Vet. Patho l. 15: 196 -207 (1978)

Pathological Evaluation of Paratuberculosis in Naturally Infected Cattle

C. D . BUERGELT , C. HALL, K. McENTEE and J. R . D UNCA N

The Theriogeno logy Section, Department of La rge A nimal Medicine , Obstetrics and Surge ry, New Yo rk State College of Veterina ry Medicine, Cornell University, It haca, N .Y .

Ab stract. T hirty-two of 51 cattle infected with paratub erculosis had chro nic e nteriti s , chronic lymphangitis or me senteric lymphade no path y , or all three , at slaughte r. G ran ulom atous inflammatory lesion s were mi ld to ad va nced and predominantly involved the dista l . Rectal involvement was see n on ly in five ca tt le . Fourteen had microgranulom as in the liver . There were three cytologica l forms o f s: hist ioc ytic, po lygonal a nd e pithelio id . T he latt er two types had engulfed moderate numbe rs of acid-fast baci lli. T he histiocytic macro phages usua lly were packed with ac id-fast bacilli. Except in th e liver and occasionally its nodes , remote lesio ns of paratuberculosis were no t found in ot her organs. One animal had endocardial a nd ao rtic ca lcifications. Most cattle with signs of had glo bule leu kocytes in or around mye nteric ganglio n ce lls . T he thymus of 3- to 8-year-o ld cattle wit h clinical signs frequently had mi ld to advanced involut ion . T he th ym us of sim ila rly aged infected anima ls without clin ica l signs, a nd of paratube rc ulos is-negative animals , had no t involuted.

Paratube rc ulosis (Jo hne's disease) , a chro nic infectio us disease of do mes ticated and wild , has been recognized thro ugho ut the world since it first was described in 1895 . Its ca usative agent is Mycobacterium paratuberculosis, a faculta tive intracellular acid-fast bacterium . T he macroscopic and histologic lesion s of paratuberculosis basically remain confined to the intes tine , mesenteric and ileocaecallymph nodes [7] . A ltho ugh M . paratuberculosis has been cultured from a variety of othe r organs , microscopic lesions have been found only in th e liver [12] . M . paratuberculosis is ass umed to spread primarily via circulati ng macro­ phages. In the liver th ese macroph ages may become foca lly trapped in sin usoi ds and with may give rise to microgr anulomas. Early inves tiga tors of par atubercul osis emphas ized its similarity to [3 , 5 , 10] . Bo th diseases are cause d by acid-fas t bacte ria th at are diff icult to culture and have lon g gro wth periods . T he inflamm at ory response in both diseases is similar. Despite invo lvement of different organ syste ms, th e micro scopic lesion s are dom inated by an d Lan ghan s' gia nt cells . Le prosy has been classified int o two po lar forms - tubercul oid an d leprom at ou s 196 Pathology of Bovine Paratuberculosis 197

leprosy - wit h ma r ked hist ol ogical differe nces [I I] . In tuberculoi d leprosy , the dermal gra nulo ma is co m posed of a well de ve lope d populat ion of epithelioid macr ophages surrounded by dense co lle ctio ns o f small lym phocytes . M . le prae o rganis ms are se ldom fo und by standard me th o ds of examinatio n. T his is in contrast to the sit ua tio n in leproma tous lepro sy in whic h small lym ph ocytes are a bs e nt o r sca nty a nd im ma tur e macrophages a re packed with le pro sy bacilli. In accordance with t he morphologic find ings a re the o bservatio ns o f immuno logic im pa irm e nt in these patie nts [1 4 ]. We ha ve classifie d parat ubercu lous lesio ns in th e in test in al tr act a nd associate d lymph nodes, usin g as a model t he mo rph ol ogic sca le of lepro sy propo sed by Ridley a nd Jopling [II] .

