*J 3‘ . . . ! .— _— —— .— —— —— —— —— —— _— —— —_ —— — Hp.) IN—4 (00003 THE EFFECTS OF ANTE PARTUM LEFTOSPIRA POMONA‘ INFECTION 0N BOVINE FETAL AND MAWRNAL BLOOD VALUES Thesis for the Degree of M. S. + MiCHiGAN STATE UNIVSRSiTY Athalie Meyer Lundberg 1960 ":1! ". ,~ 5.. -‘ Li I. I—ufiiq C. Q... LIBRARY Michigan State University M0” ' TEE EFFiCTS CF ARTE EARTUfi LEPTOSPIRA POKOEA IgszTION ON BOVINE FETAL AND MATERNAL BLOOD VALUES by l Athelie Meyer Lundberg A Thesis bubmitted to the College of Science and Arts Michigan State University of Agriculture and Applied science in partial fulfillment of the requirements for the degree of EASTBR OF bCIENCE Department of LiCPOblOlOQy and Public Health 1960 ACKJOhLfiDGEENTQ The author wishes to express her thanks to Dre. 3. L. Eorter and E. V. Morse without whose persistent efforts this work wouli never have been started, to Dr. R. G. fielding for counselling and criticism, and to a long- suffering family for their patience. TABLE OF C IIITRODIJCTICI‘I.00.00.000.00... ‘ , “1! I"' ',‘ '0 ‘ 5.2.3 LIPLLLXIULQ itJJVIAH o o o o o o o o a o o a:v;U:r-1:7 1.th uluoooooouoooooaooocoo-o TABLES, CHARTS, AND FIGURES. " “ fir ":7 UIULJUMIUHOCCOOCOOCOCOOIC... ‘ 1:15.? ' EJUril-iARIoo0000.00.90.coo-coo. blBLIOGLUK§2IiKOOOOOOOO..00.... arm‘“ .7. 't' 6;! Loo-II“! -L) '0 pa Ct: H ll 12 h) (.10 hf.- hu . .r..f.kbi . IflTRODUCTIOR Naturally acquired bovine leptOSpirosis may manifest all stages of severity from unspperent infection, detectable only by laboratory means, to an acute febrile form leading to death in a few days. Age, breed of cattle, individual re~ sistance, and the particular infecting strain are some of the factors responsible for this great variability. Ioung animals are more severely affected, w th reports of losses as high as 50; in feeder lot situations (39). Older animals, even with no history of previous exposure to the organism, exhibit fewer acute manifestations, perhaps due to a former unepparent infection or a physiologically acquired resistance. Ferguson, 33 3;. (20), in a limited study, suggested that Jersey cattle may be more susceptible than Holstein-Eriesian, and this might be extended to other breeds. Leptosgira porous is the usual infecting serotype in the United States, but even in this supposedly sero- logicelly homogeneous species, certain strs he are more virulent than others. i. sefiroe, ghhlgg, g. gagggotyphosn, t. camisole, g. icterohenorrhsgiee, and the recently iso- lated‘£. herd‘o have been implicated by serological pro- cedures (21). Some variation is found in the reports of the severity of the disease in pregnant cattle. Abortion in lute preg- nancy is given as the only symptom in most cases (2, 22); while the more severe acute febrile form of the disses has been described in other p3pers (3Q). There is a similar variation in reporting abortion rates. The figure of 5-10} is most generally found, even in herds showing 130; sero- logically positive animals. however, an occasional abortion "store" may produce fetal losses as high as 23-4Sfi in lsr5e beef cattle herds. us es, and r3); This degree of variation, its many go ossi ole c the actual mechanism of the leptospirsl abortion has been the subject of much study and debate in recent you r . fe Fungs and Bishop (#0) have suggested three sajor possible causes: 1. Pyrexia and systemic reaction in the maternal riot, resulting in abortion. 2. Localized lesions in the placentones (notern l-fet: l cotyledor ary Junction) with interru‘g ted trs.nsfer of metabolites and subsequent fetal death and expulsion. 