Brucellosis in Cows

7/29/2019 Brucellosis in Cows

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Rev. sci. tech. Off. int. Epiz., 1993,12 (3), 909-922

Successful therapeutic regimens for treatingBrucella melitensis and Brucella abortusinfections in cows

A .I . R A D W A N *, S.I. B E K A I R I *, A.M. A L - B O K M Y *, P .V . S. P R A S A D *,O.M. M O H A M E D ** and S.T. H U S S A I N **

Summary: Three therapeutic regimens were evaluated in 121 cows naturallyinfected with Brucella melitensis or Brucella abortus, using a combination oflong-acting Oxytetracycline (LA-OTC), streptomycin (ST) and OTC-intramammary infusion (IMI). Cessation of shedding of brucel la in uddersecretions and absence of Brucella in selected tissues were considered criteriafor successful treatment.

Regimen A (tested on 35 cows) consisted of LA-OTC 25 mg/kgadministered intramuscularly (i.m.) every 3 days for 42 days, ST 25 mg/kg i.m.daily for 8 days, and OTC-IMI 20 ml/teat daily for 4 days. Regimen B (tested on53 cows) was similar to regimen A, except that ST was administered every2 days for 16 days and OTC-IMI every 2 days for 8 days. Both regimens wereequally effective in eliminating Brucella organisms from all cows involved inthe tests and no relapses were recorded. However, regimen C, which was similarto regimen A, except that ST was administered every 3 days for 24 days andOTC-IMI every 3 days for 12 days, resulted in the elimination of Brucellaorganisms from only 30 (91%) of 33 cows.

Before commencement of the therapeutic regimens, B. melitensis biovar 1 or2 had been repeatedly isolated from udder secretions of 103 cows andB . abortus biovar 1 from mammary secretions of 18 cows.KEYWORDS: Antibiotics - Brucella abortus - Brucella melitensis - Cattle -Oxytetracycline - Streptomycin - Therapy.

I N T R O D U C T I O N

Several chemotherapeutic agents have been employed in recent decades for thetreatment of Brucella abortus infection in cows; however, none of these has beenentirely successful. Numerous chemical agents, general antimicrobials (phenols ordyes), trace elements, minerals and mixtures of vitamins (A and E) have been triedunsuccessfully. Fu rt he rm or e, at tempt s using antibi otics such as penicillin orsulfonamides failed to cause cessation of the shedding of B. abortus from the mammarysecretions of infected cows, or caused only temporary cessation (7,11,37).

* A n i m a l P r o d u c t i o n and H e a l t h S e c t i o n , N a t i o n a l A g r i c u l t u r e and W a t e r R e s e a r c h C e n t r e ,Ministry of A gr ic u l tu re and Water , P.O. Box 17285, Riya dh 11484, Sa ud i A ra b ia .** Na t iona l A gr ic u l tu re De ve lopme n t Co., P.O. Box 2557, R iya dh 11461, Sa ud i A ra b ia .


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910The use of broad-spect rum ant ib io t ics such as aureomycin , t e rramycin , t e t racycl inesa n d s t r e p t o m y c i n ( S T ) , s i n g l y o r i n c o m b i n a t i o n , h a s r e s u l t e d i n t h e r e d u c t i o n o fabo rt ion s in in fec ted h erd s or ind iv idual cows (20 ,2 1) . Ho we ver , the cos t o f therapy , thepresence of an t ib io t ic res idues in mi lk and the fa i lu re to cure udder in fec t ions in manycases have led to the genera l conclus ion tha t such t rea tment i s unsu i tab le for the cont ro l

of bovine bruce l los i s .Wi th t he deve lopmen t o f Oxy te t racyc l ine (OTC) and long -ac t ing (LA)-OTC, t he u seo f t h e s e a g e n t s a l o n e o r i n c o m b i n a t i o n w i t h ST h a s s u c c e e d e d i n e l i m i n a t i n g t h es y m p t o m s o f t h i s d i s e a s e a n d r e d u c i n g t h e s h e d d i n g o f b r u c e l l a e b y i n f e c t e d c o w s .Con sequen t ly , such the r ape u t i c reg imen s have bee n app l i ed in i n fec t ed h e rd s t o p reven tabort ions and reduce the spread of bruce l los i s . However , these reg imens have fa i led tore su l t in comp le t e cu re . Fu r th e rm or e , se r ious l oca l reac t ions wer e rep o r t e d in cows , dueto repea t ed in t rape r i t onea l ( i .p . ) i nocu la t i ons w i th OTC (12 ,13 ,24 ,25 ,29 ) . In add i t i on ,the reg imens prev iously t r ied involved smal l doses and few in jec t ions of the an t ib io t icsused . In one o f t hese s tud i e s , i n j ec t ions o f LA-OTC (20 mg /kg in t ramuscu la r ly [ i .m. ]every 3 days for 2 we eks ) co m bin ed w i th ST (20 mg/kg i.m. da ily for 1 we ek ) resu l ted inc e s s a t i o n o f s h e d d i n g o f B. abortus in 10 (71 .4%) of 14 cows (24) . In another s tudy ,inoc ula t io n of L A -O T C (20 m g/kg i.m. every 3 days for 2 we eks) a lon e was successful incu r ing 3 (21%) o f 14 cows . However , when LA-OTC was combined wi th ST (20 mg /kgi n t r a v e n o u s l y [ i .V.] or i .m. da i ly fo r 1 w ee k ) 14 (67% ) of 21 co ws we re successfu l lyt rea t ed (25 ) .

