Cárdenas et al. BMC Veterinary Research (2019) 15:81 https://doi.org/10.1186/s12917-019-1825-9 RESEARCH ARTICLE Open Access Risk factors for new bovine brucellosis infections in Colombian herds Liliana Cárdenas1,2* , Mario Peña3, Oscar Melo4 and Jordi Casal1,2 Abstract Background: Bovine brucellosis is a zoonotic disease that causes substantial economic losses and has a strong impact on public health. The main objective of this paper is to determine the risk factors for new infections of Brucella abortus on Colombian cattle farms previously certified as being free of brucellosis. A case-control study was conducted by comparing 98 cases (farms certified as brucellosis-free for three or more years but became infected) with 93 controls (farms that remained brucellosis-free during at least the previous three years). The farms were matched by herd size and geographical location (municipality). Information was obtained via a questionnaire completed by veterinary officers through a personal interview with the herd owners. Results: Two-thirds of the herds (67%) were dairy herds, 16% were beef herds, and 17% were dual-purpose (beef and milk) herds. After exploratory univariate analysis, all explanatory variables with a p-value of ≤0.20 were included in a logistic regression model using the forward stepwise method to select the model with the best goodness of fit. The significant risk factors were the replacement of animals from farms not certified as brucellosis-free compared to replacement from certified brucellosis-free farms (OR = 4.84, p-value < 0.001) and beef cattle farms compared to dairy cattle farms (OR = 3.61, p-value = 0.017). When herds with and without artificial insemination were compared, it was observed that farms that used natural breeding with bulls from non-certified herds had a higher risk than farms using artificial insemination (OR = 2.45, p-value = 0.037), but when the bulls came from brucellosis-free farms, farms with natural breeding were less affected (OR = 0.30, p-value = 0.004) than farms using artificial insemination, whether with frozen semen from certified brucellosis-free herds or fresh semen from uncontrolled herds. The latter is commonly sold to neighbouring farms. Conclusions: The government should make efforts to inform farmers about the risks involved in the introduction of semen and replacement heifers from farms that are not certified as brucellosis-free and to establish measures to control these practices. Keywords: Brucella-free herds, Bovine brucellosis infection, Colombia, Risk factors Background periods of –time [2, 3]. Some animals are asymptomatic, Bovine brucellosis is an important zoonosis with world- having latent infection without exhibiting clinical signs, wide distribution. The infection has been controlled or thus maintaining the disease in a herd [4, 5]. eliminated in most developed countries, but it remains Cattle become infected after the ingestion of contami- endemic in Africa, Latin America and Asia [1]. nated milk, food, water or grazing forage; close contact Bovine brucellosis is a chronic, infectious disease caused with infected animals; contact with uterine secretions or by Brucella abortus, which has developed mechanisms to aborted foetuses; and through vertical and sexual trans- live intracellularly and is able to infect cattle for long mission [6, 7]. The disease directly affects cattle and buffalo, which are the main reservoirs of B. abortus, * Correspondence: zlilianac@yahoo.es; liliana.cardenas@irta.cat although other domestic and wild species can also act as 1 Animal Medicine and Health Department, Faculty of Veterinary Medicine, Campus of The Autonomous University of Barcelona (UAB), 08193 Bellaterra, a reservoir [8, 9]. The main clinical signs are abortion, Barcelona, Spain infertility, stillbirth or the birth of weak calves, along 2 The Centre for Research into Animal Health (CReSA), Campus of The with epididymitis and orchitis in males [10, 11]. Autonomous University of Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 2 of 8 The introduction of cattle from infected herds or in opinion of veterinary services about the possible causes contact with false-negative animals can contribute to of these new infections. introducing the infection into brucellosis-free farms [12]. Transmission within and between farms has been asso- Methods ciated with different risk factors: the maintenance of Study area positive animals in the herds, large farms, communal Colombia is a tropical and agro-pastoral country located pastures, semi-intensive production systems and age (the in the northwestern region of South America and has a infection is more prevalent in adults) [13]. Brucellosis is continental area of 1,141,748 km2. The cattle population considered a complex disease due to its wide range of is estimated at approximately 