3. Actual invasion of the fetus by leotosoirse, resultiu5 in acute fetal infection, death, and eXpulsion. Several of these postulates have been the subject of further investigation. horse and hchutt (31) modify the scoot; con- tention to sts te that interference with placental and fetal circulation may be the cause or contributing factor in lepto- spirsl abortion. sorter, gt 5;. (34), su55es t that hormonal imbalance due to alterations of hormone-producing maternal crypts might be a factor. Ferguson, gt 5;. (20) pro: :ose a soluble toxin, produced by the leptospira in the dam, capable of passing the pla- cental barrier and destroying the fetal erythrocytes, there- by producing anoxis, death, and fete l e: :pulsion. Fenaestad and Borg-Peterson (13, 14, 15) support postu- late nunber three that leptospiral abortion is most often due to fetal leptospirosis of the fetus, and have denonstra- ted leptospirae in aborted fetal material by silver impreg- nation methods. They feel that autolysis of the fetus des- troys these organisms and accounts for ne5ative results reported in routine culture procedures. Further, in more recent papers (16, 17, 13), they state that acceptable evi- dence has not been presented to substantiate the toxin hypothesis. since erythrocyte destruction can be determined by simple and reasonably accurate methods, it seemed of value to explore Ferguson's proposal as to the action of a henc- lytic toxin and determine if fetal blood values were affected by maternal leptospirosis. The hematological constituents chosen for determination were total erythrocyte and leuko- cyte counts, differential leukocyte determinations, henc- elobin and hematocrit values. in addition, a selected group of chemical determinations were chosen to help clarify some of the issues advanced in the possible disturbance of maternal—fetal transfer. The blood 5ases, carbon dioxide and oxygen were naturally con- cerned in the erythrocyte destruction hypothesis; non-protein nitrogen and creatinine would indicate renal damage, the most consistent feature of leptospirosis; and glucose is a readily diffusable metabolite, reduction of which could severely ef- fect fetal development. LIThRATURE R View [. The published reports on fetal bovine blood values are exceedingly sparse. In his comprehensive compilation of blood values in 1953. Albritton (1) found only one reference to fetal cattle. This report by von Deseo (40), in 1929, dealt entirely with the erythrocytic series of blood cells, giving values for red blood cells, hemoglobin and packed cell volume. His main concern was with the relative pro- portion of total solids and fluid content of the erythrocyte at various stages of fetal life. ‘ercroft (4) combined in one volume his own extensive work on ovine prenatal deveIOpment and all material avail- able on other mammalian species. This included one short paragraph on oxygen dissociation curves in a single eight month old bovine fetus. The original article by Roos 6nd Romijn (37), however, is more detailed and reviews the work on oxygen capacity in several species. In men, the maternal oxygen capacity was higher then the fetal, but in rabbits, dogs, sheep, goats and guinea pigs, the fetal values were 15-25% higher than the maternal. These authors used oxen to establish normal values in non-pregnant cows at 10.23- 18.45 volumes per cent with an average of 14.55. Determine- tions were also made on oxen in the seventh to ninth month of pregnancy, and eight 73 to 85 month fetuses. Fetal blood in the last month of uterine life has a 2-12fi higher oxygen capacity than the maternal blood and the difference becomes smaller as term is approached. The oxygen dissociation curves mentioned in Barcroft's book show a large gap between fetal and maternal curves proving, according to the authors, the great oxygen avidity of the fetal blood. The great dis- parity between maternal and fetal carbon dioxide curves led them to theorize the now proven difference between maternal and fetal hemoglobins. Further search of the literature failed to reveal any recent publications dealing specifically with bovine fetal blood values. The economic aspect would appear to be a limiti a factor and, in fact, docs and Ronijn admit that the expense prevented further experimentation. Voluninous reports have appeared on various sepects of fetal life in other mammalian species, particularly the sheep. hany of thee publications entitled "fetal studies" are in reality on newborn animals with the blood samples being obtained