Materials and Met hod s Tissues fro m 32 Holstein cows (herd A) and fro m 19 Ang us cattle (herd B) , all infected with M . paratuberculosis, were collected at slaughter. Tissue specimens taken were from the duodenum, jejunum, , ileocaecal valve , caecum , convoluted colon, terminal colo n-rectum (last 30 centimeters anterior to the an us) , mesente ric nodes, ileocaecal node, pharyngeal tonsil. heart , lung, kidney, liver, liver node , mammary gland, supra ma mmary node , thymus, spleen, bronchia l node , ad rena l and . Major fetal orga ns and placental tissues were collected from infect ed pregnant cows. Tiss ues were fixed in 10% buffe red forma lin, embedded in paraffin and sectioned at 6 micrometers. Selected sections were stained with hematoxylin and eos in ( HE) and an acid-fast Zie hl-Neelsen stain.

Results T he necropsy a nd mi cro scopic findings of Johnes disease, the rela tive numbers o f acid- fas t bacilli a nd degree o f th ymic in vo lu tion were as shown in tables I, II a nd V . Twenty-two of t he 32 cows in he rd A had gross changes cha racte rist ic of Johne 's di sease (table I) . There were co rr ugatio ns of the m ucosa of th e dista l small in testine a nd prominent mese nt e ric a nd subserosa I lym ph a tics in 17 cows . T we lve had la rge mese nte ric a nd ile ocaeca l lymp h no des . Six cows had loss o f skeletal muscle substance o r atrophy of body fa t or both . Ten ca tt le in he rd B had gross lesions with mucosal thi c kening . O f th ese , three had pro mi ne nt subserosal lym ph a tics a nd two had mesenteric lymp hade nopathy . T he body co nditio n o f two ca tt le was poo r. Extensive a lo pecia was di agnose d in two ot her cattle . One 5-year-old cow (no. 2) had e ndocardial a nd aortic ca lcifica­ tion s (ta ble II ) .

Herd A Hi st o logic lesio ns co m patible wi t h those of pa ra we re graded as mild in nine co ws, mode rat e in 11 a nd ma rke d in 12 . In co ws wit h mi ld lesio ns (class I ) , in divid ual Lan gh an s' gia nt cells usu a lly were detected in th e lamin a propria of intestinal villi (fig . 1) o r scatt e red within th e pa racortical zo ne of mese nte ric nodes . Specific epithe lioid mac rophages were 198 Buergelt et al

difficult to identify. Acid-fast baci lli indicative of M . paratuberculosis were see n in o nly on e cow . Co ws with moderate histologic lesion s (class 2) had severa l sma ll gro ups of macrophages or severa l indi vidual Lan ghans' giant cells or both in the lam ina propria of intestin al villi, in th e int estin al submucosa, in the subca ps ular sinus, or in th e par acortical zo ne of regional me senteric nod es. Small agg regates of macrophages were seen in the livers of two cow s. In three cows of cla ss 2 the number of macrophages to giant ce lls was equal; in four cows, macrophages exceede d giant cells; in four other co ws giant ce lls were the predominant ce ll type. Cow 3 had sma ll foci of mineralisation in so me giant cells. Many macrophages and giant ce lls had infiltrated the lamina propria and the submucos a of various seg ments of the small intestine in cows with advanced lesions (class 3) .T hese macro ph ages and giant ce lls also were in the stroma l tissue of the tunica muscularis and in the serosa . T he y filled the subm ucosal, subs erosal and mesenteric lym phatics and partially occluded the lumen. A substantial number of macrophages , giant cell s and lymphocytes surrounded the se lymphatics. T he intestinal villi were short and distorted. C rypta l glands were distended and filled with ne utrophils and mucoid substa nces. Villou s lact eals were prominent becau se of dilation and some contained a few inflammatory cells. Some lacteals had ruptured and fistulati on into the gut lumen had occurred. Peyer's patches were surrounde d by infla mma tory cells, but usuall y were not infiltrated by them . The submucosa was widen ed either by infiltrating inflamm a­ tory cells or tran sud at e . In 11 cows, the myenteric ga nglion cells of Meissner's plexus eithe r were surro unde d or infiltrated by a few globule leukocytes (fig. 2) . T he gra nulomato us infl ammatory respon se tended to exte nd to th e adjacent mesenteric nodes. A ffere nt lymphatics were distended and infiltrated by macro­ phages and giant ce lls. T he subcaps ular sinuses were filled with similar cell s. The lymphoid cells within th e par acortical zone largely were replaced by macrophages and giant ce lls . Lymphoid nodules were spared. The infiltrating process usua lly stopped abruptly distally to th e ilea l part of the ileocaecal valve . T he lamina propria of the muco sa of th e large intestine , if invo lved , was on ly slightly infiltra ted by a few macrophages or by Langhans' giant ce lls. T he glan ds of Lieberkuehn remained un involved. In three cows of clas s 3 , the dominant cell type was Lan ghans' giant ce ll; in four cows, the number of macrophage s and giant cells was equal; and in five cows, macrophage s mostly were see n. Three types of macrophages could be distin guished morphologicall y. T he gra nuloma tous inflammatory infiltr at es of three cow s were composed of elonga ted, spindle-s haped macrophages, and they were arrange d in syncytial-like forma tions within the intestin al mucosa as well within mesenteric nod es (fig. 3) . Th ey also were packed with acid-fas t bacilli . Macrophages of eight cows we re polygon al with prominen t, foa my or vac uo late d cytoplasm (fig. 4) . Ten cows had macro­ ph ages th at had formed well developed epithelioid cells with deep eosinophilic Pat hology of Bovine Paratuberculosis 199

