 1960; Parker and Dade1929; Pollitzer 1967; Simpson 1929; Wat-kin et al 1974) that sheep and dogs maybe infected with Pasteurella (Francinella)tularensis. Sheep, dogs and domesticrabbits were made available through theMedical College of Ohio and the ToledoZoological Gardens for study.METHODSTwo methods are commonly used to test serafrom animals for the antibodies of tularemia:the rapid slide agglutination test, for screeningpurposes, and the tube agglutination test, todetermine the specific level of antibody ortiter in the blood.Procurement of Sera. Sterile test tubescoated with silicone (Vacutainer®, B&D) wereused to collect blood. Each tube had thecapacity of approximately 7 ml, which providedserum for at least 5 tests. Blood was obtainedby inserting the needle into the jugular veinxManuscript received October 27, 197(5 andin revised form July (5, 1977 (#76-83).after elevating the animal's head and applyingpressure over the thoracic inlet to tighten theskin over the neck and distend the vein.Blood also was taken from the central ormarginal ear vein of rabbits by means of siliconecoated Vacutainer tubes. This procedure wasdifficult since the veins in the ears of rabbitsreadily constrict. The vacuum causes therabbit veins to collapse, making the with-drawal of blood difficult, so an infrared lampwas directed toward the rabbits' cars to pro-duce vasodilation. In 2 rabbits from whichwe could not get ear vein blood, a cardiac punc-ture was used to obtain blood. The dog'sforeleg was clipped and the area was cleanedwith alcohol prior to venipuncture. A tour-niquet was placed just above the dog's 2nd jointto occlude the veins and make them easier toenter with the Vacutainer needle.Blood from all animals was centrifuged at 100rpm for 10 min. to remove the red blood cells.The clear serum was drawn into a capillarypipette, placed into a sterile test tube andstored in a refrigerator at 4° C until testscould be performed. Each tube was identifiedwith the animal's number, type, and the dateof withdrawal. The sera was recentrifugedjust before testing to insure clear serum, asscrum containing hcmolyzed red blood cells isdifficult to interpret.Slide Agglutination Test. The slide aggluti-nation test was performed as soon as possibleafter collection of blood. An aliquote of 0.04ml of serum from each animal to be tested wasplaced in one of the squares of an agglutinationtesting plate. Bacto Pasturella tularensis anti-sera (Difco Laboratories), 0.04 ml, and isotonicsodium chloride solution, 0.04 ml, were addedto additional squares to serve as positive and92Ohio J. Sci. TULAREMIA IN DOMESTIC ANIMALS 93negative controls, respectively. One drop, 0.03ml, of Biiclo Pasteurella lularensis antigen wasadded to each drop of serum and to the positiveand negative controls. Each droplet was mixed,with a separate applicator stick, and the platewas rotated gently. If a slide was used, it wasplaced on a mechanical rotator and agitated for4 minutes at 175 rpm. The plate or slide wasthen examined in a lighted viewing box.Re-actions were recorded from negative (0) topositive (4-f-). Any specimen with 2+ or morewas retesfed using 0.04, 0.02, 0.01, and 0.005 mlof the diluted positive serum, as well as positiveand negative controls. The tests were doneat dilutions of 1:40, 1:80, 1 :l(>0, and 1:320, andall samples were subsequently examined bymeans of the tube agglutination test.According to the results of Schneider et til(1950-1951), sera bearing a slide titer of 1:80 ormore were regarded, as positive. Those ex-hibiting no agglutination at a dilution of 1:40were considered negative, while those serashowing a weak reaction at 1:40 only wereconsidered weakly positive.Tube Agglutination Test. The tube aggluti-nation test was more precise in determining thetiter level. The first step was to place 9labeled test tubes in a rack and 0.05 ml isotonicsodium chloride into each. tube. A 