from the umbilical cord at birth (12, 25, 27). It is impossible to completely extrapolate work from one species to another, but the sheep and cow are fairly close physiologically, and some of the findings could be used as guideposts, at least. A volume by barclay, Franklin and Pritchard (3) on fetal circulation (1945) reviewing the work to date contains many comparative studies on many man- mals, but the sheep is still the primary example, with the human fetus second in volume of experimental work. The group of Dames, Mott and hiddicombe in various combinations (9, 10), has extensive current pUblications on fetal circulation and various blood gas relationships in I fetal lambs. The work of hetcalfe,.§t.;_. (30), on uterine blood flow and o Yy3en comsznnption, compares oxy3en diffusion in three species of animals with different types of placenta- tion. Followin3 Barron's (5) hypothesis that the 3radient varies with the thickness of placenteln membrane, they found that the values for the three layered human placenta fall between those for the sin3le layered rabbit and the five layered sheep. Likewise, certain human studies ma have is; plications, in a general way, for the values found in this study. Turn- bull and Walker (41), studying red blood cell and hemoglobin concentrations in human fetal bloods, found that the henc- 3lobin values stabilize at 13 3rans during the last tri- mester of pro3nancy, while the number of erythrocytes and i, O. ('9 the pack ed cell volun e increases as pre3nancy advanc This su33ests that the red cell becomes pro3ressively smaller as fetel e 3e increases. In fact, in s sev1uel to this arti- cle, Walker and Turnbull (43) state that any red cell over 4‘3 nine nicra in diameter is a fetal cell and the adult and fetal h- emo3loain is contained in as w: Uri to cells. The normal adult bovine values are found in such stand- ard uLjEiOlO'y texts as Du lies (11) and Coffin (3). Fer3uson, 33.33. (19). report no si3nificant relationship between pro- 3ression of pro 3: anc y vith chan3l es in nuubers of red and white cells per unit volume or the perce nta3 :e of different types of white cells. Reineke, pg 33. (56), report a series of ca hon dioxide:oxv“en ratios and reapira tory suctierfi s on a} k.) goat arterial and mammary gland blood which might likewise be an indication in bovine blood gas relationships. Unfor- tunately, Shaw's extension of this work to cattle gives only the differences in volumes per cent rather than the values themselves (33). These would have been useful criteria in the maternal studies on oxygen and carbon dioxide. QATERIALJ A33 HbTHODS Thirteen apparently mortal pure bred Holstein-Friesia heifers 24 to 30 months old were used in this project as experimental animals. They had been obtained at 8 to 12 months of age and kept free from contact :ith other animals. All were official Brucella abortus strain 1) vaccinates, and their ears were negative for leptospiral antibodies as de- termined by the modified agglutination lysis test (32) with 'g. n eons (Johnson), L, canicola, andlg. icteroheaorrhsuiig .(AB) antigens. The heifers were bred artificially with semen kindly supplied by the hichigan Artificial Breeders Cooperative. The carotid arteries were exteriorized in order to ob- tain the maternal blood samples more easily. From one to six pre-infection determinations were made on each animal to establish base normals. At stages varying from the fourth to the eighth month of pregnancy, seven of the heifers were inoculated subcuta- (strain sickard). The ”,0 T1"; 0 TIC. neously with five m1 of §. inoculun was heparinized guinea pig or hamster blood obtained during leptospirenia. This strain of g. sonona was chosen for its virulent properties and had been kept in animal pos— sage since its isolation from the urine of an infected cow. Controls received 5 ml of noana blood from guinea pigs or hamsters. 