2

4

Fig. 1: Sma ll intestine fro m cow with paratuberculo sis . La nghan s' gia nt ce ll in la mina pr opria of mu cosa (class I lesion) . H E . Fig. 2: Sma ll intestine from co w with paratuber culosis . G lo bule leukocytes (a rro w) in Me issne r's plexus. HE. Fig. 3: Mesenteric node from co w with pa ratubercu losis . Spind le-sha ped histiocytic macr ophages in bundle-like format ion s . H E. Fig. 4: Sma ll intestine fro m co w wit h pa ratuber culosis . Po lygo na l macr ophages with vacuo lic cytoplasm . H E. 200 Buergelt et a/

cytoplas m, dist inct ce ll boundari es, eccentric, oval hypoch rom at ic nuclei , and se ve ral nucl eoli of va rio us sizes (fig . 5) . Of the 32 cow s with histologic cha nges in the uppe r small intest ina l tract , six had lesions of paratuberculosis in the caecum ; three had lesio ns in the convoluted colo n; o nly two had lesions in the rectum . T he duodenum was fre e from granulomatous infi ltrations in all cows . Nine cows had sma ll gra nulo ma s in the live r (fig. 6) . Two of them had slight infiltra tio n of the he pati c nodes with La ng hans' gia nt cells. Ac id-fast sta ins were nega tive for M . parat ub erculosis. A ll ot her examined organs, incl udi ng fetal tissue s, were free from histologic evidence of paratuberculosis. In cows wit h gross lesion s in th e sma ll int estine , mo re hepat ic th an rectal gran ulom as were see n histologicall y (ta ble III ) . Similarl y, more he patic gr anulomas than rectal lesions occurred in cows wit h suc h clinical signs of John e's disease as persistent diarrh ea and weigh t loss. He patic granulo mas also occurred mo re fre quently in subclinical ca ses o f paratuberculosis (table IV). Of th e 12 cow s wit h adva nced histologic lesions, eight had clinical signs of Johne 's disease. Six of 1 I cows with moderate histologic involvement were reported to have shown clinical signs, as were three of nine cows with mild lesions (table I). In 15 no ninfected co ws from the same herd , and in all infected co ws, the thymus was exa mined histologicall y for ev idence of involu tion . T he population of lymph ocytes in the cortex and medulla as we ll as the size and number of the thymic lo bules were evaluate d . W hen correlating the histologic findings of the thymus with the age of the cow and these wit h the stage o f of M . paratub erculosis we found the following : 1) 11 of 16 cows from 3 to 8 ye ars old wit h clinica l signs had moderate to marked th ymic involution; 2) 12 of 16 3- to 8­ year-o ld cows tha t we re infected bu t free fro m clinical signs had a fully develope d thym us; 3) 11 of 15 cows betwee n 3 and 12 year s old that were histologicall y an d culturally negative for M . paratuberculosis had a we ll developed th ym us (tab le V).

