K/SEARCH (; 2~ IN

NiORRHAGIC ::TPTlCAE:· !I·\ D ITS CONri;oL

Diseas(: pos,t wn l!aerr1orrhagic septicaernia ·is taking a hea\,.,Y toU of lh1estock pc-pulation 111 .India. I'he avc·rage

annual death ra1e ,vas reported to be 1n()re tban 40,000 in 1958-:SY (I)handal 1969) .. ShH_:i:, tiien, there has been a cons.iderable fal.l in the n]te. It ca1ne do\.\'".n ~i, 8,265 ch1ri::\g· t.:h.e

11.6~1() i1: the period 19'/S ,u )9?i ar;;j 9,fi2:i in the y:::>r 1978. due to the disease has decre~1~;cd significantly i;".:- fhe last four

outl:rea1~~:: coritinues to rernain

:tear

2

4 l 97 i

5

Ou tbrc:Jks

5A65

,;J.J>58

1L356

1 1,496

22.6<\.2

l8.f-r,:, 15 .:: ·' .i

c::ct':sed by P. multocida serotv-pc h.B as

7 l

135-➔ 1

13.(H 1

9,625

fron1 other parts 0£ ~J.E:, Asia, though other types probably pla.y a ·c:1ncrc r6l.e: 13esidcJ 83 Y:/De I strains out nf J. O:? haemorrhagic .strains fron1 cattle and butcc~loe;:: India, 1t;·r;n.:~ reported to be typi IV and 13 as untypabk (Dhanrla, 1959). A fresri appr:;i:-:al of ,he ex;-icl -;t·r, !•Jpes involved and 1 :,e' • regional distribution, if any, wiii be helpful in controlling the disease.

Carrier problem The endemic nature of the disease and its seasonal incidence have been dttributed to 1.he

presence of healthy carriers. Studies revealed that the carrier rn1.e uf P. multocida (Robert's type [) wad directly related to the incidence of tbe disease with 0% in non-endemic zone2> w 1.9°k :r. a zone wiu1 moderate incidence and 5.0 to 6.C,o/" ln enzootic areas with high incider1c,0 of disease. The carrier rate was found to be higher in buffaloes :.e. 2 .. 6 10 6, 15% than in catiie Le, 1.25 to 2%. The overall carrier rate in areas where outbreaks of tlw disc1,e cccurrec1 l.o ;1 -.·,,r)~ing extent was fou,,0 to be 2.8% Le. 1 n out of 3~'ii), The examination of upper respiratory trnd ,,if cattle and buffaloes. 11

an endemic area during the outbreak, revealed 7.5')7., ,,f the animals c') L"ilrry P. rnultocida (Robert's type I). A reexa,nination of the nasopharyhx of animal;:. in ,.he sarm' viiiagc, ,10 days after the ,n,,· break wa:o controlled by vacci1ntion. rcn.'3led the absence of ;i;,y carrier (G,,;,u,, 1961'.). Similar f1 ndings on c'arrier rate have been rc:por: f(l in Sudan (Mustafa et ai., 1978).

Out rJf [he 20 isolates of P muitoc,,'fa frrw, healthy carriers, a:o many as 10 ,.,,c; '. ,,f Ro'Kn 's type I. two of type II, three of type lV a11d tlv~ remaining could not bf- typed. It was to note rhat out :A 10 type I isolates ei).;hl w:or-c c:,psulated and pathP),(Tnir· to mice 2nd hi;] b;;1b and twu

* Senior Scientist ,"l·)T;·i·;;;~--~f Standardisation, Indian \/ ~:'.·t~~~-rina~·:::~- ~itf:'."Search~·1;;·;;1·i~·;_;_t"t::: ·1zat;;:;g:;:~~-1f;;·~ifi~-:·1rr, .. India.

