I‘I‘I'I‘WHI‘ ;*‘. "Z'O‘IOoIowquO-'Q'M:OP"I' ‘U l ‘f‘“ 'T"”"" ' ‘1O“’I."": ' """"' '-‘ 7‘"; vv'"""0"w ’1' ”Igfl'O‘" , t' - ._ I O I ‘I: -/~_> MI 10 ; ‘ ' T..! l.- I 3' an -.. i-""‘ .«r. ‘0: ‘LO'I .I.”I; ""V' ..o§,.....-., "1L":"~'.H.I .' . " 'I | .h .:I-I'OI II I ‘- 9.I, ', I; "-l ‘lfvv't’o'I V‘v'w 4...... o .r- "we O'. "l 00' ... - ... g". .‘I'V' '1. Pa' |II|-II I" ,1.')\..| '| ‘ ..;;‘t “m ’3 ‘. I ‘ OIQv'r». I‘. g in. OI: “ -;..-o.".oqp- a ., ’1 I‘ :‘t I IW'I'II IH In"'I ., m: *n' u w. say -I III- . ”b- h-lv"r-1I‘09"". n .- ‘, o. ","I-nu I‘D. H.” II, . .II' I I, 'I icloI 4,-1- , “W . u -I»nu,.u..- .OI."IIV"' "II . ' .‘. II; _“I L! .l. I. II] '. I H ”E" n ‘4 " ' 1";- h-IIMIV <‘ 'I: PM)!" "'va My a MI.» I. I: I.. 'I‘.1I‘I')J."II'I I' I --~" y. 5': I; .. IIo-II Inn ~ O I.I ~ .. I w' II/I'Ia I“ I .I "if ":I , V? '- "n-III'WN . . . w” _...- - ' ' .' .V. II‘. " "' if “fi-I‘O) (O 1' 'I‘ ' 5‘. ‘I‘f'fi '7“. '"" 01' i"9"‘,l‘ ‘3' "'IO'“ ' “h \ ' """li‘ 'a' IOWIO- "LNHQ‘O ”‘3‘" '2 "f. I. ‘OI". "‘ ‘III'I' .-I' ~n' "'5 .‘.7 A”', . .. ¢ ':~ ‘\I I~$II.,:_:!’III.,.,-"."or'.¢'. 'Y' "“T: ..a .".-""' 'I.,;‘ p“ . U“. IUC'I I I 'V? " HI" I‘ .["‘1' H '4: 5 "0 “.1"! 5" I) v" ”"1“, ... l HP-f‘ 'mcvg-I .5.- 31!. ~..-..., “\ --I .Inn III-w: -II ",1 )“P‘I"l".ti3'”"l or"'~I' A N! In In ‘I 'I :,I- l"’" .I vu- :l l".‘.‘.-..' [\o-unv "'gl"“'i' - I“I'."O' N...‘ .-‘- p "I I. 1" 1' 'Iv-I’ ,( 'r'I' O A ‘gi‘n.‘-,,DOQ~-$ n- «Hp -~_1‘qu-h u 0-117!” r,--|l,. ,, .'~ “H... ‘p ",n rt,v" :“fi‘.t*‘0‘;:~ pr~.. ""““1, '. ‘0'.- II ”I' l’ ‘ ‘I‘ 7’!" ’9" II‘ :"-€;' 0 . . Q 2? ["1“ "O' 'F“!- II"'I " QQI'-1‘-”“::. glfl"('."\""" ohp’t. whova ‘1 . r .U I'rau Q“ “pv~wve t." “I -y“ ‘0' ‘0‘“! . -I III,.’ ,..I “trII' 17'" “In-"II la".- . -; "'- ) V'Hllr-fl' hyy- ”N WI’,Q‘W .;\--..:I.p-1 .....I-m uv.o.-I. — H't"? , '( It" In 'Ir-O -- y n v~-' O“?‘"l- -yu--s .-.-.-. O‘"‘l.“‘"l ~ I '."O‘"\ I .g II-v- pup-v": vQ-I ”~43 \- ”a. I-.. II~‘-~.._-‘III ‘{ . . bu I 'yI - I-y"I01\u-I~ c- you. Imp-1w a. I'nn‘ . -w9.I-, ”no...“ . n I .ul.":'I\\ ., '-- H")."'l‘xl"" v'v..”. 9» I3. ,- .I. .I “.I'. otvIov I-Iqtnvo‘.-rav' I .‘a -.~IO'Iq-k-_OO ' W')”""‘ '_‘ “-“W‘Pavz'r J'O't'iuh- ~~I .:.“‘.‘°' b...‘l"“>' Fun-f Iv. nyo~n . n ,_In_9 ,«g ‘",;vz A, 0157'..~A.Q.r' .-..- I..At‘ll" ‘u'. 09.. h a, .‘ 3"“! 31" ,1 - -‘ '. .-,~I~4.'QI.. .rIo 9v" . -.u q: ...'q I‘lug. '."f:'0|rl"’ DI. -'o-‘.:u.. '...,~u . “’I""":' 'p,.-‘ ‘.-Ig-.p'._,"... I. ' '. . . I; IIO. -- 0.\I'II'!~_ 4‘ not "o; "O n, 0“ ~" 3 .1 oI'Iytu-I ”0-” V-‘no- vine," -H 'O~.'h -¢_ 4, n ”V ,. ‘)"I~- J. .‘HDI.I~,III"I0'-ov”:,I .'l’I' WI gffID‘a-v‘ "”'."'”"‘ u gunqucpu-Ovu '5 "'0‘ ”“001 O tuna-I. I- I II""' , "r .‘I "I“ Nu“ qu- ‘90:“ n‘u-fnvw'wv ‘vg" \zprgpwv-w- - Iq-ro-xu' nu y- «yo I.- ’ I ,O X..' ""u‘o’p-I‘ - v'-HI;I-,----—u. , :— O f. Igor-1. l"."""‘ "".Y‘~' "7"" ‘F""‘ ' . . - II "0 . ,-.- . I - II'.offilQCv-'|Q.'Irv1-‘-.“ .- ‘.9I'"°"I ...9 . . .r,.o.. ""§l'|"""" >1“ ~pl..—v~..- .h-un . I I. an - )0; 3'°"I'~ .7~I"' "'- “I "-l rt! I‘O-f . Vi" “A"; o 3"! I0' I- 9""; wvrn "Q - Inn‘s; wuw-I—cu I’<~~)" I onu- - . . O -. -I.MHIII.IIII . u‘:-:H9-I Q0 --’ r p‘q‘.), I '.o‘ ‘ g. ."l .'.'I’FI“1 Ia. .2"'1"‘"“.. 0-,aI-afc I . "f-'i,_ 7‘ .p "pg-n“. ' ”I. ‘.I ’I'I'.“}'°"7 ”.1 .. u: ... . roul‘u "."9' V- f I I .- In o'usp .. 0‘. - Hal- ,1 .g~. ~qu-ov'IIooI ’ -~' tcp-unI . “v! .; ""‘(T' .a ‘ )\::“V",‘:? "7"."._.".'- ._ °"|',V?""' -:Iv,.-~‘ onus-v l;.non.r..,u ‘ ' I VII" I‘ ‘J ' '- sr‘n W"! '1‘.“ ”Iu‘Ig' '." . I I, ‘I I "1:” I. I . .p-uvaw,’ '0‘ o.-...‘I‘,’~y"- '- ‘ o- Ign u . I. . I -‘ 'I',I I I :.' .3 4. I “"""II".""X"1?*"!';0 o ,. «V m; u "I',' my.- I'IO1' - . ' “ ‘ ' . 0 wow. . "I I ,H { I ”v; -I".N~f "('1’ ' "O” “10- I‘"! ‘1" l I In‘ . . I 'Iu-.u‘;g. —_ 4....I..." ,‘.I.‘-.. 9”... ‘f' I IMO-I ulv-o :I-,-ot ooou,4o....‘;.'- .. .vuIl-uun-I.-nv.ou - H» o- I.» no I I‘I'o.---v-‘ ‘ ~ 6» -I D; 0 “>’V . ~n .I:u :O‘--!I cam .‘ . .." O . ' . a -I 3‘ '10-'00} h .1. n.- ,,”..y.-v.I-..-- ~- . ;yo.ao.v_-A.. o‘oolx- .n-I’t .v..- I I- wI—I; .o.-.h-Ip£u.I...I.- .p....y-‘|. “y..-. --,,"II . 0;J’\OI 0.1- no. .- 'I"‘I': ‘l 0" at. 'tl '-.'~—Ioa Ifi-Qo. ‘C.‘n")~kil..">‘tf' . ' u 0.0- I.‘- an.» g . O I' I . ' '. . ‘ . .5. Q . . I .1 Q-I 4.. I. .5 0-»- .- oot.--0. ’0‘" I I q oWIIIuooQ l I .1?) Vs.» Iogo .7 .-.. up N- ... ... ...¢-.",.. . U- .~Q|.w. 9" I I ~ .' ' ~I . Ina '7.""""~""'I"” ‘- 'II’ 0." . fi: ”up“ u~ no o u I.‘ . I . ' 5:... - n ”I“. vo~ ofopOO..‘-I an pqoalgu'3-u IonIau,-- “- 0.5- .l- I ~ I; .-“~.\..vra.~.b' ‘H-s-a on a. an - I .; -.:qho 1.; 9" "lurcn-I‘uOn-I -o-.-oII ”-34...” . .‘<'.." IQOIII'V... a. Ont... -. ,.. A 'Io»I-I:,-.y ..o. 9....”‘tnolfl .m~av. -I.- -q»a~h.nng “.glvt I . -.‘-.; I'. .---,- J, . . -.I 0'...’ 0.. a." .. 1—,..‘Mfiyg‘. '~.--.. 0-. -o- «s... .n‘l‘l vs ' . H-O' '"|IO'I‘ \""'.‘l §~0o'u-.o O. I . M“. 90.! 0” . a § - ‘ I ' I f I . _ - I I t I ' l . .-.. L-‘f—q-<- w... I ‘ . . .' I . . . . I . OI. l' . I l' ' , ~ ‘ o q..l-II~-|IO , J .5" on vl|'I- ' .‘00'----| I n-.u..-luo‘~ I .Izu- I... - h a.‘qu I AN-Os I“ I.. «0 --~ OMIOI.O'--'.oltg ’Ico’..‘:n- I- ~.--.Ob .‘ .. ..—_“I|.- ~oc-nI-vcp -..¢ qua. -r - It. 9'" .Il‘ I _ . I . ukI. 1|. 0 - 94' 4.. q- ..o- I.- .iD-‘O.-Il .II o- A I _ .. '~u -,¢..,....,... . . ‘ ’ OI'OO‘OMIV‘- ‘ a...‘|l'y-|Q=. Q O I ‘. ~ _ ; on -u- I'.\¢<.-v mi. II. .b-“.O..IO. I... I ' . .. ‘I-."w'§ -.. -- -..I “1\O§.-'00 an ' . 0 “.VI‘-"¢I . J .u l‘ I ‘ . . I ably-J..\|I -7|I 0‘0. .v‘.--..»‘ , an. “a. -! .nl-Itfi. I. I..u..- 0n:'..II’ yo .0: a. .Ii’~q||”. III-3....” I. u; Iii-cu do n. .U .50 u o~0-.!. w...» 4 . a 'V".“¢.7.3'.'.‘.'. .ol und- .0. a'oA..‘< It §.§.-Io. on. ..-u;-. u 7.. 1 .g q. I. \: ..'...-I,II.....\ . .. ”u. .ugl .u. .pl‘... 0- . a00..c.-~o.t...1s..5.. - Jud" h. u 0 0.9 a .- .- ~ -,.og ‘.c .0 ~ . .c qu-I O'K’Qs’vI' >4- '1 00 1 ‘n in- Inc .Ibh. «Coup-(I .-. I... “‘l...‘a.‘.p .o'M'a ”urn-Q “~- ..’OI.A' t.‘ . I I I . .. ' .. “Inn-For.“ IuHI-¢~ J , I-I . u .l a 'II - - ‘ . I' .AI.I5'~40‘ H. II... I II-.l\l.0~“- O I. A 0 III “uh I! 4.7..»- I .O O . ' _ In ||U90OIIA‘IO.‘..“ niuu I ‘ - L... ‘4. .A- |CA‘\(.-b QOMI :. I. . - .IunInuw .- 00 III “hold I. w. “”qu . . . . «tun I- huhleIN- u o u Inuuugu ”1...; .5) 0,0-0 . ‘ . .. _ I. u on, -- A3“. noun” -'u.-.;| no um I q.. )5 u 0.... c .4. In. I .uoI I1Io..~~O..OA-M‘- I II “nun-capo damn-uh ‘IJ . .I tun-In.“ .. . r 7- .o' «a: Inn-III u. ..l Ins-on“ - I-‘uu I. .- I. .n I... 1 _ .I. II I...... gt... t “Ida—ooh“ .4. mud n a. 5.. ~ .0: ..I . . -o...u -- I.............,.. . . "I. , I .4“. ....~I. ”>00. . a t- | .I-u “luau“ a \u..u'.In -- v» 0‘ .u In. H. u ”"9" and 9. you... n u - u I w -.- . . ‘ . _ unvumuv ‘OI'."“"I‘.!. o 0.44, .uunpxzuuhl annuutuuhnun 45qu ”not. .5 I, 1...“. .U\'tJ.I‘\. “uh u. n I II. .III I. "f u . 5- u. n. _ .. , II u. at |‘- t ”AIM-o“ .' ’5‘ II- cl ‘.~AO':.9UII..(.. Ila -u‘uqhd” .I. . .90. .uu.’ -II|. .l .I H. 9;. \I. .‘lnx. .U...o....I.. .... . t“. . I. .,. .. . o. .. .r .~ .. - . O '- V . I 0-. 90h. I “LO-HIM”. .OH‘. -U .IIlI l."§.\"'|..'oli .|I\§Mu\ .I'QI HA:-\::‘o L! 1‘10. . dud-purse .- .~.~v'. .‘ ...;... ..I .o...‘ '- .. .0 .. ...I. ..§ . .. h. .. - ' 'IH’ILIImn -l u. ontnuulon-nh- I’m-Iv" Pun-II. jun-.0...» III Inng.k-u\. u. .gs.“ .. . t .I . 3. I .c-u-I I I - - -Io- h.).- -n .I‘QI.A .nuo. mun-um Ion. ' on. I'- I ..u.- ‘0 -§ts .\ .0...-.I... bl u..»-...a .. ...I .35.... .. .. -. u. .... «. ... .. .. ”t I . . ‘ .IIL'QouAu ”INCH-flu. \‘I UNI-“u. ,lu‘. OI." u p. xh n 7.. .g ou.un.,«.... . cu“ m .-.c. .- a: I. u . I...“ "s.|,|. 'uvnu-Iu u 'I' In-m II -.._,-. . u. . - ... .. ’ DOD .l u- .. I . ‘ I I no I I I - - . )u Hanan . u- I II: qu IOIIIM 4-. .0 I. u . O- .1... - I . . IIIIIOIu'I‘quIu...II Ioubl .‘Iuplau’oofi...\huw $.-- urn." Il'i'uuo‘ Hun I. n0. 0 --I o I . o 'I .w s I-‘I- nu. I I “9|. C"d‘.-.II.\“II nu. 'A "4,. .II -.' .pa- CI). .nah- .-....\. ”u .. ... ...I.. o..".. I. .. .. . ..... . I , . _ .’»-Hu Quin 0...!~,‘.. ”an r .II'I‘QO 'Jfi- oI’I'V-ur oh --- 'u mun ‘Im—I-I - ,-l --‘|| I, —. I- .- b , I. o In . O _ l..\ “9' ‘-O|I'V"“-M-.\Il li'nt LL"- ' \‘ .l”-'~ .V- “on-‘0 on IO' .I- I. -..I. ‘1 -o. I. |O|~o I IO... .I ... .u 0‘ . ‘ . . I .uJQI" I ‘I"\.~I‘O-\~-)I‘b‘. Q - II kH-J In. 0 I on |-—-.. IO'QI - -Q I. u- t . .I .9...“ - I .I - I . “.5. Slh~ IIIIIH-nLIIII I'-u -I q .... -_ Luau» ' up... ....... . -OA.~. O -- . ... i .,.. .. .It- to ... , ' _ . '.- | In... “I... .In‘IlubtuuluIIL-m'lgfi. ‘gg-wvtn bl.‘ 9- ap’hb o'-.-o---IO-'n or ous I- ‘q I D.".-' nu --- v t K .4 o u I . - ' - 'I‘rbhu -I.‘-I| ‘OO1|19“1“.0-‘6)"b-0 - I-o \ v.2. “., >"‘“‘ In. I 4- I h. IO'. Mr“. -I. -‘-.~l.. .r \o--.~ .II 0-. u .. .c. I up ' . I__ 4.. 0”le 0H- "I”u-II'I ._‘."'-' MINI-HM. A. ”O ' HI u- I A.“ 90‘a.$-.0.“‘hv‘t 0 .-.. . 4",.“ ......I-.I . O -u .l .. . . II. , . ' ' . C an gun. up» Auk.“ HUI! l.§‘ I .quun \- .w I I .y II.II_ a. I .III ”13...... -n-.I.' .thu-v “I. 4 ......I-.'... .n .. I. u u lb-u “huh uldu‘ulh‘I-uulu Iauuwu II n ,0; AIIL- n»... g. I --' u'. 5.. .nlI -..« .n 9‘. u oI 4...... .I | . ..¢ ”I‘Inuuul I} nun-Nu»... \‘bob auuunv- ‘Ihi;01.u. u 'uIII. .. .n .I...n u..." H: , ' ‘ I-gu-‘Iw 'AM Inuu nu...-IyIuvnruoumuuz - 'm «Iu.I:{k.u..u-...I M'- l- .. “VIII un- "Ina. III-I.“ '0 . . nu IIN'I‘“ cl-l u .0 .,,o ,.I‘.. ...- I .. -.I ‘ . Hunt; -. A». .OI-I I ---os.a-. a .Lut Inna» I I ‘ 0 I nouns“. ”910:..qu I I. .I-o.— .u-I .4 I I 4\ .4,” "i ‘OI ‘U‘\' ‘5‘. 's'h". O ..".‘D.'b~.- IIO‘. O ..I M n9 ‘ ‘.3Q- M l||.'Q ‘Qtt' ”Duh-lulltv \‘A. "A ‘5‘ ll ' .‘b‘ I \- - . . , . Iv In. 4‘ MuIv.luqu-uvu-‘IIIIIIM .In Hu-U nu O III. M- u U...“ . \‘I no | .‘oo’l' 9-H") ‘u‘ .u u a‘\'.u .urI’I.I..u=-I.I.‘n u u ... - ‘ ' “'9“ [v °-. Hun \ I'M I .l."‘l‘i‘|‘.‘ .‘|'.:‘ k-I‘ov'IIl III no .I.:. -\ s). ...I' .I --|. ‘l'. VI. ‘0 ..I II Qt-‘d .. I ' - 1h "!‘11. ‘NIO IIIIII.‘I M‘HwJ). .InO III I O - I....\-.\1.. “~ng _.. | , . ' :n'-uw_..- quIIH-Ouuuv u I- 0 "3‘ I "‘ .I' ‘ I H. mom'u -- '93"0“"“‘ ‘ I . -'I"\ '0‘..." Q HN'.§\' Q 'A"'|.D.‘IO --, -I‘ ’ II I I u " III v-uI. gnq-u-n- Iw-v I.II.II u. .. -. O I t I: I.. up; ”I .A- .'I- Jan-Iv In OI u. ‘I H I I I .Lé "' “ 1 . ' b“ “A“ 'I‘.‘.."I {I‘d IQI- Junta) I . I... -" ‘Itl . _ ‘ . - _ . ' I'Q“.. IIIA- .. 5." HI- .‘I .‘u-' H O',“ ~ "I I I I. I- m 0 .‘ . ' . , I II“. IM-o‘. up “.0 IL... |.. ‘ I ‘I I. ‘I , I ,I, I “W . I .' " ..- .)n' 'W. .92. ' I ~,‘ ‘ ' M I ‘|.'. . - WC" .. I“ |\\"°\"aI-"I‘ ' m I‘IJ.‘ . 9' ‘ ' ‘ , I HON“ pr uH'xO uh IV“! I" d I" :OI O" II [I I'\ . II , . . 2 I a ' . ". ' 1 . a I . I . _ EVALUATION OF THE BROMSULFALEIN LIVER FUNCTION TEST IN THE RAT AND THE DOG BY E. JOHN LARSON A THESIS Submitted to the College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Veterinary Pathology 1957 ACKNOWLEDGMENT The advice and direction of Dr. C. C. Morrill, Head of the Department of Veterinary Pathology, Michigan State University, was most welcome and stimulating. Throughout the study, the author received untiring and enthusi- astic support from.many co-workers whose help was invaluable: Robert E. weber as Research Assistant; Mildred C. Prestrud and her group in Clinical Pathology; Vernon F. Baker and his group in the Tissue Laboratory. The author wishes to express his deepest appreciation to the Upjohn Company at whose facilities this investigation was pursued and to Dr. E. S. Feenstra, Head of the Department of Pathology of the company,for his encouragement and assistance. Sincerest appre- ciation for their suggestions is extended to my colleagues in the Department of Pathology and especially to Dr. R. A. Runnells for his review and criticism of the manuscript. EVALUATION OF THE BROMSULFALEIN LIVER FUNCTION TEST IN THE RAT AND THE DOG BY E . JOHN LARSON AN ABSTRACT Submitted to the College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Veterinary Pathology Ye ar 19 57 Approved: g.‘ E: 22b4£4 E. John Larson EVALUATION OF THE BROMSULFALEIN LIVER FUNCTION TEST IN THE RAT AND THE DOG (An Abstract) The bromsulfalein (BSP) liver function test has had wide acceptance as a diagnostic aid in.human medicine (Maclagan, 1956) and it has been applied to a limited extent in animals in both laboratory and field. In the studies here reported the results of the BSP test in the dog and in the rat have been correlated with various hepatopathies rather than with the results of other hepatic function tests as has often been done previously. Hepler's (1952) procedure for determining serum concentration of dye was followed in the dog but in the rat the recommended volumes were halved. Sixty-five albino rats and eighty-five purebred and crossbred dogs were subjected to the evaluation. Dye retention was determined and gross pathologic examination.made in normal rats and in those administered chloroform or pyrazolidin-B-one, l-methyl-S- phenyl.. Dye retentions were determined and gross and microscopic pathologic evaluations were made in: normal control dogs, dogs administered carbon tetrachloride, dogs receiving five other drugs, dogs given canine infectious hepatitis virus, dogs ill with canine distemper, and dogs with no other sign of disease than fever. The BSP test was unreliable in the rat under the conditions of the experiment. The normal limit of dye retention for the procedure was determined in the dog. Reciprocal agreement between retained ESP and cytological hepatic alteration in the dog was inconsistent in the lower range of E. John Larson retention (below 5%). The two factors were generally'but not uniformily correlative when the serum BSP was elevated. The BSP test was most correlative when the serum BSP was elevated. The BSP test was most correlative in acute hepatopathies. BSP test values from dogs used in drug toxicopathological investigations require cautious interpretation. The procedure appears to have questionable value in the differential diagnosis of canine infectious hepatitis and canine distemper complex because of the frequency of hepatic involvement in the latter disease. Lesions, other than.hepatic, appeared inconsequential in relation to BSP retention. Fever, peruse, in this study, had no apparent effect on the clearance of the dye from the blood stream. REFERENCES: Hepler, O. E. Manual 9£_Clinical Laboratory Methods, ed. 4, Springfield, Illinois: Charles G. Thomas, Publisher, 587 pp. 1952. Maclagan, N. F. Ch. 5. Liver Function Tests. Leon Schiff, Editor. Diseases of the Liver, Phila- delphia: J. B. Lippincott Co., 758 pp. 1956. TABLE OF CONTENTS INTRODUCTION........................................................1 REVIEW OF LITERAJURE................................................5 MATERIAL AND METHODS...............................................12 A. The bromsulfalein.Liver Function Test..........,..,,,...,.12 B. Procedures in the Rat.....................................l) 1. Normal rat...........................................15 2. Rats administered hepatotoxic agents.................15 C. Procedures in the Dog.....................................l6 1. Normal dog...........................................17 2. Dogs administered carbon tetrachloride...............l7 3. Dogs administered drugs..............................18 A. Doss with infectious diseases........................l9 5. Dogs with pyrexia....................................2O EXPERIMENTAL.RESULTS...............................................22 A. Test Values in the Rat....................................22 1. Normal rat...........................................22 2. Rats administered hepatotoxic agents.................23 B. Test Values in the Dog....................................29 1. Normal dog...........................................29 2. Dogm administered carbon tetrachloride...............29 3. Dogs administered drugs......................t.......50 h. Dogs with infectious diseases........................52 5. Dogs with pyrexia....................................3h DISCUSSION.........................................................h2 BINARY AND CONCLUSIONS...0000000000000so.00000000000000000000000005]. BIBLIOWO'...’OOOOOOOOOOOOOOOOOOOOOOOO000.000000000000000000.0053 ”MIX 1.0.0.0...OOOOOOOOOOOOOOOOCOOOO ”mm 20.00.000.00000000000000 OOOOOOOOOOOOOOOO ...... 00.00.000.062 APPWDIX 3000000 oooooo oeoos .OOIOOOOOIOOOOOOI.000.000.000.00000000068 APPEDIX hOCOOOOOOOOOOOOOOOOOOOOOOOOO 00......00.0.000000000000000092 ”MIX 500000000000...sooooooeeeooeooeooooooeoooeooeooeooeeoeoollh ”mu 6......0.0.0....OOOIOOOOOOOOOOOOOGOO...0.0.00.0000000000120 1 INTRODUCTION Hepatic dysfunction in animals, as in man, arises from.manifold causes, may or may not present a diagnostic picture, and can be sig- nificant or insignificant prognostically. It is of little moment, from the standpoint of the animal's welfare, whether the hepatopathy is primary or secondary; the principle consideration is the type and extent of the hepatic damage. This fact continues to present, as it has in the past, difficult diagnostic and prognostic problems to the clinician. The greatest obstacle to accurate prognostication lies in the phenomenal inherent functional reserve and regenerative powers of the liver. A severely damaged organ may reflect little clinically that indicates the seriousness of the condition and.then, only too late, cause the developnent of indicative symptoms when the reserve powers are exhausted. Likewise, unsuspected hepatopathy, undetected because of the reserve, occurring with systemic or primarily non- hepatic diseases, may materially alter the effects of therapy and accuracy of prognosis. Conversely, the problem is further compli- cated by diseases whose signs mimic those of primary hepatOpathy and require differentiation. Inasmuch as these problems are not confined to veterinary med- icine but are equally problematical in human medicine, an extensive battery of clinical laboratory procedures has been developed in the latter field to aid in differential diagnosis. Maclagan (1956) enumerates four factors determining the choice of tests in man: de- tection of liver damage in the absence of Jaundice; differential diagnosis of Jaundice; prognosis; and detection of excessive hemol- ysis. No single test has been developed to provide the requisite 2 information. The need has resulted in numerous laboratory manipu- lations on blood and urine for these purposes. The greatest con- centration of tests centers on hepatic activities related to the following: biliary excretion, the metabolism of carbohydrates, proteins, amino acids and lipids, and detoxification. The reasons for performing tests for hepatic dysfunction are as valid and as purposeful in lower animals as in man. Of the four factors requiring a differential test, from the practical standpoint, detection of liver damage in the absence of Jaundice has received the most emphasis in veterinary practice. The majority of tests used in practice and in the laboratory are designed for this purpose. While large animals are subject to a great variety of hepatic dis- orders induced by viruses, bacteria, parasites, poisonous plants, and chemicals, and, would seemingly have a high requirement for tests to aid in differential diagnosis, in reality it is in cyniatrics that hepatic tests find their greatest utilization. Minimal re- straint problems, economic feasibilities, and availability of lab- oratory facilities account for their frequent use in small animals. The criteria for an ideal routine clinical