Avaliação de protocolos de extração de DNA em colostro bovino e pesquisa molecular de hemopatógenos

Abstract

In tropical and subtropical regions, Anaplasma marginale, Babesia bovis and B. bigemina represent significant sanitary and economic challenges. Additionally, hemotropic Mycoplasma spp., also known as hemoplasma, have gained prominence among hemopathogens with moderate to high frequency in cattle. The vertical transmission in the intrauterine environment has been widely reported in recent decades, as mono or co-infections, with higher frequencies usually associated with individual or mixed infections of A. marginale and hemoplasmas. Bovine colostrum is the source of passive immunity for the newborn, and microbiological quality is essential. The literature reports protozoa, viruses, and bacteria that may be present in milk secretions; however, there are no reports of research on hemopathogens in colostrum. Considering the mechanisms of passage of maternal blood elements into the mammary gland during colostrogenesis and the hematogenous route as an important dissemination site for hemopathogens, we hypothesize that the DNA of hemoparasites may be detected in colostrum from positive cows. The aim of this study was to investigate the presence of DNA from A. marginale, B. bovis, B. bigemina, and hemotropic Mycoplasma (HM) in bovine colostrum. Blood (5mL) and colostrum (40mL) were collected from 104 Holstein cows from a commercial dairy farm in the southern region of Minas Gerais, Brazil. The lack of a standardized protocol and specific reagents for manipulation and processing of colostrum, the protocol was tested before the DNA extraction. The quality of the material extracted was evaluated using a microvolume spectrophotometer and PCR reaction for identification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The protocol recovered 67.03 ± 81.64 ng/µL of DNA and showed values of 1.80, 1.66, and 1.89 for the median, 1st quartile, and 3rd quartile in the assessment of the A260/280 purity ratio, as well as 100% of the samples testing positive for GAPDH. For molecular diagnosis of the agents, PCR or nested-PCR was performed on all blood and colostrum. All agents were detected with frequencies of 95.2%, 52.9%, 62.5%, and 75.3% for A. marginale, B. bovis, B. bigemina, and HM, respectively. In colostrum samples, A. marginale, B. bovis, and HM were detected at frequencies of 12.12%, 10.9%, and 6.1%, respectively. This study demonstrated for the first time the presence of this hemopathogens in bovine colostrum, highlighting the need for future investigation regarding the viability of the agents and potential for transmission through this route. The high frequency of positive cows in blood and emergence of evidence in colostrum reaffirm the importance of implementing sanitary protocols for the control and prevention of diseases caused by hemopathogens in bovine management.