22.7 million head, half of hosts and the variable signs at both the individual and which are dairy cattle, 30% of which are beef cattle, and population levels. Infected animals can remain infectious 20% of which are dual purpose (beef and milk). A after their first abortion and spread the disease [14]. On substantial portion of Colombian livestock is reared the other hand, the disease can be misclassified as other under traditional husbandry practices in rural areas with reproductive diseases, which is one of the reasons that little technology, and these herds are the basis of the bovine brucellosis is generally underestimated at the family economy. farm level [14, 15]. Only Brucella abortus has been reported in Colombia. There are no studies concerning Study design the epidemiology of brucellosis in the human population A case-control study was carried out to compare newly in Colombia, but it is assumed that in terms of human diagnosed herds with herds that remained brucellosis- health, brucellosis is an under-diagnosed and under- free. The case farms were defined as those with herds notified disease [16]. that became infected with the disease during the period The methods to control and eliminate brucellosis from 2015 to 2016, having been previously classified as a region are based on vaccination, controlling move- brucellosis-free for at least three years within the frame- ments, and testing and removing serologically positive work the official control programme. The control farms animals. Considerable effort and time are required to were those with herds that had retained disease-free achieve disease-free status. status for a period equal to or greater than three years. Control efforts against bovine brucellosis in Colombia These farms were randomly matched with case farms are led through an official programme. This programme based on herd size and location at the municipality level. is voluntary, includes 15% of farms and is based on the For five cases, we were not able to include a control, so identification of animals, compulsory vaccination of the final sample size was 98 cases and 93 controls. This calves between 3 and 8 months of age (to make this number of farms allowed the detection of differences in vaccination compatible with further serological tests), exposure of between 40 and 60% (OR = 2.25) with a con- control of animal movement, and testing and removal of fidence level of 95% and a power of 80%. The sampling positive animals older than 24 months (8 months in the frame was obtained from the Instituto Agropecuario case of males). The diagnosis of bovine brucellosis is Colombiano (ICA). made by serological serial testing with the Rose Bengal plate test and indirect ELISA and is complemented by Questionnaire competitive ELISA. A questionnaire was created to obtain information about Blood sampling is performed by the veterinary service the potential risk factors; this questionnaire was based on or by authorized veterinarians, and farms are classified existing literature and contained questions that focused as brucellosis-free if all animals are negative in two on various topics, including the bovine brucellosis health consecutive serological diagnostic tests. In 2016, 18,996 programme, the physical characteristics of the farm (pres- herds had achieved the status of officially brucellosis-free ence of fences, barns and milking facilities), animal (4% of the 514,794 cattle farms present in the country). husbandry, grazing, biosecurity, wild animals, and animal In a previous study about bovine brucellosis in movements. The questionnaire in Spanish and its English Colombia, heterogeneity was found in the control efforts translation is shown in Additional file 1: (Questionnaire and prevalence within the country [17]. In recent years, for the study of bovine brucellosis in Colombia: Electronic several farms classified as bovine brucellosis-free have Supplementary Material). become infected, in most cases without knowledge of The questionnaire was evaluated in two stages; first, the causes of these new infections. The aim of this study through a meeting with a panel of Colombian experts on is to identify the risk factors associated with new infec- the subject and, after that, by the veterinary officers of tions of Brucella abortus detected between 2015 and each affected region. Before answering the questionnaire, 2016 on cattle farms previously certified as being free of all farmers were informed that answering the questions the disease. A secondary objective is to determine the was voluntary and that they did not have to answer all Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 3 of 8 of the questions. All data were anonymously analysed. contact with neighbouring farms through water, infection The feedback to the farmers was sent by the central status of the