laboratory procedure are dependability and simplicity. A dependable test requires that normal values lie in a reasonably narrow range and are consistently reproducible. Abnormal values should lie beyond the normal range with little or no overlapping and reflect the degree of malfunction. Simplicity, both in technique and apparatus, reduces the variables inherent in complicated procedures and tends toward closer reproduc- ibility of results in different laboratories and among personnel. Such tests are rare. Many methods, however, approach the ideal and, 5 when used intelligently, are of value in estimating the over-all clinical picture. In this category are a number of methods for determining hepatic dysfunction including the bromsulfalein (BSP) liver function test. This determination measures the hepatic func- tion of biliary excretion. Since its development in 1925, the BSP function test has been modified by various investigators and has found wide acceptance in human medicine. It is considered the test of choice for demonstra- tion of liver damage without jaundice and gives a higher percentage of positive results in cirrhosis than any other single test (Maclagan 1956). As a corollary, investigators applied the test to animals, in both laboratory and field. It has proven a valuable aid in ex- perimental investigation and a body of literature has accumulated testifying to its usefulness. However, it has not found wide use in veterinary practice. Coffin (1955) probably states the concensus when he points out that the test is workable but possibly too exact- ing in its application for general use from an economic standpoint. It is equally true that it has not been applied extensively enough, especially in the dog, to fully elucidate its worth. Perusal of the pertinent literature reveals that the preponder- ance of work has been concerned with correlation of individual tests rather than relationship of an individual test to manifold hepa- topathies. A study to evaluate the BSP liver function test in rats and dogs with liver damage of multiple etiologies was deemed feasible. Further Justification for this study, besides the practical appli- cation in the dog from the veterinary clinician's standpoint, arises from the utilization of large numbers of these animals in experiment» h al projects. In the rat, a widely used experimental animal, this information would allow following an induced disease or toxicological course without recourse to sacrifice. In the dog, it would aid in: a) screening dogs for test, b) following experimental alteration without the need for sacrifice, and c) confirming or refuting sus- pected hepatic malfunction in toxicopathology or other studies. The objectives of these studies were: a) To determine whether the method to be described was adaptable to the rat as a routine procedure and to evaluate its dependability in experimental hepatopathies. b) To determine, in the dog, what correlation exists be- tween the percentage of dye retention, and the type and extent of hepatic alteration. Accomplishment of these purposes would require that the test be performed in intact rats and dogs to define the limits of normal dye retention, followed by estimation of retention in known or suspected hepatically altered animals. Correlation of retention and lesions would entail submission of the animals to a variety of known or suspected hepatotoxic agents, and the performance of the test in those with spontaneous diseases. In both instances, sacrifice of the subject, and gross and.microscopic observations of the liver would be requisite. As the work progressed, it was deemed necessary to broaden the scope of the studies to include the evaluation, in the dog, of the effect of fever and the role of pathologic alterations in other or- gans and tissues, especially the reticulo-endothelial system, on the retention of bromsulfalein. 5 REVIEW OF THE LITERATURE The superiority of the bromsulfalein liver function test over other determinations for the early detection of hepatic dysfunction in animals has been demonstrated. Casal and Olitsky (19A6), in an evaluation of bromsulfalein retention, blood bilirubin concentration, thymol turbidity, plasma coagulation time, and vaginal smear cytology in Swiss mice, found bromsulfalein retention the most dependable in indicating induced hepatic lesions. The fact that rats were unable to eliminate bromsulfalein normally even when thymol turbidity, cephalin flocculation, colloidal gold retention, serum albumin or globulin levels seldom showed change, was reported by Linder at 21. (1955). When compared to serum phosphatase, prothrombin time and intravenous galactose tests, the BSP test was the most sensitive (Drill and Ivy, l9hh) in dogs whose livers were damaged by carbon tetrachloride. Hoerlein and Green (1950) concluded that in suspect- ed clinical cases of hepatosis in dogs, bromsulfalein retention was more sensitive in measuring dysfunction than methylene blue chloride, icterus index or the van den Bergh reaction. Comparing the brom- sulfalein excretion test, serum alkaline phosphatase level and the serum protein faction levels, Gornall and Bardawell (1952) concluded that bromsulfalein retention probably reflected with fair accuracy the expected hepatotoxin-produced disturbance of function. When the results of the cephalin flocculation, thymol turbidity, van den Bergh and bromsulfalein retention tests were evaluated in dogs, Noyan (l9h9) found the latter the most sensitive in the detection of hepatic damage. In their work in dogs Svirbely gt 21. (l9h6) 6 demonstrated that of eleven different procedures the earliest indi- cation of injury to the liver from xylidine was given by bromsulfal- ein retention. The fate of the dye after injection has been investigated ex- tensively. Mills and Dragstedt (1956) investigated the role of the reticulo-endothelial system in the clearance of bromsulfalein from the blood stream in dogs. In splenectomized dogs and in dogs in which the reticulo-endothelial system was blocked by India ink and saccharated iron oxide, they reported mild transient increase in retention of the dye from.splenectomy and.moderately elevated values following single and.multiple injections of India ink and saccharated iron oxide. Continuous infusions of the two agents resulted in marked retention, especially with India ink. Their work confirmed the earlier observations of Klein and Levinson (1933). These latter investigators reported a slight but definite slowing of dye clearance from the blood in splenectomized dogs. India ink- injected dogs exhibited.Kupffer cells laden with ink but the splenic endothefial cells were not so well blocked. Bromsulfalein retention thirty minutes after the ink injection resulted in a 70 to 100% retention at five minutes and 25 to 50% at the thirty'minute sampling but normal values in three to four days after injection. Mills and Dragstedt (1938) also determined the rate of removal of the dye from the blood stream of normal dogs. Using a 2 mg./kg. dose, they found 85 to 90% was removed in five minutes and the remainder in thirty minutes. The same results were obtained with 5 mg./kg. Blockage of the reticulo-endothelial tissue resulted in 50 to 80% retention at five minutes and 10 to 50% at thirty minutes. In their experiments, 7 ether or ether and barbital anesthesia in twenty-nine dogs caused no increase in retention. Ligation of the bile ducts produced no appreciable reduction in dye clearance but administration of Decholin, a cholegogue, resulted in marked retention which was most notable in the first five minutes. Wirtz SE z_a_.l_. (1951), working with cholecystectomized dogs which also had gastric and duodenal fistulae, had been treated with carbon tetrachloride, and had re- ceived 2 mg./kg. of bromsulfalein intravenously, hypothesized that impaired capacity of the liver to excrete the dye was first man- ifested by delay in transfer from the hepatic cells to the lumens of bile canaliculi. The removal from the blood was initially un- affected but with increasing functional damage the hepatic cells become increasingly saturated with dye which resulted in inability to remove the dye from the blood. Cantarow and Wirtz (19M) reported so to 83% of bromsulfalein was excreted in the bile in the first hour and 67 to 100% within two hours in the dog and man. Cohn gt 3.1;. (191:7), employing con- stant intravenous infusion in dogs, found 31 to 65% of the dye was recovered in the bile. They calculated that in the absence of the liver, gastro intestinal tract and kidneys the peripheral tissue was capable of removing the dye 25 to 30% as rapidly as an intact animal. Using liver slices and perfused livers of rats, in the absence of bile formation, Bauer and Pessotti (l9h9a) suggested that bromsul- falein uptake in their experiments was independent of metabolic processes and that damage to the vascular bed in perfused livers re- sulted in reduction of uptake of the dye from above 90% to 10 to h0%. In the intact rat, they found carbon tetrachloride and India 8 ink readily reduced the removal of bromsulfalein from the blood stream; males cleared the dye more rapidly than females. Utilizing the continuous infusion technique in dogs, these investigators (19A9b) determined that 77 to 89% of dye leaving the blood stream of normal dogs was extracted by the liver. Carbon tetrachloride- treated animals showed extremely low bromsulfalein concentration in the bile. In all cases, they demonstrated storage of the dye in the liver with the largest amount in the parenchymal cells. However, 11 to 23% was not accountable in the liver. Bauer et 31. (1950) tagged bromsulfalein with 535 and.made nearly quantitative recover- ies of it from the liver, blood, and bile. 355 activity was re- covered in bile dye from four fractions different from bromsulfalein, none of which was colorless. The intravenous infusion of bromsul- falein into dogs with portal and hepatic vein cannulae led Pratt St Ei' (1952) to conclude that loss of dye in urine, restoration of dye to the blood stream.by intestinal absorption or lymphatic re- turn was insignificant; the dye concentration of hepatic vein plasma sample was fairly representative of the concentration of total hepatic blood flow; Recovery of 30 to 50% of the dye in the urine was reported by Giges £3 21. (1952). Linder 3’9. 1141953) adapted the mouse test of Casals and Olit- sky to the rat. The dye was administered intraperitoneally at the rate of 0.05 mg./gram.of body weight and the blood sample collected in thirty minutes by severing a carotid artery. The determination was made using a Unicam.spectrophotometer after treating the sera with 0.05 ml. of 10% HCl (blank) and 0.05 ml. of 10% NaOH, and the readings obtained at 556 mu. Using data from thirty-three normal 9 rats, they found that retention was unlikely to exceed 2.36 mg.%. 'In rats with necrosis of the liver produced by diet, they recorded retentions as high as 10.7 mg.%. 'With doses of 5 mg./100 grams of body weight, Giges gt a1. (1952) reported normal retentions of l% or less in Sprague-Dawley rats using the method of Gaebler. By the same method of determination but injecting the bromsulfalein at the level of 5 mg./kg., they observed that retention of more than 5% was abnormal in dogs. Drill and Ivy (194%) found normal retention betwee 2 and 12% in dogs when the dye was injected at 5 mg./kg. with a thirty minute serum sample, but using 2 mg./kg. standards. They regarded reten- tions over 15% as abnormal and recorded values as high as 250% in hepatic damage from carbon tetrachloride. From the spectropho- tometric method in puppies administered_7:mg./kg. of the dye and sample collection at eight minutes, McKibbin st 21. (l9hh) accepted three to eight micrograms per ml. as normal and.found values of 12 to A2 micrograms in fatty metamorphosis produced by choline de- ficiency. Svirbely et_§l, (l9h6) by spectrophotometric technique on samples from.dogs given 5 mg./kg. of bromsulfalein and sampled at five and thirty minutes, obtained average normal retentions rang- ing from 2.5 to 13.2% (average 6.2%). In xylidine-damaged livers, they recorded values as high as 65% and in fatty metamorphosis values of 1h.l% when the control reading was h.8%. In his application of liver function tests in dogs, Noyan (19h9) appraised the value of the bromsulfalein test by comparing the results in fifty normal dogs with those in dogs with.miscellaneous disturbances such as distemper, bile duct ligation, fasting and in dogs receiving pentobarbital 10 sodium, sulfamerazine, arsenamide sodium, carbon tetrachloride and fox encephalitis virus. The technique entailed injection of 5 mg./ kg. of the dye and sampling of the blood in thirty'minutes. To each cc. of serum, h cc. of distilled water, 1 to 2 drops of 10% NaOH or 1 to 2 drops of 5% H01 (control) were added. Percentage of dye retention was determined in a.Rouy-Leitz photometer. Normal dogs were always below 10% retention with the majority at 5%. The high- est retentions (6h%) resulted from hepatic damage in the form of fatty metamorphosis caused by carbon tetrachloride. In seven of twenty-four dogs afflicted with distemper, retentions up to 10% were recorded and in sera from puppies injected with fox encephalitis virus as high as 27% of the dye was retained. He found false posi- tives were attributable to fever, heart failure, ascites, obstructed bile ducts and parenterally injected dyes. He considered the test more reliable in acute than in chronic liver disorders and valuable in hepatitis and fatty degeneration but questionable in cirrhosis. Hoerlein and Greene, using the method of Rosenthal and White (1925), which utilizes the comparator block, but substituting 5 mg./kg. of the dye instead of the 2 mg./kg. dosage, concluded that no retention at 30 minutes was normal. In fifty clinical cases, twenty-two had abnormal values of 5 to 10% and three showed 100% retention. Among sixteen miscellaneous clinical cases, two of three animals with distemper had 10% retentions. They concluded that the test was of value in confirming early hepatic dysfunction, such as cirrhosis, fatty hepatosis, and other liver conditions in which the animal does not show abnormal bilirubinemia, and that it was quantitative in indicating the amount of liver damage. ll Lamson and Wing (1926b) demonstrated that as little as 0.3 cc. of carbon tetrachloride produced early cirrhosis in dogs if given every other day for fifteen weeks. They had previously reported (1926a) that threshold doses of 0.5 to 1.0 cc./kg. produced central necrosis of the liver. 12 MATERIALS AND METHODS A. The Bromsulfalein Liver Function Test The bromsulfalein used in these studies was the commercial* 5% (50 mg./ml.) aqueous solution of Sulfobromophthalein Sodium, U.S.P., (Phenoltetrabromphthalein - disodium sulfonate) . For rat use, the commercial preparation was prepared by dilution with sterile isotonic sodium chloride solution to a final concentration of 1 mg./ml. _Later .in the studies, the concentration was increased to 2 mg./ml. to reduce the volume injected. In the dog, the undiluted commercial solution was used. The dye was administered at the rate of 5 mg./kg. body weight to all animals. After collection, the blood sample was transferred to a centri- fuge tube and allowed to clot. The sample was centrifuged for ten minutes at 2000 r.p.m, for rat blood or 3500 r.p.m, for dog blood. Serum sufficient for the test was transferred to a clean test tube. If the determinations were not to be made promptly, the serum was refrigerated. The photoelectric colorimeter used in these experiments was a Beckman Model B Spectrophotometer. Hepler's (19h9) procedure, which is essentially that of Gaebler (l9h5), was followed in the estimation of bromsulfalein retention. The technique of the test is as follows: 1. 0.5 ml. of serum was accurately measured into each of two x 25 mm,, flared-lip, thin-walled test tubes. * Hynson, Westcott and Dunning, Inc., Baltimore, Maryland. l5 2. 2.5:ml. of deionized water (distilled water is recommended) was added to each tube. 3. 3.0 ml. of 0.1N NaOH was added to one tube, to develop the color, and mixed. A. 3.0 m1. of 0.1N HCl was added to the other tube, to serve as a blank, and.mixed. 5. The tubes were allowed to stand for five minutes to insure full development of the color. 6. Each of the mixtures was added to the standard curvette and placed in the spectrophotometer. The wave length was set at 580 mu. Using the blank, the galvinometer was set at 100 and the galvinometer reading was then obtained for the unknown. 7. The percentage of dye retention was obtained from a table previously prepared by calibration of a standard curve as described in the reference (Hepler, 1952). This procedure was followed on the sera from dogs. Because of the small quantity of blood collected from the rats, the volumes of the sera and reagents were reduced by one-half - 0.25 ml. of serum, 1.25 ml. of deionized water and 1.5 ml. each of 0.1N NaOH and HCl. The total volume was sufficient to fill the aperture in the carrier and not leave a.meniscus to distort the transmission of the light beam. B. Procedures on the Rat Rats of Wistar strain from the Upjohn colony were used in these studies. Their weights ranged from 129 to 392 grams. They were predominantly'males. At the time of injection of the dye solution, or prior to 1h heart puncture or surgical manipulation, the rats were anesthetized with cyclopal at 80 mg./kg. given intraperitoneally. Immediately before the test was to be performed, the rat was weighed, the dose calculated, and the injection made with a l/2-inch, 27-gauge needle. Regardless of route of administration of the bromsulfalein so- lution, blood.samples for the determination were collected by car- diac puncture. The hair was clipped over the thoracic area and the puncture made through the thoracic wall with a l/2-inch, 23-gauge needle with clean, dry syringe attached. Three to four ml. of blood were withdrawn. The animals were sacrificed within several hours after blood samples were collected if hepatotoxic agents were administered, or as late as 2h hours afterward if they were controls. Euthanasia was accomplished by severing the cervical spinal cord with heavy rib forceps. The viscera were exposed and examined for lesions with particular attention being paid to the liver. The hepatic parenchyma was examined after exposure with multiple incisions. To obtain average bromsulfalein retention values, the following methods were used. 1. Retention values were secured on eighteen animals, eight males and ten females, ranging in weight from 126 to 190 grams, by injecting the dye solution intraperitoneally and collecting the blood sample in forty-five minutes. 2. Dye retention was determined on fifteen rats, of which twelve were male and three female ranging in weight from lAO to 197 grams, after the solution had been injected into the coccygeal vein; 15 blood samples were collected at five minutes from one rat, at fifteen minutes from thirteen animals, and at thirty minutes from one. 3. In ten.ma1es, with weight range of 299 to 377 grams, the dye solution was injected into the saphenous vein which had been ex- posed by incising the skin over the vessel; the determinations were made on blood samples collected at thirty'minutes. 