Herd B Of th e 19 infected cattle , nine had mild histological lesion s of paratube rculosis, four had moderate cha nges, a nd six had seve re lesion s (table II ). Of th e catt le with mild lesio ns (class 1), five on ly had individual sca tte red Lan ghans' giant cell s. The othe r four had a few macrophage s in addi tion to giant cells . Acid-fast organi sms cou ld not be demonstrated . Two cattle with moderat e chan ge s (cla ss 2) had predominantly gia nt ce lls a nd a few acid-fast bacilli; in two cattle of this group, the number of macropha ge s and giant ce lls was equal a nd each ce ll type had a moderate number of acid-fast baci lli . Two cattle with seve re chan ge s (class 3) had mainly macrophage s th at were packed with numerous aci d-fast organi sm s. T hree cows had equal numbers of Pathology of Bovine Paratube rcul osis 201

. .. Fig . 5: Sm a ll int estin e fro m cow with paratu be rculosis . E pit helio id macr ophage s . H E . Fig. 6: Liver from cow with paratuberculosi s . Microgranulorna in he patic lobule . H E. macrophages and gia nt cell s , with a moderat e number of ingested acid-fast bacilli; o ne had mainly gia nt ce lls with a mod erat e number of bacilli . The macrophages of three cattle were spindle-s haped, hist iocytic ce lls; th ey freq ue ntly fo rme d who rls or bundles a nd were pac ked with acid-fast bacilli .T he granulo ma to us infiltrate of th ree cattle was co mposed of pleomorphic ma crophages with foa my cytoplasm . Six cattle had we ll devel oped e pithe lioid ce lls with or without La ngha ns' gia nt cell s. In the remai ni ng seven cattle, giant ce lls predomi­ nated . In three ca tt le glo bule leukocytes we re pr ominent in or around myenteric ple xus ganglionic ce lls; two had clinical signs of Johne's disease . In all M . paratuberculosis-infected cattl e, th e du oden um was free of granu lo ma­ to us in filtrates . In six cattle, the caecum was minimall y infiltrated; in fo ur of these the convoluted co lo n was infiltrated; a nd in three the termina l co lon a nd rectum also we re affected . The live rs of five cattle had microgranulomas; two hepatic lymph nod es we re infiltra te d by Langha ns ' gia nt ce lls. Acid-fast bacilli were not seen o n special stains. The pattern was simi lar to that of herd A . Microscopic hepati c lesion s we re more fre q ue nt tha n rectal mucosal infi ltrations in cattle with gross fin dings a nd in cattle without clinical signs of Johnes disease (tables III and IV) . Mos t ca tt le infected with M . paratu berculosis, but witho ut clini cal signs , and most cattle witho ut infection had a fully developed th ymus (table V) . T wo infec te d cattle fro m 3 to 6 ye ars old th at had clinical signs had an involuted thym us. 20 2 Buergelt et II I Discussion More th an half of our cattle had gross lesion s of par atuberculosis .T hese were muc osal th ickening of the distal sma ll int estine and dilat ed subserosal and mesenteric lymph atics . E nlargement of mesenteri c nodes, cac hexi a and alopecia were less fre que nt findings. Histologically, gra nuloma to us inflammat ory respon ses in int estin al and associ­ ate d mesenteric tiss ues were from mild to advance d . T he inflammatory respon ses did not exte nd beyond th e ileocaec al valve in most catt le . Lar ge intes tine seg me nts were infiltrated only in a few cattle and then on ly mild ly. This may be important in attempting to diagn ose par at ub erculosis with the help of rect al scra pings or rectal tissue tak en fo r biop sy. T he cha nces of co nfirming a clini cal diagn osis of Johne's disease with thi s method are co nsidere d low . We found infiltration of rect al mu cosa by macrophages in only a few cattle with clinical signs and with gross findings of par at uberculo sis. In co ntras t, the liver of infecte d ca ttle more fre que ntly had microgranulomas. T he re fore, liver tissue for biopsy might be mo re useful th an rectal tissue in verify ing Johne's dise ase . The finding of liver gran ulomas in par atubercul osis is paralleled by similar findings in leprosy [4] . In leprosy, th e liver was th e most freq uently affected organ othe r th an th e neurocutaneous system and lymph nod es. Hepati c granulomas were rep orted in 90 % of pat ients with lep romatou s leprosy and in 20 % of pati ents with tu bercul oid leprosy [4] . As with paratuberculosis, th ere were no signs of liver dysfunction . Many of the paratub erculou s granulo mas were composed of macrophages as well as giant cells, th e ratio fluctu ating fro m anima l to animal. Man y ca ttle had either macrophages or giant ce lls as the domina nt ce ll type. T he giant ce ll was fre que ntly th e only cell type in minimal lesion s of pa ra tuberculos is. In th ese cases, it usu all y was not possibl e to demonstrate acid-fas t orga nisms with spe cial stai ns . In more severe case s, giant cell s had ph agocytized acid-fast bacteri a , but to a con sid erabl y lesser de gr ee th an adja ce nt macrophages. Wh ere macrophages we re the dominan t cell type, th ey tended to have man y bacilli . Va rio us cytological fea tures and arrange me nts of individua l macro phages we re see n . Man y catt le had macrophages th at varied either in size and sha pe or that loo ked uniform and differentiat ed and re sembled epithe lial cells. The bact eri al index for both cell types was rou gh ly th e sa me . A few ca ttle had elongated histi ocytic macrophages, arra nge d in bundles or whorl s, th at had a comparative ly high er number of ac id-fast bac teria . Wh en th e histologic chan ges of par atubercul osis were compar ed with those of lep rosy, th e seve re ly affected dr aining mesenteric nodes look ed similar to th ose of lep rom atous lep rosy [15] . The entire par aco rtic al zo ne was repl aced by macro­ ph ages and giant ce lls, spa ring lymphoid follicles. The re we re various cyto logic for ms, fro m a histiocytic macrophage to an epithelioid ce ll. Giant ce lls were numerous. The identification of va ria tions of the gra nuloma to us respon se in th e int estin al trac t of par atubercul ou s catt le was less successful th an in the sk in of lep rosy Pathology of Bovin e Paratuberculosis 203

