Research in Veterinary Science 90 (2011) 208–211

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Research in Veterinary Science

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Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain

Julio Álvarez a, Jose Luis Sáez b, Nerea García a, Carles Serrat c, Marta Pérez-Sancho a, Sergio González a,Maria Jesús Ortega d, Josep Gou e, Lucio Carbajo b, Fulgencio Garrido d, Joaquín Goyache a,f,*,Lucas Domínguez a,f

a Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, 28040 Madrid, Spainb Subdirección General de Sanidad de la Producción Primaria, Dirección General de Recursos Agrícolas y Ganaderos, Ministerio de Medio Ambiente y Medio Rural y Marino,28071 Madrid, Spainc Sección de Ganadería y Sanidad Animal en Girona, Generalitat de Catalunya, 17001 Girona, Spaind Laboratorio Central de Veterinaria de Santa Fe, Centro Nacional de Referencia de Brucelosis Animales, Ministerio de Medio Ambiente y Medio Rural y Marino, 18320 Santa Fe, Spaine Servicio de Sanidad Animal, Consejería de Agricultura, Alimentación y Acción Social Generalitat de Catalunya, 08071 Barcelona, Spainf Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 November 2009Accepted 22 May 2010

Keywords:BrucellosisB. melitensisCattleOutbreakControlZoonosis

0034-5288/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.rvsc.2010.05.028

* Corresponding author at: Centro de Vigilancia SanUniversidad Complutense de Madrid, 28040 Madrid,fax: +34 91 3943795.

E-mail address: jgoyache@vet.ucm.es (J. Goyache)

Brucella melitensis is a major human and animal pathogen, with a wide host range that includes alldomestic ruminant species, although small ruminants are its preferred hosts. Outbreaks in cattle dueto B. melitensis have become a worldwide emerging problem particularly difficult to control due to thelack of knowledge on the epidemiology in this host species and of an effective vaccine. However, combi-nation of molecular tools and strict biosecurity measures can help to solve these difficulties and eventu-ally eradicate the disease from infected herds. In the present report, management of an outbreak in Spaininvolving four farms, more than 2000 cattle and several human cases is described. Application of MultipleLocus VNTR Analysis (MLVA) allowed identifying the most likely source of infection. Stamping out andtest-and-slaughter strategies were applied, proving their usefulness to control the outbreak dependingon infection level, and without the need of other alternative measures.

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B. melitensis, causative agent of small ruminant brucellosis, isacquiring an increasing importance in bovine as an emergent path-ogen (Corbel, 1997; Kahler, 2000). B. melitensis is also the maincausative agent of human brucellosis, one of the most serious zoo-noses all over the world (EFSA, 2007; OIE, 2008). The main sourceof infection for the population is consumption of unpasteurizedmilk and milk products, but other ways of infection such as respi-ratory or conjunctival routes have been described.

In several countries B. melitensis is the most frequently isolatedspecies from all domestic ruminants (Refai, 2002). Outbreaks incattle are often attributed to the presence of infected sheep andgoat flocks in the surrounding area (Benkirane, 2006; Samahaet al., 2008). B. melitensis infection in cattle poses a serious problemto both farmers and veterinarians, because of the lack of informa-tion regarding several aspects of its epidemiology, such as cattle-to-cattle transmission or within-herd persistence of infection(Bercovich, 1998). Control of B. melitensis infection in cattle is im-peded due to difficulties on the implementation of vaccination

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itaria Veterinaria (VISAVET),Spain. Tel.: +34 91 3943887;

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strategies: although cross-species protection between Brucella spe-cies has been demonstrated using recombinant RB51 vaccine inmice (Vemulapalli et al., 2004), it has not been proven in cattleyet; B. melitensis vaccine (Rev1) have been applied before in cattlein developing countries (Denes, 1997) and its administration inthis host species has been authorized by the European Union(Anon., 2002), but to our knowledge its usefulness to control B.melitensis outbreaks in cattle in developed countries has not beenfully assessed. Thus, slaughtering of all exposed animals is one ofthe available options to control a B. melitensis outbreak in a cattleherd. Alternatively, if the infection level is low and the outbreakis detected at an early stage, the disease might be controlled andeventually eradicated through the implementation of very strictmanagement procedures (Hamdy et al., 2008). This paper describesthe management of an outbreak of brucellosis due to B. melitensisinvolving human cases and four cattle farms (A, B, C and D) inthe north east of Spain using serological techniques, bacteriologyand molecular characterization tools. Strategies followed depend-ing on the epidemiological status of every premise are detailed inorder to identify their potential usefulness.