0n rats that sustained damage to the liver by hepatotoxic agents, bromsulfalein retention determinations were obtained utilizing the peritoneal route of injection and forty-five-minute sampling tech- nique. 1. Twelve male rats, 309 to 392 grams in weight, were exposed three times, in a closed container to ten minutes of intermittent chloroform anesthesia with three-hour intervals between exposures. Intermittency was produced by inducing deep narcosis, then allowing partial recovery, followed by deep narcosis with the cycle repeated a number of times during the ten minute periods. Respiratory fail- ure often was corrected by artificial respiration and the anhmal again submitted to the chloroform vapors during the same period. 2. Larson (1956) demonstrated that pyrazolidin-3-one, l-methyl~ 5-phenyl, in rats, induced bile stasis with inspissation and marked proliferation of bile ducts. Twenty-four rats, twelve of each sex, ranging in weight from 136 to 209 grams, were administered 100 mg./ kg. of the drug for five days and then 200 mg./kg. for the next three successive days. Retention of dye was determined on eleven males from this group. Negative findings, with few exceptions, are not recorded in this thesis. 16 C. Procedures on the Dog The dogs in these studies ranged from nondescript crossbreds to purebreds and were from a variety of sources. The majority of the beagles were from the Upjohn colony or secured for the colony from local breeders; complete histories of these dogs were available. Dogs of doubtful breeding, recognizable crossbreds and a number of probable purebreds were from dog pounds within limited geographical radius from the laboratory; histories were not obtainable on these. The animals ranged in age from several months to aged but the majority were one to two years old. Both sexes were represented. The Upjohn colony dogs were treated for intestinal parasites, inoculated against canine distemper and infectious hepatitis, and given complete physical examinations, including routine clinico- pathological tests, before being assigned to toxicological tests. The animals were weighed and the dosage calculated immediately before the test was to be performed. The hair was clipped over the radial aspect of the foreleg and the dye solution injected into the cephalic vein using a l-inch, 21-gauge needle. The hair was clipped from the ventral aspect of the neck and at #5 minutes after injection a 7 to 8 ml. blood sample was with- drawn from the jugular vein. A 1 l/2-inch, 19-gauge needle with attached clean, dry syringe was used to collect the sample. The dogs were sacrificed on the same day or not later than the third day following the performance of the function test. All of the animals were killed by electrocution. Partial exsanguination immediately followed. 17 A general necr0psy was performed on each dog but the brain and bone marrow were examined only in the animals on the drug-intoxi- cation experiments. Blocks of tissue were removed, fixed in 10% formalin solution, embedded in paraffin, sectioned at 6 microns, and stained with hematoxylin and eosin. Fat-stained sections, formalin-fixed, were prepared either by the freezingpmethod or em, bedded in Carbowax and stained with Sudan IV or oil red 0. All of the sections were examined microsc0pically. The tissues and organs routinely examined histologically varied with the different studies but any grossly pathologic tissue was saved, sectioned, stained and examined. Table 1 sets forth the organs and tissues routinely subjected to micrOSCOpic examination. The bromsulfalein liver function test was performed and necr0psy and histopathological examinations completed on eighty-five dogs: fourteen were normal controls; five had received carbon tetrachloride; thirty-five had received other drugs; twenty were exhibiting symptoms of the distemper complex; six had been inoculated with canine in- fectious hepatitis virus; and five were pyretic only. 0f the thirty~ five animals which had been administered drugs, eight had received the sodium salt of tolbutamide, nine 3,5 - dipropyl-A-allyloxy- benzoic acid, six pyrazolidin-3-one, l-methy1-5-pheny1, six the anti- biotic streptovaricin, and six methyl reserpate. The normal dogs were purebred colony beagles with normal physi- cal (two examinations) and clinico-pathologic findings. They had served as controls for toxicopathologic studies and had received only empty gelatin capsules. Tue male and three females of mixed breeding, ranging in weight 18 from 5.9 to 9.A-kilograms, composed the group which received the carbon tetrachloride. All recorded bromsulfalein retention values of 0.8% on the pretreatment determinations. A regimen of 0.25 ml. of carbon tetrachloride in a gelatin capsule administered daily, seven days a week was instituted initially for all dogs. After eight days, the dosage was increased to 0.h-ml. in Dogs. 666A and 6796, the former remaining on this dose for 12 more days and the latter for 1h days. The bromsulfalein retention was determined in Dog 666A the day after the last dose, after which she was sacri— ficed. The dose was again reduced for Dog 6796 to 0.25 ml. for the next 38 days. The function test was performed and the dog sacri- ficed on the eleventh day following the last dose. Dogs 7071, 7072, and 7073 received the initial level for 38 days, were untreated for 11 days, and then given 0.5 ml. daily for 79 days. The day follow- ing the last dose, the function tests were performed and the animals killed. Beagles from the colony were employed in all of the drug studies. The number of each sex in the studies varied from drug to drug. For one reason or another, not all the dogs in each test were in- cluded in this study. The drugs were administered in gelatin cap- sules and given in three divided doses daily with one exception. The procedural data for each drug are set forth individually below: 1. Sodium.salt of tolbutamide: Administered at the rate of 30, 100, and 160 mg./kg., to three dogs per dosage level for six months. Eight of the nine animals, two males and six females, were included in this evaluation. 2. 3,5-dipropy1-h-allyloxyl-benzoic acid: Administered at 19 approximately 30, 100, 300 and 525 mg./kg., for one month; three dogs per level for the first three dosage levels and one dog for the high~ est level were used. Five males and four females constituted the group in this evaluation. 3. Pyrazolidin-3-one, lemethyl-5-phenyl: Administered at approximately 30, 100/60, and 300/60 mg./kg., three dogs per dosage level, for 28 to 35 days. The 300 mg./kg. dosage was discontinued after five doses and the 100 mg./kg. dosage after 23 doses. The dosage was continued at 60 mg./kg for the remainder of_test. One male and five females were included in this evaluation. A. Streptovaracin: Administered at approxrmately 30, 100 and 300 mg./kg., to male dogs, three at each level of drug, for 6h days. Six.ma1es from this group were included in this evaluation. 5. Methyl reserpate: Administered at approximately 5, 15, and 50 mg./kg., once daily, for 21 days and at approximately 1 mg./ kg., once daily, for 28 days. Six (one male and five females) of the seven dogs in this group were evaluated. Fifteen male dogs, five of which were colony beagles and the remainder of pound origin, and five females, all of pound origin, manifesting symptoms of the canine distemper complex, were subjects of this study. Presumptive diagnosis was made from physical exami- nation. Eight very immature pound dogs (three males and five females) were inoculated subcutaneously on two successive days with 0.5 m1. of ICHVR (Upjohn) infectious canine hepatitis virus. This virus had proved its pathogenicity in susceptible animals*. Body temper- * E. A. Slater. The Upjohn Company, Kalamazoo, Michigan: Personal communication. 20 atures were recorded and the dogs examined daily after the fourth post-injection day. The procedures were accomplished on the twelfth day after inoculation on Dog 6h92, the eighth day on Dog 6681, and on the seventeenth day on Dogs 6697, 6760, 6761, and 6762. Five immature pound dogs (one male and four females) of mixed breeding, secured for the experimental canine infectious hepatitis study, were found on preliminary physical examination to have elevated body temperatures but no other physical signs of disease. The liver function test and euthanasia were performed on the same day as the physical examination. 21 TABLE 1 ORGANS AND TISSUES EXAMINED MICROSCOPICALLY A - Distemper Complex Dogs C - Carbon Tetrachloride Dogs B - Canine Infectious D - Dogs on other Drugs Hepatitis Dogs U Organs and Tissues A B C Cerebrum Cerebellum Pituitary Eye Lower tarsal conjunctiva M. nictitans Turbinate Tonsil Salivary'gland Thyroid Mandibular lymph node Suprapharyngeal lymph node Lung Trachea (at bifurcation) Heart Aorta Liver (left and right lateral and caudate lobes) x x Hepatic lymph node Cholecyst x x Pancreas x Adrenal Spleen x x Mesenteric lymph node x Stomach x x x Duodenum Jejunum Ileum Cecum Colon Kidney Urocyst Testis Prostate Ovary Uterus Skeletal muscle Bone marrow ><><><>< X><><>< >4><><>< ><><><>< ><>< >< ><>< ><><><><><>< ><><><>< X XX ><><><>< XNNX XXXXX xxxxxxxxxxxxxxxxxxx 22 RESULTS A. Bromsulfalein retention in the rat. 1. In the normal rat. The percentages of retention of the dye in the normal rats under cyclopal anesthesia, when it was injected intraperitoneally and the blood sample collected at forty-five min- utes, varied between 0.8 and 2.2% (Table 2). In the males, three retained 0.8%, three 2.2% and one 1.5%. In the females, one retained 1.5% and the nine others, 0.8% each. There was slight hemolysis in the sera from one male and two females. These rats were sacrificed within two hours after the blood samples were collected. Before the viscera were inspected for lesions, two m1. of 10% sodium.hydroxide solution were introduced into the abdominal cavity; the pale violet color of alkaline bromsulfalein developed distinctly in all the animals. No abnormalities of color or architecture of the liver were detected grossly and lesions in other organs and tissues were not observed in these animals. The possibility that the dye was not completely absorbed from the peritoneal cavity necessitated studies to determine the clear- ance of the bromsulfalein from the blood stream.when it was injected directly into the vascular system, A pilot study using three rats was conducted and the results suggested that a fifteen minute sample of the blood would be a sensitive indication of clearance of the dye from the blood stream. However, the results (Table 3) from the coccygeal vein injection were erratic, ranging from 0.8 to 6.8% retention. The high incidence of hemolysis of the samples in rats 2': 11 through 06 suggested that the volume of dye solution was causing hemolysis in_vizg, The concentration of dye was increased to 2 mg./ ml. in rats 07 through 012 to reduce the volume to be injected, and evidence of hemolysis was less prominent. A number of considerations led to the use of the saphenous vein as the route of injection and thirty minutes as the time of collection of the blood sample (see Discussion). Bromsulfalein retention did not exceed 2.2%, the majority retained 0.8% (Table A). 2. In rats receivingAhepatotoxic agents. Two rats succumbed during exposure to chloroform and data from them are not recorded. At least six of the animals required artificial respiration during one or more of the periods of narcosis. 0n the day following, which was the day the function test was performed, all the rats were mildly depressed. Post-mortem.examination revealed that five of the eleven animals had demonstrable liver lesions (Table 5) indicative of de- generation of the parenchyma. Ten rats had dye retention values of 0.8% and one retained 1.5%. The sera of the animals receiving pyrazolidin-3-one, l-methyl- 5-phenyl were intensely icteric but otherwise clear. A retention of 0.8% bromsulfalein (Table 6) was exhibited by ten rats and 3.0% by one rat. Ten of the eleven anrmals had grossly visible defects in the liver (Table 6). The lesions were similar to those observed in rats on a subacute toxicity test which included gross and.microscopic examinations of the liver. Continuation of the procedures in the females was considered useless in view of the results obtained from the males. Rat No. 11 12 15 1A 16 20 2A TABLE 2 CONTROL RATS A5 MINUTE SAMPLE Weight (gmsQ s35 190 M 166 M 172 M 177 M 180 M 129 M 1A1 M 1A3 M 155 F 138 F 155 F 1A1 F 126 F 165 F 1140 F 159 F 1A7 F 1A7 F 1+ trace of hemolysis slight hemolysis % Retention 0.8 2.2 0.8 2.2 2.2 0.8 0.8 1.5 1.5 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 1+ 1+ 1+ 1+ Rat No. 11 2i 5i 01 02 05 01+ 05 06 07 08 09 010 011 012 25 TABLE 3 CONTROL RATS COCCYGEAL VEIN Weight (gms.) Sex Sample/Minutes 166 171 197 158 165 1A0 1A1 160 1A5 169 165 172 1A6 168 150 1+ F F I-rj I: I: I: I: {Z {Z :Z {Z 23 I: 2: {Z 5 15 3O 15 l5 15 15 15 l5 15 l5 15 15 15 l5 trace of hemolysis slight hemolysis % Retention 5.1 0.8 A.3 1.5 5.0 1.5 0.8 6.8 0.8 2 2 0.8 0.8 0.8 5.6 0.8 1+ 1+ 1+ 1+ 1+ 1+ 26 TABLE A SAPHENOUS VEIN 3OMINUTE SAMPLE §§£_§9; weight_(gms.) sgg’ %Retention B1 556 M 0.8 B2 570 M 1.5 B5 299 M 0 .8 B6 556 M 0.8 B7 577 M 2.2 B8 509 M 0.8 B9 5A6 M 0.8 B10 5A6 M 0.8 B11 521 M 1.5 B12 505 M 0.8 27 TABLE 5 CHLOROFORM,INTOXICATION A5 MINUTE SAMPLE Gross Appearance Rat No. Weight (gms.) Sex % Retention of Livers 55 316 M 1.5 1+ Essentially normal. 57 309 M 0.8 Faint yellow cast. 58 592 M 0.8 Slight yellowish mottling. 59 3A2 M 0.8 + Slightly swollen but normal in color. 60 565 M 0.8 Essentially normal. 61 561+ M 0.8 Essentially normal. 62 526 M 0.8 Essentially normal. 63 377 M 0.8 Essentially normal. 6A 366 M 0.8 1+ Slight yellowish mottling. 65 387 M 0.8 Essentially normal. 66 515 M 0.8 Slight yellowish mottling. + II trace of hemolysis 1+ slight hemolysis 28 TABLE6 PYRAZOLIDIN-3—ONE, l-METHYL-S-PHENIL RATS A5 MINUTE SAMPLE Rat No. weight (gms) Sex % Retention* 51 201 M 0 .8 52 191 M 0.8 55 22A M 0 .8 5A 190 M 0 .8 55 177 M 0.8 56 159 M 5 .0 57 205 M 0.8 58 209 M o .8 59 226 M 0 .8 A0 187 M o .8 A2 181 M 0 .8 * - all sera icteric. Gross Appearance of Livers Essentially normal. Yellowish tan in color; central veins or triads very conspicuous. Reddish tan in color with dark tan mottling through- out;loss of architecture. Left lateral lobe tan in color; median lobe reddish tan with circumscribed whitisrl areas near poste- rior border; right lobe reddish tan with multiple whittish areas; caudate lobe tan in color. Major parts of left later- al and median lobes tan with red mottling - re- mainder of lobes normal. Light tan diffuse areas disseminated throughout. Normal in color but lobu- lation indistinct. NMltiple, tan areas throughout. Multiple, tan areas throughout. All lobes tan in colorvdfla loss of lobular architec- ture except in several areas which were normal structurally but pale pink in color. Dark reddish tan areas with loss of lobular archh tecture interspersed with lighter tan fields with normal structure . 29 B. Bromsulfalein retention in the dog. 1. In healthnyuntreated dogs. Dye retention of 0.8% was re- corded for thirteen and 1.5% for one of the fourteen control dogs which were free from clinical signs of disease and were not recipients of drugs or infectious agents. 0n necropsy, however, eight of the fourteen had grossly discernible lesions in various organs and tissues. The livers, both grossly and.microscopically, were lesionless. The histopathological examination of various other organs revealed a wide variety of spontaneous lesions. The results of the liver function test and the gross and.microscopic examinations are in detail in Appendixul. 2. In dogs administered carbon tetrachloride. .All the dogs receiving this agent lost over a kilogram.of initial body weight during the test period. Dog 666A was killed after 21 doses of the chemical because severe stomatitis developed. Marked jaundice was present in Dog 6796 after 10 days on the 0.A m1. regimen and 5 days later the dose was reduced. Body weight loss, poor condition, and, later, slight loss of appetite were the only physical manifestations displayed by Dogs 7071, 7072, and 7073. Dog 6796 was kept for 11 days following the last administration of carbon tetrachloride to allow the acute phase of the intoxication to subside before authanasia. Bromsulfalein retention ranged from 0.8 to 3A.0% in the carbon- tetrachloride~intoxicated dogs (Appendix 2). Dog. 6796, with a dye retention of 0.8%, had residual hepatic damage consisting of hepato- cellular swelling,:mild cirrhotic change and centrolobular bile stasis. Severe hepatocellular degeneration and disturbed circulation were prtmary'histologic alterations in the liver of Dog 666A which had a 30 21.8% retention. In Dogs 7071, 7072, and 7073, the dye retention of 3A.0, 29.8 and 23.6%, respectively, varied inversely to the degree of cirrhosis encountered and directly with acute damage in their livers. 