Open Veterinary Journal, (2023), Vol. 13(8): 1012-1020

Original Research

10.5455/OVJ.2023.v13.i8.7

Risk factors associated with enzootic bovine leukosis in Boyacá and Cundinamarca municipalities, Colombia

Deisy J. Lancheros-Buitrago¹, Diana M. Bulla-Castañeda¹, Julio C. Giraldo-Forero^2,3 and Martin Orlando Pulido-Medellin^1*

¹Grupo de Investigación en Medicina Veterinaria y Zootecnia—GIDIMEVETZ, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia

²Grupo de Investigación en Parasitología y Microbiología Tropical, Programa de Biología, Universidad INCCA de Colombia, Bogotá, Colombia

³Grupo de investigación en Ecoepidemiología y Salud Colectiva, Facultad de Medicina y Ciencias de la Salud, Universidad Militar Nueva Granada, Bogotá, Colombia

*Corresponding Author: Martin Orlando Pulido Medellin. Grupo de Investigación en Medicina Veterinaria y Zootecnia—GIDIMEVETZ, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia. Email: martin.pulido [at] uptc.edu.co

Submitted: 02/05/2023 Accepted: 25/07/2023 Published: 31/08/2023

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Abstract

Background: Enzootic bovine leukosis (EBL) is a lymphoproliferative disorder caused by the bovine leukemia virus (BLV), a virus of the Retroviridae family. The infection is distributed worldwide, and a high percentage of animals infected by the BLV are asymptomatic and act as carriers of the virus in many cattle populations.

Aim: To identify the risk factors associated with EBL in the municipalities of Boyacá and Cundinamarca (Colombia).

Methods: A simple descriptive cross-sectional study with random sampling was conducted. A total of 1,140 blood samples were taken from cattle (females and males) from the municipalities of Chiquinquirá, Ubaté, and San Miguel de Sema of different breeds and age groups. The samples were processed using the commercial ELISA SERELISA® BLV Ab Mono Blocking kit (sensitivity 97%, specificity 98%). The data were processed with the statistical programs WinEpi and Epi Info® version 7.2.4.0, estimating the prevalence ratio, implementing the chi-square test (p ≤ 0.05) and logistic regression.

Results: A true prevalence (TP) and apparent prevalence (AP) of 23.61% and 22.7% in Ubaté, 19.22% and 18.1% in Chiquinquirá, and 15.61% and 14.3% in San Miguel de Sema, respectively, were established. Bovines 2–4 years old were the most prevalent in Ubaté and Chiquinquirá (37.5% and 21.21%, respectively), while in San Miguel de Sema individuals >4 years had the highest percentage of antibodies (18.3%). The Holstein breed had a higher prevalence in Ubaté and San Miguel de Sema (26.02% and 19.67%), and crossbreeds were more BLV-seroprevalence in Chiquinquirá (20.20%). In Ubaté, re-use of needles was identified as a risk factor, contaminated blood in needles is considered one of the main routes of transmission. On the other hand, manual milking was identified as a risk factor in San Miguel de Sema.

Conclusion: The non-implementation of an individual needle per animal in Ubaté; the Holstein breed and manual milking in San Miguel de Sema were identified as risk factors for the presence of antibodies against the disease. EBL prevention and control plans should be established that focus on the implementation of management and sanitary practices based on herd biosecurity.

Keywords: Cattle diseases, ELISA, Leukosis, Risk factors.

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Introduction

Enzootic bovine leukosis (EBL) is a chronic lymphoproliferative disorder caused by bovine leukemia virus (BLV), of the genus Deltaretrovirus and family Retroviridae (Benitez et al., 2019, 2020; Kobayashi et al., 2020; Bulla-Castañeda et al., 2021; Cândida Ramalho et al., 2021; Nakada et al., 2023). BLV generates a chronic, progressive, and contagious disease characterized by malignant lymphoid cell hyperplasia and systemic lymphadenopathy, which affects almost all countries and regions where cattle are raised (Ma et al., 2021). The disease is considered an economically important infection of dairy cattle and the common mode of virus spread is horizontal or iatrogenic transmission, through direct and indirect exposure of susceptible animals to infected lymphocytes from blood, milk, colostrum, and by natural mating (Benitez et al., 2019; Kuczewski et al., 2021; Jiménez Sánchez et al., 2022).