Wi th regard to bruce l los i s in sheep , 9 sheep exper imenta l ly in fec ted wi th B. abortuswere in jec ted da i ly for 6 days wi th 1 g Chlor te t racycl ine , then 20 days la te r wi th 1 .4 gt o g e t h e r w i t h i m m u n e s e r u m f o r 3 d a y s . O n l y o n e s h e e p w a s f o u n d t o b eb a c t e r i o l o g i c a l l y p o s i t i v e a t s l a u g h t e r , 4 6 d a y s a f t e r i n f e c t i o n ( 3 0 ) . F u r t h e r m o r e ,R a d w a n a n d c o l l e a g u e s ( 3 4 ) c o n d u c t e d a l o n g - t e r m t r e a t m e n t t r i a l u s i n g a v a r i e t y o fdoses o f OTC, a lone o r i n combina t ion w i th ST , on 118 sheep na tu ra l l y i n fec t ed w i thB. melitensis. In th i s t r ia l , g roups of in fec ted Najd i sheep were in jec ted wi th 250 ,500 or1 ,000 mg OTC dai ly for 6 weeks , by the i .p . rou te . In the respect ive groups , 52%, 69%and 100% o f t he shee p were found to be Brucella-re-c at the en d of th e t r ial . In addi t ion,t re a tm en t wi th O T C (250 m g i.p . da i ly for 6 we eks) c om bine d wi th S T (1 g i .m. da ily for3 weeks) showed a synerg is t ic e ffec t , increas ing the percentage of Brucella-tree sheep to8 2 % . Moreover , when 8 i n fec t ed Na jd i sheep were i nocu la t ed w i th 1 ,000 mg LA-OTCi .p . every 3 days for 6 weeks , 6 sheep were Brucella-iiee. at necropsy (34). In this t r ial ,the major i ty of the t rea ted sheep developed subcutaneous s te r i le abscesses in the f lankat the s i te of repea ted i .p . inocula t ion wi th OTC. Recent ly , Radwan and co l leagues (35)eva lua t ed s ix d i f fe ren t l ong - t e rm t rea tmen t reg imens on 480 sheep and goa t s na tu ra l l yi n f e c t e d w i t h B. melitensis, u s i n g h i g h d o s e s o f L A - O T C c o m b i n e d w i t h ST . O n e o ft h e s e r e g i m e n s ( L A - O T C 2 5 m g / k g i .m . e v e r y 2 d a y s f o r 4 w e e k s c o m b i n e d w ithST 2 0 m g / k g i . m . e v e r y 2 d a y s f o r 2 w e e k s ) p r o v e d t o b e t h e m o s t p r a c t i c a l , s a fe ,e ffec t ive an d leas t exp ensiv e mea ns of ach iev ing com ple te e l im inat ion of B. melitensis inthe 80 she ep and goa ts t re a te d (35 ) .

In view of the encouraging resul ts in cows (24,25) and the successful resul ts in sheepand goats (35) , the present s tudy was under taken to eva lua te the eff icacy of th reelong - t e rm the rapeu t i c reg imens u s ing a combina t ion o f LA-OTC, ST and OTCin t ramammary in fus ion ( IMI) i n e l imina t ing B. melitensis o r B. abortus from natural ly-infec ted cows. The a im of the s tudy was to ob ta in a therapeut ic reg imen which i seffec t ive , p rac t ica l , wi thout s ide-effec ts and re la t ive ly inexpensive , in order to maket re a tm en t of Bri ice l la - infec ted cows wi th super ior genes a v iab le a l te rn a t ive to s laughter .


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911M A T E R I A L S A N D M E T H O D S

AnimalsA tota l of 121 cows were selected, which gave positive results for brucellos is after

serological and bacteriological tests. These animals were selected to provide a moreconclusive evaluation than that obtained in previous studies . Th e cows orig inated fromthree large commercial dairy herds raised in the central and eastern provinces of SaudiArabia. Th e animals belonged t o two br eed s (108 Hols tein x Friesian and 13 Jers ey).The cows were aged 3-10 years and weighed between 450 kg and 750 kg. The th ree herdswere composed of highly valuable breeding cows originally imported from the UnitedStates of America (USA) and Eur ope. Herd records indicated that none of the cows hadbeen vaccinated during calfhood.

In two herds, B. melitensis was isolated from udder secretions of 103 cows (biovar 1from 97 cows and biovar 2 from 6 cows) . These two herds originally tes ted seronegat iveon arrival in Saud i Ar ab ia , app roxima te ly four yea rs pr io r to this study. Th e cowscontracted brucellosis from sheep and goats infected with B. melitensis (biovars 1 and 2)which had been introduced to the two farms 8-12 months before bovine brucellosisoccurred in the two cattle herds (unpubl ished findings).

In the third herd, B. abortus biovar 1 was isolated from the mammary sec retions of18 cows. This her d conta ine d few seroposi tive cows on arrival in Saudi Ara bi a, eightyears prior to the present study.

Shortly before initiation of the presen t tre atmen t regimens, 89 cows were found notto be pr egn an t (having already abor ted or calved 4-8 mon ths befo re tr ea tm en t) and32 cows were in various stages of pregnancy. Milking was ceased in lactating cows. The121 cows were divided into three treatment groups (A, B and C), without regard to age,number of previous pregnancies, date of recent pregnancy and number of previousabortions due to Brucella. The reason for the division into thr ee tr eat men t scheduleswas to choose the most practical and effective regimen, particular ly because LA-OTC isa long-acting antibiotic, in contrast to ST and OTC-IMI which are not. Since the numberof avai lable cows infec ted with B. abortus was small, these were placed in group Btogether with 35 cows with B. melitensis. All cows in the study were isolated from otheranimals. Non-treated cows were not included as controls, as several previous studieshave indicated that Brucella infections persist in cattle and spontaneous recovery isuncommon (10,22,28).

One mon th after trea tme nt, all non -pr egn ant cows were artificially inse minat ed.Three months later, a total of 12 pr egnant cows (4 from each tre at me nt g roup) weresacrificed, and selected tissues were collected for bacteriological examination. Inaddition, immedi ate ly after b irth, a total of 3 calves (1 from a cow in each tr ea tm en tgroup) were sacrificed, and selected tissue samples were cultured.Therapeutic agents

The pharmaceutical products used in this study were as follows:- LA -O TC injectable solut ion (from Fia nc e) conta ining 200 mg O T C base pe r

millilitre- ST sulphate (from Giza, Egypt) supplied in vials, each containing lg or 4 g,

dissolved (just before use) in 3 ml or 12 ml sterile distilled water, respectively


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912- O T C - I M I ( f ro m A t h e n s , G r e e c e ) in 1 0 m l s y r i n g e s , e a c h c o n t a i n i n g 2 00 m g O T Ch y d r o c h l o r i d e , 1 00 m g n e o m y c i n s u l p h a t e , 1 00 m g o l e a n d o m y c i n p h o s p h a t e , 5 m gP re dn is o lo ne a nd 10 ml spec ia l ex c ip ien t .