V\/hen the Grganisrn :.s transfecrc:d VJ a p;;._rtia1hr irrnnu.niz(·d anirna·1 .. the 1~1tter bt·:ron:.e~·:. a potential danger for- ·d1e initiation of thr: rJE:;xt outb1<-:ak. 'fJv-~ prt~fjtr!.Ce oi resistant anirnals as reported by· Bain in vther SJ~ ... 4sian eountrie:~ ·in cornn1only encountered iYl India. ra.pid slide aggh1t111aJ:Ion test de\~elo,ped Ilhand::-i tl al. has bet ;.-1 fnund t.J

v-ery nseful ar:d is bein\t extensive1\ ~J::;ed to dei..t:ct t.he'.>:: ~, HJtura.nv

Rrservn;,· ,~tato-'> Pa:,,r,,.pf:a :w~!: , dn R ,~,.. typ ; ;-,;1s beE'l, ,,-,,ortec

.~Toup fa .·:cc..n setc ~Jtbe-r than

'.,o be~·;:, 1974)

St'i'lll~-~-nre and couk\ possibly affect a \~_,ider host role in. transrnission of ,J!;-:.;c-ase fr "t!·i one hc•s"( sr>ecies

India also i::lucidate their role a~; reservoir for !1aen1-orrbagic septicaen:.!_id ~ n_ cattle.

'Vaccine:~

.:iCt o[

{-T1f' Of

Mainly three types of Y~t<c.i.nes e.g, i1) oil-ad.!1.n'ant ([Yh.anda et td.~ 19[~6) \2) ahn1· (Iyer el al .. , 195~)) and (:3) bnJf ,. bacterin are produced usincr, hi~{hly i1nn1uHCgf:nic local ;~,;train P-f:12 .. st<rnty-pe 6:R. '"I'he aveta.g(: yearly production lhe la~t

of \Vhich about 62% consisted o! alum pn·upiiated vac c;ne, 31c2e,,, of h,m:1 bact;'rin ;;r1d 2.8% t,J oil-adjl:, ,\;,: vaccine. ·T'he oil-adjuvant vaccine is produced frorn_ agar~vash su.spe!·~sior1 of the orgel.nisn)5< ~:Che yield is. therefore, very srnall. Son1t} trials have beer:_ conducted ·vlit.t the produ.ction h:-1 ·vortex aerated tanks (Ahc,oja ef ai. 1 1979), but the rnethod is yet to be· adopted for lnr,ge scak~ 111rinufacture. rt: is essential to step up the production of :~Jil-adjuvant vaccin.e harnessing the 'le.test technology,

Oi!­·\djuvant

Alum Prccipit>it:d

Broth B~lc1e.r!,1

Tota.l

1974 1975

2,?Rh.966

13,911.,460 ; ; .898,?S(·

12,440.970

754.48•) 999.2'" 6,'! 500363

2CL402,9i0 19.S4H .. 780 22 .342.650 94.404.550

l 2.POH.15U 6 1 ,.5 9 ! .S 1 U

afford

rv:: ::essary

"\/arinl1~: uti'·ter vacci.1:e~~ ;;_;=_~en as 195f1b), sodiurn "Xtracl found U-~:e-:f11..l 1-Srlvast.ava ,"1a.h; are a ls/~ "''.r--·_h;r \Vay to assess ~-:he usefulne

va,. ,:,, d,·scn\. :Jy TF,' .{nd Cart<::: , l '3:'8J tor in catt:k~

I11 ?-.ccordancc \Vit:b the of Ind::,. potency of vaccine te-sred by cI1allengr-, b\:tffalo-cal\'f'-': ;:tudies ha-\;•f~: tH::'r:"'.n undertr1.ken to devel<YO a testing procedure for this \-'"accine rr:-Ict.'. (Nagra):1!·; 1972, tJ and Sar,:·~,-~n., -.l.976)1 and rabbits et al,1 I96f) I\.f.lttal and 1970). and Mu.:.:,:rr:'.itf·:- (1968:) h2.r-c: s-cggest:ed that a vacci:ne giving tv-;··o

in m.ict:~ C()uld be cc,rv:~idered satistaci:oty flowever, all the batches of vac-cine\ \Vhich protected buff-calves r,::,; (~ffective ,,-,.hen tested thi~ rnethod in n-1.ice have given prn,ectioll grearer •haTJ four wgs. St::-:Jies ate undf'r war to deternune wherher li•e hatches ~•f ·vaccines V{hich ~~ive n1ic~~ prcit.ection bet\?('f~n to 4 logs are.- protective for buff-calves