013 animal was intra-utsrinely infected as des- cribed by Horter (33). The one remaining animal was found to be not pre5nant, so was des i5ns ted as a norgsal, non-pre5- nant, non-11f ected cor trol. See Table l for animal numbers, period of 5e sta ation, route of inoculation and fix;al disse- sition. Bacteriological and serological evidence of infection has been reported elsewhere (53% since these animals were used for several different as? sets of e.s perimental lepto- Spirosis. The day of necropsy, a maternal arterial sample was ob- tained, then a hysterotony was performed after tranquilize- 1® of the left ps rslumear fossa. Fetal venous and arterial tion with Dique and anaesthetization by a procaine T block samples were obtained from the umbilical vein and artery for comparative studies, especially of the blood 5ases. Due to the lar3e nu2sber of separate determinations desired, it was necessary to procure at least 20 ml of blood from each site and divide this into 4 separate samples. The method of treatment of these samples, the determinations for WfllCh each was employed, and the test procedure used in obtaininu values are as follows: 1. 5 ml of blood added to dried 20X potassium oxalate under a layer of mineral oil, for the blood 53 s deternina— tions using the Von Slyrhe and Eeill method for si UlUJUb us .3 determination of carbon dioxide er u 0}: ’,J 4 2." ‘LJ. if‘-.LE.3 a." CC 5en in a °ample (35). 2. Approximately 3 ml of blood combined with 0.1 ml of 135 pota.ssiun oxsls te for nos t of the rraoiriiw chemical and hematological determinations. Total erythrocyte and lO leukocyte values were calculated by standard methods, and the new cyann,them05lobin procedure was used for the hemo- 5lobin (7). For efficiency, the three renaining chemical values were all determined on Folin—Wu filtrates; glucose by the method of Folin and hug non-protein nitrogen by the Koch and Mcfieekin modification of the ori5inal'Folin and Wu method; and the creatinine by the Folin and Wu alkaline picrate aethod (24). 3. 2-3 ml of lood added to the dried residue of 0.5 ml of a combination potassium and ammonium oxalate for the hematocrit estimation after the method of hintrobe (7). 4. Two blood smears were made on glass slides, stained by Wright's method, and examined for the differential leuko— cyte determination (5). ll RESULTQ The hematological and chemical values obtained in the various determinations on materne and fetal blood at vari- ous stages of geststion are sumnsrized in Tables 2 through 6. The sstornal values for both non—infected and intrsperi- toneslly infected animals compare well with the non-pregnant, non-infected control heifer, and are well within the normal limits as given in Dukes (11) and Coffin (8). There is no basis for comparison of the fetal values, other than the erythrocytic series, and the values given by von Jeseo are closely paralleled. Chart 1 compares the changes in the fetal erythrocytic series produced by the two routes of infection. Figures 1 and 2 depict these changes pnotogrsphicellj. The other values for the fetus from the intrs-uterinely infected dam were not outside the range established on the remaining fetal samples. Table 7 gives the respiratory quotients of the fetuses from the 6 month infected, 7 month infected and 9 month non- infected heifers. Table 1. EXperimental Animal statistics. Stage of Gestation Animal at Termination fioute of Number in months Infection 7 6 Intraperitononl 7 Intraperitoneal 10 7 Intrsperitoneal 83 7 Intrsperitoneal l 3 Intraperitoneal 5 3 Intraperitonecl 03 8 Intraperitoneal l} 7 Intra-uterine 17 6 Hon-infected 3 3 non-infected 9 9 non-infected 07* 4a- * Normal Controls 1/ 1‘ .mmmepo>s easuno oa new: massage mo Loosen opooaozd mononucou new Ga messes: asooa\mszao> Haoo noxomm I o .Hs ooa\m:spa 2a :Hnoamoso: I n .890\m:OHHHHE ma m u:oosspmpn I a Cfiwb HfiOHHHQED HQQWN I ha .hth .QH< 4J)44H$:\ H.3um4 I t; «H11H5904 HiJQQJn‘ I W nfiHfSHiHH§Hd 3% II ~.m am. >@ II s. OH ON. m db mm 0. HH Oo.h x m.mm H.ma on.m mm c.ma ow.w m a noasuzoo negeou Hwy haw H.¢m n.w wa.h (.mm >.w mm.¢ >m o.mm ¢.m om.o O.nm o.m m+.m ea m.mm ¢.oa NH.> 0.0m 0.,H om.m m m.wm m.HH me.s m.em Q.HH mm.» s m Hmv new ma ¢.m an. o.mn o.m mo.u 5m ma H.m Hm.m 0.Hm m.