Dairy and beef cattle breeds are susceptible to BLV infection; however, the disease is the most prevalent in dairy herds, mainly due to management practices (Gutiérrez et al., 2020). Proximately 70% of animals appear clinically healthy for the first few years’ post-infection, becoming asymptomatic carriers of the virus and developing persistent lymphocytosis (Benitez et al., 2020; Kobayashi et al., 2020; Jiménez Sánchez et al., 2022). The main transmission via is the horizontal way, this occurs by direct contact with infected leukocytes. Transplacental transmission could occur in less than 10% of infections in cattle (Tsutsui et al., 2016; Ruiz et al., 2018).

BLV infection leads to decreased immune function, making animals more vulnerable to other diseases, which could shorten their productive life (Benitez et al., 2020). On the other hand, could be a potential zoonotic disease, Ma et al. (2021) and Olaya-Galan et al. (2021) reported that BLV has been found in the blood and mammary tissues of women with breast cancer, and an association between this and the virus was confirmed in Colombia, as an intermediate risk factor; however, the link between BLV and human breast cancer is somewhat controversial. Moreover, there is no specific treatment for the disease or commercial vaccine that allows the prevention of the disease (Bulla-Castañeda et al., 2018).

Consequently, in the absence of control measures, the virus spreads slowly among the animals in the herd, which is why it is considered necessary to prevent the spread of BLV to reduce economic losses related to cattle infection (Gutiérrez et al., 2020; Cândida Ramalho et al., 2021; Wu et al., 2023). Until now, there have been few seroprevalences of BLV studies in Colombia (Corredor-Figueroa et al., 2020). Therefore, the objective of this study is to determine the risk factors associated with EBL in the municipalities of Boyacá and Cundinamarca (Colombia).

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Materials and Methods

Study area

The Altiplano Cundiboyacense comprises the valleys of Ubaté, Chiquinquirá, and the valleys of Tunja, Duitama, and Sogamoso. Villa de San Diego de Ubaté, Chiquinquirá, and San Miguel de Sema are municipalities localized in the valleys of Chiqunquirá and Ubaté (Castrillón Franco, 2014; ICA, 2016). The municipalities are part of the high tropic zones where most milk is produced, with 75,731 heads of cattle registered in the region (ICA, 2022).

Sample size

The municipalities of Chiquinquirá, San Miguel de Sema, and Villa de San Diego de Ubaté have 33,398, 23,057, and 19,276 heads of cattle, respectively (ICA, 2022). Based on the number of bovine animals in the municipalities, a sample size of 385, 378, and 377 cattle was established for each of the municipalities, correspondingly. The sample size for each testing municipality was determined by considering a 95% confidence interval (CI), an accepted error of 5%, and an expected prevalence of 50% since there are no studies in the area, implementing the WinEpi statistical program and following the following equation:

where: n=sample size; E=accepted error; p=expected value of the proportion; α=tail probability.

Sample collection and preparation

Blood samples were taken from females and males of dairy cattle breeds belonging to different age groups (<2, 2–4, and >4 years). The biological material was obtained via coccygeal venipuncture, using a 16 and 18- gauge, 3-inch needle. Approximately, 14 ml of blood was extracted in tubes without anticoagulant, previously labeled. The samples were transported to the Veterinary Parasitology Laboratory of the Universidad Pedagógica y Tecnológica de Colombia for processing. The tubes were centrifuged at 2,500 revolutions per minute (rpm) for 10 minutes to separate the cells from the serum (Figueiredo Marques et al., 2017). The serum samples were processed via an Enzyme-Linked Immunosorbent Assay (indirect ELISA) technique with the commercial ELISA kit SERELISA® BLV Ab Mono Blocking (sensitivity 97%, specificity 98%), according to the manufacturer's instructions.

Variables

An epidemiological survey was conducted in each cattle herd, and a descriptive form was filled out to propose possible risk factors associated with EBL seropositivity. Is a form that retrieves information from the farms and that was designed considering those variables that, according to the literature, have some relationship with the positivity of the virus. The information was taken from questions whose answers were based on yes/no. The variables to be evaluated were classified into four categories: a) reproductive variables: artificial insemination, natural mating, certified semen, uncertified semen, and shared bull; b) management variables: livestock owned by other owners, pasture leasing, re-use of needles, attendance to livestock expositions, mechanical milking, and manual milking; c) herd size: large herd, and small herd; and d) animal variables: gender, age group, and cattle breed.

