A PCR for differentiate between Anaplasma marginale and A. centrale

Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...]

A PCR for differentiate between Anaplasma marginale and A. centrale

Background: Anaplasma marginale ssp. centrale (A. centrale) exhibits low pathogenicity and therefore is used as a live vaccine against bovine anaplasmosis in several countries. During production of the vaccine, accidental contamination with Anaplasma marginale (A. marginale) is a risk that can jeopardize the entire batch of vaccine. Due to limitation of microscopic examination to detect low levels of parasitaemia, the present study aims to standardize a polymerase chain reaction assay using primers for the msp4 gene of Anaplasma sp. for detecting and differentiating with greater sensitivity and specificity the species of A. centrale and A. marginale in blood samples from experimentally infected cattle.Materials, Methods & Results: The DNA extraction was performed from frozen blood. Erythrocytes infected with known A. centrale, A. marginale served as positive control and the erytrocytes infected with Babesia bovis and Babesia bigemina served as the negative control polymerase chain reaction. PCR was standardized from annealing temperature variations of the primers, magnesium chloride concentration, amounts concentration of primers and DNA concentration of rickettsiae. By PCR method, it was analyzed the DNA from blood samples of 13 cattle positive to A. marginale by microscopic examination from smear stained with Giemsa. The PCR assay was specific for A. centrale and A. marginale, presented 100% identity without presenting cross-reactivity with other bovine hemoparasites. The detection limits of the PCR were 0.25 pg and 0.125 pg of DNA for detection of A. centrale and A. marginale DNA respectively.Discussion: A. marginale is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia being considered the main agent of bovine anaplasmosis.[...](AU)

Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus / Antimicrobial peptides in Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by flies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale. Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the first line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire of antimicrobial peptides (AMPs). However, the Anaplasma sp. have developed evolutionary mechanisms to be able to adapt and survive in this arthropod which is the main biological vector this pathogen. The AMPs can be expressed constitutively by the immune system, induced by infection, or by the recognition of surface components of microorganisms such as lipopolysaccharides (LPS) and peptidoglycan (PNG). However, through evolutionary events, the Anaplasma marginale lost genes encoding these components characteristic of the cell wall of Gram-negative bacteria, and thus, is likely that the major surface proteins (MSPs) are involved in its strengthening as the resistance to AMPs. Although the mechanisms of action of AMPs have not been fully elucidated, models are proposed to demonstrate how the interactions between lipid bilayer and AMP happen. More than 1,000 AMPs have been described in several groups of eukaryotes. In particular, amphibian peptides account for 592 of total AMPs representing a rich source of these molecules. Additionally, another 166 AMPs were isolated from insects. However, in R. microplus, few studies have described the existence of AMPs. The known R. microplus antimicrobial peptides are defensin and ixodidin (both isolated from hemocytes), the microplusin (isolated from female hemolymph and eggs), VTDCE ( isolated gut and ovary), and other two peptides characterized as fragments of bovine hemoglobin, Hb 33-91 and Hb 98-114, (isolated from engorged female gut). Conclusion: Since the silencing of genes encoding AMPs expressed in R. microplus decreases the number of A. marginale, it is suggested that this bacteria could adapt to support the tick immune defense mechanisms generating a symbiotic relationship, a evidence that the expression of AMPs can be manipulated by the pathogen to assist in its multiplication by a mechanism not yet defined, thus the Anaplasma sp. and the tick vector can live together allowing the bacteria transmission by the host. More studies about antimicrobial peptides expressed in R. microplus against invading microorganisms are necessary in order to improve the comprehension of its immune system and its competence for bovine anaplasmosis as a vector.