Cont:tot l'n,ventive inoculations in ,!nckm;c regions anc1 containment of rmt.ureaks by vaccination in

affected herds are 11sur.dly strategy to control and e1.in1inate the focus of infection frorn <0 ndemic area 'Nas carrifi~ 0\1, m Madhya Pradesh wit;, ~,pectacular results (Gupta. 1974). It (TE·

c;isted of loc;ning priority viJ!ages l. lI all(] f!f :1aving di;..laiit." c,utbredr,s tor three or lnore, two ;md one times respectively, m the preceding Jive years, in a pamcular emiemic focus, Mass vaccination w,.1s c;i tried out •,vii:h HS oii-adjuY;.-1• vaccine covermg the maJmit:,· of fot· cattle population before the bn,ak of mo11soon every yeilr in JJriority I villages and viilages comiguous to them within five­miie radius, Priority II and III viliages were also covered with oil-adjuvant vaccine while the 0·illages contiguous to them were vaccinated v,ith alum precipitated ,-accine. District Ujjain having had the hight>st average incidence of HS in Madhya Pradesh of more than 80 outbreaks annually from 1960 to 1965 with a cattle population of 1,000,000, was covered under this programme. The disease incidence, ,,1onality and vaccinations canie,.1 OLlt in the district are shown in Table 3. In the year 1965-66 the vaccination was concentrated in tlw outbreak area and was increased from 28,433 to 45.59:1. wifr a resultant fall m number of outbreak:, f:-om 88 to 41. The planned vaccination following ti',e c::raregy Wils i:;nried out in Apri11hrough June 1966 and then followed every year. Jt ii~ noticed that there was a sudden fall in number of outbreaks from 41 ru 9 in the first :,-ear and to 3 in the next year. The number of outbreaks rose to 1 i in the year 1968-69. Thereafter the incidence remained at a low ievel of 3 to 4 annually with a rise ro 11 in the year 1971-72, till it became nil in the vear 1974-7G. The rise in incidence of outbreaks in the year 1968-69 and 1971-72 was due to attacks in new foci, In the first year of the programme 88 . .i 95 e.g. 8.8'Vo of the total cattle population of the district was vaccinated. In the year 1968-69 when there were some new foci of infection _the number of vaccinations was increased to 111,92:3 to cover the new villages. About 15% of the vaccinated animals H'L:eived oil-adjuvam vaccine while the rest rhe alum precipitated vaccine. Implementation oi similar programme in other endtmic areas may prove effective in combating the ,.

a1sease.

, ,<o. 67

196 7 .• ,.:;

1%:<• \96<,.·;,,

19)0 --71

197 l ·- 72

4

l l

44 88.195

\U

81 .684

61

7t)O

Recent studies on toxin'- and toxic fractions Attempts to det,,ct the produui0:1 o! soltlh1P t:,xin durir,g in vi!'() ur in vitro giowtb of P

>nultocida havf' not been fruiiful (Dhanck i 959a. l9fi0 R:in. 1%3). Recent studies revealed the r•resence u:.· :,r1 aggressivF• factur in the c·.1ln:1·e iiitrates of :i pathogenic avian st:-ain of P multocida. A. non pathogenic strain inoculated along Yl.·it:b the fiitra.tf frorn. 6-hour broth cu]ture of pathogenic strain killed 13 m.ice {J"l~.t of a total o-f 18 in 24 hours. while none 1Jut nf three when inoculated alone. The frnction was prnhal)!y ident!c:1. v,rit½ the bet.a·?.r1tigen described by Prince (1969) ,vhich was a li;:lnpniysaccharide a;,s,wiated wii.1·1 p,·,,tein.

In vin· gro,vth of P. ,,,m!t,.:dda in diffus;on chambers implanted in the periV•nea] ,:avity did not ,psult in the dc:atb of chick:, nr mice. Thi::: 'Nas ir1 conformity with the observations of Bain (1963) who failed 1o demonstra,P aay lethal activity in the cultmc filtrate or plasma and tissue exudates from animals dyinr d h;1emorrhagic septicaemia.

A soluble toxi.c fraction lethai to mice released in normal saline suspension during the process ,:>f harvesting bacterial growth from lhe surface of yeast extract agar and subsequent over night storage at 4 °C has been described (Gupta. 1973). The gel diffusion and immunoelectrophoretic 2tudies reve2.1ed that it crmtai,.ed at least two fractions. It could be purified by centrifugation at 105,000 x G or bv fi1t ;;tion 1.hro111<h sephadex gel (G200). The purified fraction formed a line of identity with "Pa1.ticulate antigen'' prepared as described by Rebers et a! .. (1965). against P rnultor:ida whole cell antiserum in agar gel. It gave positive reaction for tests for protein and car· bohydrate and was highly imrnunogenic.