3. In dogs receiving drugs. The results of the liver function test and the gross and.microscopic examinations are given in detail in Appendix 3. To simplify the discussion of findings, both under this heading and Discussion, the drugs will be assigned an alpha- betical designation: Drug A Streptovaricin Drug B Tolbutamide, sodium salt Drug C Dipropyl-allyloxy-benzoic acid Drug D Pyrazolidin-3-one, l-methyl-S-phenyl Drug E Methyl reserpate Bromsulfalein retentions ranged between 1.5 and 9.0% in dogs receiving Drug A. At 30 mg./kg., retentions in Dogs 592A, 5925, and 5926 were 2.2, 2.2, and A.8%, respectively, but the gross and histologic examinations of the livers disclosed no abnormalities. In the dogs receiving 100 mg./kg., Dog 5927, with 3.6% retention, had no gross or microscopic hepatic lesions. Dog 5928 retained 1.5% of dye in the serum and showed focal lymphocytic infiltration or proliferation in the hepatic parenchyma. With a 9.0% retention, Dog 5929 showed an early state of an acute purulent hepatitis micro- scopically. At 30 mg./kg. of Drug B, the retentions in the three dogs were 0.8, 0.8, and 1.5% without hepatic alteration. At 100 mg. /kg. of the drug the dye retention was 2.2% (Dog 6078) and 1.8% (Dog 6080), 31 without discernible liver damage, and A.3% in Dog 6079 which had diffuse hepatocellular vacuolization. In the two dogs (6081 and 6082) administered 160 mg./kg. of Drug B, A.3 and 1.5% values for the function tests were recorded; mild diffuse subacute hepatitis was detected.microscopica11y in the livers of both dogs. Gross pathologic hepatic change was seen in Dog 6081 but not in Dog;6082. The livers were essentially normal macroscopically and.micro- scopically in the subjects on 30 mg./kg. of Drug C; dye retentions were 2.2, 0.8, and 0.8%. Retentions were 0.8% in each of the three dogs on 100 mg./kg. of the drug. Dog 619A, however, displayed an allergic hepatic response, histologically, characteristic of parasitic injury; the livers in the other two dogs were essentially normal. One dog (6198), at 300 mg./kg., retained a serum.concentration of dye of Al.2% and showed hepatic congestion and degeneration of hepatic cells at necropsy and microscopically. There was no apparent hepato~ pathy in the other two dogs and retention of bromsulfalein in both dogs was 0.8%. Hepatopathy of varying degrees occurred in all the dogs included in the study from the Drug D evaluation. Likewise, elevated dye re- tentions were recorded for all the subjects. Hepatotoxic manifes- tations observed at necropsy consisted principally of alterations in the color of the organ and less consistently in changes in texture. Histologically, the lesions were characterized.by pigment (probably hematogenous) accumulation and damage to the hepatic cells and vas- cular bed. The intensity of these changes paralleled somewhat the level of the drug and was reflected, generally, in the concentration of bromsulfalein in the sera. In Dog 62A7, with 6.2% retention, 32 the tissue alterations were relatively mild whereas in Dog 62A3 the hepatic damage was marked, the pigment abundant and blood clearance of the dye was delayed, resulting in a 21.8% retention. Extending the sampling of the blood to an hour and forty-five minutes resulted in a 7.0% retention at that time in Dog. 62A1 which sustained severe hepatic damage from the drug. Bromsulfalein retentions of 0.8% were found in the six subjects receiving Drug E. With one exception, the livers of these dogs were free of abnormal change. Microscopic examination of the liver of Dog. 6A70, on 1 mg./kg. of the drug, disclosed a very mild, focal, subacute hepatitis. A. In dogs affected by infectious disease. a. In dogs with clinical_signs of the distemper complex. The results of the liver function tests, and the clinical, post- mortem, and histopathological examinations are in detail in Appendix A. A definitive diagnosis of canine distemper based on the presence of intranuclear or intracytoplasmic inclusion bodies in tissues was made in six of the twenty dogs (6557-h, 6557—6, 6557-9, 6557-10, 6558-c, and 6AA5-6). Eight dogs had bromsulfalein retentions of 0.8%; retentions of 1.5 to 9.8% were recorded for the other twelve dogs. In the former, one liver showed early degenerative changes and one contained lesions of mild.focal subacute hepatitis. The livers of the subjects having retentions A.O% and over contained, principally, lesions indicative of focal hepatitis, centrolobular necrosis or centrolobular hemorrhage and congestion. In the cases of retentions below A.O%, degenerative 55 changes predominated in the livers but there was one case of mild focal hepatitis and one of larval granulomatosis which was identified by its similarity to larvae-containing lesions observed in dogs in previous studies at this laboratory. Both antmals had a 2.2% of dye. b. In dogs injected with infectious canine hepatitis virus. In the first series, three dogs were recipients of the virus. Two of the three developed persistent low-grade fevers beginning on the fifth day and subsiding on the eleventh day after injection. The other puppy was afebrile for six days after injection, then sustained a rise in body temperature to 10A.6°F. with serous ocular and nasal discharges and died unexpectedly two days later. The function test was executed in the two remaining dogs on the eleventh day after in- jection. The dye retentions were 0.8% and 5.9%, respectively. Only the dog with the elevated retention was evaluated pathologically (Appendix 5). Evidence of a systemic infection was apparent grossly and microscopically in this antmal but the typical hepatic intra- nuclear inclusion bodies were not evident. The liver sections were characterized by mild focal subacute hepatitis, moderate centrolobular fatty metamorphosis, and inexplicable tinctorial nuclear changes which were chacterized by reversal of staining affinity or mauve coloration of the nucleoli. Febricity was not a characteristic sign in the second series of dogs receiving the virus. All the subjects, however, lost condition repidly after the seventh post-injection day. Dog 6681 was in such debilitated condition, but with no other discernible signs, as to require sacrifice, after the function test was performed, on the eighth post-injection day. Debility was the only clinical sign of 3A disease evident in Dogs 6760, 6761, and 6762 during the observation period but Dog 6697 was emaciated, weak, and presented an ocular and nasal discharge, terminally, on the seventeenth day. Dog 6681 had a A.3% retention of dye. Grossly the liver had a yellowish cast and there was a very mild acute focal hepatitis histo— pathologically. Dogs 6760, 6761, and 6762 had shown dye retentions of 0.8%. The livers were not abnormal grossly and, in Dogs 6760 and 6761, the organ was essentially normal microscopically. Moderate focal, subacute (bordering on chronic) hepatitis was detected in Dog 6762. The hepatic lesions in Dog 6697 were characterized macro- scopically by irregular yellowish mottling and microscopically by disseminated acute focal hepatitis; bromsulfalein retention was 12.0%. Intranuclear inclusion bodies were not engendered by this virus. 5. In dogs with pyrexia. The bromsulfalein retention in each of the five dogs was 0.8% (Appendix 6). The livers were essentially normal on macroscopic and microsc0pic examinations, with two ex- ceptions: Dog 6AA6-l had mild hepatic larval granulomatosis and Dog 6AA6-5 had very mild hepatocellular cloudy swelling. In the former, the lesions were focal, confined and two in number. The cause of fever could be determined with certainty in only two of the animals. Dog 6AA6-1 was in the early stages of bronchopneumonia and Dog 61116-2 had severe unilateral tonsillitis and lymphadenitis of the suprapharyngeal lymph node. The lesions (hemorrhage, lymph- ocytic depletions, and/or lymphoid hyperplasia) in several of the lymphoid tissues detected grossly or in the tissue sections from Dogs 6AA6-2, 6AA6-3, 6AA6-A, and 6AA6-5 suggest that they were in 55 the prodromic stage of the systemic infection, conceivable viral. Seventy-two of the eighty-five dogs exhibited lesions in one or more organs or tissues, other than the liver and major reticulo- endothelial organs (spleen and lymph nodes). Sixty-one animals had involvement of one or more of the major reticulo-endothelial organs. Lesions were found in other than hepatic loci in anrmals which had minimal levels of retained bromsulfalein in their serums and in those which retained a high percentage of dye in the blood stream. A summary of the percentage of retained dye and the histopathol- ogical alterations are set forth in Table 7. 56 - m.H z m.aa a cmmm - w.o z o.ma a memo - m.o z m.m a omen - m.o z m.aa 2 mass - m.o z w.oa 2 mwam - m.o z H-m a neon - w.o z m.m a meow - m.o z m.m z meow - w.0 z :.w z mmmm - w.o z a.» a mmmm - m.o z m.» z swam amoaosenaopaam asunder concea>o .eoa soom< A.wxv wow .oz macaw mam .p: moo mfipfipmmos mSOflpooon ooflowo a mHo mneoama< mwdan u naoams< moofiaoasooapcp donate u so “dogwooo n z mUZHQZHh A mxoamaoo homaopmfio osflomo u 090 "modems 0p hog 57 .mflpfiadomo>flamm aflo> Hoopooo .mophoosaEhH mo Hoom cosmopoom .GOprH:E:ooo newsman com soapmuomfiaoaa pose mafia odes “amorphoaon use mnmonnflm amaspoaoaoomanom opmaoooa “soapouooomou our oo«pwuoomwoo uoHoHHoUOpwaom .mamoupwm Hdpaomfinom one headboafiaom odes “soapwasadooo pooswwm mowmnhuoaoo headboaoapsoo «soapouooowoo oao>om .sofipmhooowoh UHHE mooaenaouaaoaa pose page ease anemones“ acaanoaaaca sass mowonaaosoo «mamoaooo awHoHHmUOpwmoo mcofipwaooowoo oao>om .mammpw mafia mnemonnfim UHHS mmofiaaozm awadaaooOpmmom .mHmooQHQSMpoa appom amadnoaoapooo oao>om mmwmoaooo woodwohmow msoHpmaoommoU omadnoaoapsoo o.m m.a m.m w.: m.m N.N m.mm (Q 8? o.nn ®.o w.am w.0 w.0 w.0 '¢ '< '< '¢ '< .¢ Bo 80 HO HO BO 2 z z N.® H.m o.» m.~ m.: m.ma m.® E: E: 21 21 21 22 mmmm mmmm emmm mmmm mmmm swam n>o> meow awow omen some acne meet ammo 38 soapstooomoo soadaaooopmmoz was soapmowsoo omHSQOHoaoooo .Aoflpfimwaomv ooflpoooa aflwaoaam Hmoom .pooswfim was Hoom haooofifioamofl ooawppwom .Hwow won or amassed saanaeaonnm .mwmonmhoa looms spasm aradnoaoaobmwhom «mapfipmmos opooondm opwaoooz .Apom poov soapmNflHoooo> adeHHmOOpmamm 1*1 w.o N.H: m.0 w.0 w.0 ®_O w.0 m.0 N.N m.a m.a m.a m.: m.m m.a w.o w.o w.w :.w a.a H.> m.w w.s m.m m.m o.w o.w m.» m.> H.w o.m o.w w.e a.m 2: 21 k. [In oaaw woaw emaw woaw moaw aaaw noaw moaw Hmaw mwow Hwow owow meow meow show weow meow .oowpmnooomoo aoadaaooooomon was: .mmwoomo o>fipwaooowoo magma «case ham> .mwpwpomon opoooQSm awoom mafia ham> m7 .mamooaaoswpoa haven opwaooofi , ooufiamuooom Hoow ofiponooo Hmoowmoooo poo Gowpmaosomoo hoadaaoo newsman morass .GOfipoHdadooo pooswfim opoamooa "headboaoapomo .mHmoomaoswpoS appom cams snoflpwaooowoo amasaaoooumaon cornea asogwadoudoom pooswwa uadeQOHOApoco , .mfimooaaosspoa humom owners moo sowpmamsomoo Hofioapo «oofipmadaooow pooswwm .mHmOpoms one sowpoaooowmo awadaaooOPoaoo “headboaoapsoo .hnmoapaomhm Howamnpflmm pose mafia mnemonmaoa noose hears was pooswwm mo soapoaofiooow noaopoaoapooo was: .mflmoompoe looms appom wees mowosaaoaon oafie one homoaoaoamo Hoflaoopoooo amadpoaoaoooo mpooemwm mo soapdeEdooo amaoooaoapooo opmaoooz m.m m.0 m.0 w.0 m.0 m.0 m.o ®.o o.» m.oa m.m m.o UQU Ono o.mm o.» m.w o.» m.w w.m :.ma .1. m.> m.» m.> m.» m-amow H-annw wwew mwew awew mwaw Heaw oesw Admm :zmw newt semw wamw m .QOfipmaonommo amadaamoOpomon paws ham> .mfipfipmmon opdoo Hooom mpohoooz E - .mfipfipomon opooondm Hooom mafia hao> .mfipfipoaon opdoonsm Adoom paws hao> .mfipfipomon .mpdompdm op spoon .Hsoom oafiz .mwmopoaoasnoaw Ho>hoa «mapapoaos canonso op monowpsm Hooom washroom “soapmaooowoo amazomm> onshore: .mfipfipomos mason Hooch woman: .oOapwaooomoo amasaaooooomos oouaamaoooo .mflpfipomon monounsm on spoon «Hooom sopoaoooa 0p was: .GOHpoaosmmoo awasaaooopomoo oafiz .ooflpwnmoowoo aoHdHHoUOPomon one oofipmowooo o>Hmmom was: .oofipoaoommoo amaoaamoopwaoo oaflz .oofipmomooo amafiooaoapooo our soapmaosmmoo goddaaooOpmmmm m.o m.a 0.: ®.0 w.a m.a m.o N.N m.m N.m w.0 w.0 N.H m.0 m.0 p.: ODD 090 090 090 Ono 090 090 U90 we. as o.oa m.HH me 0.0H n.0H is wé ~.m o.» w.m m.» :.w n.m w.aa o.> :1 E: I: mummmw enmemw m-m:mw m-anmw Huszmw o-wmnw n-mmnw o-wmnw OHuwmmw muwmnw muhmmw Puwmnm wuvmnw muemnw :uhmmw nsm-ewmw nuemmm A1 .mQOHoo>aompo mmoam Eaflmooo poo oflo Qoflpooflsoxo HoOHwoaoomeOpmflm A*v .mmemeoadooam Hm>pwq .mwpfloomoz oozes Hooom woman: .mflpflpmmmn aofiooano OP opdomnsm Ndooom morocco: va - .sofimaoomwmo amadaamoOpomon endowed mmfipflpomoo spoon odes hao> .SMHmeoamohHom amaooaods mmflpwpomon monsoQSm Hooom mafia «soapoaooowoo amHSHHoOOpmmos oumamooz .mHmOPmandooaw Ho>aoq w.0 m.0 m.0 m.0 w.0 O.NH m.o w.0 w.0 n.: m.m N.m mHo mHU mHo mHo MHU mHo UGO m.w w.w m.m e.w m.» s.a w.o w.oa m.m m.» o.w N.® m-weaw a-wenw m-waaw m-weew H-waew eoww mwew Hwew owew waw maaw w-naaw r..— u.»— o 9 Ca 1 9.4 ’7‘ 2y -q til \vd F‘1 uh A2 DISCUSSION The application of the BSP test to normal rats revealed a number of factors that require clarification. The indication that at least part of the bromsulfalein injected into the peritoneal cavity was still present therein places a questionable value on the results obtained from the forty-five—minute blood sampling. If the amount of dye in the abdominal cavity was within narrow limits in all animals the measure of blood clearance would be more valid. This could be readily ascertained if it were the only problem inherent in the application of the test to rats. Intra- venous injection of the dye solution via the coccygeal vein gave such erratic results in retention that, under conditions of these experiments, this injection site should not be considered. The cause of these results is only speculative. While the saphenous vein route with thirty—minute serum sampling, gave the most uniform results in the limited number of animals, the requisite surgical manipulation precludes this method from routine use in a large number of animals. However, the percentages of dye retention found in these animals and those injected intraperitoneally corre- lated rather closely. The lack of increased dye retention, even slight, in mild and severe grossly detectable hepatopathy indicates that the method, under conditions of this experiment, was unreliable and would be of questionable value as a routine procedure in rats. A5 A.major objective of this study in dogs included the production and detection of a variety of hepatopathies. Acute to chronic pro- cesses were produced. Inflammatory, degenerative, necrotic, vascu— lar, and pigment metabolic changes, sometimes occurring as relative- ly pure entities but more often in combination, were observed. Hepatitis as acute, subacute or, infrequently, chronic, and very mild to marked in intensity and extensiveness affected seventeen subjects. Hepatocellular degeneration, usually centrolobular, was a frequent histologic finding and it was generally of chemical etiology. Congestion, hemorrhage and other vascular alterations were found only in combination with other lesions. This was also true of pigment accumulation, necrosis, and fatty metamorphosis. Lacking from this evaluation are results of the function test in passive congestion, massive necrosis, either infractive or chemically induced, or fatty metamorphosis uncomplicated by other processes. Although a definitive diagnosis of canine distemper we possible in only six of the twenty dogs diagnosed clinically as such, this does not necessarily negate the clinical definition of the majority of the sick animals. Unfortunately, inclusion bodies are not present in every case of distemper. There was hepatic involvement in three of these animals strongly suggestive of infectious canine hepatitis but, again, typical inclusion bodies were not evident. Experimental infection with infectious canine hepatitis virus is difficult even under the most ideal conditions. All eight dogs injected with the virus demonstrated some type of reaction indicat- ing their susceptibility. Only two dogs had liver lesions that AA approached the classical description but no inclusion bodies were detected. The remaining dogs were infected as evidenced by the clinical observations, and gross and microscopic examinations but did not show typical hepatic damage or inclusion bodies. Dog 6697 was also afflicted with distemper. It is possible that handling the virus reduced its pathogenicity and/or the susceptibility of the test subjects was reduced by previous exposure or inherent resistance. The route of administration, likewise, may have affected the pathogenesis. All previous work with the BSP function test using photoelec- tric methods was based on thirtyeminute blood sampling and, there- fore, direct comparison between the results and those reported previously is not possible. If the findings obtained from several of the dogs administered drugs A and B are~e1iminated, tentative ranges of retention emerge and allow speculative normal and ab- normal limits for interpretation. Under conditions of this experiment, the upper limit of retention in apparently normal Beagle dogs, without hepatopathy, was 1.5% and it can be considered the upper normal limit. Retentions between 1.5 and A.0% should be considered in the range of suspicion or indicative of at least functional, if not mild cytological, alteration in the liver. Retentions above A.0% are indicative of definite hepatic dysfunction in which cytological evidence can be expected. Table 8 points up the fallibility of the function test in the lower retention bracket, i.e., below five percent. Whereas dogs normal by both clinical and necropsy standards did not retain serum dye above 1.5% (and this in one animal only, the majority having A5 0.8%), fifteen animals presenting mild to moderate hepatic altera- tions histopathologically retained 1.5% or less. In a percentage evaluation, of fifty-two dogs retaining 1.5% or less of bromsulfa- lein, 29% demonstrated very mild to moderate microscopic hepatic lesions. Three, or 33%, of nine dogs having retentions between 1.8 and 2.5% had mild lesions. Of the five animals with serum dye values between 3.6 and A.3%, four, or 80%, had mild to moderate cytologic changes in the liver. In the total evaluation, sixty- eight dogs in this study had bromsulfalein retentions of less than 5.0% and of these, twenty-three, or 3A%, sustained very mild to moderate hepatic alterations. The remaining seventeen subjects had retentions over 5.0% and all, or 100%, had moderately to markedly affected livers. Thus, the correlation between bromsulfa- lein retention and hepatic defects was inconsistent in these studies. In carbon tetrachloride-intoxicated dogs, in which uniform correlation could be expected, there were inconsistencies between dye retention and intensity of the morbid hepatic state. Dog 6796, although sustaining a moderate degree of hepatic damage, had dye retention in the normal range. The high retentions from the four other dogs was a more accurate indication of the acute cyto- logic alteration than a measure of the developing chronic reaction because the dog with the most advanced cirrhotic state had the lowest retention. Dogs receiving Drug A (Streptovaracin) manifested notable difference between retention and the cytologic condition of the liver. Retention was uniformly slightly elevated and lesions were A6 evident in the liver of only two of the six dogs. Retention of 1.5% in Dog 5928 was not inconsistent with the very mild focal subacute hepatitis but the moderate perivasculitis in Dog 5929 hardly justi- fied the 9.0% dye retention recorded for him. NOr was there cyto- logical evidence for the slight retentions in Dogs 592A, 5925, 5926, and 5927. It is conceivable that this drug, which is an antibiotic, functionally altered the excretory activity of the liver without reflecting visible cellular change. This would further verify the conclusion of previous investigators as to the sensitivity of this test in detecting even subcytological differences in hepatic func- tion. Five of the eight dogs receiving Drug B (Tolbutamide, sodium salt) were free of microscopic hepatic lesions and the degree of retention in all the animals varied with the dosage level. Dye retentions were not excessive. However, at 160 mg./kg., two dogs which demonstrated hepatic lesions very similar in extent and in— tensity showed a rather wide variation in the dye retained; one was within the normal limits and the other had a value somewhat compat- ible with the injury. In the Drug C (Dipropyl-a11yloxy-benzoic acid) group of animals there was good correlation of the two evaluation factors with one exception. Retentions were within normal range and the livers were not abnormal but in Dog 6198 the retention of dye was Al.2%, the highest recorded for any animal in the study. The hepatic lesions would not appear to justify the high retention and it was more likely the result of the severe generalized circulatory collapse. There was a more reciprocal agreement between retention and A7 liver injury in the dogs administered Drugs D and E. Definite excretory failure was evident