Statistical analysis

The apparent prevalence (AP) and true prevalence (TP) were determined with the WinEpi statistical program, considering the sensitivity and specificity of the test. Subsequently, the database was consolidated in Excel and processed using the statistical program Epi Info® version 7.2.4.0. The proportion of animals and herds affected by EBL and exposed to the factors was evaluated with the same proportion of a population not exposed to that factor to estimate prevalence ratios (PR). This PR was used to measure the association between EBL and the hypothesized causal factors, as well as the significance of these associations using a Chi-square test (Thrusfield, 2005). PR values greater than 1 (lower 95% CI < 1) and with p < 0.05 were considered risk factors, while PR values less than 1 (upper 95% CI < 1) and with p < 0.05 were protective factors. Once these factors were established, a stratified logistic regression was performed to test for confounding variables and to identify the simultaneous interaction between variables significantly associated with EBL (Martin et al., 1997).

Ethical approval

This study was conducted in accordance with Resolution 8,430 of the Colombian Ministry of Health and Social Protection and Law 84 of 1989. These establish the standards that are suitable for the welfare of the animals during the research. Written informed consent was obtained from cattle owners before sample blood collection.

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Results

An AP and TP of 23.61% (89/377) and 22.7%, respectively, were determined in Ubaté, with a positive predictive value (PP+) of 93.5% and a negative predictive value (NP−) of 99.1%. In Chiquinquirá, the AP was 19.22% (74/385), while the TP was 18.1% (PV+ 91.5%; NP− 99.3%), and in San Miguel, AP of 15.61% (59/378) and TP of 14.3% (PV+ 89%; NP− 99.5%) were found. Females were more prevalent than males in the three municipalities (Table 1).

According to the study, individuals between 2 and 4 years had the highest seropositivity in Ubaté and Chiquinquirá with 37.5% and 21.21%, respectively, while in San Miguel de Sema bovines >4 years had the highest percentage of antibodies (18.3%) (Table 2). The Holstein breed had a higher prevalence in Ubaté and San Miguel de Sema (26.02% and 19.67%, respectively), although crossbreeds were more seroprevalent in Chiquinquirá (20.20%) (Table 2).

No significant statistical association was found between seropositivity to the disease and variables gender, age, and breed of cattle in the municipality of Chiquinquirá (p ≥ 0.05). Nevertheless, statistical significance was found between antibodies presence and bovines 2–4 years old in Ubaté ( p=0.04742422), while in San Miguel de Sema the Holstein ( p=0.02443112) and Normande ( p=0.00103089) breeds presented significant statistical association with the presentation of BLV antibodies (Table 3).

In relation to management practices and reproductive variables evaluated in the epidemiological survey, the purchase of animals and natural mating was statistically significant in Chiquinquirá (p ≤ 0.05). In Ubaté, livestock owned by other owners, pasture leasing, re-use of needles, attendance to livestock expositions, purchase of animals, and implementation of uncertified semen were statistically significantly correlated with antibodies presentation. Also, the variables shared bull, natural mating, manual and mechanical milking, purchase of animals, pasture leasing, and livestock owned by other owners were statistics significant to the seropositivity disease in San Miguel de Sema (p ≤ 0.05) (Table 4).

The variables of natural mating; in Chiquiquirá, re-use of needles and uncertified semen in Ubaté; and Holstein breed and manual milking in San Miguel de Sema were established as possible risk factors for the antibodies presentation (Table 4). Re-use of needles in Ubaté, Holstein breed, and manual milking in San Miguel de Sema were identified as true risk factors for the disease in the mentioned municipalities (Table 5).

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Discussion

EBL is the most frequent tumor disease in cattle. Even though a high percentage of animals infected by BLV are asymptomatic, the infection can go unnoticed when the viral load is relatively low (Gutiérrez et al., 2020). Despite this, there are different investigations on EBL worldwide. Seroprevalences of 0.36% have been reported in the Zona Deprimida del Río Salado (Buenos Aires, Argentina) (Panei et al., 2017), 2.27% in three provinces of Ecuador (Vásconez-Hernández et al., 2017), 10.8% in Paraíba (Brazil) (Ramalho et al., 2021), 10% in China (Ma et al., 2021), and 17.7% in Egypt (Selim et al., 2020), with seropositivity below those found in the municipalities evaluated in the present study, except in Ubaté with a seroprevalence lower in contrast to Selim et al. (2020).

Table 1. Seroprevalence of EBL by gender in cattle in the municipalities of Chiquinquirá, Ubaté, and San Miguel de Sema.

Table 2. Seroprevalence of EBL by age group and breed in cattle in the municipalities of Chiquinquirá, Ubaté, and San Miguel de Sema.