Therapeutic regimensT h e t r e a t m e n t r e g i m e n s o f t h e t h r e e g r o u p s o f i n f e c t e d c o w s a r e s h o w n i n T a b l e I .B o t h S T a n d O T C - I M I w e r e a d m i n i s t e r e d w i th L A - O T C a t t h e c o m m e n c e m e n t oft h e r a p y . T h e L A - O T C a n d S T w e r e i n j e c t e d i. m . i n t h e c e r v i c a l , t h i g h a n d s h o u l d e rmus c les , a t a dos age o f 20 -3 0 ml pe r s i t e . T he da te o f p rev ious abor t ions in r e la t ion tothe in i t i a t ion o f t r ea tmen t was no t r eco rded on the fa rms invo lved .

Serological testingA l l a n i m a l s w e r e s u b j e c t e d t o t h r e e s u c c e s s i v e s e r o l o g i c a l e x a m i n a t i o n s a t m o n t h l yi n t e r v a l s . A t e a c h e x a m i n a t i o n , a n i m a l s w e r e i n i ti a l ly s c r e e n e d fo r t h e p r e s e n c e of

Brucella a g g l u t i n i n s b y t h e r o s e b e n g a l t e s t ( 3 ) . T h e r o s e b e n g a l t e s t a n t i g e n w aso b t a i n e d f ro m t h e C e n t r a l V e t e r i n a r y L a b o r a t o r y , N e w H a w , W e y b r i d g e , U n i te dKingdom. A n imals t e s t ing pos i t ive to the ros e benga l t e s t were r e tes ted by the s t andardU S p l a t e a g g l u t i n a t i o n p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f Brucella ant ibody t i t res (3) .T h e s t a n d a rd Brucella p l a t e a n t i g e n ( m a d e o f B. abortus s t r a in 1119 -3 ) was ob ta inedf r o m t h e U n i t e d S t a t e s D e p a r t m e n t o f A g r i c u l t u r e i n A m e s , I o w a , U S A . A g g l u t i n a t i o nat 1 :100 or greater was cons idered pos i t ive .Fo l lowing the th ree s e ro log ica l ex am ina t ion s , mi lk s am ples f rom a l l s e ropos i t ive cowswere s ub j ec ted to bac te r io log ica l ex amina t ion fo r iden t i f i ca t ion o f Brucella s hedder s .Fu r th e r m or e , s e ra f rom th e s e lec ted 121 s e ro log ica l ly - an d bac te r io log ica lly -pos i t ivecows were s e ro log ica l ly t e s ted aga in immedia te ly be fo re in i t i a t ion o f the p res en tt r ea tmen t r eg imens and a t mon th ly in te rva l s a f t e r comple t ion o f t r ea tmen t ( fo r 4 mon thsin 12 sacr if iced cow s an d for 16 m on th s in the re m ain ing 109 cow s) . In add i t ion , sera fromall ca lves bo rn f rom t r ea ted cows we re ex am ined im m edia te ly a f t e r b i r th .

Bacteriological examinationT h e m e d i u m f o r Brucella c u l t u r e w a s p r e p a r e d a s d e s c r i b e d p r e v i o u s l y (3 5).I m m e d i a t e l y b e f o r e i n i ti a t io n o f t h e t h e r a p e u t i c r e g i m e n s , c u l t u r e s o f u d d e r s e cr e ti o ns

w e r e m a d e f r o m t h e s e l e c t e d 1 2 1 p o s i t i v e c o w s t o c o n f i r m s h e d d i n g of Brucellao r g a n i s m s . S e p a r a t e 3 0 m l s a m p l e s of u d d e r s e c r e t i o n s f ro m e a c h q u a r t e r w e r e a lsoc u l t u r e d e v e r y w e e k d u r i n g t r e a t m e n t , e v e r y m o n t h a f t e r c o m p l e t i o n o f t r e a t m e n t u n t i lc a l v i n g a n d a g a i n e v e r y 2 w e e k s a f t e r c a l v i n g f o r 6 m o n t h s . I n a d d i t i o n , f r o m th e12 p re gn an t sac r if i ced cow s (4 cows f rom eac h t r ea tm en t g rou p wh ich w ere s l augh te reda t 4 m o n t h s p o s t - t r e a t m e n t ) t h e f o ll o w i n g t i ss u e s p e c i m e n s w e r e a s e p t i c a l l y c o ll e ct e df o r b a c t e r i o l o g i c a l e x a m i n a t i o n : s u p r a m a m m a r y , p r e s c a p u l a r , i l i a c , p r e c r u r a l ,m e d i a s t i n a l , m e s e n t e r i c , p o p l i t e a l a n d h e a d l y m p h n o d e s , u d d e r s e c r e t i o n , a n d s ec ti on sof udder , u te rus , ovary , b ra in , l ive r and s p leen . In add i t ion , s am ples o f s tom ach con ten t s,l i v e r , l u n g a n d s p l e e n o f f o e t u s e s f r o m t h e s a c r i f i c e d a n i m a l s w e r e c u l t u r e d . S i m i l a rs e lec ted t i s s ue s pec im ens w ere cu l tu red f rom 3 s ac ri f iced ne w bo rn ca lves (1 f rom a cowi n e a c h t r e a t m e n t g r o u p ) .

A l iq uo ts f rom each udder s ec re t ion s ample were s p read wi th s t e r i l e co t ton s wabs on2 - 4 f r e s h l y p r e p a r e d p l a t e s o f c u l t u r e m e d i u m . E a c h t i s s u e s p e c i m e n w a s i n d i v i d u a l l yh o m o g e n i s e d i n a t i s s u e g r i n d e r a n d a l i q u o t s w e r e s p r e a d w i t h s t e r i l e c o t t o n s w a b s o ns e v e r a l ( 4 - 8 ) f r e s h l y p r e p a r e d p l a t e s o f c u l t u r e m e d i u m . T h e p l a t e s w e r e i n c u b a t e d a t3 7 C fo r 7 d a y s i n t h e p r e s e n c e a n d a b s e n c e of 1 0 % C 0 2 a t m o s p h e r e . T h e i s o l a t e d


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914Brucella cultures were identified morphologically, microscopically, biochemically andserologically (3). The biotyping of the identified isolates was performed at the CentralVeterinary Laboratory, New Haw, Weybridge, United Kingdom.

When Brucella organisms could no longer be recovered from udder secretions norfrom any of the selected tissue specimens collected at necropsy, the respective cow wasconsidered to be cured (successful treatment). Continued or resumed shedding ofBrucella organisms in udder secretions or the isolation of Brucella organisms from anyof the tissue specimens obtained at necropsy were considered as conclusive evidence oftreatment failure.