in the livers of dogs receiving Drug D (Pyrozolidin—3-one, 1-methyl-5wphenyl) but lowered dye clearances occurred in those animals sustaining greater hepato- cellular alteration in the form of degeneration and necrosis. Conversely, there was no evidence of hepatopathy in the dogs receiving Drug E (Methyl Reserpate) and their test values were within the normal range with the one exception of Dog 6A70 which had a mild focal hepatitis but an 0.8% dye retention. In the distemper complex dogs, correlation of test and hepato- pathy was poorest at the border line of retention. Mild to moderate lesions without elevated retention were common in these dogs. Likewise, the type and, to a degree, the extent of hepatic lesions were not reflected in the clearance of dye from the blood stream in several of the animals. Dog 6337-7 had focal hepatitis which contained necrotic foci yet had 0.8% retention whereas Dog 65A5-5 had similar defects but a A.0% retention. In other dogs, however, there was reasonably good correlation between retention and either the intensity of the liver damage or the extensiveness. Only one dog injected with infectious canine hepatitis virus, and with notable hepatic defects, recorded a false negative function test; in the others, lesions correlated with retention. Fever, per gs, in the dogs tested did not cause elevated retention. These findings are not in agreement with N0yan's (19A9). There was no definite evidence that lesions in other organs, and especially the primarily reticulo-endothelial tissues, caused increased retention of bromsulfalein. A number of dogs had from A8 infrequent to frequent involvement of other organs and tissues with no hepatopathy and with normal retention. However, in those dogs with marked liver 1esions,high retention, and nonhepatic lesions this fact is less evident and there is no irrefutable evidence that at least part of the retention was not caused by lesions in the nonhepatic tissues. When all facets of this study in dogs are considered, these facts pertaining to the function test emerge: (1) Correlation of dye retention and hepatic lesions was not consistent when both were mild. (2) Dye retentions belowELO% were least correlative with the state of the liver; elevated bromsulfalein levels without detectable parenchymatous alterations were most commonly encountered (false positive) but the reverse also occurred (false negative). This was particularly true in those cases where evaluation was made in dogs either receiving drugs or afflicted with infectious diseases. (3) The test did not reflect the circumscribed granulomas of para— sitic origin or allergic (eosinophilic) reaction in the liver. (A) Correlation between more severely affected livers and reduced blood clearance of the dye was only partially reciprocal and was not uniformily quantitative as suggested by Gornall and Bardawell (1952)and Hoerlein and Greene (1950). The results in this study essentially confirm Noyan's (19A9) conclusions that (5) the brom- sulfalein function test is most reliable and valuable in acute hepatopathies. (6) The test would appear to be of little value in the differential diagnosis of canine distemper and infectious hepatitis because secondary toxic hepatitis apparently is a common finding in dogs affected with canine distemper. (7) In drug toxico- A9 pathology the test cannot stand alone as indicative of liver injury although retention of dye suggests alteration of hepatic function. This evaluation of the BSP function test in the dog waspxxsibly too critical and the histopathological findings, divorced from appraisal of the total clinical and clincopathological evidence, places its usefulness in a questionable position among the clinical laboratory procedures. It merely re-emphasizes the common fallibil- ity of a single biological test in appraisal of a complex patholog- ical state. % retention O. 1 10. 12. Al. 43' 43‘ 4:” \N 8 .2 50 TABLE 8 SUMMARY Lesions 10 1 Milesions 55 0 Total A5 51 SUMMARY AND CONCLUSIONS The bromsulfalein liver function test, following the procedure of Hepler, was performed in sixty-five rats and eighty-five dogs; the competencies of the livers were appraised by necropsy and histopathologic examination. In the normal and hepatically altered rat, in spite of variation in procedure, the test was unreliable under the conditions of the experiment. Dye retention was determined and pathologic evaluation made in: normal dogs; dogs administered carbon tetrachloride; dogs receiving other drugs; dogs given canine infectious hepatitis virus; dogs exhibiting clinical evidence of distemper complex; and dogs with fever but without other readily detectable signs of disease. Under conditions of the study, the normal limit of retention was 1.5% or under; retentions between 1.5 and A.0% were consid- ered suspicious and above A.0% as frankly abnormal. Reciprocal agreement between retained dye and cytological hepatic alteration was inconsistent, especially at the lower range of retention. Twenty-nine percent of the dogs with 1.5% retention or less had detectable liver defects of varying degree. The two factors were generally but not uniformily correlative in the higher or abnormal range. The test was most sensitive and correlative in acute hepatopathy. When the test is used in toxicopathological evaluation of drugs, the results should be interpreted with caution. Excessively high dye retention, without correlative liver damage, gunfire wen-hr“ u- ' W \W~Y-~t 10. 52 occurred in cardiovascular collapse. Pathological states in other organs and tissues, including reticulo-endothelial, appeared inconsequential in relation to bromsulfalein retention. Pyrexia, per se, apparently was not a factor in reduced clearance of dye from the blood stream in this series. 55 BIBLIOGRAPHY Bauer, R. W. and R. L. Pessotti. The removal of bromsulfalein from blood plasma by the liver of the rat. J. Pharm. and Exp. Ther. 97:558-570. 19A9a. ----. BSP uptake and excretion in the dog. Am. J. Physiol. 162:565-57A. 19A9b. Bauer, R. W., J. S. Krebs and R. L. Pessotti. Bromsulfonphthalein as a tool for study of liver physiology. Fed. Proc. 9:259. 1950. Cantarow, A. and C. W. Wirtz. Excretion of bromsulfalein in the bile. Proc. Soc. Exper. Biol. and Med. A7:252-25A. 19A1. Casals, J. and P. K. Olitsky. Tests for hepatic dysfunction in mice. Proc. Soc. Exper. B101. and Med. 63:383-390. 19A6. Casals, J. and P. K. Olitsky. Tests for hepatic dysfunction in mice. Proc. Soc. Exper. Biol. and Med. 63:383-390. 19A6. Coffin, D. L. Manual of Veterinary Clinical Pathology, ed. 3, Ithaca, New York: Comstock Publishing Associates, 332 pp. 1955. Cohn, C., R. Levine and D. Streicher. The rate of removal of intravenously injected bromsulfalein by the liver and extra- hepatic tissues of the dog. Am. J. Physiol. 150:299-303. 19A7. Drill, V. A. and A. C. Ivy. Comparative value of bromsulfalein, serum phosphatase, prothrombin time and intravenous galactose tolerance tests in detecting hepatic damage produced by carbon tetrachloride. J. Clin. Invest. 23:209-216. 19AA. Gaebler, C. H. Determination of bromsulfalein in normal, turbid, 5A hemolyzed or icteric serums. Am. J. Clin. Path. 15:A52—A55. 19A5. Giges, B., J. D. Mann and W. S. Sharon. Bromsulfalein retention in obstructive jaundice. Proc. Soc. Exper. Biol. and Med. 79:575-576- 1952- Gornall, A. G. and C. J. Bardawell. The study of liver function in dogs. Canad. J. Med. Sci. 50:256-271. 1952. Hepler, O. E. Manual 9: Clinical Laboratorprethods, ed. A, Springfield, Illinois: Charles C. Thomas, Publisher, 387 pp. 1952. Hoerlein, B. F. and J. E. Greene. The bromsulfalein liver function test as an aid in the diagnosis of canine hepatosis. North Am. Vet. 51:662-665. 1950. Klein, R. I. and S. A. Levinson. Removal of bromsulphalein from the blood stream by the reticu!b-endothelia1 system. Proc. Soc. Exper. Biol. and Med. 31:179-181. 1933. Lamson, P. D. and R. Wing. Early cirrhosis of the liver produced in dogs by carbon tetrachloride. Jour. Pharm. and Exp. Ther. 29:191-202. 1926a. ----. Blood fibrin and levulose tolerance in acute and chronic carbon tetrachloride intoxication. Jour. Pharm. and Exp. Ther. 28:599-AO8. 1926b. Larson, E. J. The Upjohn Company, Kalamazoo, Michigan: Unpublished data. Linder, G. C., R. G. F. Parker and G. Selzer. Bromsulfalein retention in young rats on diets producing liver necrosis. Brit. J. Exper. Path. 3A:656-660. 1955. 55 Maclagan, N. F. Ch. 5. Liver Function Tests. Leon Schiff, Editor. Diseases of _tlg £1123, Philadelphia: J. E. Lippincott Co., 758 pp. 1956. McKibbin, J. M., S. Thayer and F. J. Stare. Choline deficiency studies in dogs. J. Lab. Clin. Med. 29:1109-1122. 19AA. Hulls, M, A. and C. A, Dragstedt. Bromsulphalein dye retention test as a measure of functional activity of reticulo-endothelial system. Proc. Soc. Exper. B101. and Med. 3A:228-231. 1936. ----. Removal of intravenously injected bromsulfalein from the blood stream of the dog. Arch. Int. Med. 62:216-221. 1958. Pratt, E. B., F. D. Burdick and J. H. Holmes. Measurement of liver blood flow in unanesthetized dog using the bromsulfalein dye method. Am. J. Physiol. l7l:A7l-A78. 1952. Noyan, Fethi. Observations on some of the liver function tests in dogs. Doctoral Dissertation: Ohio State University. 19A9. Rosenthal, S. M. and E. C. White. Clinical application of bromsul- phalein test for hepatic function. J. Am. Med. Assn. 8A:lll2- 111A. 1925. Svirbely, J. L., A, R. Monaco and W. C. Alford. The comparative efficiency of various liver function tests in detecting hepatic damage produced in dogs by xylidine. J. Lab. Clin. Med. 31: 1153-11A5. 19A6. Wirtz, C. W., A. Cantarow, WL J. Snape and B. Delserone. Bile volume and excretion of pigment and bromsulfalein in dogs receiving carbon tetrachloride. Am. J. Physiol. 165:680-687. 1951. 56 APPENDIX 1 CONTROL DOGS - BEAGLES DOG 5921 Male BSP retention: 0.8% Gross Pathology: Consolidation of antero-dorsal aspect of right diaphragmatic lobe of lung; hemorrhage in left suprapharygneal lymph node; moderate trichuriasis. Histopathology: Liver - essentially normal. Lung - organization of bronchopneumonia. Prostate - three large retention cysts and mild hyperplasia. Suprapharyngeal lymph node - congested. DOG 5922 Male BSP retention: 0.8% Gross Pathology: No gross lesions. Histopathology: Liver - essentially normal. Prostate - subacute interstitial prostatitis. DOG 5925 Hale BSP retention: 0.8% Gross Pathology: Focal pneumonia, right diaphragmatic lobe of lung; scar, .5 cm. in diameter, dorsum of right apical lobe of lung. 57 Histopathology: Liver - essentially normal. Lung - subacute focal suppurative bronchopneumonia and pleuritis; circumscribed areas of squamous metaplasia, with fibrous stroma, assuming glandular form and having polymorphic cells in some areas but generally differentiated and exhibiting intercellular bridges. Mesenteric lymph node - medulla congested. DOG 6072 Male BSP retention: 0.8% Gross Pathology: No gross lesions. HistOpathology: Liver - essentially normal. Prostate - cystic dilatation of acini in one lobule, remainder of gland in early stage of hyperplasia. DOG 6075 Female BSP retention: 0.8% Gross Pathology: Mild inflammation of vagina. Histopathology: A11 tissues essentially normal - section from vagina not examined. DOG 607A Female BSP retention: 0.8% Gross Pathology: Two slightly raised, white, subcapsular foci, 1.5 mm. and 1 mm. in diameter, in the kidney. 58 Histopathology: Liver - essentially normal. Kidney - foci of lymphocytes, plasma cells and numerous eosinophiles in the cortex adjacent to the capsule; focal area of interstitial infiltration with lymphocytes. Pituitary - one large and many small cysts in pars nervosa. DOG 6188 Male BSP retention: 0.8% Gross Pathology: White, firm.mass, 3 cm. in diameter, on posterior border, upper 1/3 of spleen. HistOpathology: Liver - essentially normal. Tonsil - mild acute tonsillitis. Prostate - focal subacute prostatitis. Spleen - lymphocytoma. DOG 6189 Male BSP retention: 0.8% Gross Pathology: No gross lesions. Histopatholggy: Liver - essentially normal. Submandibular lymph node - mild hemorrhage in medulla. DOG 6190 Female BSP retention: 0.8% 59 Gross Pathology: No gross lesions. Histgpathology: Liver - essentially normal. Kidney - five circumscribed, spherical foci, randomly distributed in cortex, ranging from acute to chronic reaction, resulting from larval migration - remnants of a larva in one focus. Pancreas - small focus of densely packed eosinophiles, lymphocytes, fibroblasts and a few macrophages with a surrounding zone of pro- liferating capillaries in the peripancreatic connective tissue. DOG 62A9 Female BSP retention: 0.8% Gross Pathology: Marginal fibrosis and numerous small red infarcts, 2-3 mm. in dia- meter, irregularly scattered along the posterior border of spleen. Histopathology: Liver - essentially normal. Spleen - hemorrhages in some Malpighian corpuscles. Kidney - larval granulomas. DOG 6250 Female BSP retention: 1.5% Gross Pathology: No gross lesions. Histopathology: All tissues essentially normal. 60 DOG 6251 Male BSP retention: 0.8% Gross Pathology: Slight petechiation of mucosa of urocyst; marginal red infarcts in spleen; mild roundworm infection. Histopathology: Liver - essentially normal. Spleen - small hemorrhages in several of the Malpighian corpuscles. Prepuce - mild follicular balanoposthitis. DOG 6A68 Male BSP retention: 0.8% Gross Pathology: Liver dark red in color; two small uroliths in urocyst; area of sub- peritoneal edema, 7.5 cm. long, in lumbar region just posterior to right kidney. Histopathology: Liver - essentially normal. Spleen - capsular siderosis; mild lymphocytic hypoplasia. Kidney - infrequent sudanophilic inclusions in glomeruli; fat in convoluted tubules in several areas. DOG 6A69 Male BSP retention: 0.8% Gross Patholggy: No gross lesions. Histopathology: Liver - essentially normal. 61 Lymph nodes - Section 1: acute eosinophilic lymphadenitis; Section 2: moderate infiltration of medulla with neutr0philes and eosinophiles and mild reticulodendothelial proliferation; Section 3: accumulation of eosinophiles in medulla: Section A: erythrocytic phagocytosis. Spleen - capsular siderosis; mild lymphocytic hypOplasia. Prostate - scattered foci of subacute prostatitis; several areas of vicarious lymph node formation with definitive germinal centers (mitotic figures numerous). Lung - single focal area of eosinophilic infiltration with pro- 1iferation of septal cells; several foci of mild hemorrhage; fibri- nous exudate in alveoli. 62 APPENDIX 2 CARBON TETRACHLORIDE DOGS DOG 666A Beagle Cross Female BSP retention: 21.8% Gross Pathology: Emaciated; two circumscribed, necrotic foci, 3.0 cm. in diameter, one on the buccal surface of each cheek, covered by a fibrinonecrotic membrane; focal hepatization and generalized edema of the lungs; liver yellowish brown with irregular mottling (nutmeg appearance); spleen small and pale. Histopathology: Liver - severe centrolobular degeneration characterized by disruption of cordal architecture with vacuolization of the hepatocellular cytoplasm, bizarre nuclei, occasional necrOtic foci and congestion involving 2/3 of the lobule; minimal inflammatory response; peri- pherolobular cloudy swelling and frequent vacuolization; diffuse congestion; severe centrolobular fatty metamorphosis. Lung - acute focal bronchopneumonia and edema. Kidneys - cloudy swelling of the convoluted tubules; vacuolization of the collecting tubules; numerous casts. Lymph nodes - medullary edema and lymphoid hyperplasia. DOG 6796 Beagle Cross Male BSP retention: 0.8% £1038 Pathology: IMarked obesity; multiple, elevated red or gray nodules, 2-3 cm. in (diameter, on the posterior border of the spleen; thyroids enlarged ‘ 'ar , ‘ . ,A“'J‘- Fig. 1. Liver. Dog 666A. Carbon tetrachloride toxicosis. Severe centrolobular congestion, disruption of cordal architecture, and pycnotic nuclei; periphrolobular fatty metamorphosis, cloudy swelling, and focus of regenerating hepatic cells. XAOO. his. ' "a C (4-. ‘5 \:\ ‘0. L‘. 6A and gray on cut surface. Hispgpathology: Liver - generalized hepatocellular swelling with distortion of the cells in several areas; frequent mild to moderate jproliferation of fibroblasts and infrequent lymphocytic cuffing at the central veins; generalized bile stasis in the small bile canaliculi around the central veins; numerous individual cells with sudanophilic inclusions, centrolobularly. Spleen - lymphoid hyperplasia with many of the germinal centers re- placed by epithelioid cells or small hemorrhages; focal capsular hemorrhage; abundant pigment, free and phagocytized. Thyroid - unilateral, diffuse subacute thyroiditis. DOG 7071 Tan Mongrel Male BSP retention: 3A.O% Gross Patholggy: Numerous, small, white scars or bullae and several small hemorrhages on both lungs; spleen rough with multiple marginal white nodules; moderate ascites; numerous white subcapsular scars, 2 mm. in diameter, in both kidneys; gastric mucosa congested; hepatic lymph nodes juicy and dark red in color; mucosa of duodenum and anterior segment of jejunum moderately congested; liver slightly atrophic, yellowish ‘with deeper yellowish mottling, with a finely pebbled appearance, cuts with resistance; Cholecyst frosty in appearance on.mucosal surface; fine petechiae in neck of urocyst. Histopathology: Liver - distinct lobulation delineated by early perilobuler fibrosis; 65 hepatocellular necrosis with hemorrhage - the latter predominating in extent over the fibrotic reaction; slight infiltration with lymph- ocytes, macrophages and occasionally neutr0philes; mild proliferation of the endothelium; occasional new bile duct formation; all hepatic cells unusually acidophilic; cordal architecture deranged with dila- tation of sinusoids and bile canaliculi; several scattered areas of phagocytized pigment (probably hematogenous); scattered foci of re- generating hepatic cells. Hepatic lymph node - medullary sinuses dilated and filled with erythrocytes and erythrophagocytotic macrophages; cortex slightly atrophic with areas of phagocytized pigment similarly found in several large trabeculae. Mesenteric lymph node - moderate lymphoid hyperplasia. Stomach - mild congestion of mucosa. Spleen - capsular siderosis; abundant free and phagocytized hemato- genoid pigment; increase in stromal cells of red pulp. Urocyst - moderate hyperemia in proximal part of submucous layer. Kidney - abundant intracellular pigment in tubule cells; abundant acidophilic and basoPhilic amorphous material in lumens; many glo- meruli contained acidOphilic granular material. Cholecyst - adherent polychromic, fibrinoid material on the mucosa. DOG 7072 Black and Gray Mongrel Female BSP retention: 29.8% Gross Pathology: Two large hemorrhages on left suprapharyngeal lymph node; marked congestion of urethra; vagina congested; duodenal mucosa congested; DC ‘1'. 