~ oo.n es en ¢.OH m0 0.4m m. CH Hm.m r new m.mm m.flH oe.m m N Hwy! on o.» ~s.o em m.m mm.n so on m.» mm. mm m.® om.m am pm w.aa mm.b an n.0H mo.h 2 en m.HH mm.» m m Adv “Ho unannouee pawn song ouHAOOpesom nan: nomm oaauoouoeom non: sum: condos menses :M oHossm moaaspmos coagoomsH seapooezH mo ommum seahouanmnpna amocoudaodmhacm douoomcHlnoz .mnoapwofispoaoo oaumoopnpman 0.9 edema l4 .omx 00H\QqH 0mm @moooHodn L0H oopomhpou I *s “om OQH\0m cam woes oHos: soH oopoopnoo I a .omx 00H\mepH00M5oH mSOHLe b no m I 3 “ago o\mHHeo QOOHD ouHHfi I o .mophooeo: I a “mouHOOJAJHH I H ”noumoowseH LooHozcoomaosmHoH I H “HaJH oudeH epr: money I c “HHnmonHmos I e N mm mm H H Ommm >h H mm mm 4 m QmHOH n m an Hm m m Done H om 0H m ammo as e em wm H mH Odom m on #m H m ommCH m m Ns Om m nH ommHH m ww mH H mH ommHH m m E :v m mm m H abeeo H He eH a ommm . . en H em m e arena m es NH m m 0mm) ea a mo mm m m_mHm m 5w m 0 CH omOCH E n mw em e b omHoH m w HS L; mm mm m n OOHN m m» MH H m come be He mm s m QOHm mm NH m m ones as w mm mm m m Omwm N mm Pm m mm memo E m om om H l ome m u H3 H3 Erin H slim some 4 A H a a some n 4 m nnIM sue? HmeeonemmHm h HnHosonemuHm a 9w HsHacopouuHc cannon menses QH n oHann QOHuopmoe QQHpoeHoH SOHuoommH mo ensue oanoueIhHusH HnoCOQHAoaspuoH wouowumHI20u I radoHossHshoaom ophoomsoq .m oHnoe III. I." . . I... . H.¢¢ o.m es m.m¢ m.H d; e.me m.mH ; m.m¢ H.¢H ¢.¢¢ m.mH H m 0.530 in. ) HS :3 H.om qu o.ee 5.0 u m.on m.m H.Hn 0.0 em o.J¢ m.nH m.m¢ 0.0H m m.Hm m.> 9.0m w.mH m m 5 H3 H3 HHV H.¢¢ Q.H m.w¢ N.M >H mums m.n 4.3 moo en e.me m.eH m.me H.mH : ¢.ee Q.Hm n.0H m h TS HHV :3 E i m.me m.¢ >m m.nm 0.0 as m.om m.0H WM L.m¢ ¢.QH H o E i; moo No moo NO was NO 009303 mzaflos CH W mosSHo> s mms5H0> e mmsdHo> mosSHo> a mosoHo> mossHo> onssn soHpoumee . . no m®nun COHuoomcH eoHooeHnH oanopaIesonH Heonoanoaoaan wouoomcHIno: .mGOHaocHsLmqu mow vOOHm .¢ oHQoB 15 mm; Aenaaoo Jesse: Hmv >.mm ms.e Hm.e om.m NH.N Hmv a. om him/kw +~omu .u o UV :5 O tHCsonns\ H V C‘ IKEU‘ILTW HRH-1T 0 one 8:... cum In IOJ CHO HHV .SocosoHs FWKD O O 0 {‘3 H C ‘3 r" (“J Us {A I‘W {1 -l- a» v 0.3 [\— (WK I Cu 0.} C 4 .~ g“. HHV :HmsonsIeos mH raw s >b d m. s:> .14 1-114 ~ ..4 r-é-t (1.9 3 0 now we oo.¢ H.mm >s s.m em om.m m.em em m.H n.¢m NH.m e.Hm m ow.H m.om m e HHVI. HHV Hmv va om.m II m.¢ em >m oo.¢ II om.¢ Ts em mm.m II mm.m Am m om.H m.om . m m HHV HHM HHV R as I m me e me e R we a an w ms common Janos 1H ochHpeopU seas oanHeeoho emmw oanHusvo exam mHossw :oHeoMmoc no mason CQHuooucH COHpocmnH ecHsopaIenan Hoocoqu oopHCH wepoowcHlmoa .monpds esopon momopan .m eHnea Table 6 o 17 Glucose Determinations. Stage of Non-Infected Intra- Intra- Peritoneal Uterine Infection Infection AFIOCL:'&‘; mm 1 Pi} Eal‘lfito- ‘- Gestation Sample in months Source Glucose mg % Glucose m3 % Glucose mg g (1) T1) 6 P 69 Li 60 130 FA 350 212 FV 316 212 ~(3) (l) 7 P 80.5 M 75.3 100 FA 212 250 W 216 2:30 (1) (37 8 P 72 8 1-1 72 107 FA 139 204 FV 134 193 (1) (2) 9 P 74 1‘3 ORE-EAL M 71 COETROL FA 147 71 FV 170 Table 7. fetal Respiratory guotiente. samgle Volumes fl Volumes 3 § Age of Fetus Louroe 02 CUB R.Q. 6 months Umbilical infected Artery 0.0 53.8 Umbilical 1'00 V9111 4.3 4905 7 months Umbilical infected Artery 0.25 49.4 Umbilical 0°704 V9111 3.7 4609 9 months Umbilical non—infected Artery 1.8 48.5 Umbilical - 1'05 Vein 5.0 44.1 Venous-Arterial C02 Volumes % *ROQO 3 ~——— Arterial-Venous 02 Volumes E 12-280 l0 SQUARES 10 THE INCH Cl TART I .l_T..T77T... 1‘ ,.7l_T. .+.. TTTT lTITT Turn UT i {TE} NBISON. (DP FEELL TERY'THBOCY I‘E GMTIGES PTEODUCETD T BE 2 RIE'ITPTUDS_._QF M0 I l I _._.7f 77. . 7T . 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T A T I T . ‘77T T 7 T . . Hf , LT T .T . T—Tr .I . . . 7.I ‘ . . ‘1 7. f . . 7 -7 +7 T % Tia . . 4» q 7—. . :1 T_ :Tfir .’.‘. . . . - .. . . 1T; . . [T T .7+. ‘.7< . . .+‘ g~+ TTTT T 7 . lfi+ T 10' T e . 2‘ BOT .1. 1Z0. 3 TT 7 7 “.7? . . . .- . .. .. . .T , . ‘3 ... . . . . . i . J. ’Tfl —T+‘T—. 77. . . . . y . A.7. . . .7. .. . . ‘77, . .7T. ‘ . l . 