ANALYSIS OF FIELD DATA

Introduction

The experiments and concep ts developed in thi s the s i s have

l arge l y evo lved in re sponse to the fund ame n t a l que s t i on "wha t i s

the s i gni ficance o f anima ls whi ch are repeatedly card te s t posi tive

and comp lement f i xa t ion,t e s t negat ive , in the New Ze a l and

brucel lo s i s eradicat ion scheme ? " T o enab l e a proper appreciation o f the re lationship be tween the-card and comp lement fixation tests nat ional and regional data was ana lysed to assess the way in which the two te sts interacted as the scheme progre s sed . T imbs � �· (1978a) inve stigated the exi stence and eventua l fate o f animals that were repeatedly BCT+ and CFT- . Thi s s tudy examines the prevalence of BCT+/CFT- animal s in herds wi th varying ini tial CFT reactor rates.·

Mater ials and Methods

Certain bas i c info rmation concerning details o f all tests carr ied out during the N . Z . bruce l lo s i s eradicat ion scheme i s s tored wi thin a centra l computer f i le .

Unti l August 1 9 7 7 a l l sera from ini tial dairy herd te s t s were card te sted and card test positive sera were then comp lement

fixation tes ted . Init ial bee f herd tests were treated in thi s way

unti l August 1975 . At tests o ther than these the card test was

not nece s sarily per formed and the computer fi les did no t

di fferentiate herd s whi ch were card tested and had no BCT pos i t ives from those which were not card tes ted . Thus data from a l l repeat herd te s t s and some ini tial bee f tests could not be used .

106 . Resu lts

Tab l e XVI l i s ts detai l s of BCT and CFT reactor rates for dairy and beef herds at ini tial herd tests .

( a ) For da iry herd s

Linear regre ss ion y a + bx

y No BCT+ No CFT+ _{X lOO} No animal s - No CFT+ X No CFT+ lOO No anima l s X a = 2 . 5 b 0 . 225 F l , 5 = 60 . 7 1 ·k·-k ( li.) R2 9 2 . 4 i.

(b) For beef herds

a 3 . 7 b 0 . 346

F l , 5 _{1 8 . 1 0} ";'('•/( _{( l} i.) _R2 = 7 8 . 47.

Thus a s igni f i cant corre lation exi s ts between the overa l l herd infect ion rate and the proport ion of animals wi thin herds that are BCT+ but CFT- .

107 .

Tab le XVI . BCT and CFT reactor rates a t ini tial herd t e s ts

( a ) Dairy herds

Herd CFT No . herds No . animals No . BCT+ No . CFT+ i. CFT -

reactor rate that are

BCT+ 0 4895 5 20 1 3 7 17903 3 . 44 0- 1 1435 244045 5 9 24 1586 l. 79 1-5 4147 5 33 9 9 2 2 7 9 2 7 1417 3 2 . 64 5 - 10 2439 343042 3834 1 249 7 7 4 . 20 10- 15 1 305 183006 3 149 6 2 240 1 5 . 6 6 15- 20 608 8 3 1 2 2 18898 1429 1 6 . 69 > 20 418 49090 1 5 2 3 1 1 2 35 7 7 . 8 2 ( b ) Beef herds

Herd CFT No . herds No . anima ls No . BCT+ No . CFT+ i. CFT-

reactor rate that are

BCT+ 0 6 207 8 3 2 1 84 33904 4 . 07 0 - l 8 2 9 2 7 7 155 9 434 1 1 98 2 . 9 7 1 - 5 1 27 4 2 20418 1 6 1 88 5 3 19 s . os 5 - 10 406 807 29 1 1427 5 7 7 9 7 . 5 3 10- 15 155 30945 5 8 7 8 367 3 8 . 09 1 5 - 20 5 8 8 3 7 1 1 842 1 4 2 6 6 . 00 > 20 6 3 5 0 10 1 85 4 1 3 1 8 14 . 5 2

108 .

Discuss ion

Fo l lowing the inab i l ity to cul t ure s i gnificant organi sms from 20 repeat BCT+/CFT - cows the theme o f this proj ect swi tched to an inve s tigati on of the means by whi ch animal s might react to the card test b u t not to the comp l ement fixat ion tes t .

are : -

Pos s i b l e exp lanations for the o ccurrence of BCT+/CFT- animal s ( i ) I f the animal i s infected an unusual ly high s p e c i f i c

I gG1 : I gG2 rat io may cau s e the comp lement f ixa t ion t e s t t o pro zone t o s uch an extent that i t i s erroneous ly cons idered to be negat ive .

( i i ) An animal , previou s ly s ens i t i sed by ca l fhood s train 19 vaccinat i on , may come into contact wi th ki l l ed

( i i i )

� · abortus organi sms o r wi th low doses o f l ive

organisms , such that an immune res ponse is generated wi thout infection becoming e s tab l i shed .