In this study, aborted foetuses from the three cattle herds involved were cultured forBrucella isolation, in addition to routine bacteriological examination for listeriosis,salmonellosis, mycoplasmosis and mycotic abortion.

R E S U L T S

Breeding performanceA comparison was made between breeding performance in the three herds prior to

identification of the infected cows and that in the treated animals in the breeding seasonfollowing treatment. Before treatment, the cows had been living in an infectedenvi ronment for periods of between 1 and 8 years. The abortion rat e in the th ree herdsran ged betwe en 5 % and 15% and the infertility rate was app roxi mat ely 15% . Inaddition, there were unusually high rates of metritis, retained placenta and mastitis,with milk produc tion reduced by 10-15%. However, in the breedi ng season followingtreatment, the cows were living in an uninfected environment. Of the cows treated withregimens A, B and C, 94% became pregnant and experienced normal calving. Noabortions were observed among the treated cows. On the whole, the treated cowsexpelled placenta from the womb without requiring any special manual manipulation,or the use of chemotherapeutic measures. Furthermore, the 32 cows which were invarious stages of pregnancy at the time of initiat ion of the the rap eu tic regimens A, Band C ex peri enced nor ma l calving within 4 mo nths following compl et ion of thetreatment. Moreover, there were no cases of metritis or mastitis in the treated cows, andmilk production was within the normal range for uninfected animals of the same breed.Side-effects of the therapeutic agents

In 8 cows, there was very light swelling and signs of painful sensations at the site ofrepeated LA-OTC injections, particularly in the thigh muscles. These slight localreactions disappeared within 36 hours. Meat inspection of the twelve cows which wereslaughter ed (4 mont hs pos t-t rea tment) revealed comple te absence of any detectableabnormalities in the muscles at the site of intramuscular inoculations of LA-OTC andST. Furthermore, no systemic reactions were observed in any of the treated cows duringthe treatment period.Cost of therapeutic agents and animals

The weight of the treated cows ranged between 450 kg and 750 kg, and the averagecost of the the rapeut ic agents alone used (regimen A, B or C) in treating a cow weighing600 kg was US$186 (SR700).


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91 5Th e t r e a t e d c o w s w e r e im p o r t e d a f e w y e a r s p r e v io u s ly a s p r e g n a n t h e i f e r s , a t a c o s tof U S$3,600 each , inc lu d ing th e cos t o f a i r f re igh t to S aud i A ra b i a .

Serological findingsA m o n g t h e t r e a t e d a n i m a l s , t h e r e w a s a d e c r e a s e o f o n l y o n e d i l u t io n in t h e U S p l a t ea g g l u t in a t i o n t e s t ( f ro m 4 0 0 t o 2 0 0 ) f r o m t h e b e g i n n i n g of t r e a t m e n t u n t i l 4 m o n t h sp o s t - t re a t m e n t i n 4 o f 12 s l a u g h t e r e d c o w s . T h e r e m a i n i n g 8 s l a u g h t e r e d c o w s d id n o tshow a n y d e c r e a s e in t i t r e o v e r th e sa m e p e r io d .T h e Brucella a g g l u t i n i n t i t r e s w e r e a l s o c o m p a r e d w i t h r e s u l t s o b t a i n e d p r e -t re a tm e n t a n d 1 6 m o n t h s p o s t - t r e a t m e n t i n t h e r e m a i n i n g 1 0 9 t r e a t e d c o w s . T h e r e w a s ad e cr ea s e o f t w o , t h r e e a n d f o u r d i l u t i o n s in 4 2 % , 4 3 % a n d 1 0 % o f t h e t r e a t e d c o w sr espe ct iv e ly . I n a d d i t io n , o n ly 5% o f th e 109 c o w s b e c a m e se r o n e g a t iv e 16 m o n th s p o s t -t r e a tm e n t , w h i l e a l l c a lv e s b o r n f r o m t r e a t e d c o w s w e r e se r o n e g a t iv e fo r b r u c e l lo s i s .

Bacteriological findingsI n th e p r e se n t s tu d y , n o Listeria, Salmonella, Mycoplasma o r p a t h o g e n i c f u n g i w e r ei s o l a t e d f r o m t h e a b o r t e d f o e t u s e s e x a m i n e d i n t h e t h r e e h e r d s ; o n l y B. melitensis o r

B. abortus w e r e i s o l a t e d . T h e e f f i c a c y o f t h e t h r e e t r e a t m e n t r e g i m e n s a p p l i e d i ne l imina t ing B. melitensis o r B. abortus f r o m 1 2 1 n a t u r a l l y - i n f e c t e d c o w s i s s h o w n i nTable I.I n r e g im e n A , th e se l e c t e d t i s su e sp e c im e n s f r o m 4 sa cr i fi c e d c o w s ( a t 4 m o n th s p o s t -

t r ea t m e n t ) a n d a l l r e p e a t e d u d d e r s e c r e t i o n s a m p l e s f r o m t h e r e m a i n i n g 3 1 c o w s ( fo r16 m o n th s p o s t - t r e a tm e n t ) w e r e a l l fo u n d to b e f r e e f r o m Brucella o r g a n i s m s .W hen 53 cows (of which 35 were na tu ra l ly in fec ted w i th B. m elitensis an d 18 infected with

B. abortus) w e r e t r e a t e d w i th r e g im e n B , a l l s e l e c t e d t i s su e sa m p le s f r o m 4 s l a u g h te r e dcows a n d th e r e p e a te d u d d e r se c r e t io n sa m p le s w e r e fo u n d to b e Brucella-free.H o w e v e r , w h e n 33 c o w s (n a tu r a l ly in fe c t e d w i th B. melitensis) w e r e t r e a t e d w i t hregimen C, B. melitensis b i o v a r 2 w a s i s o l a t e d ( 1 -5 c o l o n i e s p e r p l a t e ) f r o m m a m m a r yse cr et io n s, u d d e r t is su e a n d su p r a m a m m a r y ly m p h n o d e s of o n ly 1 o f 4 sa c r i fi c e d c o w s

(at 4 m o n t h s p o s t - t r e a t m e n t ) . I n a d d i t i o n , B. melitensis b io v a r 1 w a s i so l a t e d fr o m th er ep e a te d u d d e r se c r e t io n sa m p le s f r o m o n ly 2 o f 29 c o w s . T h e a v e r a g e n u m b e r o fBrucella c o lo n ie s r e c o v e r e d p e r p l a t e f r o m th e u d d e r se c r e t io n sa m p le s o f t h e se 2 c o w sprior t o t r e a t m e n t w a s a p p r o x i m a t e l y 3 0 t i m e s h i g h e r t h a n t h e n u m b e r r e c o v e r e dfo llow ing t r e a t m e n t , w h i c h r a n g e d b e t w e e n 2 a n d 8 c o l o n ie s p e r p l a t e . H o w e v e r , w h e nthese 2 Brucella-shedding c o w s w e r e r e - t r e a t e d w i t h r e g i m e n B , t h e y c e a s e d s h e d d i n gBrucella.