Other studies Cytophiiic and opsonin-adhering antibodies against P multorida have bee,, reported in the sera

of vaccinated buffalo-calves. Highest titres were found in sera from vaccinated animals after challenge infection with P. multocida (P-52 str.) (Sarmah, 1976).

Cytophilic antibodies against P. multocida have also been produced in calf, rabbits, g. pigs and :nice. Sera of these animals showed cytophilie antibodies to peritoneal macrophages and spleen cells of mice. Rabbits and mice sera did not show cytophilic antibodies for neutrophils of g. pigs by direct test but indirect test showed cytophilic antibodies. Treatment of mouse peritoneal macrophages with proteolytic enzymes viz. trypsin and papain increased the subsequent uptake of

1dentical treatrTierit did not

shovved that thf arc ·::roteolytic enzyn)(:· ;·. ar,:.: :10-c sensiti\·e to thc:se enzymes for an· ti bodies.

Biochernical the blood sarnples frc,ro tnxifa.loes in the febrile stage of r.he disect~2 r:;:;vealed an increasi·,· bii.ittJl)irT and other biie ::.;_;.11-t·-· '.J de< rca:3e in cltoL:>~;rerni and changes in t

iJattern of scrurn aU corrsistent V."ith hepatic · · ,.,, ·.,,.(Gupta , 1977). i\. signific:ant ;ncrease in plate kt adhe::;,venp,;:,, and plasma clonu~.g ::rne ii; ::,heep in:r,xtc,1 'Nith P. multocida iw 0

been report.eel (Sara ct I 9·'/8}. }\1rther studies on the bioche1nical changes in the blood of affected arnrnals wili be heipful in throwing ligl1t 011 foe r:mse of death in hae010rrhagic sepricacmia disease.

\7 ,,: ious serniogical techniques l:ave been u~;ed for typing ot P. rnuilocida isolatf's. ·with v,,rying degree of sensitivity. Gupta (1962). whiie ,omparing varimw serological techniques, found ha,,rnagg1utinarion inhibition test using cn~(k polysaccharide extract as anugen to be highly specific, StT1siiiv,~ and free from cross-reachms. Baxi (1966) while r.vping 138 str;;ins or P ;nuliocida "uund indirect hae'.,;ogl(lu1:,nd1ion usmg alkalme aut,,ciaved c:-.tract as antig,.~n 1 o be a most sensil:,•.: ~<:St.

References, 1) AHO0JA, M.L. l ·'D'.'i. MITTAL. K.R. and :~J>:11 ,, K.C. (1\11'.i): Improvements of H 5.

Oil-Acljm·,mt Va,-c,:,'t' aeration techniqrn:. lndwnj. An£m. Sci. 49, 113-117. 2) BAIN. R. V .S. (1963): Haemorrhagic ~.,:p:ic;wmia. Food and Agriculture Org;rnisat [on of \; eited

Nations, Rome. 3) BAXI. K .K (1966): Thesis suh;r1irced l c A.(("ra University ( 1966). 4) DHANDA, M.R. (1959a): Proceedin~s ,,)f the First F.A.O. lvieeting on Haemorrhagic sep­

ticaemia. Manila, Philippines, Hl69. 5) _____ (195%): Immunisation of cattle against haemorrhagic septicaemia with purifiec:

capsular antigens. Indian Vet. I :36, 5 .. 8, 6) _____ (1959c): Punfication and properties ot ,he soluble antigens of Pasteurella set,hca

type LA prelimina! y z:ommunication. /ndian I Path. B,zct. 2, 59-62. n _____ O 960): Studies on immunochemistry L Isolation and char::1.cterization of soluble

antigens of P. septica. Indian], Vet. Sci. 30,114. 8) ................................ DAS, M.S., LALL, J.M. and SETH, R.N. (19;',6): Immunological studies on

Pasteureila septiw. L Trials on adjuvant vaccine. Indian.]. Vet. Sci. 26, 273-284. 9) ____ , SEKERlAH, P.C.. LALL, J.M. and SEN GUPTA, B.R. (1959): Immunological studies

on Pasteurella septica !IL Indirect te;c:ts as indicators of immunity in haemorrhagic septicaemia. Indian], Vet. Sci. 29: 30-46.