Farms management system: Farms A, B and C were large dairyfarms (>550 animals) that shared feed and milk transport andthe veterinary services located in an area were no brucellosis

J. Álvarez et al. / Research in Veterinary Science 90 (2011) 208–211 209

vaccination had been performed since 2002. All three farms rou-tinely send new born heifers to farm D (916 animals at the begin-ning of the outbreak), where they were served. These animals werekept in farm D for most of the pregnancy and approximately oneweek before calving the pregnant cows were sent back to theirfarms of origin. Before moving back, all animals were segregatedwithin the previous 30 days before transportation to a separatedpen (‘‘pre-movement yard”) and subjected to Brucella testing usingRose Bengale Test (RBT) (OIE, 2008). The pre-movement yardwould hold approximately 18 animals.

Outbreak: On September 14th 2007 brucellosis was confirmedin a man that worked in farm A. Almost at the same time, routinetests performed as part of the Spanish brucellosis eradicationprogram detected three positive reactors at the complement fixa-tion test (CFT) with titers equal or above 20 international comple-ment fixation test units (ICFTU)/ml (OIE, 2008) in the same farm(Table 1), located in an area where no bovine brucellosis had beendetected for more than 6 years. Positive animals were slaughteredand sampled for bacteriology, and serological analysis were re-peated on day 19 post-detection (p.d.) of the outbreak, revealing53 reactors at CFT and/or RBT (Table 1). Epidemiologically con-nected farms B, C and D were also tested; in farm D only two posi-tive reactors were found; both have just arrived four days beforefrom farms A and B respectively, and consequently infection intheir farms of origin was the most likely hypothesis. No more po-sitive animals were detected in this farm on subsequent analysis.In farms B and C 131 and 23 reactors were detected in the firstanalysis (Table 1). Once the extent of the outbreak was determined,periodical testing of all animals from the four farms were per-formed (Table 1). Samples of animals from the three infected farms(A, B and C) that had been culled on the first tests were collected atabattoir to culture the Brucella strain involved in the outbreak. B.

Table 1Results of serological analysis in farms A, B, C and D using Rose Bengal andcomplement fixation techniques.

Farm Daya Analyzed samples Positive samplesb (%) RB+c CF+d

A 0 579 3 (0.5) 3 319 574 53 (9.2) 48 5053 656 28 (4.3) 25 2597 387 64 (16.5) 60 15

127 272 24 (8.8) 23 12B 34 596 131 (22) 123 106

60 509 79 (15.6) 72 5496 344 121 (35.2) 120 38

133 294 53 (18) 50 37C 39 642 23 (3.6) 18 20

55 528 6 (1.1) 3 567 636 0 (0) 0 090 515 0 (0) 0 0

105 506 2 (0.4) 2 2125 493 0 (0) 0 0147 628 0 (0) 0 0

D 26 916 2 2 243 913 0 (0) 0 054 67e 0 (0) 0 070 917 0 (0) 0 080 8e 0 (0) 0 086 29e 0 (0) 0 0

100 59e 0 (0) 0 0121 12e 0 (0) 0 0131 945 0 (0) 0 0139 29e 0 (0) 0 0148 17e 0 (0) 0 0

a All dates at referred to the day of first detection of the outbreak (14thSeptember).

b Number of positive samples to the Rose Bengale and/or complement fixationtests.

c Number of positive samples to the Rose Bengale Test.d Number of positive samples to the complement fixation test.e Includes tests performed on heifers 8 and 30 days post-calving.

melitensis was recovered from all samples, and cultures were iden-tified as belonging to biotype 3 by agglutination with A and M-spe-cific antisera (OIE, 2008). In addition, seven workers in total (fourfrom farm A and three from farm B) were also found to be infectedby B. melitensis biotype 3. No increase in the abortion rates in allfour farms had been reported before the outbreak was detected.