pa tients , in whom nodular lesions a re easier to e va lua te . Well defined gra nulo mas with ce ntra l macropha ges and peripheral lymph ocytes as see n in th e skin of tube rculoid lepro sy patie nts were not found in the intestinal tr act of infect ed a nima ls [10 , II , 13 , 14] . Indi vid ua l macrophages, howeve r , va ried in the ir cyto log ica l appea ra nce. Man y were polygo nal with vacuolate d cyto plasm, but wit ho ut a te ndency to evolve to epithe lio id ce lls . La ngha ns' gia nt ce lls were or were no t present. Eos ino phils, plas ma ce lls a nd lym phocytes we re int e rmingled in varying numbers. The fo rma tio n of e pithelio id ce lls was recogn izable in o ther insta nces . Co ntrary to tuberculo id lepro sy, many of the epithe lioid ce lls co uld be sho wn to have ingested va rio us numbers of acid-fast . Sim ilar to the ir a ppeara nce in lepro mato us lepro sy, the macropha ges in a few cattle with paratu­ berculos is were undiffere ntiated . T hey also were filled with acid- fas t bac illi . T he e pithe lioid ce ll has been descr ibed as th e e nd stage of stim ulated, phagoc ytic macropha ges [1, 91. T hes e macropha ges are derived fro m circ ulati ng mon ocytes of bo ne marrow o rig in. Macrophages turn into e pithe lioid ce lls when they become im mo bilize d at the site of infla mma tio n [91. E pithe lioid ce lls a re active in ph agocytosis, degradati on of ingested subs ta nces a nd de struction of microbes. T hey have secreto ry function s as we ll. Multinucleated gia nt cells arise by fusion o f mat ure macro ph ages. T he giant ce lls in itiall y formed are of the fo re ign bod y typ e and convert late r to Lan gh an s' type [1] . T he glo bule leukocyte is consid ered a mast cell that is in the pro cess of dischargin g its contents of ph armacologica lly ac tive arnines [6] . G lo bule leu kocytes in o ur investi gation we re detected in man y cases wit h cli nical signs of Joh nes disease and in the absence of infestati on with helminths. T heir close associa tio n with myente ric ga nglio nic ce lls may be mo rph ol ogic ev ide nce of an immunologic mecha nism by whic h diarrhea is medi at e d . It has bee n suggeste d that diarrhea in the init ial ph ase of Johnes disease is pr ecipitat ed by im mediate hype rsen sitivity [8 ]. It is e licited by histam ines release d from mast ce lls a nd is res po nsive to a ntihista mi nes. The histology of parat uberc ulosis did not diffe r betwee n breeds. Necro sis, caseatio n and ulce rat ion as th e y oc cur in tubercul osis we re a lways a bse nt. T his observation can be hel pful in dist ing uishing par atuber cu losis fro m tu be rcul osis in ca tt le. With rar e exceptio ns (o ne a nima l ea ch) int estina l a nd cardiovascular min eral isation were a lso a bse nt, the la tte r being in co ntrast to the re po rted high incidence of a rteriosc lerosis in cattle with Jo hne's disease [2 , 5] . The histol ogic evalua tio n of the thymus with regard to its po ssib le ro le in th e pat ho ge nesis of parat ub erculosis led to the observation th a t in age-matc hed a nima ls with clinica l signs, there is a te ndency to lymphocytic deple tion . Catt le without clinic al signs a nd paratuberculosis-negat ive anima ls, be have d simila rly in that o nly a few of th em had sig ns of thymic invo lut ion . Age-de pe nde nt invol ution co uld no t be demonstra ted . T he occure nce of th ymi c depletio n in clin ica lly ill cattle ca n be int e rpr eted as contributi ng to the ca use, o r as bein g a co nseq ue nce of the clinical condition . It co uld be a n additio na l mo rphologic co rrelate of the