origin of replacement heifers and bulls, veterinary service, which reported the results to field number of cattle introduced into the farm, and status veterinarians, who in turn informed the farmers. of the mother (for internal replacements). The questionnaires were completed by local veterinary The final multivariable logistic model (Table 2) officers through personal interviews with the farm owners. included three risk factors: Herds that bring in heifers Additionally, following completion of the questionnaire bought from farms with an unknown brucellosis status and considering all the findings, the veterinary officer had a higher risk of infection than those that obtain chose the most likely cause of re-introduction of the replacements from brucellosis-free herds or that replace disease according to his/her opinion (only one cause). All from within (OR = 4.84, p-value: < 0.001). Beef cattle farms variables obtained through the epidemiological question- were more affected (OR = 3.61, p-value = 0.017) than dairy naire were categorical. or dual-purpose cattle farms. Finally, farms that use bulls The information obtained via this questionnaire was from farms with positive/unknown Brucella status had a complemented with other data, such as farm identi- higher risk (OR = 2.45, p-value = 0.037) than those that fication, census, vaccination status, location, and the use artificial insemination (AI). Farms with natural brucellosis status of the interviewed farm and related breeding that use bulls from brucellosis-free farms were farms (neighbours or origins of replacement heifers and less affected (OR = 0.30, p-value = 0.004) than those bulls), which were obtained from the ICA database. The that use AI. data were recorded in a Microsoft Office Excel 2017 file. The questionnaire included a question asking the opinion of the veterinarians that did the interview about Statistical analysis the causes of infection. Table 3 shows the most cited Bivariate analysis was performed with the cases and con- answers; the three most frequent were contact with trols using the chi-square test. Variables with a p-value of other domestic species (18%), contamination through ≤0.20 were retained and included in the logistic regression water or feed (15%) and the introduction of infected analysis. In the case of collinearity among independent cattle (14%). For most of the cases (80%), it was assumed variables evaluated with the generalized variance inflation that the new infection was exogenous; the reasons factors (GVIF) using a covariance matrix, the variable with proposed for re-infection of the other 20% included the most biological sense was retained. resurgence due to keeping animals diagnosed as positive A stepwise logistic regression model was applied to (7%), the absence/deficiency of biosecurity on the farm the selected variables using forward selection. The (5%) and the re-circulation of the bacteria (8%). Only the analysis was started with an initial model without introduction of infected animals coincided with the explanatory variables, and variables were introduced one results of the model. by one, selecting those that best defined the dependent variable and gradually removing variables with no statis- tical significance (p-value > 0.05) in the model. The Discussion models were compared by the Akaike information cri- Bovine brucellosis is endemic in Colombia [17] and, over terion (AIC). All statistical analyses were performed the last 20 years, has been subject to an official control using R Project software [18]. programme based on the compulsory vaccination of all bovine and bubaline females between 3 and 8 months of Results age and on a voluntary programme of testing and During the last two years, 98 previously brucellosis-free removing positive animals. Despite this programme, the farms were infected and included in the study. These incidence of brucellosis has remained stable, with the farms were located in fifteen departments, representing percentage of positive farms and positive animals 60% of the Colombian cattle census (Antioquia, Arauca, averaging 22–23% and 4.7–4.6%, respectively, between Bolivar, Boyacá, Caldas, Cesar, Córdoba, Cundinamarca, 2006 and 2012 [17]. One of the reasons for the lack of La Guajira, Nariño, Putumayo, Quindío, Risaralda, progress in brucellosis control is the detection of new in- Santander and Tolima), and the 93 controls were from fections in herds that had previously been brucellosis-free. the same departments. Between January 2015 and December 2016, 98 farms be- Nineteen binary categorical variables had a p-value of came infected. In this case-control study, we determined ≤0.20 when they were compared between the cases and the possible factors related to these new infections. When controls (Table 1). These variables were included in the these farms were compared with 93 herds that remained logistic regression model. brucellosis-free, three factors were statistically significant: The significant variables in the bivariate analysis the origin of replacement animals, type of animal (dairy, (p < 0.05) were the type of livestock, direct contact, indirect beef or dual purpose) and use of AI or natural mating. Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 4 of 8 Table 1 Categorical variables included in the bivariate analysis (chi-squared test with p < 0.2). Controls: 93 farms that remained brucellosis-free for at least three years. Cases: 98 herds previously free of brucellosis that became infected Variables Control Case OR 95% CI Lower Upper p-value Livestock type Dairya 68 (73.12%) 60 (61.22%) 1 Beef 8 (8.60%) 22 (22.45%) 3.12 1.29 7.52 0.009 Dual purpose (beef and milk) 17 (18.28%) 16 (16.33%) 1.07 0.5 2.29 0.869 Equipment shared with other farms Noa 81 (87.10%) 77 (78.57%) 1 Yes 12 (12.90%) 21 (21.43%) 1.84 0.85 4 0.12 Cleaning and disinfection of clinical equipment and clothing Yesa 86 (92.47%) 85 (86.73%) 1 No 7 (7.53%) 13 (13.27%) 1.88 0.72 4.94 0.197 Presence of facilities to shelter animals Noa 41 (44.09%) 31 (31.63%) 1 Yes 52 (55.91%) 67 (68.37%) 1.7 0.94 3.08 0.077 Farm fencing Completelya 91 (97.85%) 91 (92.86%) 1 Partially 2 (2.15%) 7 (7.14%) 3.5 0.71 17.3 0.105 Contact of the herd with other neighbouring farms Noa 78 (83.87%) 67 (68.37%) 1 Yes 15 (16.13%) 31 (31.63%) 2.41 1.2 4.83 0.012 Infected animals in the neighbouring herd Noa 7 (7.53%) 2 (2.04%) 1 Yes 86 (92.47%) 96 (97.96%) 3.91 0.79 19.32 0.074 Mixed farm (farms that have species other than buffaloes or cattle) Noa 80 (86.02%) 72 (73.47%) 1 Yes 13 (13.98%) 26 (26.53%) 2.22 1.06 4.648 0.032 Water access points shared with other farmsb No farms or brucellosis-free farmsa 59 (63.44%) 48 (48.98%) 1 Non-brucellosis-free farms 34 (36.56%) 50 (51.02%) 1.81 1.01 3.22 0.045 Presence of drainage from other farms Noa 58 (62.37%) 48 (48.98%) 1 Yes 35 (37.63%) 50 (51.02%) 1.73 0.97 3.07 0.063 Animal movement: Brucella statusb of other herds at the destination Brucellosis-free farms or no movementa 89 (95.70%) 87 (88.78%) 1 Non-brucellosis-free farms 4 (4.30%) 11 (11.22%) 2.81 0.86 9.17 0.076 b Status of the origin of replacement animals (internal or brucellosis-free farms vs calves from non-brucellosis-free farms) Brucellosis-free farms or internala 85 (91.40%) 67 (68.37%) 1 Non-brucellosis-free farms 8 (8.60%) 31 (31.63%) 4.92 2.12 11.39 < 0.001 Internal replacement with calves from the same farm whose mothers were positive Noa 89 (95.70%) 78 (79.59%) 1 Yes 4 (4.30%) 20 (20.41%) 5.71 1.87 17.41 0.001 Introduction of heifers < 24 months old into the farm No admittancea 79 (84.95%) 72 (73.47%) 1 Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 5 of 8 Table 1 Categorical variables included in the bivariate analysis (chi-squared test with p < 0.2). Controls: 93 farms that remained brucellosis-free for at least three years. Cases: 98 herds previously free of brucellosis that became infected (Continued) Variables Control Case OR 95% CI Lower Upper p-value Admittance 14 (15.05%) 26 (26.53%) 2.04 0.99 4.2 0.052 Number of cattle introduced into the farm (per year) Nonea 66 (70.97%) 53 (54.08%) 1 Ten or fewer 19 (20.43%) 23 (23.47%) 1.51 0.74 3.06 0.255 Between eleven and thirty 6 (6.45%) 10 (10.20%) 2.08 0.71 6.08 0.178 More than thirty 2 (2.15%) 12 (12.25%) 7.47 1.6 34.85 0.004 b Infection status of the origin of the cattle (of all ages) Tested negative for Brucella or brucellosis-free farma 71 (76.34%) 58 (59.18%) 1 Not tested, unknown status of the farm, positive herds 22 (23.66%) 40 (40.82%) 2.23 1.19 4.16 0.012 Introduction of other domestic species into the farm Nonea 88 (94.63%) 85 (86.74%) 1 More than twenty 0 (0.0%) 3 (3.06%) 7.25 0.37 142.37 0.13 Less than or equal to twenty 5 (5.37%) 10 (10.20%) 2.07 0.68 6.31 0.194 Brucella statusb of the origin of the bulls Artificial inseminationa 36 (38.71%) 34 (34.69%) 1 Free herds 44 (47.31%) 16 (16.33%) 0.39 0.18 0.81 0.011 Unknown/positive status 13 (13.98%) 48 (48.98%) 3.91 1.81 8.46 < 0.001 Access of dogs and cats to/contact with aborted foetuses and reproductive discharges No (or no dogs)a 66 (70.97%) 59 (60.20%) 1 Yes 27 (29.03%) 39 (39.80%) 1.62 0.88 2.95 0.119 a Category considered as reference. Variables with more than two categories have been pairwise compared with this category b The brucellosis status of other farms was obtained from the tests performed within the framework of the control programme One of the more important causes of the introduction of 4.8 (32% of the herds that became infected had brought of diseases into non-affected populations is the introduc- in animals from non-brucellosis-free farms, compared tion of infected animals: The acquisition of replacement with 9% of the