7. . -7. .7. 1‘ TT ~o 7—.7. :— 7- . HEIMATTDCRIT Vol-vine *pa'cked ‘ceT ls/lfllmln + 4 4 : + ;' «T. ' 3: 7 ¢ 1 . . A7. . .17. .7. .? T.:. T: 7 . 7.7. ,1. UTT , . . . . T‘. '. 7. . .7 711 I r, . . T l , . .‘._. '71 TTTTTJ 4.; TT ll...+_; T; 7; .: .JTF I,;.7T:TT_. __._l 1 1L ”(“7 T“ ;;‘T W1: . 7: T.:;ii 71‘: HT1 4T .T I. 4 ‘ . . ‘ 71T8+ ~ .4 ._. T'..; i“; ; T .1 T' :Li; 11%th .LHFVT m .‘ .y. .,1.J. .T :1- ile 1 AT T if f 11% A T '- T..T t . .4 e 7. . . _. 7i*“““.:m: fl 1' ..T L ; .. T7; T H - 7. .3133. To . 7. .7] l .l . T" Tl...‘l,f_ T :FA- 7 -:..T‘T A i 74 ‘T NW ._fi_+_.*lr.T - . T T T. .7: 1L- . - _ n h: . .7 +77 -m. : T .TTTT. ”V r .7. . + , . T . . . . . ._ - 7 . . . A—4 . . +.7.77. . 7 fr' . .T I. L . — .k. ITLT . .JT TT‘TT T11; 39474 1.? ,1t1.‘ ;:2T. ‘37,:4 :‘5TT:TT€>_6.,:7,: 38% 29m: 6 :T i He+ . T .77 4 :aemarmocqus Mglliene/cul ms- ~ I .1 <7 4 T T m T AMT}? .7‘T T T, 7x..;7.. ‘1 TTTTTT IE? “1+? ”L71 6 ' f ‘ : f ‘ *" T’T :“ ‘ ‘T; 41; i : ‘ 1. . .T—T—T .‘.~ .. 1T1 . _.7T r , T.T . l. T ,T L T ..7 .7 7 , 3F T m lair! THTWJT. nh' WWWW 7 77 777 W 77WWW 7 7WW W W7W 7 .W 777 . 7 7 77 77 7 77 7 7W_ 77 7 77 77 W 7 7 7 7 7 77 _ 7 7 7 _ _ 7 _ 7 W 7. W 7 7_W_7 _ 77 77 7 7 W7 747 _ ,7 7 7 . W7 _ W7 W7 WW7777 77 77 7 __ _777 7 _ 7 7 77 7 7 77 7 7 W “7 W77 WW 77 __77 77 77. 77 7 W 7 77 7 77 7 7 7 7 7W7 W 7 7 W _ 7 WW7 _ 7 W 77 77 7 77 W 7 _ 77 7 7 7 W 777W 7 77 ,7 7 _ 7 7 W77 7 7 7 WW7 7 7 777. 7 7 +77» 7.»W»7 W .7 kW. 7 7 7777 7 .7 7 __ 7 _7 7 W _ 7 7 W 77W W7 7, _ 77 W _ W 7 W _ 77 77 _ 777W 7 _ 7 7 7 W7W77 7 W 7777 W W 7W 7 777 7 7 7 _ 7 7WW7 W77 7 7 7 W 7W _7 7W 7W 77 W7 W777 7W W7 7 77 W7 77W 777 W 7 _ 7 7 _ 7 7 7 7 7 l 7 7.77 k 'tl t Figure 1. Blood smear from the sixth month fetus of a dam infected intra— peritoneally with Legtosgira mmona. Cells appear normal in size and shape. ’ . Figure 2. Blood smear from the sixth month fetus of a dam infected intra- uterinely with Laptosgig mmong. Note anemia, poikiloc sis and aniso— cytoslis. Arrow indicates normoblast (nucleated red cell . TO 5...: DISCUSSIOfl In general, the values obtained would seem to substan- tiate observations made by workers in several different fields. No s 5nificant change in levels between non-infected and intraperitoneally infected pregnant heifers was found. \ These values, in turn, are not outside the range establi hed ' . ‘. 3"!“ .J' for normal non- reenant cattle. This agrees with 'ublished 3 o .. P .. *1 reports on blood values in pregnant cattle usefi in eXperi- ‘ mental work on other bacterial diseases. It also would ap- pear to support the workers who found few clinical symptoms in cattle infected with p. noaona in the last trimester of pregnancy. The variations between samples obtained from the umbil-' ical artery and the umbilical vein are either too slight or too inconsistent to be of any significance except in the case of the blood gases. The main purpose in collecting both arterial and venous samples was, in fact, to compare these blood gases so as to assess the effect of a possible leptospiral hemolytic toxin. It must be kept in mind that the fetal circulation is the reverse of the normal adult circulation, with oxygenated blood carried in the umbilical vein and the arter al blood actually containing reduced hemoglobin and carbon dioxide which is being returned to the maternal circulation. The values given in standard clinical texts for most animal species have very wide ranges which may be due to a “r 4!! '2 ' f.