Among possible sources of disease, infected sheep flocks locatedin the vicinity of farm D were considered as the most likely source.However, due to the fact that no positive animals were detectedduring the whole outbreak in this farm (with the exception ofthe two cows that were sero-positive only two days after arrival)this possibility was ruled out in the beginning. Nevertheless, athorough investigation revealed that on the first week of Mayone sheep of unknown origin was kept in farm D for four days,showing a lack of appropriate biosecurity measures. On May25th one cow that was about to return to farm C before calvingaborted in farm D (in the pre-movement yard), and was then trans-ported to farm C without any supplementary analysis followingabortion. This animal was later positive in the first analysis per-formed in farm C, although it was negative in the pre-movementBrucella test carried out before the abortion. Most animals sharingthe pre-movement yard with this animal were later positive inboth RB and CF tests in the first analysis performed in their desti-nation farms (A, B and C), even though they were also negative inpre-movement test. Therefore, the cow aborting on the 25th ofMay could have been the first case; animals infected in farm Dwould have spread the infection on their destination farms oncethey had been transported just before parturition.

Molecular epidemiology: In order to confirm the suspected linkbetween cattle, human and sheep strains, five strains from farmA, three from farm B and one from farm C were submitted to theSpanish National Reference Laboratory for Animal Brucellosis formolecular characterization using multiple locus variable numbertandem repeats analysis (MLVA) on 15 polymorphic loci (MLVA-15) as previously described (Le Fleche et al., 2006). In addition, iso-lates from two infected farm workers and from several positivesheep flocks located in the vicinity of farm D were analyzed. Com-parison of the results observed in the eight conserved loci includedin panel 1 by Le Fleche et al. (2006) with those available in theMLVA bank (http://minisatellites.u-psud.fr/MLVAnet) revealedprofile 51 was present in all strains. Patterns obtained in the sevenhighly variable loci included in panel 2 are summarized in Table 2.MLVA analysis revealed four different patterns in panel 2 due topolymorphisms on loci Bruce09 and Bruce16: profile I was identi-fied in two cattle isolates from farm A, and profile II was present inone sheep strain, cattle isolates from farms A (n = 3), B (n = 3) and C(n = 1) and in the two human isolates (Table 2).

Outcome: due to the large number of animals involved in thisoutbreak several options for its control were discussed. Finally,strategies were applied depending on the B. melitensis infectionprevalence detected on every premise: the high numbers of reac-tors detected in farms A and B, together with the uncertainty aboutthe possible evolution of the outbreak and sanitary and socio-eco-nomical reasons, made stamping out the most feasible controloption, and all animals in both farms were culled 140 days afterdetection of the outbreak. A different approach was adopted onfarm C, which had a lower seropositivity rate (Table 1): all animalswere tested every 15–20 days using CFT and RBT, with immediateremoval of positive reactors. Besides, biosecurity measures weremaximized regarding handling of pregnant animals, disinfectionof all material in contact with positive reactors, control of move-ment of all animals, etc. A similar strategy was established in farmD: all pregnant cows held in farm D were not moved back to theirfarms of origin before calving to avoid entering animals in the in-fected farms; instead, all cows calved in farm D in a separated area.Special care was taken to avoid any contact of these animals with

Table 2Number of repetitions in tandem detected by multiple locus variable number tandem repeats analysis (MLVA) in highly discriminatory loci described by Le Fleche et al. (2006) inBrucella melitensis isolates from sheep, cattle and human samples.