A l l se l e c t e d t i s su e sp e c i m e n s f r o m th e 3 n e w b o r n c a lv e s (1 c a lf f r o m a c o w in e a c ht rea tment g roup) were found to be f ree f rom Brucella o r g a n i s m s .I n a l l su c c e ss fu l ly t r e a t e d c o w s in th e p r e se n t s tu d y (118 c o w s t r e a t e d w i th r e g im e nA , B o r C ) , sh e d d in g o f Brucella o r g a n i s m s i n m a m m a r y s e c r e t i o n s h a d c e a s e d w h e nthese s e c r e t i o n s w e r e c u l t u r e d ( 1 w e e k a f t e r i n i t i a t i o n o f t h e a n t i b i o t h e r a p y ) a n d d i dnot r e c o m m e n c e . H o w e v e r , i n t h e 3 c o w s o f r e g i m e n C w h e r e t r e a t m e n t f a il e d , t h ea m ou nt o f s h e d d i n g d e c l i n e d a f t e r t h e i n i t i a t i o n o f t r e a t m e n t a n d r e m a i n e d l o w u n t i l4 m o n th s p o s t - t r e a tm e n t in 1 s l a u g h te r e d c o w , o r a ft e r c a lv in g in th e o t h e r 2 c o w s ( th e s ean im als w e r e th e n r e - t r e a t e d w i th r e g im e n B ) .


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916D I S C U S S I O N

In order to increase milk production, commercial dairy producti on was introduced toSaudi Arabia through the importation of valuable and highly productive exotic cattle ata very high cost. These cattle were originally imported as pregnant heifers from theUSA and some European countries, namely: the Netherlands, Germany and France.On arrival in Saudi Arabia, the cattle were raised in large numbers under intensivemanagement systems and sometimes together with small ruminants.

Brucellosis has been reported and confirmed in both livestock and humans in SaudiArabia. B. melitensis biovars 1, 2 and 3 were responsible for all infections in sheep,goats, camels and dairy cattle, except cows in one dairy herd which had B. abortus.Consequently, B. melitensis accounted for 92% of human cases and B. abortus for 8%,while B. suis was not found in animals or humans (1,2 ,4,5,6 ,16,18,19,23,31,32,33,34,35,39).

In the present study, two of the cattle herds contracted B. melitensis infection fromsheep and goats raised on the same farms. Transmission of this infection to cattleproduced an infection similar to that produced by B. abortus. Such infection wasdirectly responsible for the high rate of abortions, infertility, metritis, retained placentaand mastitis with reduced milk production, leading to serious consequences for cattleperformance and cattle handlers. However, in B. abortus infection, it is recognised thatthe economic consequences of infection are encountered principally during initialinfection and become less prominent in further pregnancies.

In a campaign against brucellosis, it is impossible to apply only one method whichwould suit every country. The "test and slaughter" method combined with calfhoodvaccination no doubt contributes to considerable reduction in the number of infectedherds; however, for economic reasons this method cannot be applied in all cases. Aneradication campaign through slaughter and compensation is not within the economicscope of developing countries, especially where it is difficult to change agriculturalcustoms or social habits. On the other hand, mass vaccination of infected herds protectsonly uninfected animals without altering the course of infection. The infectedvaccinated animals continue to present a serious public health risk. In addition, oneworker assessed the average cost of an infected cow at approximately US$3,200, not justfor abortion or stillbirth, but from the subsequent problems or irregular breeding, lossof milk production, and the reduced human productivity resulting from a spread ofinfection (38). Moreover, with the development and widespread use of techniques suchas artificial insemination and embryo transfer, cattle with superior genetic potentialhave become increasingly valuable. Slaughter of infected animals with superior geneticpotential has serious economic and genetic consequences (24). Livestock producers inSaudi Arabia and in many other developing countries cannot afford the traditional "testand slaughter" approach used in developed countries. An effective, practical and safeant ib iot her apy wou ld be of enormous benefit to producer s in the se count ries as analternative to slaughter of infected animals of high value. Although this has been thegoal of several workers , none has been fully successful.

The following explanations were given for the previous treatment failures anddifficulties:

a) Brucella organisms are known to survive within phagocytic cells of thereticuloendothelial system, particularly in lymph nodes, liver, spleen, bone marrow, the


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91 7m a m m a r y g l a n d s , r e p r o d u c t i v e o r g a n s a n d ti s s u e s w i t h a w e a k b l o o d su p p l y , a n d a r et h er e fo r e p r o t e c t e d fr o m a n t i b o d i e s , c o m p l e m e n t a n d a n t i b i o t i c s . T h e a b i l it y o fB ruce l l a o rgan i s m s t o s u rv i ve i n t r ace l l u l a r l y can res u l t i n l ong- t e rm ch ron i c i n f ec t i on .In a d d i t i o n , r e l a p s e s a f te r a n t i m i c r o b i a l t r e a t m e n t w e r e a l s o e x p l a i n e d b y t h ei nt ra c el lu la r l o c a t i o n of b r u c e l l a e ( 9 , 2 4 , 2 5 , 2 6 ) .