“ R) R) great variation among different breeds. host of the values determined in this work fall in a much narrower range as would be expected with animals of the same breed. The blood gas determ nations were the most variable, and since these are the samples most affected by external factors, this is not too surprising. Reineke, ££.El- (36), found that exci- tation, which increases the carbon dioxidetexygen ratio could be prevented by anaesthetizing the aninal before ob- taining the blood samples. On the other hand, xcess anaes- thesia must also be avoided since this leads to apparently reduced respiratory quotients. Dawes and hett (9) likewise list anaesthesia among the causes of the great variation they observed in their oxygen values. Shaw (38) found large fluctuations in both blood gases when the samples were taken within 15-20 minutes after anaesthetization. Since our fetal values had to be obtained on viable animals, it was necessary to obtain both maternal and fetal samples long before this desirable time lag could occur. Some diffi- culties were also encountered with certain heifers who were refractory to tranquilizers and anaesthesia. fiaturally, too, a few blood samples met with such seemingly unavoidable ac- cidents as clotting or even leakade or droppage during the trip from barn to laboratory. This accounts for the blank spaces found in the tables or for certain averages being computed on a smaller number of tests. The respiratory quotients given in Table 7 are the only three sets of fetal values which fall in a range permitting this analysis. The fact that even these few comparisons 23 could be made is encouraging and proves the techniques are workable if more and better samples could be obtained. The mall number of animals available limits ti1e possible inter- pretations of many of the determinations. These quotients are within the limits found by other workers on maternal blood samples. Unity is an auproximate med ian for these quotients with some workers reporting from 0.5 to 2.0. The 5r at disparity in maternal m1d fetal oxygen values indi- cates that the much lower fetal oxygen value contributes to what one author calls the oxygen avidity of the fetus, and the very low values in certain cases (6 and 7 month infected and 8 month n n-infected) argues for almost complete utili- zation of. all aveila‘ole oxygen by the fetus. The fetal glucose and nitrogen values are higher than the maternal values in all but one case (non-protein nitro- gen in the 8th month non-infected heifers). There is no published data to account for the nitrogen results, but bovine glucose concentrations may follow the pattern dis- cussed by Huggett, et a1 .(9 6), in their sleep studies. In the connonly used methods of glucose determination, includ- ing the Folin and ‘nu pooced sure used in this payer, a com- bination of reducing substances is actually being measured. This includes a variable amourt of fructose. They feel that the maternal and fetal glucose concentrations should be ap- prox mately equal, with the apparent exce s in the fat a blood representing the amount of fructose present. Glucose is diffusable through the placental membranes in either direction, but fructose can only go from mother to fetus. It can, however, be reeu ily produced by the fetus from glu- cose and may be utilized better in this form by the fetus. Goofiwin (23). who has divided most manrssls (in -cludin3 wholes) into two groups according to the concentration of fructose in the blood of the fetus, finds appreciable fructose in the calf at birth and shortly after. This work illustrates again the use of tile word fet J8 when the expeanentel anime 1 is sctusll3 ne~g-born. The tie “tolo mic values substantiate certain predic- tions anfi observations. The leucocyte counts showed no ap- precicble change n either total numbers per unit volume or in the pore nts;3os of the different types of white cells for :13 of the £zpecimens: fetal or maternal, infected or non- infected. This would indicate that the infection was not of the severe, acute type which causes 31est13 incr seed numbers of cells with an obs ol uto increase of immature poly- morphonuclce r leuho c3 tea. The maternal erythrocyte, hemoglobin, and heustocrit values likewise showed little sex vistion in infectefl and non- infected heifers. The fetal values seemed uneffcc tel by the ir ctre.perit0hs wel crnsl infection, but in the fetus of the 3 LlOWIl 02 intrs~uterinely infected heifer, the dramatic chm 3e in Chart 1 occurred. This deeonstretes that when the lepto- f. rel or3enism actually enters 'hc uterus, the 1's tus is cefinite13 and sewerely effected, with