A chronica l ly infe cted anima l may have only l ow leve l s o f spec i f i c I gG1 and IgG 2 . Such low l eve l s may cause the card test to react but may no t cause a comp l ement f ixat ion react ion e i ther because o f the very low antibody l ev·e ls or because o f an unsuitab l e I gG 1 : I gG 2 ratio.

( iv ) I n the e a r l y s tage s o f infect ion an IgM response

genera l ly precede s that of I gG . The greater s ens i t iv i ty o f the card te s t to specific I gM (Al lan � � 1 9 7 6 ) may resul t in a card test reac tion in the abs ence o f any comp l ement f ixat ion t i tre .

In Case ( iv ) i t i s to be expected that at a subsequent s amp l ing the anima l wou l d have become BCT+/CFT+ whi l s t in cas e s ( i ) , ( i i ) and ( i i i ) i t i s conceivab l e that an animal could cont inue to be BCT+/CFT - for some t ime .

In thi s the s i s an attempt has been made to inve s t i gate some of the c i r c ums tance s l eading to the exis tence of such BCT+/CFT ­ animal s par t icularly those which may be explained by po ints ( i ) and ( i i ) above .

109 .

Analys i s o f the data pre sented in T ab l e XVI i l lus trates

that anima ls in heavi ly infec ted herds are more l ikely to be BCT+/ CFT - than animal s in non- infec ted or l ight ly infected herds . Thus

it is evident that the card test reactiv i ty may be s t imu l ated by exposure to - b u t no t nece s s ar i ly infe c t i on b y - B ruce l l a

organi sms .

l t h a s b e e n obs erved by some worke r s that a card t e s t r e a c t ion deve lops ear l ier than a CFT t i tre in infected animal s (Nico l e tt i , 1 9 6 7 ;

19 7 7 ) .

Morgan � � . , 1 9 69 ; Davis , l 9 7 l ; Fens terbank , l 9 7 3 ; P i e t z , I t cou l d be that some o f the BCT+/CFT- anima ls inve s t i ga ted

above be long to this group . Resu l t s of exper iments repor ted by Fens terbank ( 1 9 7 3 ) and P i e t z ( 19 7 7 ) ind i cate that the average per iod be tween deve lopment of a pos i t ive BCT reac t ion and a po s it ive CFT reaction i s o f the order o f 10- 20 days . I t is unl ikely therefore that this phenomena contributes s igni ficant l y t o the overa l l numbers o f BCT+/CFT - animals detected . T imbs

� � . ( l978a) found tha t approximately . 1 6% of BCT+/CFT- anima l s , in infected herd s , detected at any par t icular herd test migh t be expected to become CFT+ at a later test . Inc luded i n this number wou l d be animal s in the ear ly s t ages o f t i tre deve lopment which had deve loped a BCT t i tre prior t o producing a CFT t i tre .

1 10 . CHAPTER 7 . GENERAL DISCUSSION

In choos ing a t e s t on whi ch to base a bovine bruce l lo s i s contro l scheme i t has been cus tomary for e ach country t o carry out p i lo t s tudies to determine not only the preva l ence and d i s tribut ion

of the d i sease but a l so to inve s t i gate the relative mer i t s of the various d iagno s t i c te s t s . Unt i l recently i t has been the SAT

that ha s invar iably been cho sen for use in routine te s t ing whi l s t var ious modi f i cations o f this t e s t such a s mercapto-ethano l , rivano l , heat- l ab i l e and Coomb s t e s t s have been used a s supp l e ­ mental t es t s . Among the deve loped count r i e s New Zealand was relative ly late in formu lating an erad i ca t ion programme .

Techno l ogical improvements did , however , a l l ow the deve lopment o f an automated comp l ement fixation test which was chosen a s the de finit ive test for u s e in the s cheme .

A par t i cu lar prob lem me t with in j us t i fy ing the use o f the Auto-Analyzer adap tat ion of the CFT was the theore t ical argument

that a s the test is e s senti a l ly carried out at one d i l u t ion , i t may no t detect sera which caus e a strong pro zone react ion . Inve s t i gat ion o f t h i s aspect has shown that in practice such sera are in fact d e t e cted , and examinat ion o f the serum d i lut ion gradient wi thin the t e s t sys tem has exp lained the ab i l i ty of the au tomated te s t to detect such sera .

Examinat ion o f pro zoning sera re covered from known infe cted anima l s revealed that i t is the Bruce l l a specific I gG 1 : IgG 2 ratio that determine s the degree o f pro zoning o f a serum in the CFT . I t was a l so s hown that a high ant i gen concentrat ion can

suppre s s the extent to which such pro zoning occur s . The re su l t s

obtained confirm the f ind ings o f P lacke t t and Al ton ( 1975 ) and o f McNaught � �· ( 19 7 7 ) that sufficient quant i t i e s o f Bruce l l a spec i f i c I gG 2 can cause pro zoning and abnorma l reactions in the CFT or prevent comp l ement fixa t ion by I gG 1 altoge ther .