Table 3. Analysis of breed and age groups as possible risk factors associated with BLV infections. Results are presented as PR and 95% CI.

Table 4. Analysis of management practices and reproductive variables as possible risk factors associated with BLV infections. Results are presented as PR and 95% CI.

In Colombia, percentages of antibodies against BLV have been determined in 13.5% of cattle from Toca (Boyacá) using Göttingen hematological keys and ELISA (Pulido-Medellín et al., 2017), in 21.8% of individuals from the department of Santander (Vargas-Niño et al., 2018), in 79.17%, 53.74%, and 46.67% in the Oriente, Norte, and Valle de Aburrá subregions (Antioquia), respectively (Úsuga-Monroy et al., 2018a), 31.1% in Sotaquirá (Boyacá) (Bulla-Castañeda et al., 2021), 14.64% in Puerto Boyacá (Naranjo et al., 2022), and 31.1% of the animals sampled in Paipa (Boyacá) (Jiménez Sánchez et al., 2022). Variations occur due to the number of animals sampled, management practices, and production intensification, as well as the geographical location of the farms since it has been reported that there is a greater tendency to seropositivity to the disease in farms with temperate climate and higher altitude, that is mainly associated with sanitary and management practices such as re-use of needles, rectal palpation gloves, natural matting, among others (Vásconez-Hernández et al., 2017).

As regards the age of the cattle evaluated, the highest seroprevalences were established in the older age groups in our study, this finding is similar to that reported by Ma et al. (2021); Bulla-Castañeda et al. (2021) and Jiménez Sánchez et al. (2022), who also found increased antibody percentages in animals in production, this may be due to the fact that the time of infection by the virus is an important factor in the pathogenesis of EBL at an early age since commonly BLV requires a long latency period to cause the disease, this is why bovine leukosis in calves is more likely to present as a sporadic form (Oguma et al., 2017). Individuals 2–4 years of age in Ubaté had a significant statistical association with BLV seropositivity, which is related to that established by Jiménez Sánchez et al. (2022) who reported this association with animals 3–4 years old but differs from Bulla-Castañeda et al. (2021) because in their research the relationship was determined with the age groups of 1–2 and older than 4 years.

Taking this into account, it should be mentioned that the prevalence in cattle increases with age (Ma et al., 2021). Research reports that adults are four times more likely to be seropositive than heifers, leading to the virus being widely distributed in herds (Luciani et al., 2022) and that the disease seropositivity was associated with the number of calvings, where there is an 80% higher probability of being BLV-positive when calvings are ≥5 (Selim et al., 2020). So timely prevention and control during the calf period can reduce the spread of the disease, which is why it is recommended to strengthen detection in cattle older than 1 year and timely isolate those that are seropositive (Ma et al., 2021).

Table 5. Analysis of variables as possible risk factors associated with EBL infections.

Females were more prevalent than males in the three municipalities sampled, which agrees with Bulla-Castañeda et al. (2021), who also found that gender is a risk factor. On the other hand, the Holstein breed was the most seroprevalent in Ubaté and San Miguel de Sema, while the crossbreeds were more seropositive in Chiquinquirá, and Holstein and Normande individuals were statistically significant in San Miguel de Sema; dairy and beef cattle breeds are susceptible to virus infection; however, the disease is more prevalent in dairy production, mainly due to management practices (Gutiérrez et al., 2020); hence, individuals of the Holstein breed have been considered a risk factor for antibodies against disease presentation.

Úsuga-Monroy et al. (2018b) mention that the infection percentage was 55.9% for the Holstein breed while for Creole and crossbred cattle this value was lower, which according to what the researcher indicates can be attributed to the presence of resistance genes in the Creole breed, they also report that the level of infection is lower in crossbred dairy cows than in the Holstein breed. Recent research establishes that BLV infection leads to a decrease in immune function, which makes animals more vulnerable to other diseases, which could shorten their productive life and increase economic losses in the cattle industry, also according to reports, BLV seropositive dairy cows are more likely to be culled earlier compared to other uninfected animals in the herd (Benitez et al., 2020).