a drop in hemoglobin ccncen ration oi alznost 505 from an averc3e venous and erte- rial concentration of 8.5 11‘sms/lOO ml of blood to 4.5 Trams/100 ml. file home tocrit, in like menzcr, drop )Gd from h) Ul a packed cell volume of 33 ml/lOO ml of blood to only 16 ml/ 100 ml, again averaging the fetal venous and arterial values. The total erythrocyte value, dropping from an average of 6,530,000/cu.mm. to 2,080,000/cu.mm. appears to be the most severe change of the three determinations, but this esti- mation is also the one with the greatest inherent error. Therefore, the hemoglobin and hematocrit values are a truer representation of events. In Figure l, a blood smear from the 7 month fetus of intraperitoneally infected heifer #10, the erythrocytes appear normal in size and shape. Figure 2, a similar smear from the 7 month fetus of intra-uterinely infected heifer #13, exhibits marked poikilocytosis and anisocytosis. The arrow indicates a normoblast (nucleated erythrocyte), an in- mature cell released by the bone marrow into the peripheral circulation because of hypoxia caused by the great destruc- tion of circulating erythrocytes. As indicated in Table 3, the two fetal samples from heifer #13 averaged 133 hormo- blasts per 100 leukocytes. To draw the obverse correlation, since the fetal blood concentrations of these various constituents were not af- fected by the intraperitoneal infection of the dam, it would appear that no toxin crossed the maternal barrier. naturally, an abortion, or abortions, would have pro- vided answers more on the positive side, but the small num- bers of animals, and the low eXpectancy rate of abortions in leptospiral infections did not favor this occurrence. Sauer's (6) work with an henolytic exotoxin in lambs. h.) ‘\ and further studies with this henolysin in our laboratory in pregnant ewes, strongly suggests that this substance is the factor responsible for the erythrocytic destruction leading to Jaundice, hematuria and renal damage of the classic, acute forms of leptospirosis. Strains of g. pomona have been shown to vary in their henolytic activity and this, along with individual physiological differences as previous- ly discussed, may account for the wide variations in symp- tons in leptospiral infections due to tho ponona serotype. Our next step, when animals are available, will be to. try this henolysin on pregnant heifers to see if they follow the same pattern as the ewes, or if this marked red cell destruction has some species specificity due to increased erythrocyte fragility. 27 Emil-LARK ‘he intraperitoneal infection of seven pregnant heifers with‘g. ponona failed to produce any significant change in fetal blood values in a selected group of ten chemical and hematological determinations. The intra-uterine infection of one pregnant heifer with the same strain resulted in a definite reduction in the fetal erythrocyte count, hemoglobin content and volume of packed erythrocytes. The remaining blood values were not affected in this fetus. These results would appear to negate the hypothesis that leptospiral bo- vine abortion is produced by death of the fetus due to a toxin which is capable of crossing the placental membrane. This small series of determinations establishes a range of bovine fetal blood values during the sixth to ninth month of uterine life. 10. ll. BIBLIOGRAPHY Albritton, Erret C. (1953) Standard Values in Blood. W. 8. Saunders Co. Philadelphia. Alston, J. K. and J. C. Broom (1953 Leptospirosis in tan and Animals. E a 3 Livingstone Ltd, sdinburgh. Barclay, A. 3., K. J. Franklin and H. M. L. Pritchard (1945) The Foetal Circulation. Charles C. Thomas, Sprlmfield o Barcroft, J. and D. H. Barron. (1942) Circulation in the Placenta of the Sheep. J. Physiol. Proceedings. 100 20F. Barron, D. H. (1944) he Changes in the Fetal Circu- lation at Birth. Physiol. Rev. 24:277-295. Bauer, D. C. (1959) Virulence Factors of Laptogpira omens. Ph. D. Thesis. Kichigan state University. Cartwright, George E. 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