Table III. Co mpa riso n between gross changes of par atubercu losis and histologic lesions in the rect um a nd liver , herds A a nd B Gross Changes No Gross Chan ges

He rd A Herd B He rd A Herd B Rectal lesio ns 2 3 o o

Hep ati c lesion s 9 5 o o 206 Buergelt et a/

Table IV. Co mpa rison between clin ica l signs of Jo hnes disease a nd histo logic lesio ns in the rec tum a nd liver, he rds A and B Clinica l Signs No Clinica l Signs

He rd A Herd B He rd A Herd B Rect al lesion s 2 2 o

Hepat ic lesion s o 2 3 3

Table V. Herds A and B . Co mpa riso n between thy mic status , pr esen ce of M . puratuberculosi s, wit h and without clini cal di sease , a nd age of ca tt le ~~~- lnrected ca tt le with Infected ca ttle with o ut Un infe cte d ca tt le Age , clinica l signs clinica l signs Numbe r ye ars o f catt le 4 + 3+ 2+ 1+ ), , 4 + 3+ 2 + 1+ 4 + 3+ 2+ 1+ 0- 2 ( 10) ( I) ( 10) (2 1) 3- 4 2 2 3 ( I ) 10 ( I) 1(1 ) 4 24 (3) 5- 0 ~ 2 2( 1) 1 2 ( 1) 11 (2) 7- X 1 ( 1) 4 ( l ) 9-1 0 ( I ) ( 1) ( 1) (3) 11- 12 1 2 1 5 Over 12 2 1 3 Total 5 5 5( 2) 12 ( 11) 2(2 ) 2 (3) 11(11 ) 2 ( 1) 47(30)

J H isto logic thymic invo lution . 4 + = nor mal ; 3+ = mild invol utio n ; 2 + = mod e rat e involution ; 1+ = mar ked invo lution . , Nu mbers in pa re nthes es re presen t cattle fro m herd B.