b) I n c o r r e c t c h o i c e of a n t i b io t i c s ( 7 , 1 1 , 3 7 ) .c) I n s u ff ic i en t d o s e s o f e ff ec ti ve a n t i b i o t ic s ( 1 2 , 1 3 , 2 4 , 2 5 , 2 6 , 2 9 ) .d) I n su ff ic ie n t d u r a t i o n of t r e a t m e n t ( 8 , 1 2 , 1 3 , 2 4 , 2 5 , 2 6 , 2 9 ) .e) U s e o f t h e i n t r a p e r i t o n e a l r o u t e f o r a d m i n i s t r a t i o n o f a n t i b i o t i c s c r e a t e d s e r i o u sreact ions in some cases (17 ,34) .f) U s e o f o n l y o n e a n t i b i o t i c ( 2 0 , 2 1 , 2 4 ) .g) H i g h c o s t o f a n t i b i o t h e r a p y ( 2 4 , 2 5 , 3 4 ) .h) F a i l u r e t o c u r e u d d e r i n f ec t io n s i n m a n y c a s e s ( 2 4 , 2 5 , 2 6 ) .i) P r e s e n c e o f a n t i b i o t i c r e s i d u e s i n m i l k a n d m e a t o f t r e a t e d a n i m a l s , a n d t h eposs ib le har m ful ef fect on co ns um er s (27) .T h e p r e s e n t s t u d y a i m e d t o o v e r c o m e t h e a b o v e p r o b l e m s a n d i n v o l v e d w o r k u n d e rpar t i cu l a r l y d i f f i cu l t cond i t i ons wi t h r egard t o t he l eve l o f i n f ec t i on am ong t he cowsu sed , w h i c h w e r e a ll s e r o l o g i c a l l y a n d ( p a r t i c u l a r l y ) b a c t e r i o l o g i c a l l y p o s i t i v e .C o n s e qu e n tl y, t h e f ol lo w i n g p o i n t s w e r e t a k e n i n t o a c c o u n t w h e n a t t e m p t i n g t o o b t a i n

m ore s ucces s fu l r e s u l t s . S i nce s uccess f ul t h e ra py i n hu m an be i ngs app ear s t o de pe nd onthe pe rm ea bi l i ty of ce l l wal l s to drug s (24) , the O T C ba se was used be ca us e i t i s ca pa bleof pen e t ra t i n g i n t r ace l l u l a r l y and i nh i b i t s bac t e r i a l p r o t e i n s y n t hes i s a t t he leve l of t her ib o s o m e s. T h e L A - O T C v a r i a n t w a s s e l e c t e d t o s a v e t i m e a n d e ff o r t a n d t o p r o v i d el on g -l as ti ng O T C c o n c e n t r a t i o n i n b l o o d p l a s m a , a s o n e in j e c t i o n g a v e a n e f f e c ti v econcent ra t ion of 0 .6 ug/ml for three days (26) . ST was a l so used , because i t i s known toin hib it p r o t e i n s y n t h e s i s o f G r a m - n e g a t i v e b a c t e r i a . F u r t h e r m o r e , S T a c t ss ynerg i st ica ll y wi t h O T C t o i nh i b i t g r ow t h o f B. abortus w i t h i n b o v i n e c e l l s c u l t u r e din vitro. O T C a t 0 . 5 - 1 . 0 g / m l , p e r m i t t e d g r o w t h o r w a s b a c t e r i o s t a t i c f o r b r u c e l l a ewithin t i ss ue ce l ls . H ow ev er , O T C p r ov ed t o be e ff ec ti vel y b ac t e r i c i d a l wh en co m bi n edwith 1 0 g / m l o f S T ( 1 5 , 2 5 , 3 4 , 3 6 ) . I n a d d i t i o n , a c o m b i n a t i o n o f L A - O T C a n d S Tp r o v e d v e r y e f f e c t i v e i n c u r i n g s h e e p a n d g o a t s n a t u r a l l y i n f e c t e d w i t h B. melitensis(35). H o w e v e r , e a r ly st u d i e s ( 2 0 , 2 4 , 2 5 ) o n t h e t r e a t m e n t o f b o v i n e b r u c e l lo s i s i n d i c a t e dpoor s u cc e s s r a t e s w h e n L A - O T C o r S T w a s u s e d a l o n e . F o r t h e s e r e a s o n s , L A - O T Cc o m b in e d w i t h S T w a s u s e d f o r t h e t r e a t m e n t of c o w s n a t u r a l l y i n f e c t e d w i t hB. melitensis o r B. abortus.

F u r t h e r m o r e , in i n f e c te d c o w s , d e s p i t e t h e u s e of a c o m b i n a t i o n o f L A - O T C a n d S T ,severa l au t h o rs f a i led t o cu re u dd er i n f ec t i ons i n m an y cas es ( 2 4 , 2 5 ,2 6 ) . Fo r t h is r e as o n ,b e f o r e i n i t i a t i o n o f t h e p r e s e n t t r e a t m e n t r e g i m e n s , m i l k i n g w a s s t o p p e d i n l a c t a t i n gcows and udd ers wer e com pl e t e l y ev acu a t e d be f o re each i n j ec t ion , i n o r de r t o de c re as ethe n u m b e r o f Brucella o r g a n i s m s in u d d e r t i s su e s a n d s e c r e t i o n s . C o n s e q u e n t l y ,O T C- IMI was us ed l oca l l y i n com bi na t i on wi t h s ys t em i c ( i .m . ) i nocu l a t i on o f L A- O T Cand S T . T h i s c o m b i n a t i o n p r o v e d t o b e h i g h l y e f fe c t i v e i n e l i m i n a t i n g Brucellao rg an is m s f r o m a l l i n f e c t e d c o w s i n r e g i m e n s A a n d B . H o w e v e r , O T C - I M I a n d S T ,when adminis tered every 3 days in reg imen C, were less ef fect ive in e l iminat ing Brucellaorgan is m s i n i n f ec t ed cow s . T h i s m ay be due t o t he ' f ac t t ha t bo t h S T and O T C - IM I a re


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91 8n o t l o n g - a c t i n g a n t i b i o t i c s . A l s o , i t s e e m s u n l i k e l y t h a t O T C - I M I a l o n e w o u l d b eef fect ive for the t rea tement of l ac ta t ing cows due to the cont inuous f lushing of OTC bym i l k i ng , and t he p ro t ec t i on o f Brucella organ i s m s i n t he p res ence o f m i l k .

P r e v i o u s s t u d i e s in c o w s a n d s h e e p ( 1 7 , 3 4 ) c o n c l u d e d t h a t i. p . i n o c u l a t i o n o f O T Cw a s n o t sa f e , d u e to th e d e v e l o p m e n t o f s e v e r a l s e r i o u s l o c a l r e a c t i o n s a t t h e s it e ofi n j e c t i o n . I n t h e p r e s e n t s tu d y , i .m . i n o c u l a t i o n o f L A - O T C d i d n o t r e s u l t i n h a r m f u lsys temic ef fect s or any obvious las t ing local react ions a t the s i t e of inocula t ion .

