Sero-prevalence of brucellosis (Brucella abortus) in bovines from Caquetá state, Colombia

Brucellosis is a disease with implications in the public health due both to its zoonotic character as its easy transmission trough of dairy products contaminated with the microorganism Brucella sp. The aim of this article was determinate the prevalence of bovine brucellosis in nine municipalities that carrying the 75% of bovine population in the Caquetá state, Colombia. Blood samples were collected in 100 herds dedicates to dual-purpose cattle, obtained blood serum from 1000 animals, of which 882 corresponding to cows. Rose Bengal Plate Test was performed and the confirmations of positive cases were made by competitive Elisa. The sero-prevalence of bovine brucellosis in Caquetá state was of 3.23% (95%, CI: 0-6.53%), in males 2.6% and females 3.29% respectively. At the municipal level, the sero-prevalence varied of 0 to 6.06%. The prevalence of brucellosis at herd level was of 22% (95%, CI: 12.24-31.76%). In conclusion, the sero-prevalence is low in bovines but moderate at the level of herds, therefore, it is necessary to increase the performance of the official control entities at the level of herds to reduce the high prevalence of the disease for guaranteed the safety of milk and thus the health of consumers.


I. INTRODUCTION
Brucellosis is a highly contagious bacterial infection of livestock and continues to be of great health concern and economic importance in worldwide (Maadi, Moharamnejad, & Haghi, 2011); (Pathak et al., 2016). This disease is caused by aerobic gram-negative bacteria of the genus Brucella, which affect domestic animals, wild animals and human, being an important public health problem in many parts of world (Aguirre et al., 2008;Reyes, Sánchez, Lotero, Restrepo, & Palacio, 2010;Akbarmehr & Ghiyamirad, 2011), and the brucellosis is considering a re-emerging zoonosis (Seleem, Boyle, & Sriranganathan, 2009). According to Aguirre et al. (2008), Matope et al. (2011) and Astaiza, 2012, brucellosis in Colombia has caused high economic losses in the livestock production, estimates are 7 to 12 million of dollars each year according to evaluations between 1993 and 1999.
The bovine population in the Caquetá state amounts in the 1,686,852 animals, where 374,317 are females older than 36 months, this state is the fifth dairy basin more important of Colombia (Beltrán- Barreiro & Torrijos, 2013), with a milk production higher to 1,215,833 kg per day, the which obtains 90% in nine municipalities. Until the year 2013, 32 municipalities were certificated free of bovine brucellosis, however, none from the Caquetá state (FEDEGAN, 2013). The objective of this research was determinate the prevalence of bovine brucellosis in the nine municipalities that produce 90% of milk in the Caquetá state.

A. Area of Study
The research was made in nine municipalities from Caquetá State, located to the south of Colombia and the northeast of the Colombian Amazonia, between the 00°42'17" of latitude south and 02°04'13" of latitude north, and the 74°18'39" and 79°19'35" of longitude west of Greenwich. Borders to the North with both Meta and Guaviare states, to the east with both Vaupes and Amazonas states, to the south with the Putumayo and the west with both Cauca and Huila states; the Caquetá state has an extension of 88,965 km 2 and is politically divided into 16 municipalities (Caquetá Gobernación de, 2012 In the Caquetá state has three thermal floors (cold, temperate and warm) (Corpoica, 1998;García, Cipagauta, Gómez, & Gutierrez, 2002). In the warm climate that covers the greatest area to state, the average precipitation is of 3800 mm/year, without dry station well defined, (nevertheless, the lower precipitation is registered between December to January and the greater precipitation between March to November), the rainfall erosivity index (R or EI30) multiannual is the 2750 tm/cm/ha/h, value three times greater than other regions of country, the relative humidity is greater than 80%, but can fluctuate between 64 to 93%, with temperatures range between 18 to 36 °C with an average annual of 25 °C characteristic of a regime isohyperthermic. The evapotranspiration potential is 1,435 L/m 2 /year, the average of solar radiation is of 1,800 hours/year and the intensity of 268 cal/m 2 /day. The climatic characteristics in the study area are of tropical rainforest (Holdridge, 1978).

B. Sample Size
Using the information of the agricultural assessment 2016 of the secretary of Agriculture of Caquetá state, the total of bovines females older to 36 months were of 374 317 (Caquetá, 2016). Was calculated the sample size from a known population, using the equation (1).
Where n = sample size, N = population size (374,317), Z = confidence level (95%), p = probability of success (0.5), q = probability of failure (0.5), and d = precision (maximum allowable in terms of ratio error) (3.5%), thus was estimated a minimum sample size of 782 females older to 36 months.
For determinate to sample size for the dual purpose herds was used the previous equation with the following assumptions: N = population size (11,128), Z = confidence level (95%), p = probability of success (0.5), q = probability of failure (0.5), and d = precision (maximum allowable in terms of ratio error) (10%), thus was estimated a minimum sample size of 95 herds of dual purpose.

C. Selection of Herds
The herds were selected from the database of project Implementación y validación de modelos alternativos de producción ganadera en el departamento del Caquetá, taking into account the 500 herds of dual-purpose cattle distributed in nine municipalities of state, and the criteria proposed by Yamamoto et al. (2007), were used, but modified for this research: a) the farm size (50-180 hectares), b) farms with more than 10 cows in milking (older to 36 months), c) availability to cooperate in the project, and d) accessibility and roads in good conditions.