Acknowledgements We thank Mrs. D . Bell a nd Mr s . C. Lear y for technical assista nce . Supported in part by Ha tch Fund ad ministe re d thro ugh Cornell U nive rs ity A griculture E xpe rime nt Sta tio n . Patho logy of Bovine Par atu berculosis 20 7 References ADAMS, D .O .: T he gra nulo ma to us in flam mat o ry resp o nse . A review . A m J Pat hoi 84 :1 64 -1 9 1, 19 76 2 A LIBASOGLU, M .; D UNNE, H .W.; Guss , S. B .: Na turall y occur ring a rte rioscle ro sis 111 cattle infected wit h Johnes disease . A m J Ve t Res 23 :49- 57, 196 2 3 BERGMAN , A .: E inige Wa hrn e hmunge n bezu eglich de r chron ische n spe zifische n D ar­ mentzuendu ng beirn R ind vieh . D tsch Ti erae rzt l Wschr 21:8 26-827 , 191 3 4 C HEN, T .S .N .; DRUTZ, DJ .; WHELAN,G .E.: He pa tic gra nulo ma s in leprosy . T he ir re la tion to bacteremia . A rc h Pathol Lab Med 100:1 82- 185 ,1 9 76 5 H AGAN, W .A .: Jo hn es di se ase or parat ub ercu losis of cattle wit h a no te o n the di se ase in shee p ill T ube rc ulosis a nd Lepro sy, th e Mycobacteri al D ise ases , Sy m p Scr Am A Adv Sci , part 1; pp . 69- 79 , 1938 6 H ER BERT, WJ .; W ILKI NSON, P .C.W. : AD ictionary of Im munol ogy , p . 73; 2nd ed ., Blackwe ll Sc ie ntific Publicat io ns , Oxfo rd , 1972 7 J UBB, K .; KENNEDY, P .: Pathol ogy of Do mestic A ni ma ls, pp. 119- 122 ; 1st ed ., vo l. 2 , A cademic Pr ess , New Yo rk , 1963 8 M ERK AL, R .S .; KOPECKY, K .E .; LARSE N, A .B .; NESS, R .D .: Im m unol ogic mec han ism s in bovine parat uberculosis . A m J Vet Res 3 1: 4 75- 4 85 , 1970 9 PAPAD IM ITRIO U, J .M . ; S PECTOR , W .G .: T he o rigin, pro pe rt ies a nd fat e o f e pit he lio id cells . J Pat hoi lOS: 187- 20 3 , 197 1 10 PALLASKE, G .: Z ur ve rg leic he nd e n H istol ogic der Para tu be rkul ose des Rind es un d der Le pra des Mensche n . Vi rc h A rc h 263 : 189- 204 , 192 6 11 R IDLEY, D .S .; J OPLI NG, W .: Classifica tio n of le prosy acco rd ing to immunity . A five­ gro up syste m . Intern at J Leprosy 34:255- 273 , 196 6 12 SCHAA F, J .; BEERW ERTH , W .: Die Bed e utung der Generalisa tio n de r Paratube rkul ose , de r A ussche idun g des E rregers m it der Milch un d der ko ngc nita le n Uebertra gung fue r die Be kaempfun g der Seuche. Rinde rt uberkulose an d Br uce llose 9: 11 5-1 24 , 1960 13 SKINSNES, O .K .: Co mpa rative pathoge nesis of mycobacterioses . A nn N Y Aca d Sci IS4: 19- 3 1, 1968 14 T URK , J .L.: Ce ll med iate d immunological processes in le pro sy . Bull W HO 41 :779­ 792 , 1969 15 TU RK, J .L. ; WATERS, t\1.F.: Im m uno logical significa nce o f cha nges in lym ph nodes acro ss the le prosy spect ru m. C lin Exp Im munoI 8 :363- 376 , 19 71

Request re prints from Dr. C. D . Bu erge lt , Depart me nt of Pathology , D ivision of Co mpa r­ ative Pa tho logy, Bo x J-275 , JHMH C ,U niversity of Flo rida , Gainesville, F L 32 6 10 (USA) .