1:15 dilu-tion of the serum to be tested was prepared byadding 0.2 ml of serum to 0.8 ml of isotonicsodium chloride. Then 0.05 ml of the serumdilution was added to the first tube and mixedwell. From the first tube 0.05 ml of fluid wastransferred to the second tube and mixed. Thesame amount of fluid, 0.05 ml, was seriallytransferred to the remaining tubes. BactoPasteurella tularensis antigen (0.5 ml) was addedto each tube and mixed by shaking the rack.The resultant dilutions were: 1:20, 1:40, 1:80,1:160, 1:320, 1:640, and 1:1280 for tubes 1 through7 respectively. Tubes 8 and 9 wrere positive andnegative control tubes. The tubes were incu-bated in a 37° C water bath for 2 hours. Afterremoval from the water bath, the tubes wereplaced in a refrigerator at 2-10° C.Similar agglutination tests with Brucellaabortus antigen were performed on the sameserum of the sheep and dogs to determine ifthere was a cross-reaction or false-positivereactions with Brucella antibodies.RESULTSSheep. The sheep with the highesttiters (1:320) for tularemia were numbers64 and B2 (table 1). A Brucella test wasconducted to determine the degree ofcross-reaction with Brucella. Sheep #64had a Brucella titer of 1:80, which wasdiagnostic, and sheep #B2's Brucella titerwas 1:40, weakly positive. If the Bru-cella titers, which might be concluded asnonspecific, were subtracted from thePasteurella titers, 64 and B2 still wouldhave diagnostic titers for tularemia.Rabbits. The 8 rabbits (New Zea-land's from Risingsun, OH) used in thisstudy were from a controlled environmentwhere the animals were kept in separatecages which were cleaned and disinfectedregularly. Rabbits arc not known to besusceptible to brucellosis, which accountsfor the negative results from the Brucellatests (table 1). No rabbits had titersover 1 :4() by means of the tube aggluti-nation test.Dogs. One of 8 dogs from FultonCounty had a positive titer for P. lularen-sis. The dog with the highest liter oftularemia in the tube agglutination testwas #3412, whose titer was 1:1 (>(). A Bru-cella test also was done to eliminate thepossibility of a false-positive or a cross-reaction (table 1). The Brucella titerwas subtracted from the Pasleurella titermaking it 1:8(), which may be considereddiagnostic for tularemia.DISCUSSIONTularemia is a disease which is com-monly thought to be found only in rab-bits. Most veterinarians do not con-sider tularemia as a serious disease insheep and dogs. Philip et al (1935) re-ported an outbreak in Montana affecting1320 ewes with the death of about 200animals. They also isolated the causa-tive micro-organism and incriminatedticks as the vector. In 1974, Watkin etal studied an outbreak of tularemia inAlberta, Canada, in a band of 850 ewesfrom which they succeeded in isolatingPasteurella tularensis from both animalsand ticks. From the work of theseauthors and other reports of losses undersimilar circumstances, it is believed thattularemia can be a serious disease ofsheep, causing heavy losses.Jellison and Kohnls (1955) reported189 human cases of tularemia in workersassociated with sheep from 1934 to 1952.The source of infection was attributed tocontact with sheep by skinning deadanimals, shearing sheep, or by bites of in-fected ticks {Dermacentor andersoni) in 46cases. Two sheep in our study appearto have had tularemia. This is over 20%,but the number of sheep was too small toreach a meaningful percent of infection.The sheep tested were from MonroeCounty, MI.Unfortunately in our studv no wild94 M. M. LONG AND D. H. CLIFFORD Vol. 78TABLE 1Tube tests* on serum from sheep, rabbits and dogs tested for antibodies associated withtularemia and brucellosis.SheepNo.6264668589B1B2B3B4_ c * * *RabbitNo.1.2.3.4.5.6.7.8.