10) Gl'PTA, B.K. (1962): Studies on the carrier problem in haemorrhagic septicaemia. Thesis submitted to Punjab C niven°ity, Chandigarh ] %~~-

11) _____ (1973): Studies on the Pathogenicity of Fasteurella multocida and the development of fowl cholera vaccine. Tlwsis submitted to Agra U mversity, Agra 19TL

12) ____ (1974): Unpublisheddata. 13) ____ and KUMAR, S. (1978): Serotyping of Indian isolates of Pasteurella multocida from

poultry. Indian], Anim. Sci. 48: 301-304. 14) GUPTA. G.C .. JOSHI, B.P. RA!. P. (1977): A note on some biochemical studies in haemorrhagic

septicaemia in buffaloes (Bubalus bubalicus). Indian I Anim. Sci. 45. 503-504. 15) GUPTA, K.C. (1969): Studies on carriers of Pasteurella multocida in pigs. Thesis submitted to

J awahar Lal Nehru Krishi Vishwa Vidyalaya, Jabalpur. 16) GOPTA, M.L. and SAREEN, R.L. (1976): Evaluation of haemorrhagic septicaemia oil-adjuvant

vaccine by Mouse protection test. Indian Vet. I 53, 489-492. 17) IYER, S.V., GOPALKRISH.'iAN, K.S. and RA~.lANI, K. (1955): Studies on haemorrhagic sep ..

adji.rvanL:::; ~1))011 the l-;asteurel/(1 buvf,epti-.·11 01·g'ac1isn1s. Ind/(/n ;,/(:!. J. 31,

.8) MiT: ·"· 1<,.R. «nci ',L. T.N. (!:o'; :~,;: Porenc: o.f h.aen1or1·ha~:.-i<·: ;:._;cpticae1n1.a oJJ,. adjuvant vaccine in

AL. 'T.~t. ser,-•ticae1nia vvater in oil-and l'et. ]. 54~ 7?:+-778.

,;1d ---adju;-r;1nt ~In.d n1ultiple en-;ulsion calve,. lndw1 Fet I. 56. :149-454

:.·rial in .R2btiits. Indian

(1979): ~)t.ndies on hden10rrhag{c scpticaen1ia 011:· 1/accines. LL lr11munity trials in 1.-nlce, rabbitr.: and

)J l½USTAFA,. ,,;;\,_;-\, .. , C~HALIB~ fJ ··vv. -~Ind SHJt;II..!L \:L~T, (1978): (:arrier rate of l)asteurella tnultocida rn :·attle herd ,bs,:-ciated wit'• rm outbreak Gl H .S. in Sudan. :3rir ·ve1. ! 1 ~-M. 375-378

?2) NAGRAJAN, V .. St:NDARS/\1', S. and F<AY,.\:-JJ, K ( 1972): Evaiuat ion of the potency of H.S. alum precip1tated vaccine the n1ouse protection test . .Iruiian Vl.·t. }~ •-19, 1080-1 (Hr).

'.'3) NAN.li\. S.S., BAXI, K.K., Gt:LR:-:.;-..:; T.S. ai1d Sr:F:T,IARAI\L,N, C (19fif): [Iaemorrhagi( septicaemia oil-·adjuvant v:1ccine~ study· oi. potency test in rabbits~duration of irnrnunity and ke-eping qu'111ry Indian llet. J, ~t:il 279.

E.E. a.nd l\:1t:ENSTER, () .. A., (1968):· \ rnethod of ;~~\t2duatiun vaccinet1 roEtaining Pasiearrdfo multocidu.. 1111,'t.]. ·vet. Rrc_, 29, 1863· 186/i.

'.?5) PRI~<CE. <;.H. (196!-J): Prociuction of antigens of Pa.~t1•1ffella rnultocidi1. anri cletectioIJ or an tibodic,; against them in bovine sera.]. Comp. Path. 79, 173-186.

2fi) RAMIAH, V.R (1977): The production and detection of Microphage i:ytophilic antibooies toP multocida antigen', in diffeff•nr li,horatory aum:,is. Thesis ;:;ubrnitted 11., Agra UnivnsiLy, Agra.

2'.71 REBERS PA., HEnlJLFs·1ot;, K.L. and GANFIELL, D.].11966): Isolation and characterization of a toxic particulate and protective antig::'nic fraction trom P. mulfocida. Fedn, Proc. 24,698.

;'.:8) SARA, 1 VYAS, D\\ARK:'.l.',.\TH P.K. Wi7S): Influence of Pasteureila infection on plarelets, platdet adhesiveness piasma clotting- time and fibrinogen in sheep. Indian Vet.]. 55, 282-284.