T h e d o s e s a n d d u r a t i o n o f a p p l i c a t i o n o f s e l e c t e d a n t i b i o t i c s i n t h e p r e s e n t s t u d yi n d i c a t e t h a t t h e l o n g - t e r m t h e r a p y a n d t h e d r u g c o m b i n a t i o n s u s e d y i e l d e d b e t t e rr e s u l t s t h a n s e v e r a l o t h e r r e g i m e n s ( 8 , 1 2 , 1 3 , 2 4 , 2 5 , 2 6 , 2 9 ) .I n h u m a n s , a l t h o u g h O T C h a s b e e n c o n s i d e r e d t h e m o s t ef fe c t iv e a n t i b i o t i c fort r ea t m en t o f b ruce l l o s i s , a s m al l num ber o f r e l aps es do occu r . Re l aps es have a l s o beenrep or t ed i n cows ( 1 4 ,2 4 ,2 5 ) . In t he p res e n t s t udy , r e l ap s es oc cu r r ed i n on l y 3 o f 3 3 cows

t rea t ed wi t h r eg i m en C . However , r e l aps es d i d no t occu r i n any o f t he 8 8 cows t r ea t edw i t h r e g i m e n A o r B ( p r o v i d e d t h a t t h e t r e a t m e n t p r o t o c o l is s t ri c tl y o b s e r v e d ) . I n th ep r e s e n t t r i a l , t h e m a j o r i t y ( a p p r o x i m a t e l y 9 0 % ) o f t h e c o w s t r e a t e d w e r e m o n i t o r e db a c t e r i o l o g i c a l l y f or 1 6 m o n t h s p o s t - t r e a t m e n t i n o r d e r t o d e t e c t p o s s i b l e r e l a p s e s.E v e n in r e l a p s e d c o w s (3 a n i m a l s i n r e g i m e n C ) , o n l y a s m a l l n u m b e r o f Brucellaco l on i es were r ecovered f rom l a rge t i s s ue s am pl es .I n t h e p r e s e n t s t u d y , a l l t r e a t e d c o w s w e r e d r i e d b e f o r e i n i t i a t i o n o f tr e a t m e n t ;c o n s e q u e n t l y , t h e r e w a s n o r i s k o f h a r m f u l e f f e c t s o n h u m a n b e i n g s f r o m a n t i b i o t i cres i dues i n t he m i l k f rom t hes e cows du r i ng and a f t e r t r ea t m en t . However , t he r e s i dues

o f an t i b i o t i c s i n m i l k and m ea t o f t r ea t ed an i m al s s hou l d be i nves t i ga t ed t o de t e rm i neaccu ra t e l y when m i l k and /o r m ea t can be s a f e l y cons um ed f o l l owi ng com pl e t i on o f t het r e a t m e n t r e g i m e n s .T h e p r e s e n t r e s u l t s o f s e r o l o g i c a l e x a m i n a t i o n s w e r e o f s o m e u s e i n e v a l u a t i n g t h ee f f e c ti v e n e s s o f t h e t r e a t m e n t r e g i m e n s . H o w e v e r , t h e r e s u l t s w e r e n o t c o n c l u s i v e , asa n i m a l s w e r e o n ly m o n i t o r e d fo r 1 6 m o n t h s p o s t - t r e a t m e n t , a n d t h e r e w a s n o n on -t r e a t e d c o n t r o l g r o u p f o r c o m p a r i s o n . T h e r e s u l t s o b t a i n e d w e r e i n a g r e e m e n t w i t hf i nd i ngs on s ucces s f u l t r ea t m en t o f b ruce l l o s i s i n s heep and goa t s when m on i t o red f o r8 m o n t h s p o s t - t r e a t m e n t ( 3 5 ) . O t h e r i n v e s t i g a t o r s , h o w e v e r , r e p o r t e d t h a t s e ro lo g ic a l

f i nd ings we re no t u s e fu l in eva l u a t i ng t he e ff ec ti venes s o f t r e a t m en t r eg i m en s ( 2 4 ,3 4 ) .In S aud i Arab i a , t he t r ea t ed cows were o r i g i na l l y i m por t ed as p regnan t he i f e r s a t anaverage cos t of US$3,600 , including the cos t of a i r f re ight . The cos t of an t ib io t ics usedwas ca l cu l a t ed acco rd i ng t o sa l e p r i ces i n t he S au d i m a rk e t a t t he t i m e o f t he ex per i m en t( U S $ 1 = S R 3 . 7 5 ) . T h e a v e r a g e c o s t o f t h e a n t i b i o t i c s u s e d i n r e g i m e n A , B o r C f o rt r ea t i ng a cow of an av erag e we i gh t of 6 00 k g was U S $ 1 8 6 ( S R 7 0 0 ) . T h i s d i d no t i nc ludet h e c o s t o f e q u i p m e n t o r i n t e r v e n t i o n s , b e c a u s e a l l s e r o l o g i c a l a n d b a c t e r i o l o g i c a le x a m i n a t i o n s w e r e p e r f o r m e d f r e e of c h a r g e a t G o v e r n m e n t i n s t i t u t i o n s . H o w e v e r , ino t h e r s i t ua t i on s , t he cos t of s upp l i e s and i n t e r ven t i on s ho u l d be co ns i de red . T h e cost of

s ucces s f u l t r ea t m en t o f a cow rep res en t s app rox i m at e l y 5 % o f t he o r i g i na l p r i ce o f t hean i m al , wh i ch i s cons i de red econom i ca l l y f eas i b l e i n S aud i Arab i a .I n c o n c l u s i o n , w h e n t r e a t m e n t p r o t o c o l s w e r e s tr i c tl y o b s e r v e d , r e g i m e n s A a n d Bp r o v e d t o b e e f f e c t i v e , p r a c t i c a l , w i t h o u t s i d e - e f f e c t s a n d r e l a t i v e l y i n e x p e n s i v e i nc a u s i n g c e s s a t i o n o f t h e s y m p t o m s o f b r u c e l l o s i s a n d e l i m i n a t i n g Brucella organ i s m sf r o m i n f e c t e d c o w s . C o n s e q u e n t l y , a t t h e p r e s e n t t i m e , t h i s t r e a t m e n t w o u l d p r o b a b l ybe l i m i t ed t o va l uab l e b reed i ng an i m al s . However , f u r t he r r e s ea rch m ay y i e l d a r eg i m en


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919which could be used for the treatment of all infected animals in a herd and forprophylaxis of those animals recently exposed to Brucella infection. Furthermore, it isconcluded that the failure of a small number of cows to respond to regimen C cannot beaccounted for by the development of resistance by Brucella. The treatment ofseropositive animals should be performed together with vaccination of all seronegativeanimals in the same herd to achieve effective control and minimise public health risks.