A l though a s er ious imbalance or the Bruce l l a speci f i c I gG 1 : I gG2 ratio wi l l l ead to obvious pro zoning·, a l e s s serious imb a l ance wi l l no t be man i fe s t in this way but may b e re f lected in sub­

o p t imal comp l ement f ixat ion l eading to a reduced t i tre . The extent to whi ch a t i tre may be reduced i s d ependent upon the degree to whi ch the ant i gen concentrati on u sed var i e s from the optimal ant igen concentration required for the p ar t i cu lar serum.

T o ensure that the comp l ement fixat ion te s t system i s made a s s en s it ive as po s s ib le t o prozoning sera , i t i s es s enti a l that

1 1 1 .

adequate care i s taken i n the t it rat ion o f ant igen . A ser i e s o f

known po s i t ive sera mus t be used i n antigen t i trat i ons t o enab l e a n appreciation of the r e l at ive range o f op t imal value s l ike ly to b e obtained for a wide r ange o f sera . Thi s princ i p l e s app l i e s

equal ly t o t i trat ions for the automated o r manual te s t s .

The exi stence o f prozoning s era can exp lain why some sera are

BCT+ yet apparent ly CFT- . S uch sera can be tho s e wi th an

unusua l ly high proportion o f spe c i fi c IgG2 which comp l e te ly blocks

any comp l ement fixat ion . The frequency _wi th whi ch s uch sera

o ccur and their l ikel y s i gn i f i cance in an erad icat ion programme i s

unknown . Animal s y i e l d ing prozoning sera have o ccas iona l ly been

r e - b led after 2 - 6 weeks and the p rozone e ffect has u s ua l ly been very much reduced - and o ften non- exi s tent - in the repeat samp l e . B r andon e t a l . ( 197 1 ) and Wi l l iams and Green ( 19 7 6 ) found that at

about the t ime of parturi t ion serum I gG1 l eve ls fa l l as thi s immuno­ g l obul in i s selective ly concentrated in the udder . The serum IgG 1

I gG2 ratio therefore undergoes cons iderab l e change . A revers ion

to a more normal rat io i s achieved some 4-6 weeks after partur i t ion . B e cause o f the repet i t ive nature o f t e s t ing in an eradi cat ion

p rogramme the exi s tence o f a sma l l number of cat t le with low IgG1 I gG2 rat i o s at any par t i cular t ime i s unl ikely to unduly affect t e s t ing progre s s . However , care mus t be taken in the interpretat ion o f test r e s u l t s from r ecent ly calved or abor ted cows , and the

exi stence of potenti a l ly fa l s e negative CFT res u l t s shoul d be r e cogni sed .

Inve s t i gations o f the way in which the IgG 1 change s in bovine s are practi ca l ly non- exi s tent .

I gG2 ratio B e cause of the

e ffect a change in the B ruce l la specific IgG 1 : I gG2 ratio may have on re sul t s o f the CFT , e xamination o f the physio logical changes in this ratio parti cu l ar ly during the incubat ion phase o f infect ion and a l so at about the t ime o f partur i tion , is required .

Upon examinat ion o f scheme record s , i t was apparent that mor e animal s were l ike ly to b e BCT+/CFT-- i n infected herds than i n non­ infected herd s . Thi s l e ad to .the-hypothe s i s that animal s

previous l y vaccinated with l iving s train 1 9 B r . abor tus a s calve s , then exposed to , but no t infected by , the viru l ent organ i sm as

1 1 2 .

adu l t s , may deve lop a BCT t i tre in the abs ence of any t i tre to the CFT . A lthough studies u s ing l i ght cha l lenges of viru l en t organi sms were no t carr ied out , inoculation wi th ki l l ed organ i sms did

s t imul at e sero logical response s and proved that such exposed

animal s could in fact be BCT+/CFT- . On the other hand i t was a l so shown that o ther anima l s s imi l ar ly exposed could be BCT- /CFT+ .

Another mechani sm by whi ch the BCT may produce fa l se posi tive

react ions but which has not b een inve s t igated in this thes i s i s the pre sence of res idual s train 19 vacc inat ion titres . As I gM is the p r inc i p l e immunog lobul in s t imu lated in re sponse to Br . abortus s train 19 vac cina t ion , it has been pos tu l ated that BCT+/CFT­ reaction s in non- infected cat t l e may be due to the e f fe c t o f a residual vaccinat ion t i tre (Al ton � �. , l975b ) . A l l an � �·

( 19 7 5 ) demons trated that spec i f i c IgM ant ibody i s up to ten t ime s more e ff i cient on a we ight bas i s at producing a BCT reaction than

i s e i ther IgG 1 or I gG2 .

S train 19 vac cination o f a l l female ca lve s was compul sory in

New Zea land between 1966 and 1 97 6 . The pre sent vo l untary

vaccination programme wi l l eventua l ly be phased out when the