66 liver mildly congested and with very distinct interlobular septa; Cholecyst wall markedly edematous; gastric mucosa congested. Histopathology: Liver - microscopic picture primarily that of degeneration; glycogen laden cells especially prominent periportally; focal hemorrhage with residumm of hematogenoid pigment; slight increase in periportal fibrosis; centrolobular hemorrhage, mild fibrosis and pigment accumu- lation (chiefly in Kupffer cells); scattered areas of interlobular fibrosis in perilobular connective tissue; inflammatory reaction scant and consisting primarily of lymphocytic infiltration; cloudy swelling and acidophilia of remaining hepatic cells; disruption of cordal architecture from dilated sinusoids and bile canaliculi. Spleen - capsular siderosis at hilus; abundant pigment, free and phagocytized. Stomach - mild congestion of mucosa; mild edema Just below the mucous glands. Cholecyst - marked intra-adventitial edema. Kidneys - abundant amorphous material in lumens of tubules; mild hypertrophy of Bowman's capsule. Suprapharyngeal lymph node - single focal hemorrhage in cortex. Hepatic lymph node - moderate medullary hemorrhage. Spleen - splenic corpuscles depleted; lymphoid exhaustion in parenchyma. Urocyst - marked, distinct, hyperplasia of fibroblasts in submucous layer; luminal surface of transitional epithelium flattened and with a thin hyperkeratotic band. 67 DOG 7073 Tan Mongrel Female BSP retention: 25.6% Gross Pathology: Moderate ascites; liver small, uneven, with numerous, almost clear, elevated nodules on the surface - yellow and red mottling - firm and cuts with resistance; hepatic lymph node congested; edema of the mesentery near the hilus of the liver and involving the pancreas. Histopathology: Liver - distinct lobulation from perilobular fibrosis, and vascular dilatation and hemorrhage in interlobular connective tissue; diffuse glycogen infiltration involving practically the entire lobule leaving only an incomplete border of regenerating or albuminous degenerating hepatic cells; frequent bile duct proliferation not necessarily con- fined to the islands of Glisson; inflammatory reaction scant and primarily lymphocytic; abundant phagocytized hematogenoid pigment in areas of congestion and hemorrhage; moderage widespread fibrosis. Pancreas - moderate peripancreatic edema involving also the inter- lobular connective tissue. Cholecyst - moderate hyperplasia of the mucosal epithelium. Kidneys - marked glomerulo-tubular nephrosis. 68 APPENDIX 5 DRUG TOXICOPATHOLOGY DOGS - BEAGLES STREPTOVARICIN 50 mg.[kg. DOG 592k Male BSP retention: 2.2% Gross Pathology: Moderate whipworm infection. Histopathology: No significant lesions. DOG 5925 Male ESP retention: 2.2% Gross Pathology: Mild, focal congestion of the urocyst; localized area of petechiation in mucosa of the gastric cardio-fundic junction. Histopathology: Liver - essentially normal. Prostate - subacute focal prostatitis. Pituitary - solitary cyst in pars anterior. DOG 5926 Male ESP retention: h.8% Gross Pathology: Hepatization of left diaphragmatic lobe of lung; moderate whipworm infestation. Histopathology: Liver - essentially normal. n «- 69 Lung - subacute bronchopneumonia. lOO mg./kg. DOG 5927 Male BSP retention: 5.6% Gross Pathology: Mild, widely distributed, petechiae in ileal mucosa. Histopathology: No microscopic lesions (section from ileum normal). DOG 5928 Male BSP retention: 1.5% Gross Pathology: Scar on left apical lobe of lung. Histopathology: Liver - excessive number of'micro-foci of lymphocytes, not vascularly associated. Lymph node - marked histiocytic erythrOphagocytosis. Adrenal - multiple, discrete, old hemorrhages in zona reticularis. Lung - multiple foci of cicatrization and two areas of hyperplasia of metaplastic alveolar epithelium; adjacent pleura thickened by fibrosis. Kidney - several inear foci of interstitial infiltration with lymph- ocytes and plasma cells in the vicinity of the thin loops. DOG 5929 Male BSP retention: 9.0% Gross Patholggy: Phlegmon, approximately 10 mm. in length, ventral aspect of neck; 70 cervical lymph nodes enlarged and edematous; suprapharyngeal lymph nodes greatly enlarged and edematous; multiple ecchymotic hemorrhages in Cholecyst; moderage hyperemia of gastric fundus; several patches of petechiae in duodenum near pylorus; mild whipworm infection. Histopathology: Liver - moderate pavementing in and perivascular infiltration of neutr0philes at the central veins. Cholecyst - massive hemorrhage in the muscle layer in two areas and perimuscular coat in one area. Lymph node - diffuse medullary edema. Pituitary - two large cysts in pars anterior. Skin - acute suppurative dermatitis and phlegmon of the subcutis. Thyroid - large areas of follicles with hypertrophic epithelial cells but with an occasional normal follicle within the areas. TOLBUTAMIDE :0 Ins-1kg. DOG 6075 Male BSP retention: 0.8% Gross Pathology: Entire length of external surface of small intestine reddish tan in color; liver pale tan in color. Histopathology: Liver - essentially normal. Lymph node - focal deposition of hematogenoid pigment and numerous eosinophiles in the sinuses. Pituitary - pars anterior polycystic. 71 Prostate - focal chronic prostatitis. Stomach - amorphous bas0philic material probably calcium surrounded by a thin wall of fibroblasts at irregular intervals mid-zonally in mucosa . DOG 6076 Female BSP retention: 0.8% Gross Pathology: Mild pneumonia of antero-ventral border, right diaphragmatic lobe of lung; liver yellowish in color with focal areas of fine congestion on the surface. Histopathology: Liver - no abnormalities in sections examined. Spleen - capsular siderosis. DOG 6077 Female BSP retention: 1.5% Gross Pathology: One focal area of red hepatization, 5 cm. in diameter, and a diffuse area of induration and compensatory emphysema in right diaphragmatic lobe of lung. HistOpathology: Liver - essentially normal. Lung - subacute bronchopneumonia. Lymph node - hemorrhagic. 72 100 mg./kg. DOG 6078 Male BSP retention: 2.2% Gross Pathology: Two and a half by one cm. scar on perietal surface, ventral aspect of spleen. Histopathology: Liver - essentially normal. Prostate - chronic focal prostatitis. Spleen - focal area of trabecular hyperplasia containing small islands of congestion, phagocytosis, abundant pigment and remnants of lym- phatic follicles. Thyroid - marked hypertrophy and hyperplasia of follicular epithelium and infiltration of lymphocytes. DOG 6079 Female BSP retention: h.5% Gross Pathology: Edema of right vocal cord; mild tapeworm infection. HistOpathology: Liver — diffuse hepatocellular vacuolization (not fatty metamorphosis). Kidney - single larval granuloma. Thyroid - multiple small foci of interstitial infiltration of lymph- ocytes and plasma cells; epithelium of several follicles slightly hypertrophic. 73 DOG 6080 Female BSP retention: 1.8% Gross Pathology: Cataract, left eye; liver slightly enlarged but otherwise normal. HistOpathology: Liver - essentially normal. Thyroid - follicles small wflh hypertrophic epithelia, reduction in colloid, and.marked lymphocytic infiltration and proliferation to point of formation of germinal centers. 160 mg.[kg. DOG 6081 Female BSP retention: h.5% Gross Pathology: Spleen enlarged, congested and the capsule pebbly; hepatic lymph nodes enlarged and edematous; liver enlarged with surface uneven, yellowish.mahogany in color and containing yellowish white foci throughout; cholecyst filled with viscid granular bile. Histopathology: Liver - widespread micro-foci of distorted hepatic cells, lymphocytes and fibrOblasts or of a few necrotic hepatic cells and infiltrating neutr0philes; clusters of lymphocytes throughout the section with dense accumulations around the large bile ducts; hepatocellular vacuolization; peripherolobular fatty metamorphosis. DOG 6082 Female BSP retention: 1.5% Gross Pathology: Moderately widespread, white, raised, subcapsular foci, 2 mm. in Fig. 2. Liver. Dog 6081. Tolbutamide toxicosis. Focal infiltration with lymphocytes and neutr0philes and atrophy of hepatic cells. XhOO. 71; \ ..O 75 diameter, in cortex of kidney; restricted area of hyperemia and petechiation, proximal 8 cm. of ileum. Histopathology: Liver - hepatocellular swelling, otherwise lesions essentially similar to those in Dog 6081 but more neutr0philes present. Lymph node - medullary congestion. Kidney - single larval nodule. Thymus - cystic involution. Thyroid - several small foci of lymphocytic infiltration; follicles smaller than normal with columnar epithelium. DIPROPYL-ALLYLOXY-BENZOIC ACID 30 mg./kg. DOG 6191 Female BSP retention: 2.2% Gross Pathology: No gross lesions. Histopathology: Liver - a small focus of lymphocytes, angioblasts and regenerating hepatic cells; scattered pigment. Ileum - neutrophilic infiltration of Peyer's patches and focal acute enteritis. Lung - early pneumonia. Pancreas - focal acinar degeneration and occasional necrosis adjacent to or confluent with the islets. Spleen - capsular siderosis; mild to moderate hematogenoid pigment in the red pulp. 76 DOG 6192 Male BSP retention: 0.8% Gross Pathology: Translucent, hard, verrucous mass on left atrio-ventricular valve. Histopathology: Liver - essentially normal. Spleen - mild hematogenoid pigmentation. Mandibular lymph node - medullary sinuses markedly infiltrated with eosinophiles; several areas showing cords of proliferating reticulo- endothelial cells. DOG 6193 Male BSP retention: 0.8% Gross Pathology: No gross lesions. Histopathology: Liver - essentially normal. Tonsil - small hemorrhages in many of the germinal centers of the lymphoid follicles. Spleen - mild hematogenoid pigment accumulation. Adrenal - small focal hemorrhage in the zona fasciculata. 100 mg./kg. DOG 619u Female BSP retention: 0.8% Gross Pathology: Moderate roundworm and severe whipworm.infestation. 77 Histopathology: Liver - mild perivascular eosinOphilic infiltration and relative increase in eosinophiles scattered throughout the parenchyma; rectangular area, 500 x 250 microns, consisting of eosinophiles, lymphocytes and fibroblasts. Kidney - two larval nodules in cortex. Lymph nodes - moderately infiltrated with eosinophiles; one node, dense accumulation of eosinophiles in medullary sinuses (one of three sections). Small intestine - notable increase of eosinophiles in the lamina propria of all three sections. Spleen - abundant eosinophiles and relative increase in neutr0philes. DOG 6195 Female . BSP retention: 0.8% Gross Pathology: Two small, white, elevated foci in cortex of kidney; central congestion of the mesenteric lymph nodes. Histopathology: Liver - essentially normal. Mesenteric lymph nodes - sinuses packed with pigment-laden macro- phages and eosinophiles; hyperplasia of the lymphoid follicles with dense accumulations of lymphocytes in the sinuses; a few megakaryo— cytes observed; abundant hematogenoid pigment. Lymph node - medullary congestion and hemorrhage; moderate hemato- genoid pigment. Spleen - large, canalized thrombus, rich in pigment, and calcifica- 78 tion of capsule, at the hilus. DOG 6196 Male BSP retention: 0.8% Gross Pathology: No gross lesions. Histopathology: Liver - essentially normal. Prostate - purulent exudate in lumen of two alveoli. 500 mg /kg. DOG 6197 Male BSP retention: 0.8% Gross Pathology: Mild roundworm infestation. Histopathology: Liver - essentially normal. Prostate - diffuse subacute nonsuppurative prostatitis. DOG 6198 Female BSP retention: hl.2% Gross Pathology: Emaciated; generalized peripheral congestion; blood very viscid; adrenals enlarged; lungs edematous with frothy, free-flowing exudate in the trachea - partial hepatization: apical lobes and left diaphragmatic lobe, complete in: cardiac lobes, azygos lobe and right diaphragmatic lobe; stomach markedly dilated (stony), con- gested and containing a large quantity of gelatinous exudate; pancreas intensely congested; edema of the external surface of the intestines; entire tract was empty; spleen shrunken; congestion of 79 mesenteric lymph nodes; perirenal edema of right kidney and kidneys slightly congested with yellowish cast and dark cortices; liver purplish cast with yellow mottling and very fine, dark, subcapsular, foci without a further pattern of distribution; cholecyst engorged; all viscera mildly congested and faintly yellow in color. Histopathology: Liver - disruption of hepatic cords from congestion of the sinusoids, cloudy swelling peripherally and fatty metamorphosis centrolobularly. Adrenals - mild congestion. Cholecyst — severe autolysis of mucosa. Brain - dilatation and perivascular edema of the fissural veins. Kidney - mild passive congestion with tubular cytoplasm granular and vacuolated; areas of cloudy swelling. Lung - diffuse alveolar congestion; perivenous edema; mild infil- tration of neutr0philes whose distribution is diffuse and not that of typical bronchopneumonia; areas of edema and compensatory emphysema; basophilic granular material suggesting aspirated foreign material in a number of alveoli in one section. Suprapharyngeal lymph node - marked depletion of fellicles; sub- capsular sinuses stuffed with neutr0philes that also infiltrated the medullary sinuses; edema. Mesenteric lymph node - venous congestion; germinal centers of fOllicles practically abolished; proliferation of macrophages; sinuses packed with lymphocytes. Spleen - trabeculae prominent; paucity of red pulp; moderate hypo- plasia of follicles; focal hyperemia; moderately abundant hemato— genoid pigment. 80 Stomach - muscular coat thin, mucosa diminished in depth and the mucous glands dilated with secretion; parietal cells swollen and cytoplasm of chief cells disrupted. Pancreas - moderate venous congestion. Tbnsil - germinal centers replaced, centrally, by'lymphoblasts or macrophages and collagen or hyaline-like material - young and mature lymphocytes present in gland. DOG 6199 Male BSP retention: 0.8% Gross Pathology: No gross lesions. Histopathology: Liver — essentially normal. Cerebellum - focal area of encephalomalacia involving molecular layers in two apposing gyri with proliferation of microglia and astrocytes; some glial cells undergoing karyolysis as well as those in the glanular layer. Kidney - moderate, focal, subacute pyelitis. Prostate - mild, focal, subacute prostatitis. Spleen - capsular siderosis; mild pigment accumulation. Urocyst — mild, focal, subacute cystitis. PYRAZOLIDIN—B-ONE, l—METHYL-S-PHENIL 5O mg.[kg. DOG 62h6 Female BSP retention: 9.8% Gross Pathology: Liver mottled throughout (nutmeg appearance) with greenish cast 81 and increased firmness; small cyst in anterior lobe of pituitary; severe tapeworm infestation. Histquthology: Liver - diffuse, moderately intense and predominately centrolobular, deposition of hematogenoid pigment in small to large globular masses; Kupffer cells in area pigment-laden; slight increase in perivascular cells (lymphocytes and neutr0philes); commonly, hyper- trophy of endothelial cells of central veins or, less commonly, rhexis of the vein with small, confined hemorrhage; mild, centro- lobular, fatty metamorphosis; lesions involved approximately 25-35% of section. Bone marrow - abundant pigment not hemosiderin; mild decrease in myelocytes. Lung - solitary calcifying granuloma. Pituitary - small mucoid cyst in pars anterior. Spleen - moderate hematogenoid pigment accumulation; reticulum cell hyperplasia at periphery of corpuscles. DOG 62h7 Female BSP retention: 6.2% Gross Pathology: Liver mahogany colored; areas of congestion scattered throughout the small intestine; small focal hemorrhages in mucosa of urocyst. Histopathology: Liver - extensive but mild, predominantly centrolobular, accumula- tion of pigment as fine to large globules; Kupffer cells not pig- ment bearing to any significant degree; hypertrophy of small bile duct epithelium almost obliterating the lumens at the triads; 82 perivascular accumulations of lymphocytes and neutr0philes slight to normal; mild,centrolobulan fatty metamorphosis; approximately 10% of section area involved. Bone marrow - abundant pigment (not hemosiderin); relative decrease in myelocytes. Lymph node - small follicular hemorrhage; two confluent foci of eosinophiles with numerous mitotic figures in lymphocytes in the medulla. Pituitary - small mucoid cysts in pars anterior. lOQ/6O mg./kg° DOG 62h} Male BSP retention: 21.8% Gross Pathology: Emaciated; generalized muscular atrophy; edema of the subcutis; liver nutmeg in appearance, slightly bronzed and cuts with increased resistance; spleen enlarged with mottled surface and irregular areas of congestion; lymph nodes edematous; kidneys pale, friable, and with a few white subcapsular foci (B-h mm); myocardial hypertrophy and dilatation of the heart; testes atrophic; catarrhal enteritis and bile-staining of the mucosa of the small intestine; cyst in anterior lobe of pituitary; marginal consolidation (h-S mm) of the lungs. Histopathology: Liver - generalized dilatation of the sinusoids, centrolobularly especially, with atrophy of adjacent hepatic cords and fatty degen- eration and necrosis at the central vein; scattered foci of lympho- cytes and neutr0philes in the dilated sinusoids; scattering of fine 83 pigment, intra-and extracellularly, throughout and larger particles located centrolobularly - in these areas, infiltration of lympho- cytes and fibroblasts, and disorganization of reticular fibers of stroma; marked fatty metamorphosis centrally with involvement of 1/2 to 2/5 of the lobule; approximately 50% of the section area involved. Spleen - corpuscles indistinct and showing marked atrophy, paucity of lymphoid cells with frequent pycnotic nuclei, hyalinization of intercellular substance, moderate hematogenoid pigment accumulation; and areas resembling pale infarcts. Lymph nodes - loss of follicular pattern in some nodes; accumulation of hemosiderin in cortical follicles. Testis - spermatogenesis retarded chiefly at secondary spermatocyte stage; numerous mutinucleated giant cells in lumens of tubules. Pancreas - atrophy of peripheral acini. DOG 62% Female BSP retention: 10.5% Gross Pathology: Liver faintly greenish in color but mottled throughout and firm; adrenal slightly enlarged; spleen enlarged; consolidation of right cardiac lobe of lung; mild roundworm infection. Histopathology: Liver - predominantly centrolobular deposition, intra- or extra- cellular, of pigment and commonly in large droplets; in associated areas, hepatocellular atrophy, vacuolization and hydropic degener- ation and dilatation of the sinusoids; inflammatory cell infiltra- tion infrequent and consisting essentially of eosinophiles, with 8h lymphocytes and a few fibroblasts; edema around the portal and hepatic veins; 2/5 of lobule involved; approximately 50% of section area involved. Spleen - mild lymphoid hypoplasia and moderate pigment accumulation. Adrenal - moderate cortical hyperplasia. Lung - perivascular edema and hemorrhage around larger pulmonary vessels. Lymph node - pigment in follicles; erythrophagocytosis. Bone marrow - abundant pigment (not hemosiderin) and moderate myelo- cytic hypoplasia. Pituitary - mucoid cyst, 1 mm in diameter, in pars anterior. DOG 62u5 Female BSP retention: 16.2% Gross Pathology: Spleen enlarged and congested; few white subcapsular foci in both kidneys; liver congested, slightly mottled, firm and with wrinkling or pitting of the surface; pancreatic and gastric lymph nodes con- gested; small cyst in dorsum of anterior lobe of pituitary. Histopathology: Liver - lesions essentially similar to those in Dog 62hh but hepato- cellular degeneration more intense, bordering on frank necrosis, at the central vein; mild centrolobular fatty metamorphosis; approxi- mately 50% of section area involved. Spleen - congested and moderate pigment accumulation. Bone marrow - abundant pigment (not hemosiderin) and moderate myelo- cytic hypoplasia. Fig. 5. Liver. Dog 62h5. Pyrazolidin-5-one, l-methyl-5-phenyl toxicosis. Centrolobular cloudy swelling and deposition of pigment in fine and large droplets, both intra- and extracellularly. XMOO. G; 86 DOG 62in Female BSP retention: 7.0% (l:h5/hrs.) Gross Pathology: Small organized hemorrhage on tricuspid valve of heart; liver firm with dark pebbling; kidneys congested; adrenals enlarged; mucosa of stomach stained with bile; spleen congested and firm. Histopathology: Liver - passive congestion, moderate centrolobular accumulation of pigment, both fine and large particles, with marked atrophy of cord cells many of which showing degeneration of cytoplasm and necrosis; mild edema; generalized foci of fine sudanophilic material but larger and more numerous at the central vein; approxi- mately 75% of section involved. Spleen - moderate accumulation of pigment and reticulum cell hyper- plasia. Kidneys - marked congestion of glomeruli and calcific plugs in some collecting tubules in medulla. Heart - deposition of finely dispersed fat in myocardial fibers. Pituitary - small mucoid cyst in pars anterior. METHYL RESERPATE l mg.