D. Ethics Aspects
The samples were taken by Veterinarians and endorsed by the inspectors of the entity of inspection authorized by the Colombian Agricultural Institute (ICA by the acronyms in Spanish) in the Florencia branch, following the ethics, technical, scientific and administrative regulations for the research with animals, according to the law 84 of 1989. Throughout the research, the confidentiality of farms positives to brucellosis was maintained. The project was approved by the resolution 005 of 2013 by the Science, Technology, and Innovation Fund (FCTeI) of the National Planning Department (DNP) of Colombia with the advice of Colciencias as technical secretariat.

E. Diagnosis
Between the months of January to March of 2016 blood serum sampling were carried out in 100 herds dedicated to the dual purpose cattle system, in each were sampled ten livestock (9 cows and 1 Bull) older to 36 months for a total to 1000 bovines.
To obtain blood samples, venipuncture was performed in the coccygeal vein preliminary cleaning and disinfection of the area with ethyl alcohol at 70%, the blood samples were deposited in sterile tubes without anticoagulant (red top) to ensure that the serum obtained, all samples were properly labeled the animal information: identification, sex, age, identification to herd and date of sampling, Subsequently, the blood samples were stored in thermos conveyors maintaining refrigeration at 4-8 ° C. All samples were sent to the laboratory of veterinary Diagnostics of the Colombian Agricultural Institute (ICA) in the Florencia city.
In the laboratory of Veterinary Clinical Diagnostics was conducted the tests established by the ICA for the diagnosis of brucellosis in Colombia (Resolution 0840, 2011), blood serum was obtained and was carried out with Pink Bengal agglutination technique as a test sieve, with a particulate Antigen consisting of whole strains 1119-3-cell or C99 of B. abortus that interacts with serum producing a network of agglutination dependent on optimal proportions of the reactants. Antigen dampens pH low, by the addition of pink Bengal dye, buffered usually 3.65 ± 0.05, since the non-specific agglutination of smooth Brussels boils down to pH low, favoring the activity of type IgG1 specific antibodies, testing has shown high although confirmatory test because of its specificity it requires relatively low sensitivity, therefore the positive Rose

III. RESULTS AND DISCUSSION
In total were sampled 1000 bovines distributed in 100 herds of dual-purpose cattle, of which 882 blood serum corresponding to females, 77 to Bulls and 41 to hemolyzed blood serum.
Sero-prevalence of bovine brucellosis in the Caquetá state was of 3.23% (95%, CI: 0-6.53%), with the highest prevalence in females than in males (Table 1). At the municipal level were found percentages higher than the sero-prevalence in the state, as in the case of the San Vicente del Caguán, San José del Fragua, Milán, and Cartagena del Chairá municipalities, for La Montañita and El Doncello the seroprevalence was of 0%, however, statistical difference was not found (Figure 1). Of the 77 bulls analyzed only two (2) were positives for brucellosis, being a seroprevalence of 2.6% respect to all male's samples. In the females, the seroprevalence of bovine brucellosis was of the 3.29% obtaining 10 positives livestock by competitive Elisa test. Sero-prevalence (%) The positives cases for bovine brucellosis were meet in 22 herds of seven municipalities, thus the prevalence of disease at farm level was of 22% (95%, CI: 12.24-31.76%) ( Table 2). Different researchers have found minor's values of sero-prevalence of bovine brucellosis respect to this research, for example, in a study conducted in Montería, Córdoba, Colombia (González, Rios, & Mattar, 2007), found that in 384 bovine females with infertility problems, the seropositive for brucellosis were 3.1%. Likewise, found in 206 blood serum analyzed in the municipalities of Roscio and Ortiz a sero-prevalence of 2.9% (Tamasaukas, Purroy, Rodríguez, Ruiz, Roa, & Labrador, 2002). In the same way, reported a sero-prevalence of 0,639 in livestock from Machiques de Perijá municipality in Venezuela, being the lowest for Latinamerica (Sánchez-Villalobos et al., 2009). In the municipality of La Plata, Huila, Colombia, found a sero-prevalence in livestock of 2.11% (Tejada & Ayala, 2008). In the same way, found an overall sero-prevalence of 1.90% in the Huila state, neighbor to the Caquetá state, nevertheless, the same authors report at municipal level a maximum sero-prevalence of 4.52%, value less than those found in municipalities in this research (Dussan-Embus, Castañeda-Repizo, & Durán-Bautista, 2012).

Sero-prevalence of bovine brucellosis in Caquetá state
Nevertheless, a research performed in the municipality La Cañada de Urdaneta in Venezuela, found a sero-prevalence of bovine brucellosis of 9.1%, which is greater than this research (D´Pool et al., 2004).  Poveda & Carrillo (1981), found prevalence in herds of 11.7%.

In bulls located in
Then, the sero-prevalence of bovine brucellosis in Caquetá state has gone from 0.33% in the year 1980 to 12% in 1999, nevertheless, the sero-prevalence has decreased for the efforts of the official control Institutions as ICA, being the seroprevalence in livestock low with the 3.23% for the year 2016. However, is necessary more effort by eradicating this zoonotic disease.
In this regard and taking into account the epidemiological implications of the brucellosis disease, it is necessary strengthen of a programme of control and eradication of bovine brucellosis in a coordinated and sustained manner (Seleem, Boyle, & Sriranganathan, 2009), affects people in contact with animals and derived products such milk, being that the animal reservoirs control allows a reduction of the incidence of the disease in humans.

VI. CONCLUSIONS
Bovine brucellosis in the Caquetá state has a low sero-prevalence being of 3.23% in cows older to 36 months and moderate prevalence at farm level with 22% of the herds samples, which indicates that despite a low incidence of the disease in animal and moderate in herd, it is necessary a greater performance of official control entities at farm level to reduce and eradicate the disease for guarantee the safety of milk and therefore the health of consumers.