29) SARMAH, A.K. (1976): Studies on cytophi!ic and opsonin adhering antibodies in ca::tle, buffalo and rabbit sera. Thb;i.s suhmitted to Agra Universi1y, Agra, 1976.

-~O) SIN<;H.K.P (1974): Pa,,tflire!losisinpig:s. UttarPradesh Vet.I 2, 1-5. 31) SINGH, T K.J. (196:3) L11mpa,ative '.,tHdie:; on the antigenicity of whole cell and crude fractions

of P multocida (Roberts type I). Thesis »ubmii ted to Agra U nivers1ty. Agra, 1963. 32) SRhASTAV,\, N.C., HARBOLA, P.C. and Ki-LERA. S.S. (1976): Preliminary observations on

combined vaccination against haemorrhagic septicaemia and black quarters. Indian Vet.]. 53, 168-174.

;;;3) \V:El. B.D. and C,w: 1:F, G. R. (1978)· Live streptom;cin-dependent Pasteurella multocida va,· cine for the preventwn of haemorrhagic septicemia. ilmer. I Vei. Res. 38, 1534-1537.

Discussion HiranmrK T. (Japan): ln your paper you stated that you have isolated 4 strains of Pasteurrlla

multocida helonging to 6:B group from pou!tr:1 1) What ()fgan were the strains isolated from? 2) Do you have any experience with experimental infection \Virh 6:B strain in chickens? Can you indicate its pathoge!1icity in chickens?

Answer: 1) I did not make the isolations but I had serotyped three strains of poultry origin from the stock strains maintained at IVRI and one strain from an outbreak of chronic fowl cholera in Maharashtra St.1.te. The last isolate was from heart blood. 2) I am sorry. I have no experience of transmission of 6:B strains experimentaliy.

Gatapia S.L. (Philippines): You mentioned the production of toxins in Pasteu.rella multocida. Don't you think that it would be desirable to carry out studies on media to enhance toxin production for toxoid preparations in immunizing the animals against haemorrhagic septicaemia (HS)?

.\nswer: ft !:actor and its n1edia and growth cur~dit:ion

Watanabe 1\'l,., (Japan,: y·ou 111e:ntioned that :'.,;;,n·1ers to propagc.1 te t1e ci1sfa.c:;et

75

1\ns,vcr: i\nimals becorne rL:.!:inira,JJ~d in Lile dry season (sununer) and foilcY',:ing Lhe stress of inclernent \·veat:her in the beginning of tl-H:: rncnsoon they becon1e rnore susceptible. 'I'he -vvarrn ttnd hun1id cli:nate is conducive to the and spread of urganisn1s. i\.t that ti1ne, the earners tr;1n.srnit' the infection tc, the '.·,ontricts which then b,?corne ill and further :spread the disease. Th<' carriers are usually resis:ant t<j tne disease. Also panial immunity can lead to the carrier status. Iridescent type of organisms ,vhich are harboured hy anin1als \i\·ith lov,,7 resistance ;::,rn to the blue typt winch remains for a long tirnf' i;] the. ;,,nirnal (carrier).

de Alwis M.C.L. Lanka) Comment: ln Sn Laak,, the carriers have been found tc harbour strains of Lhi:c virnl,·i;t rype producing c1ini,·a, These st:·ams di,,appear 6 weeks after tr,e outbreak.

Hanafi l\:t. (Indonesia): I.) ~VVhat is the total \"accination coverage e,ach year in India? 2) '{ou n1entioncd tha-t the oil~adjuvant vaccir1e g-a-.7":: the best results. ll0Vr1 e'-."er, ,vhy is it Lhat )''OU lJSe it less than the o;:hers:' 3) How about haemorrhagic sq,ticaemia \HS) carriers among wild a.nimab?

,.4-rHlV.;er: 1 'T'he overall coverage is below· 17cr1o (bovine population: 200 .rnillion; doses of vaccine: 35 rrdlicn). However, tiK: overad cu,;e;age is irrelevam beca;1se vaccinat.irm is confmcd to endemic pl1ckets where the disea3,, 1, found. 2) The production of oi1-adjuvant is very luw because of the iimitations associated with agar· wash method of produniun employed in India at present Attempts are being mack to adopt the growth in vortex t.1!iks and cor:tinuous cuiture methods. 3) No authentic reports of ,viid animals as HS carriers are mailable, to my knowledge.