[kg. DOG 6h70 Female BSP retention: 0.8% Gross Pathology: Lateral ventricles enlarged indicating moderate hydrocephalus. Histopathology: 87 Liver - section 1: one small focus of lymphocytes, eosinophiles and fibroblasts; section 2: several triads exhibit infiltration with lymphocytes, eosinophiles, macrophages and a few plasma cells - lesions small, confined, and very mild. Kidneys - section 1: one focus of lymphocytes, with some fibro- blasts, around regenerating tubules in the cortex; section 2: two foci of lymphocytes at the junction of the medulla and pelvis. Lymph node - medulla filled with eosinophiles; localized accumula- tion of pigment in large masses, with many pigment—laden macrophages. DOG 6h71 Female BSP retention: 0.8% Gross Pathology: In estrus; mucosa of neck of urocyst congested; low breaking strength of ribs. Histopathology: Liver - essentially normal. Lymph node - medullary sinuses profusely infiltrated with eosino- philes and proliferating reticulo-endothelial cells; masses of pigment at one pole. Spleen - moderate accumulation of pigment. Uterus - degenerating endometrium. 5 m . k . DOG 6L6} Female BSP retention: 0.8% Gross Pathology: Obese; epidermis of rear paws thin; complete consolidation of 88 azygos lobe and partial consolidation of cardiac and diaphragmatic lobes of right lung; bronchial lymph nodes swollen and congested; spleen swollen and plumecolored; intracapsular cysts in both kidneys; rickets. Histopathology: Liver - essentially normal. Kidneys - mild subacute interstitial pyelitis in one and moderate subacute pyelonephritis in the other kidney; in one section, area approximately'J.mm. in diameter comprises 5 defective glomeruli and a small focus of lymphocytes and proliferating fibroblasts along the tubular tracts. Heart - very fine sudanophilic particles in tail-like conformation directly behind or in front of the nucleus of the myocardial cells involving a rather extensive area beginning at the endocardium and extending deep into the myocardium. Tonsil - mild purulent tonsillitis. Lung - section 1: scattered areas of inflammatory cells concen- trated primarily peribronchiolarly although several foci are located in alveoli — eosinophiles primary cell with macrophages secondary and lymphocytes scanty - many single eosinophiles randomly distribe uted throughout the lung parenchyma; section 2: one focus of lymphocytes, macrophages, eosinophiles and neutr0philes around a foreign body. Lymph node - subcapsular, circumscribed, eosinophilic granuloma. Spleen - marked congestion. Stomach - multiple solitary lymph nodules with active germinal centers in the mucosa. 89 DOG oueu Female BSP retention: 0.8% Gross Pathology: Thyroids slightly enlarged; spleen swollen and plum-colored; urocyst slightly congested. Histopathology: Liver - essentially normal. Lymph node - section 1: mildly hyperactive germinal centers; section 2: mild hyperplasia of follicles with mild infiltration of medullary sinuses peripherally (near the cortex) with eosinophiles. Tonsil - mildly hyperactive germinal centers. Thyroid - lymphocytic proliferation, primarily at the periphery of the organ but moderately abundant in the central portion, consisting almost exclusively of young lymphocytes with only a few scattered concentrations of mature cells; follicles generally not affected except in areas of lymphocytic proliferation causing them to under- go pressure atrophy leading to complete obliteration. Spleen - marked congestion and mild hypoplasia of Malpighian corpus- cles. Adrenal - excessive sudanophilic inclusions in zona glomerulosa. 15 mg./kg. DOG 6h65 Male BSP retention: 0.8% Gross Pathology; Sacrificed i2 extremis; emaciated; generalized venous congestion; fundament smeared with tan fecal material; liver slightly shrunken with yellowish cast and congested areas; heart slightly enlarged 9O flabby; pancreas congested; spleen smooth and congested; mild infestation with whipworms; irregular congestion in extreme anterior segment of duodenum; linear congestion of colon; muscles atrophic; posterior parathyroids enlarged. Histopathology: Liver - essentially normal. Colon - mild venous dilatation in mucosal vessels at apex of several plicae. Lymph node - sinuses stuffed with reticulo-endothelial cells. Pancreas - pancreatic islets appear smaller than normal. Prostate - chronic interstitial proliferation. Spleen - intense congestion with moderate follicular hypoplasia. Tonsil - acute purulent tonsillitis. DOG 6h66 Female BSP retention: 0.8% Gross Pathology: Excoriation of rear foot pads; epidermis of forepaws thin but intact; hind parts matted with dried, black, fecal material; diarrhea; vulva relaxed; spleen smooth, large, swollen and plum- colored; pieces of paper carton in stomach; slight congestion of mucosa of urocyst; rickets. Histopathology: Liver - essentially normal. Kidneys - heavy, almost exclusively intracellular, deposition of orange pigment in convoluted tubules of both kidneys - collecting tubules and glomeruli spared; several glomeruli swollen with clumps of sudanophilic material within. 91 Lymph node - mild follicular hypoplasia; extensive erythrophago» cytosis. Spleen - marked congestion; moderate hypoplasia of Malpighian corpuscles. 92 APPENDIX u DOGS WITH CANINE DISTEMPER DOG 655h—1 Black Cocker Cross Female BSP retention: 0.8% Clinical: Temperature, lO5.h°F; serous nasal discharge; slight ocular dis- charge; sclera injected; induced cough; slightly increased vesicular murmur. Gross Pathology: Pulmonary lymph nodes congested; spleen pale and thick but of normal length and width. Histopathology: Liver - sinusoids slightly dilated; hepatic cords slightly atrophic. DOG 655h-2 Balck and White Collie Cross Male BSP retention: 2.5% Clinical: Temperature, lO5.8°F; profuse mucopurulent nasal discharge; harsh cough. Gross Pathology: Spotty red hepatization, right cardiac lobe; multiple,fine, white foci in the kidneys; yellowish cast to the liver; no food in stomach; intestines empty. Histopathology: Liver - frequent individual cells undergoing karyolysis adjacent to the central vein and mid-lobularly; scattered Kupffer cells pigment- laden; intranuclear salt inclusions conspicuous near central veins. 9‘) DOG 6554-5 Brindle Mbngrel Male BSP retention: h.8% Clinical: Temperature, th.h°F; profuse mucopurulent nasal discharge; sclera injected; induced cough. Gross Pathology: Diffuse pulmonary congestion and hepatization, right cardiac lobe; liver congested, but with large yellowish areas; mucosa of urocyst congested; stomach and intestines empty. Histopathology: Liver - dilatation and congestion of sinusoids centrolobularly with atrophy of adjacent hepatic cords and small nidi of proliferating Kupffer cells; hepatic cells undergoing cloudy swelling and fatty metamorphosis in more severely affected areas; throughout sections, infrequent cells exhibiting karyolysis or pycnosis of nuclei in areas adjacent to central veins. Spleen - splenic lymphoid follicles indistinct and germinal centers practically abolished; mild, diffuse infiltration with neutr0philes and reticulo-endothelial cells prominent. Lung - acute nonsuppurative bronchopneumonia. Kidney - mild dilatation of the collecting tubules. DOG 6397-5h9 Beagle Male BSP retention: CL8% Clinical: Temperature, 105.69F; profusefipurulent ocular and nasal discharge; increased vesicular murmur; sclera injected. Gross Pathology: Fig. A. Liver. Dog 655h-5. Canine Distemper Complex. Centrolobular dilatation and congestion of the sinusoids and atrOphy of adjacent hepatic cords. The fine pigment in the cells is artifact. XAOO. 95 Nares occluded with dried exudate; mucopurulent exudate in conjunc- tival sac; gastrointestinal tract empty. Histopathology: Liver - sinusoids slightly dilated in some but not all lobules at central veins and infrequent individual hepatic cells exhibiting karyolysis in some areas; mild cloudy swelling; few scattered Kupffer cells are pigment-laden. Lung - healing pneumonia with fibrosis prominent. Stomach - gastric pits distended with mucus. Prostate - mild interstitial fibrosis. Turbinates - mild purulent rhinitis. DOG 6557-h Black Cocker Cross Male BSP retention: 0.8% Clinical: Temperature, 105.h°F; harsh tracheal sounds; sclera injected; purulent ocular discharge; serous nasal discharge; sneezing. Gross Pathology: Emaciated; tonsils slightly congested; mandibular and suprapharyn— geal lymph nodes enlarged, edematous and irregularly congested; nasal mucosa congested and covered with purulent exudate; liver congested; roundworms, tapeworms and whipworms in intestines; gastrointestinal tract devoid of ingesta. Histopathologyi Liver - mild passive congestion and cloudy swelling. Lymph nodes - several cortical hemorrhages; germinal centers in- distinct and cortex and medulla solidly cellular with lymphocytes and proliferating reticulo-endothelial cells. 96 Spleen - acute splenitis. Nictitating membrane - multinucleated giant cells present. Trachea - a few intracytoplasmic inclusion bodies. DOG 6557-5 Black and White MOngrel Male BSP retention: 1.2% Clinical: Temperature, lO5.l°F; slight purulent ocular discharge; sclera in- jected; moderate purulent nasal discharge; mild diarrhea. Gross Pathology: In poor flesh; hemorrhages in left tonsil; consolidation of part of right cardiac and posterior edge of right apical and entire left apical lobes; liver congested; congestion of prostate; small varicee in urocyst; gastro-intestinal tract empty; light infestations with hookworms, tapeworms and whipworms. Histopathology: Liver — cloudy swelling of hepatic cells and scattered Kupffer cells pigment-bearing. Lung - subacute purulent bronchopneumonia. Urocyst - a solitary, diffuse hemorrhage in submucosal layer. Kidney - bilateral subacute pyelitis, mild in one kidney and moderate in the other. Prostate - focal subacute prostatitis. DOG 6557-6 Beagle Female BSP retention: 0.8% Clinical: Temperature, 105.h°F; mucopurulent nasal discharge; cough; sclera 97 injected; dull areas on auscultation; mucoid diarrhea. Gross Pathology: In fair flesh; mucopurulent nasal exudate; mucoid exudateimitrachea; consolidation of lower half of right cardiac lobe of lung, postero- ventral portion of left apical lobe and entire left cardiac lobe; one mature Dirofilaria immitis in right ventricle of heart and one small dirofilaria in pulmonary artery; solitary diffuse hemorrhage in urocyst; congestion of gastric mucosa; multiple, subcapsular, White foci, 2 mm in diameter, in both kidneys; mild infestation of roundworms and whipworms; nasal cavity occluded with mucopurulent exudate and congestion of mucosa. Histopathology: Liver - scattered pigment—bearing Kupffer cells in all sections; one small oval area of fibrosis in one section. Lung - diffuse, acute, purulent bronchopneumonia. Trachea - intracytoplasmic inclusion bodies. Spleen - acute nonsuppurative splenitis. Kidneys - unilateral subacute nonsuppurative pyelitis and one small larval granuloma . Urocyst - mild acute cystitis with mild erosion of the mucosa; intracytoplasmdc inclusion bodies distinct. Lymph node - subacute lymphadenitis. Membrane nictitans - subacute proliferative conjunctivitis. Stomach - mucosa flattened and mucous glands abbreviated. DOG 6357-7 Black and Brown Mongrel Male BSP retention: 0.8% Clinical: 98 Temperature, lO2.l°F; sclera slightly injected; dried exudate on external nares; cough. Gross Pathology: Liver paler than normal; multiple, diffusely distributed small white foci, located subcapsularly and in cortex of kidneys; gastro-intes- tinal tract empty except for small tapeworms, a few roundworms and many whipworms. Histopathology: Liver - lst section: one fine focus of lymphocytes, fibroblasts and eosinophiles; one focus, irregular in outline but sharply circum- scribed, composed of lymphocytes, eosinophiles, fibroblasts and a few neutr0philes surrounding several new capillaries; lumen of adjacent small vein completely obliterated by proliferating endo- thelium and circumscribed by thick layer of lymphocytes, eosino- philes, degenerating hepatic cells, macrophages and new capillaries; increased perivascular cuffing; approximately 1/500 of total section involved. 2nd section: one area containing multiple adjacent foci, either circumscribed of spreading, consisting of focal necrosis, infiltration of lymphocytes, eosinophiles and macrophages with few neutr0philes and proliferation of fibroblasts; approximately'l/ho of total section involved. Kidney - moderate bilateral subacute pyelonephritis; larval granu- loma; several areas of chronic nephritis. Membrane nictitans - subacute conjunctivitis. DOG 6357-8 Black and Tan Mbngrel Female BSP retention: 6.2% (mild hemolysis) 99 Clinical: Temperature, lO5.6°F; mucopurulent ocular and nasal discharge; sclera injected; serosanguinous exudate from mouth (severe bilateral necrotic stomatitis); emaciated. Gross Pathology: Emaciated; fetid, sanguinopurulent exudate on the lips and circum- oral hair; at angle of the jaws, on the buccal surface of the cheek, an ulcerated area (2h mm in diameter) with raised, ragged edges and with fibrinonecrotic membrane; manidibular lymph nodes muddy gray in color and irregularly congested; heart enlarged, flabby and with thin myocardium; irregular areas of atelectasis, gray in color, in cardiac lobe of lung; slight yellowish discoloration of liver; blood viscid; gastro-intestinal tract devoid of ingesta. Histopathology: Liver - loss of cord structure with loss of cell architecture and karyolysis common,especially centrolobularly; frequent vacuolization of hepatic cell cytoplasm; many solitary polymorphsin arisinusoids; clusters of two or three Kupffer cells frequentztithe same location; peripholobularly, cloudy swelling and vacuolization; lesions present throughout section. Spleen - acute splenitis. Lymph node - lymphocytic depletion; node solidly cellular. Kidneys - mild bilateral pyelitis. DOG 6557-9 Black Cocker Cross Female BSP retention: 9.8% Clinical: Temperature, 105.0°F; emaciated; sibilant rales on auscultation; lOO mucopurulent ocular and nasal discharge; sclera injected; pupils dilated; diarrhea, with fluid, green, blood-tinged feces; cough. Gross Pathology: Emaciated; external nares occluded with dried exudate; mucopurulent exudate in conjunctival sac; red hepatization of lower 1/5 of right apical and diaphragmatic lobes of lung, entire right cardiac lobe and lower third of all left lobes; pulmonary lymph nodes enlarged and congested; yellowish color to entire liver with areas of more distinct yellowish mottling; kidneys slightly congested; nasal mucosa congested; tonsil congested; gastro-intestinal tract empty. Histopathology: Liver - multiple lesions throughout section, consisting of two ‘distinct entities: a) at the central vein, rhexis of the vein with hemorrhage, congestion of adjacent sinusoids, accumulation of hematogenoid pigment and degeneration of adjacent hepatic cells; b) well demarcated, approximately round or oval in shape, foci of necrosis with infiltration of neutr0philes and macrophages and hemorrhage, not necessarily centrolobular; approximately fifty percent of the total area of sections involved with both types of lesions. Urocyst - intracytoplasmic inclusion bodies. Tonsil - depletion of lymphoid elements; replacement of germinal centers by epithelioid cells. Bronchial lymph node - depletion of mature lymphoid elements; pro- liferation of reticulo-endothelial elements. Lung - diffuse, acute, purulent bronchopneumonia. Spleen - lymphocytic depletion. Fig. 5. Liver Dog 6557-9. Canine Distemper Complex. Focal necrosis with infiltrating lymphocytes, macrophages, and neutr0philes. XAOO. lOl . a. d. f p _- i I , ‘~ I c v o .. I ' \ I \ . C A .D ‘ § 6 Q ~ . i s Q J ‘ . u . 4 . I I! .Q. ' a n u u I . .0 O . . v. o it A 0 . . . - . . p . x . 4 . v tn I n .. . . . ‘ I C t . I u . f . ' . 0 n 1 I n I D .3. a. a. .\ ..a 4 WV \ d‘ I. 01M \. :f. 3. J... 31 in .\ I!“ ..?\f It L . a. c. a 2x ., a... \ JG, . , .s.‘ 0 .fl. . H. . 1...