controls). animals from farms with an unknown status or where Beef cattle farms had a higher risk of becoming brucellosis is present carries a significant risk of introdu- infected than farms with dairy or dual-purpose herds cing the infection [12, 19]. In Colombia, the origin of (OR = 3.6, using dairy cattle as the reference). In general, replacement animals was also the most significant beef cattle producers are more reluctant than dairy factor associated with the new infections, with an OR producers to apply control measures because they do Table 2 Risk factors associated with new brucellosis infections in cattle farms compared with farms that remained brucellosis-free for at least three years based on multiple logistic regression model Variables B (SE) OR 95% CI OR p-value (Intercept) −0.35 (0.26) 0.71 (0.43; 1.18) 0.184 Brucellosis-free farms or internal replacement 1 Replacement from farms with unknown/positive brucellosis status 1.58 (0.47) 4.84 (1.92; 12.20) < 0.001 Type: Dairy 1 Type: Beef 1.28 (0.54) 3.61 (1.26; 10.35) 0.017 Type: Dual purpose (beef and milk) 0.18 (0.47) 1.20 (0.48; 2.99) 0.696 Artificial insemination 1 Brucella-free status of the origin of the bulls −1.21 (0.42) 0.30 (0.13; 0.69) 0.004 Positive/unknown Brucella status of the origin of the bulls 0.90 (0.43) 2.45 (1.06; 5.68) 0.037 B coefficient estimated by the model, SE standard error, OR odds ratio, 95% CI OR confidence interval of the OR (lower limit of the 95% CI; upper limit of the 95% CI) Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 6 of 8 Table 3 Opinion of the veterinary officer that completed the questionnaire concerning the most likely cause of the introduction of the brucellosis infection into the cattle farms Causes of new infection % Exogenous origin of the new infection 80 Contact with other domestic species 18 Contaminated water/feed sources 15 Introduction of infected animals 14 Admittance of animals that have been in contact with seropositive animals in the originating farm 10 Transhumance/pasture sharing 8 Contact with infected people 4 Contact with wild species 4 Artificial insemination or embryo transfer 3 Movement of animals without official control 2 Endogenous origin of the new infection 20 Recirculation 8 Maintenance of animals diagnosed as positive 7 Absence/deficiency of biosecurity on the farm 5 not appreciate the benefits deriving from a brucellosis- sanitary control is the reason that farms using AI have a free herd: The disease does not affect prices, and they do higher risk than those that use natural mating with bulls not understand the importance and the cost of repro- from brucellosis-free herds. Unfortunately, we could not ductive disorders. Additionally, animals are distributed differentiate between insemination with refrigerated over large, extensive grazing areas that are usually diffi- semen and insemination with frozen semen. Frozen cult to access, where the control measures are more semen comes from insemination centres certified as difficult to apply, and the animals are prone to greater brucellosis-free by veterinary services. contact with uncontrolled contaminated sources. In this study, the role of neighbouring herds was not In contrast, dairy cattle producers are fully aware of observed except for the use of fresh semen, as men- the loss of production due to abortions and infertility tioned above. Brucella infection has been associated with and the subsequent important milk losses [20]. Further- the spread of infection through direct contact with cattle more, brucellosis-free herds can obtain more competi- from neighbouring herds and through the exchange of tive prices for milk than non-certificated farms, because bulls between farms for mating [21]. Indirect contact brucellosis is a concern for the milk industry and with neighbouring farms, such as through shared drinking customers pay a bonus to brucellosis-free herds. Thus, water access points and grazing lands, also constitutes dairy farmers are more prone to adopt the official a risk for the transmission of brucellosis between control programme and to apply prevention and control herds [7, 21]. measures more strictly than beef producers do. The presence of mixed farms, including interactions The third variable that was significantly different with small ruminants, has also been associated with high between the cases and controls was the origin of the prevalence of brucellosis [22] and with a resurgence of the bulls. As expected, the entry of bulls from herds with a disease in cattle herds [14]. A relationship between these positive or unknown brucellosis status was a risk factor species and brucellosis was not observed in our study, for the introduction of the infection in previously despite traditional husbandry practices in Colombia, brucellosis-free herds. Surprisingly, the use of AI carries