ACKNOWLEDGEMENTSThe au thor s wish to express their sincere tha nks to M.S. Bin Salamah , Di re ct or

General of the Saudi National Agriculture and Water Research Centre and NationalDirector of Project 016/UTFN/SAU/FAO, for his encouragement and co-operation. Theauthors also acknowledge the technical assistance of A. Mukayel and A. Al-Taher.Thanks are also extended to the owners and veterinary staff of the three dairy farmsinvolved, for their support and co- operat ion in the impl emen ta ti on of the pr es en ttreatment regimens.

** *

DES PROTOCOLES THRAPEUTIQUES EFFICACES POUR LE TRAITEMENTDE L'INFECTION PAR BRUCELLA MELITENSIS ET BRUCELLA ABORTUSCHEZ LES VACHES. - A.I. Radwan, S.I. Bekairi, A.M. Al-Bokmy, P.V.S. Prasad,O.M. Mohamed et S.T. Hussain.

Rsum : Trois protocoles thrapeutiques ont t valus chez 121 vachesatteintes d'une infection naturelle par Brucella melitensis ou Brucella abortus,combinant la fois l'Oxyttracycline action prolonge ( l o n g - a c t i n gO x y t e t r a c y c l i n e : LA-OTC), la streptomycine (ST) et l'infusion intramammaired'OTC ( O T C - i n t r a m a m m a r y i n f u s i o n : OTC-IMI). Deux critres ont t jugsdterminants pour conclure au succs du traitement : l'arrt de l'excrtion debrucelles dans les scrtions mammaires et l'absence de brucelles dans des tissusspcifiques.

Le protocole A (appliqu 35 vaches) consistait en une administrationintramusculaire (i.m.) de 25 mg/kg de LA-OTC tous les trois jours pendant42 jours, de 25 mg/kg de ST (i.m.) quotidiennement pendant huit jours et en uneOTC-IMI quotidienne de 20 ml par trayon pendant quatre jours. Le protocoleB (appliqu 53 vaches) tait similaire au prcdent, sauf pour la ST qui taitadministre un jour sur deux pendant 16 jours et pour l'OTC-IMI, pratique unjour sur deux pendant huit jours. Les deux modes de traitement se sont rvlsaussi efficaces l'un que l'autre pour l'limination totale des brucelles des vachesobjet de l'exprimentation et aucune rcidive n'a t constate. Quant auprotocole C, similaire au protocole A, hormis pour la ST qui tait administretous les trois jours pendant 24 jours et l'OTC-IMI, pratique galement tous lestrois jours pendant 12 jours, il n'a permis l'limination des brucelles que sur 30(91 %) des 33 vaches traites.

Avant l'application de ces trois protocoles thrapeutiques, les bio types 1 ou 2de B. melitensis ont t rgulirement isols dans les scrtions mammaires de103 vaches et le biotype 1 de B. abortus dans celles de 18 vaches.


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920M O T S - C L S : A n t i b i o t i q u e s - B o v i n s - B r u c e l l a a b o r t u s - B r u c e l l ameli tensis - Oxyttracycline - Streptomycine - T h r a p i e .

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P R O T O C O L O S T E R A P U T I C O S E F I C A C E S P A R A E L T R A T A M I E N T O D E L AIN F EC C I N P O R BRUCELLA MELITENSIS Y BRUCELLA ABORTUS E N VACAS.- A.I. Radwan, S.I. Bekairi, A.M . Al-Bokmy , P.V.S. Prasad, O.M. M oham ed y S.T. Hussain.Resumen: Se evaluaron tres protocolos teraputicos en 121 vacas infectadas porva natural por Bruce l l a mel i t ens i s o B r u c e l l a a b o r t u s , a partir de unacombinacin de oxitetraciclina de accin prolongada ( long-ac t ingO x y t e t r acy c l i n e : LA-OTC), estreptomicina (ST) e infusin intramamaria deOTC ( O T C - i n t r a m a m m a r y i n f u s i o n : OTC -IMI). Para considerar eltratamiento exitoso se tuvieron en cuenta dos criterios: el cese de la excrecin debrucelas en las secreciones mam arias y la ausencia de brucelas en tejidosespecficos.

El protocolo A, que fue experimentado en 35 vacas, consista en laadministracin intramuscular (i.m.) de 2 5 mg/kg de LA-OTC cada tres dasdurante 42 das, de 2 5 mg/kg de ST (i.m.) diarios durante ocho das y en unaOTC -IMI diaria de 2 0 ml porpezn durante 4 das. El protocolo B, que seexperime nt en 53 vacas, era similar al anterior ex cepto en cuanto a la ST, quese administr cada dos das durante 1 6 das, y ala OTC -IMI, que se administrcada dos das durante ocho das. Uno y otro tratamiento se mostraronequivalentem ente eficaces para la eliminacin total de las brucelas en las vacastratadas y no se constat ning una recidiva. Por ltimo, el protocolo C, similar alprotocolo A salvo en que la ST se administraba cada tres das durante 2 4 das yla OTC -IMI tambin cada tres das durante 1 2 das, slo permiti eliminacinde brucelas en 30 de las 33 vacas tratadas, es decir, un 91%.

Antes de aplicar estos tres protocolos teraputicos, losbiotipos 1 o 2 deB. mel i t ens i s fueron regularmente aislados en las secreciones mam arias de103 vacas y el biotipo 1 de B. abor tus en las secreciones mam arias de 1 8 vacas.P A L A B R A S C L A V E : A n t i b i t i c o s - Bovinos - Brucel la abor tus - Brucel lamelitensis - Oxitetracicl ina - Estreptomicina - Teraput ica .

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