}. . ~ 0 —7 I. C . u I U I V o . .. Hues J/G. \ 102 Suprapharyngeal lymph node - depletion of lymphocytes, mild hemorrhage and edema in medulla; proliferation of reticuloendo- thelial elements. Submandibular lymph node - medullary hemorrhage and edema; loss of follicular architecture and replacement of several germinal centers by epithelioid cells. I Nasal passage - mild acute purulent rhinitis. DOG 6557-10 Black and Brown MOngrel Male BSP retention: 5.9% Clinical: Temperature, lO5.l°F; mucopurulent ocular and nasal discharge; tonsils congested; coughing; diarrhea with tan, mucoid feces. Gross Pathology: Dried exudate at medial canthi; mucopurulent discharge from external nares; heart pale and flabby; multiple, small, white foci, both subcapsular, and deep in the cortex of the kidneys; heavy infestation with ascarids and tapeworms, and a few whipworms; pancreas slightly enlarged with gelatinous, depressed, grayish areas; liver enlarged, diffusely mottled with yellow, red and tan areas - lobules distinct throughout with circular area l-l/2 cm. in diameter, yellow in color, granular and friable, on the parietal surface of the right central lobe near the hilus of the diaphragm and a lesion similar = but approximately .5 cm. in diameter on the parietal surface of the the left lateral lobe; wall of gall bladder slightly thickened and edematous; hepatic lymph node congested, enlarged and edematous. Histopathology: Liver - frequent rhexis of the central veins with mild hemorrhage 105 and congestion concomitantlywith hypertrophy of the venous endothe- lium and degeneration of the adjacent hepatic cells; multiple small foci of proliferating endothelial cells, fibroblasts and infrequent neutr0philes or eosinophiles; several sections having three to four larval granulomas, one containing an entrapped larva. Approximately 1/2 of section areas involved. Urocyst - intranuclear inclusion bodies in epithelial cells. Pancreas - diffuse, subacute pancreatitis. Kidney — two larval granulomas in cortex; edema of the renal crest and frequent cellular casts in lumens of collecting tubules in this area. Heart - single larval granuloma in myocardium. Nasal passage - acute rhinitis. Suprapharyngeal lumph node - depletion of lymphocytes; sinuses packed with epithelioid cells; germinal centers replaced by epithee lioid cells. Cholecyst - edema of the wall. Spleen - acute splenitis; lymphoid hyperplasia and depletion of mature cells. Tonsil - depletion of lymphoid elements; many germinal centers re- placed by epithelioid cells. Hepatic lymph node - essentially same as tonsil; sinuses edematous and contain epithelioid cells in moderately large numbers. Membrana nictitans - subacute conjunctivitis. Lung - early focal acute pneumonia. DOG 6558-a Beagle Male BSP retention: 2.2% Fig. 6. Liver. Dog 6557-10. Canine Distemper Complex. Rhexis of the central vein and centrolobular congestion; adjacent hepatic cord cells are atrophic and vacuolated. thO. 101+ 105 Clinical: Temperature, 102.h°F; mucopurulent ocular and nasal discharge; soft, painful cough; increased.vesicu1ar murmur and areas of dullness on auscultation; diarrhea; depression; sick for one week. Gross Pathology: Purulent discharge in nasal cavity; purulent material expressed from bronchi; bilateral red and gray pulmonary hepatization; apical and cardiac lobes adherent; bronchial lymph nOdes enlarged and congested; pancreatic lymph node hemorrhagic; gastrointestinal tract empty except for a few small roundworms in small intestine. Histopathology: Liver - randomly distributed, relatively few, very fine nidi of predominantly neutr0philes but occasionally lumphocytes and fibro— blasts and, in several areas, perivascular cuffing with neutr0philes; one large focus of periductal infiltration with eosinophiles, lymph- ocytes, a few macrophages, and fibroblastic proliferation. Lung - diffuse, acute bronchopneumonia and pleuritis. Urocyst - edema and congestion of the submucous layer. Membrana nictitans - subacute conjunctivitis. Spleen - germinal centers frequently replaced by epithelioid cells; a focus of capsular hemorrhage. Lymph nodes - depletion of lymphoid elements. DOG 6558-b Beagle Male BSP retention: 0.8% Clinical: Temperature, lO5.h°F; mucopurulent ocular and nasal discharge; sclera injected; left Harderium gland enlarged; increased vesicular 106 murmur; depressed. Gross Pathology: In good flesh; slight yellowish discoloration of liver; Harderian gland of left eye enlarged; patchy pneumonia of both apical lobes, red hepatization involving 2/5 of right cardiac lobe along ventral aspect. Histopathology: Liver - each section contained several very small foci of lymphocytes and fibroblasts. Lung - acute purulent bronchopneumonia. Kidney - unilateral subacute interstitial nephritis. DOG 6558-c Beagle Male BSP retention: 1.5% (slight hemolysis) Clinical: Temperature, 105.0°F; tonsils congested but not appreciably enlarged; thick mucopurulent ocular and nasal discharges; slightly increased pulmonary lung sounds; sclera moderately injected. Gross Pathology: In good flesh; small patches of pulmonary consolidation, ventral areas of both apical and cardiac lobes; gastrointestinal tract empty. Histopathology: Liver - slight dilatation of the sinuoids at central vein with very small, scattered foci of lymphocytes, few neutr0philes and an occa- sional plasma cell or fibroblast. Stomach - intranuclear inclusion bodies in epithelial cells. Eyelid - mild subacute conjunctivitis. 107 Nasal passage - mild acute rhinitis. Membrana nictitans - mild subacute conjunctivitis. Tonsil - mild acute tonsillitis. Spleen - hemorrhage in the germinal centers of the splenic follicles. Lymph node - mild lymphocytic depletion and moderate amounts of pigment. DOG 65hh-l Beagle Female BSP retention: 1.8% Clinical: Temperature, lOl.l°F; very emaciated; anorexia; sclera injected; mild keratitis with slight serous ocular discharge from right eye; mucopurulent nasal discharge; pulmonary sounds indefinite. Gross Pathology: Body fat depleted; subcapsular, minute white foci in the cortices of the kidneys; partial mid-lobular consolidation of right apical and cardiac lobes of the lung; slight yellowish discoloration of liver; linear, ecchymotic, multiple hemorrhages in the urocyst; heavy whipworm infestation. Histopathology: Liver - no significant lesions. Kidney - single larval granuloma. Lung - diffuse, acute bronchopneumonia; one bone spicule deep in lung parenchyma(heterotopic osteogenesis). Urocyst - early acute cystitis. Bronchial lymph node - cortical atrophy; abundant pigment. Suprapharyngeal lymph node - abundant pigment both free and phagocy- tized. 108 Tonsil - mild acute tonsillitis. Spleen - mild acute splenitis; abundant pigment. DOG 65hh-2 Springer Cross Male BSP retention: 0.8% Clinical: Temperature, lO2.5°F; sclera moderately injected; mucopurulent nasal discharge; induced cough; bilateral increased vesicular murmur and area of dullness over right lung. Gross Pathology: In good flesh; pancreas congested; few roundworms in intestine; cryptorchidism, right side. Histopathology: Liver - Kupffer cells frequently pigment-laden. Pancreas - venous congestion. Spleen - marked lymphocytic depletion; infiltration of red pulp with neutr0philes. Testis - atrophy and tubular degeneration. Tonsil - moderate lymphocytic depletion. Prostate - mild subacute interstitial prostatitis. Lung - interalveolar congestion; small foci of subacute pneumonia confined to perivascular areas. DOG 65h5-5 Black, Long Haired Mongrel Male BSP retention: h.0% Clinical: Temperature, lO5.h°F; slight mucopurulent ocular discharge; profuse mucopurulent nasal discharge. Gross Pathology: 109 Two small (5 mm) subcapsular plum-colored areas in the cortex of left kidney with one focus extending into medulla; several slightly raised plum-colored areas in diaphragmatic lobes of lungs; testes and prostate extremely small for size of dog; moderate whipworms infestation; pituitary slightly enlarged and congested. Histopathology: Liver - small, rather widely separated, lesions consisting of centers of necrosis or degeneration surrounded by slight hemorrhage, fibrin, loosely arranged lymphocytes, neutr0philes, and a few macrophages; centrolobularly, moderate dilatation of sinusoids; frequent individual neutr0philes occupying sinusoids throughout the section and occasional mild perivascular cuffing with these cells; approximately 1/55 of section involved. Spleen - moderate lymphocytic depletion; acute splenitis. Mesenteric lymph node - abundant pigment; germinal centers indis- tinct; sinuses filled with macrophages. 1 Prostate - juvenile. Testis - juvenile. Kidney - bilateral mild acute pyelitis; focal subacute interstitial nephritis, left kidney. Lung - congestion and hemorrhage. Pituitary - venous congestion of pars anterior. DOG 65h5-h Beagle Cross .Male BSP retention: 1.5% Clinical: Temperature, lO5.h°F; mucopurulent nasal and ocular discharge; sclera injected; induced cough. 110 Gross Pathology: Lymph nodes at thoracic inlet enlarged and congested; two adult 2, immitis in right ventricle of heart. Histopathology: Liver - individual neutr0philes in the sinusoids; mild generalized cloudy swelling; scattered Kupffer cells pigment-laden. Kidneys - bilateral, moderate subacute pyelitis. Prostate - mild, focal subacute interstitial prostatitis. Spleen - abundant pigment, free and phagocytized. Submandibular lymph node - lymphocytic depletion; folliclesenropic; medullary hemorrhage. Tonsil - lymphocytic depletion. Suprapharyngeal lymph node - lymphocytic depletion. DOG 65h5-5 Black Cocker Male BSP retention: 0.8% Clinical: Temperature, lO2.h°F; emaciated; mucopurulent ocular and nasal dis- charge; left tonsil swollen and congested; induced cough; dull areas on auscultation of lungs. Gross Pathology: Few scattered hemorrhages in cecal mucosa. Histopathology: Liver - Kupffer cells frequently pigment-laden. Kidney - two linear foci of subacute interstitial nephritis. Lung - healing pneumonia (healing by resolution). Testicle - early tubular degeneration. Eyelid - focal subacute blepharitis. lll Urocyst - mild congestion of the submucous layer. Membrana nictitans.- mild subacute conjunctivitis. DOG 6hh5-6 Beagle Male BSP retention: 2.2% Clinical: Temperature, lO5.0°F; congested sclera; abundant mucopurulent nasal discharge; feces semisolid; harsh rales on auscultation; soft, moist cough when excited. Gross Pathology: Nasal mucosa markedly congested with adherent thick mucopurulent exudate; thick mucopurulent exudate in trachea and larynx; right bronchial lymph nodes slightly enlarged, rough with red mottling; liver pale with yellowish cast; cholecyst frosty and wall slightly thickened; hepatic lymph node irregularly congested; moderate roundworm infestation; multiple, elevated hemorrhages ranging from petechial to ecchymotic in mucosa of urocyst. Histopathology: Liver - multiple, small, randomly distributed, circumscribed lesions of larval migrans occuring as practically pure granulomas with in- frequent eosinophiles or as mixed granulomas with abundant eosino- philes; approximately 1/50 of sections involved. Kidneys - focal chronic interstitial nephritis, left kidney; focal subacute interstitial nephritis, right kidney. Lymph nodes - a) multiple larval granulomas (several larvae in sections); lymphocytic depletion; b) lymphocytic depletion and edema; c) lymphocytic depletion and edema; c) lymphocytic depletion and medullary'hemorrhage; multiple larval granulomas. 112 Lung - scattered areas of subacute interstitial pneumonitis with inclusion bodies in septal cells. Nasal mucosa - acute purulent rhinitis. Urocyst - mild active congestion. Trachea - eosinophilic intranuclear inclusion bodies. Spleen - marked lymphocytic depletion and marked reticulo-endothe- lial proliferation. Fig. 7. Liver. Dog 61+h5-6. Canine Distemper Complex. Larval migrans granuloma without eosinophilic infiltration. xuOO. 115 ’ . in" ' a! O .9 n ‘0' ‘ ."”‘Q .. ' :é‘.’ - . . are? a. ' 9‘ .A _l . ,k. . .L c. a v a. n it, x... . l c a A u ‘ o 5 j ‘0 p .. W‘ ' . .¥."" . _ ’ a. d & o - ‘J .A . I. ‘ O ‘ ' 'tfi. 11h APPENDIX 5 EXPERIMENTAL INFECTIOUS CANINE HEPATITIS DOGS DOG 6&92 Shepherd Cross Female BSP retention: 5.9% Gross Pathology: Multiple ecchymotic hemorrhages in submaxillary lymph nodes; Suprapharyngeal lymph nodes slightly enlarged and edematous with clusters of petechiae near the anterior pole of right node; slight yellowish discoloration of heart; liver light reddish brown in color with large, irregularly distributed, yellowish areas - on incision, yellowish color with regularly distributed, very fine, yellowish-white foci throughout parenchyma; gall bladder distended and wall edematous; mild roundworm infestation; pancreas reddish' brown in color. Histopathology: Liver - infrequent intranuclear salt inclusions; in severalseetiOns,_ change in stain affinity of hepatic cell nucleoli from basophilic to acidophilic or polychromatic resulting in a mauve coloration; widely scattered, very fine nidi of lymphocytes with few macrophages or larger foci of fibroblasts and fibrin, located.peripherolobularbg centrolobular fatty metamorphosis involving approximately'l/5 of the lobule. Heart — diffuse fatty metamorphosis. Kidney - cloudy swelling of thick part of descending loops of Henle. Suprapharyngeal lymph node - mild acute lymphadenitis. Submandibular lymph node - multiple small hemorrhages in cortex. 115 DOG 6681 Collie Cross Male BSP retention: h.5% Gross Pathology: Slight yellowish discoloration to liver; cholecyst enlarged from bile retention; spleen small and pale. Histopathology: Liver - few and widely scattered, small foci of neutr0philes, a few lymphocytes and several proliferating Kupffer cells; diffuse cloudy swelling; dilatation of sinusoids at the central vein with cytoplasm of adjacent hepatic cells granular and condensed; lesion that of very mild acute hepatitis. Cholecyst - edema of submucosal layer. Tonsil - lymphocytic depletion. Spleen - mild acute splenitis. DOG 6760 Tan, White, and Black MOngrel Female BSP retention: 0.8% Gross Pathology: Emaciated; left cornea opaque; blood bright red, thin and watery; all visceral lymph nodes enlarged; liver slightly pale; excessive fluid in abdominal cavity; moderate infestation with roundworms and hookworms; spleen small and pale. Histopathology: Liver - essentially normal. Eye - iridocyclitis and edema of cornea without vascularization. Lymph node - slight lymphocytic depletion and increased macrophages. DOG 6761 Retriever Cross lee BSP retention: 0.8% 116 Gross Pathology: Superficial inguinal lymph nodes enlarged with fleshy streaks within and pigment at corticomedullary junction; bronchial lymph nodes congested; longitudinal tract of edema and hepatization on dorsal aspect of left diaphragmatic lobe of lung; mild hookworm infestation; cholecyst pale and dull with very light-colored bile; multiple, widely dispersed, lesions occurring either as small circumscribed ‘ hemorrhages flush with the surface or as very small, white foci with a hemorrhagic border in both kidneys - on incision, white glistening streaks primarily in cortex but extending also into cortico-medullary tissue. Histopathology: Liver - essentially normal. Kidneys - subacute interstitial nephritis and descending subacute pyelonephritis. Pancreas - single sclerotic lobule with scattered areas of subacute (few areas of acute) interstitial pancreatitis. Prostate - juvenile. Cholecyst - mild subacute cholecystitis. Testis - prepubescent. Lung - two spherical cysts just beneath the visceral pleura; alveolar interstitium.thickened by cellular infiltration consisting of lymphocytes primarily but with abundant proliferating endothe- lial cells, some fibroblasts and neutr0philes; mild generalized congestion. DOG 6162 Black Mongrel Male BSP retention: 0.8% 117 Gross Pathology: Thick, mucopurulent material in the gastro-intestinal tract but most abundant in the pylorus, duodenum, and jejunum; one roundworm in intestine; several subcapsular, white, glistening foci in liver; several white, glistening foci in the cortex of both kidneys. Histopathology: Liver - section 1: centrolobularly, tinctorial change in which hepatic cells stain lightly and cytoplasm.more granular than at periphery; sinusoids dilated centrolobularly; widely scattered but relatively numerous small foci either of lymphocytes and prolifer- ating endothelial cells with granular acidophilic material or areas of proliferating fibroblasts suggesting replacement fibrosis in the peripherolobular position; section 2: single large perivenous lesions consisting of eosinophiles, lymphocytes, proliferating endothelial cells, fibroblasts and mild edema, otherwise essentiaDjr the same as above; section 5: two relatively large foci of lymph- ocytes, endothelial cells and fibroblasts, otherwise essentially the same as section 1. Spleen - depletion of lymphocytes of the follicles but very prominent germinal centers indicating reactive hyperplasia. Prostate - several circumscribed accumulations of lymphocytes. DOG 6691 Beagle Cross Female BSP retention: 12.0% Gross Pathology: Emaciated; purulent ocular discharge; all visceral and cervical lymph nodes enlarged and edematous; massive edema at thoracic inlet; liver pale with irregular yellowish mottling; mild infesta- 118 tion with roundworms and tapeworms; spleen pale and slightly en- larged; tissues pale. Histopathology: Liver - numerous, diffusely distributed, small, necrotic foci with infiltration of neutr0philes and proliferating endothelial cells; marked perivascular infiltration with neutr0philes and lymphocytes; hepatic cells slightly swollen. Spleen — marked lymphocytic depletion, increase in reticulum cells and mild neutrophilic infiltration. Lung - early bronchiolitis. Lymph nodes - marked lymphocytic depletion; marked increase in epithelioid cells almost completely obliterating the sinuses. Lymph node - serous lymphadenitis. Stomach and urocyst - intranuclear inclusion bodies. Fig. 8. Liver. Dog 6697. Experimental Canine Infectious Hepatitis. Perivascular lymphocytic infiltration. XAOO. 119 120 APPENDIX 6 PYRETIC DOGS DOG 6uu6—1 Black Mongrel Female BSP retention: 0.8% Body temperature: 105.9°F. Gross Pathology: Small intestine congested, especially the duodenum; severe tapeworm infestation; single, isolated, mesenteric lymph node congested. Histopathology: Liver - two small larval granulomas; moderate amount of hemato- genoid pigment, randomly distributed; lesions involved approximately 1/100 of the area in the sections. Lung - early focal bronchopneumonia. DOG 6hh6-2 Retriever Cross Female BSP retention: 0.8% Body temperature: 105.6°F. Gross Pathology: Right tonsil enlarged and congested with proliferation of lymphoid tissue posterior and downward in confluency with the right supra- pharyngeal lymph node which was enlarged to three times its normal size - dorsal surface hemorrhagic or congested entire length of node; mild infestation with tapeworms and roundworms; Peyer's patches prominent; surface of spleen rough. Histopathology: Liver - essentially normal. Tonsil - diffuse suppurative tonsillitis. Suprapharyngeal lymph node - marked lymphoid hyperplasia and reticu- 121 lo-endothelial proliferation. Spleen - lymphocytic depletion but with hyperplastic germinal centers of Malpighian corpuscles. DOG 6hh6-5 Beagle Cross Female BSP retention: 0.8% Body temperature: 105.l°F. Gross Pathology: Suprapharyngeal lymph node enlarged and with fine hemorrhages sub- capsularly; ileum congested; heavy infestation with hookworms and tapeworms. Histopathology: Liver - essentially normal. Ileum - mild congestion of the mucosa. Suprapharyngeal lymph node - small, focal hemorrhages in cortex. Spleen - mild congestion. DOG 6hh6-h Terrier Cross Female BSP retention: 0.8% Body temperature: lO2.8°F. Gross Pathology: Suprapharyngeal lymph nodes congested and containing multiple small hemorrhages; mesenteric lymph nodes congested centrally; mild in- festation with tapeworms and whipworms. Histopathology: Liver - essentially normal. Suprapharyngeal lymph node - multiple, small focal hemorrhages in cortex and medulla; marked lymphoid hyperplasia. Mandibular lymph node - mild medullary hemorrhage. 122 Spleen - marked lymphocytic depletion.' DOG 6hh6-5 Collie Cross Male BSP retention: 0.8% Body temperature: 104.6°F. Gross Pathology: No significant lesions; essentially normal grossly. Histopathology: Liver - slightly granular cytoplasm. Testis - prepubescent. Spleen - marked lymphocytic depletion. Urocyst - edema of submucous layer. 303 |l| II‘I I |||||l ‘- I |I| ll Illl I'll Il|| a I‘ ‘4 I I'll I|l6 ||I| |I ||| ||I| I‘. 5 I Ill ‘II '8 || Illo |I| I‘ll IIII ll | I I | l || Ill ‘I II‘I' I9 I'll || || T I2 l |I|I1 ‘I|| I'll! I \‘3 || ‘II |I||II| | III‘ IIII | II|I|I