Number of isolates Host Farm of origin Bruce 18 Bruce 21 Bruce 04 Bruce 07 Bruce 09a Bruce 16a Bruce 30 Profile

2 Cattle A 7 8 7 6 9 10 3 I3 Cattle A 7 8 7 6 8 10 3 II3 Cattle B 7 8 7 6 8 10 3 II1 Cattle C 7 8 7 6 8 10 3 II2 Human – 7 8 7 6 8 10 3 II1 Sheep – 7 8 7 6 8 10 3 II4 Sheep – 7 8 7 6 7 10 3 III1 Sheep – 7 8 7 6 7 11 3 IV

a Loci were polymorphisms among isolates were found.

210 J. Álvarez et al. / Research in Veterinary Science 90 (2011) 208–211

the rest of the cattle in farm D, as it could not be discarded thatthey were infected, in order to minimize the risk of spreadingthe infection. Handling of these high risk animals by human work-ers was performed with extreme care. The heifers were testedeight and 30 days post-birth to maximize the possibilities ofdetecting remaining infected animals after calving, in order toovercome difficulties in the diagnosis of Brucella infections inprimiparous heifers, especially difficult to detect before calving.

The evolution of results of diagnostic tests confirmed the viabil-ity of a conservative approach to control the outbreak in farms Cand D: on farm C only eight more reactors were detected onfollowing tests (Table 1), and since then no more positive reactorshave been detected by any diagnostic test, confirming the eradica-tion of the disease. In farm D only two positive animals weredetected and both were likely infected in their origin farms,although the origin of the outbreak could have taken place there;segregation of cows on the pre-movement yard would have madepossible that only animals in that particular group would havebeen affected. Nevertheless all animals in this farm were also sub-jected to regular testing for more than one year. The managementof the pregnant cows in this farm avoided the spread of the disease,therefore saving the heifers that were later used to restock farms Aand B.

This B. melitensis outbreak affected seven workers and fourcattle farms, involving more than 2000 animals. The absence ofevident clinical signs together with a possible lack of detectableimmune response to routine bovine brucellosis diagnostic test ininfected primiparous heifers before abortion or calving make thiskind of outbreaks an emerging problem difficult to handle. How-ever, it was identified and controlled by minimizing the numberof animals that had to be culled thanks to several key actions:

– Immediate control measures adopted when the first cases werereported allowed, through a complete epidemiological analysis,the detection of the most likely source of infection, an infectedovine flock near farm D.

– Culture and molecular characterization of the etiological agentfrom all host species involved confirmed the possible origin ofthe outbreak.

– Measures adopted on the different farms were based on theinformation available, avoiding the use of alternative measuresas vaccination. An exhaustive diagnostic strategy was success-fully implemented in farms C and D, removing all infectedanimals in approximately two months.

The application of a highly discriminative characterization tech-nique found two profiles in the B. melitensis isolates from cattle,with subsequent repetition of the analysis confirming this result.Difference between the two patterns were focused on locusBruce09, which had been described as the most polymorphic lociamong MLVA-15 targets in different panels of B. melitensis strains

(Garcia-Yoldi et al., 2007; Tiller et al., 2009). The two most likelyexplanations are a possible mutation in the outbreak strain leadingto a different profile (with only one more TR repetition) or theinvolvement of two different strains in the outbreak, althoughthe second profile could not be identified on other sheep strainsfrom the same area, and therefore would be of unknown origin.Limited number of sheep strains typed in this study cannot ruleout this possibility. However, although loci included in theMLVA-15 have demonstrated their usefulness for epidemiologicalpurposes, occasional one-step increase or decrease changes inone or more loci have been reported before after in vivo andin vitro passages of Brucella strains (Whatmore et al., 2006; Kanget al., 2009). Therefore, the difference found in highly polymorphiclocus Bruce09 cannot exclude a possible common origin of all cat-tle strains.

The present reports highlights the importance of maintainingstrict biosecurity measures and regular testing of cattle herds,and routinely report cases of abortions for investigation in areasfree of bovine brucellosis but where B. melitensis is present in smallruminants, as in some cases clinical presentation of the disease incattle can go unnoticed.

Conflict of interest statement

None.

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