Regarding the management practices implemented in the evaluated farms in this study, a significant statistical association was established between the purchase of animals and the presence of animals from other farms. Although those variables were not considered as risk factors, that differs from Bulla-Castañeda et al. (2021) and Ramalho et al. (2021) who did report these variables as risk factors for the presence of antibodies against disease presentation. In addition, Nekouei et al. (2015) mention that herds that purchased animals with unknown infection status had a higher seroprevalence. Therefore, it is considered important to know the origin and health status of the animals prior to entering the farm. Besides, the re-use of needles had statistical significance in Ubaté, Hutchinson et al. (2020) report that the reuse of needles is associated with a higher incidence rate of the virus, this may be due to the fact that the main route of transmission of the virus is iatrogenic through the transfer of blood containing infected lymphocytes (Benitez et al., 2019); hence, the re-use of needles was determined as a risk factor for disease.

Methods of milking had a significant statistical association with seropositivity disease in San Miguel de Sema. In this sense, it is important to mention that BLV proviral DNA has been identified from milk and colostrum (Benitez et al., 2019). Hutchinson et al. (2020) mention that there is an association between milking frequency and increased incidence of EBL, suggesting a possible mode of transmission that has been largely overlooked, as BLV-infected lymphocytes are known to be present in the milk of seropositive cows, highlighting the link between increased milking frequency and BLV incidence (Ferrer et al., 1981; Hutchinson et al., 2020). In addition, if residual milk containing infected lymphocytes remains within the teat lining and a negative cow with minor teat damage is subsequently milked, it may be exposed to BLV-infected cells. Probably residual milk may reflux into the mammary gland of the next cow during mechanical or hand milking (Thompson and Miller, 1974; Hutchinson et al., 2020). It has also been mentioned that among the factors associated with the prevalence of EBL at the herd level is the exclusive use of hand milking (Hutchinson et al., 2020), which would explain why this practice is a risk factor for EBL in San Miguel de Sema.

Regarding the natural mating, the use of uncertified semen and shared bulls was significantly associated with the presentation of antibodies to BLEV (p ≤ 0.05). However, Jiménez Sánchez et al. (2022) have reported no significant statistical association with these variables. Artificial insemination has been established as a risk factor for the presentation of EBL (Bulla-Castañeda et al., 2021; Ramalho et al., 2021) but this was not found in the herds evaluated; it should be kept in mind that proviral DNA of BLV has been identified in semen and smegma (Benitez et al., 2019), so not knowing the origin and sanitary status of semen implemented on farms should be considered as a point to be taken into account for the design of LEB prevention and control plans.

BLV infection is present in Colombia, so the establishment of prevention and control plans should be implemented based on herd-level measures, focusing on good hygiene in manual and mechanical milking, sound practices and standards for artificial insemination and serological testing of animals prior to purchase (García and Olivera-Angel, 2019; Ramalho et al., 2021). Additionally, it must be kept in mind that the eradication of BLV in herds should be based on the implementation of good livestock practices, where the importance of biosecurity measures such as the establishment of a closed herd is highlighted, thus avoiding the introduction of BLV in herds (Kuczewski et al., 2021).

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Conclusion

A moderate seroprevalence of EBL was found in the herds of the three municipalities evaluated. Nevertheless, general knowledge about the asymptomatic disease presentation, and identification of the health status of livestock, would allow the establishment of control and prevention plans for the implementation of biosecurity measures based on management practices. Adult cattle and dairy breeds were the most seroprevalent, which is mainly due to intensive herd management practices and prolonged exposure to the virus that older animals may have had. Non-implementation of individual needles, Holstein breed, and hand milking were determined as risk factors for EBL, which is related to the susceptibility of dairy breeds and the importance of implementing proper management practices to prevent iatrogenic transmission of the virus.

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Acknowledgments

The authors would like to thank the Universidad Pedagógica y Tecnológica and the producers who participated in the study.

Conflict of interest

The authors declare that there is no conflict of interest.

Author´s contributions

Diana M. Bulla-Castañeda, Julio C- Giraldo-Forero and Martin O. Pulido-Medellin were responsible for the design of the study; Diana M. Bulla-Castañeda and Martin O. Pulido-Medellin performed the experiments; Diana M. Bulla-Castañeda analyzed the data; Deisy J. Lancheros- Buitrago and Diana M. Bulla-Castañeda wrote the manuscript; Deisy J. Lancheros-Buitrago, Diana M. Bulla-Castañeda, Julio C- Giraldo-Forero, and Martin O. Pulido-Medellin reviewed and editing the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This investigation was funded by the call for proposal number 02-2023 of the Research Department of the Universidad Pedagógica y Tecnológica de Colombia (UPTC).

Data availability

All data supporting the findings of this study are available within the manuscript. Any extra data needed are available from the corresponding author upon reasonable request.

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