+c***DogNo.14153411341234133414341834192808+C***-C***1:40++-j-+-1:40_————-1:40—-|-4--j-—-f-_(--\-—Tularemia1:80 1:160 1:320**+ ++ + +_ _ __|_ _|_ —_ _ _+ — —+ + ++ + —— _ _+ + +— - -Tularemia1:80 1:160 1:320**_ __ _ __ _ __ _ __ _ __ _ __ _ __ _ _+ + +_ _ _Tularemia1:80 1:160 1:320**_ _ _+ + —_ _ __ _ __ _ __|_ _ ___ _ _+ + +— — —Titer1:1601:3201:401:1601:401:801:3201:1601:40Pos.Neg.TiterNeg.1:401:40Neg.Neg.Neg.1:40Neg.Pos.Neg.Titer1:40Neg.1:1601:401:40Neg.1:801:40Pos.Neg.1:40+-f.-f_)--|--f--|-+—-1:40_———————-j-—1:40+———-|-_p.-)-—Brucellosis1:80 1:160+ ++ —— —— __ _— —— —4- +_ —- -Brucellosis1:80 1:160— —— —— —_ _— —_ _— —-|- -|-- -Brucellosis1:80 1:160— —— —— —_— —— —_ _+ +— —1:320"_—__—_——-1:320_———————-1:320_———_———_1-—Titer1:1601:801:401:40Neg.1:401:401:160Neg.Pos.Neg.TiterNeg.Neg.Neg.Neg.Neg.Neg.Neg.Neg.Pos.Neg.Titer1:40Neg.1:40Neg.1:40Neg.1:40Pos.Pos.Neg.*Similar results were obtained by means of the slide test.**Positive results were not obtained in greater dilutions.***Control with positive antisera included with the test mater ia l=+C; saline control = - C .rabbits were captured so domestic rabbitsfrom a commercial rabbitry were testedinstead. Medical historians have evi-dence that tularemia as a disease occur-red at the latter end of the Miocene orearly Pliocene period (Olsufieun 1963)and that rabbits contract the diseasefrom common wood ticks (Bell 1945).Tularemia is rarely diagnosed in dogs.Although most mammals are susceptibleto tularemia, dogs are not infected unlessthey are exposed to infected ticks. Dogsthat live outside for longer periods oftime in wooded areas are apt to be in-fected and dogs who tend livestock maybe infected by the ticks that live onOhio J. Sci. TULAREMIA IN DOMESTIC ANIMALSfarm animals. The dogs whose bloodwas tested were from rural FultonCounty, OH. Dogs should be examinedregularly for ticks, and infected animalsand their quarters should be treatedwith an acaricide.Acknowledgments. The authors wish to thankDr. Edward O'Donnel and Mrs. LorettaLorenzo, Clinical Laboratory, Medical Collegeof Ohio, for counsel and assistance in perform-ing the serological tests, Dr. Charles Hardin,Toledo Zoological Gardens, for providing therabbits and the Medical College of Ohio atToledo for the other animals tested. Mr. PatBehan, Mr. Gary Forrest, Ms. Barbara Groes-beck, Mrs. Ann Hansen, Mr. Gary Hochmuth,Mrs. Jody Kallich, Mr. Randy Melich, and Mr.Thomas Puhl assisted in making "live traps,"in obtaining blood samples, and in procuringsupplies and equipment.LITERATURE CITEDBell, J. F. 1945 Infection of ticks (Derma-centor variabulis) with Pastenrella tularensis.T. Infect. Dis. 76: 83-95.Jellison, W. L. and G. M. Kohnls 1955 Tula-remia in sheep and in sheep industry workersin western United States. U.S. Public HealthService, Public Health Monogr. 28: 1-17.Olsoficu, N.G.and O. S.Rodneva 1900 Tula-remia. Moscow, Medgiz. 1-458.Olsuficun, G. 1903 On paleogenesis of thecaustic agent of tularemia. Czechoslovak.Acad. Sci. Prague 1: 109-175.Parker, R. R. and J. S. Dadc 1929 Tularemiain sheep in nature. Reprint #1202, U.S. Pub-lic Health Dept. 44: 120-130.Philip, C. B., W. L. jellison and H. F. Wilkins1935 Epizootic tickbornc tularemia in sheepin Montana. J". Amer. Vet. Mcd. Assoc. 80:730-744.Pollitzer, R. 1907 History and incidence oftularemia in the Soviet Union. FordhamUniversity, Institute of Contemporary Rus-sian Studies, New York. 300 pp.Schneider, N., R. Mitchell and A. V. Hardy1950-51 A rapid slide agglutination test forPasteurella tularensis infection. Public HealthLab. 8: 33-35.Simpson, W. 1929 Tularemia: history, pa-thology, diagnosis and treatment. Paul B.Hoeber, New York. 102 pp.Watkin, R., R. H. Painter and W. I. Moynihan1974. Tularemia in sheep. Canadian J.Comp. Med. Vet. Sci. 0: 103-108.

A Serological Study of Tularemia in Domestic Animals and the Potential Threat to Humans

https://kb.osu.edu/bitstream/1811/22521/1/V078N2_092.pdf

A Serological Study of Tularemia in Domestic Animals and the Potential Threat to Humans

Abstract

Similar works

Full text

Available Versions

KnowledgeBank at OSU