including rearing different species in the same habitat. a higher risk than natural mating with bulls derived from The results of this study contribute to understanding herds certified as brucellosis-free (p = 0.004); these dif- new infections of previously infection-free herds and ferences are probably due to the common practice on may help decision makers in Colombia and other tro- some farms of using fresh semen obtained from bulls on pical countries with a similar situation to improve neighbouring farms. The use of artificial insemination control strategies for bovine brucellosis. with refrigerated semen obtained from a farm’s own The factors described here are well known. Indeed, bulls is a common practice in Colombia, and some veterinarians recommend the need to introduce only farmers sell semen to neighbouring farms without any animals from brucellosis-free herds and to require a previous sanitary control. In our opinion, this lack of brucellosis-free certification for semen, but veterinary Cárdenas et al. BMC Veterinary Research (2019) 15:81 Page 7 of 8 officers believe that many farmers do not comply with Acknowledgements such recommendations. Consequently, it is important to The authors would like to thank the Colombian veterinary service for providing information and for supporting the development of this study. emphasize to cattle owners the risk involved in the introduction of animals and semen from farms of Funding unknown health status. Furthermore, control measures Zaida Liliana Cárdenas Contreras holds a Ph.D. grant from the Ministry of Education in Colombia (COLCIENCIAS 617/2013 scholarship). for the introduction of animals into brucellosis-free herds, including a quarantine of newly acquired animals Availability of data and materials to assure that these animals are negative, should be All data supporting these research findings are included within the manuscript. The databases (without personally identifiable information) are strictly applied. available from the corresponding author upon request. As the prevalence of Brucella is relatively low, cul- ling positive animals should be mandatory and re- Authors’ contributions Conceived the study: JC and LC. Collected the data: LC and MP. Generated inforced with an educational programme for farmers the databases: LC. Performed the analysis: LC, OM and JC. Wrote the first to highlight the importance of brucellosis as a zoo- draft: LC. Reviewed the paper and gave final approval: All authors. notic disease [17, 21]. This campaign should also include Ethics approval and consent to participate the importance of biosecurity measures to reduce the Written consent forms were signed by the farmers for the brucellosis control spread of brucellosis between and within herds. programme, which includes the animal health status monitoring and Finally, when the risk factors were compared with the epidemiological investigations related to this disease. In addition, all people were verbally informed before completing the questionnaire that answering veterinary officers’ opinions, there was substantial agree- the questions was voluntary. ment on the consequences of introducing infected ani- mals into the herd but not on other significant variables Consent for publication Not applicable. such as the role of artificial insemination with semen from non-controlled herds. Additionally, other variables Competing interests that can also play a role in reintroducing brucellosis The authors declare that they have no competing interests. were cited. This analysis reflects the fact that there are distinct perceptions of possible causes for the introduc- Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in tion of brucellosis into herds and highlights a lack of published maps and institutional affiliations. knowledge regarding some of these causes. Author details 1 Animal Medicine and Health Department, Faculty of Veterinary Medicine, Campus of The Autonomous University of Barcelona (UAB), 08193 Bellaterra, Conclusions Barcelona, Spain. 2The Centre for Research into Animal Health (CReSA), Three risk factors for the re-introduction of brucellosis Campus of The Autonomous University of Barcelona (UAB), 08193 Bellaterra, into previously brucellosis-free herds were identified: the Barcelona, Spain. 3Animal Health Department, Colombian Veterinary service, Instituto Colombiano Agropecuario-ICA, 11161 Bogotá, Colombia. 4The introduction of replacement animals and bulls from farms Statistics Department, Faculty of Science, National University of Colombia, with unknown or positive brucellosis status, the use of AI 11001 Bogotá, D.C, Colombia. with semen from neighbouring non-brucellosis-negative Received: 17 October 2017 Accepted: 26 February 2019 herds and the identity of a herd as a beef herd. 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