Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.     

Bovine abortion associated with Anaplasma marginale.

During the period from June 1974 to June 1975, five bovine fetuses between seven to nine months old were received for necropsy from four different counties of the S. Paulo State, Brazil. All of them were from brucellosis-free herds. Necropsy revealed slight liver enlargement generally accompanied by capsular petechial hemorrhages. Enlargement and congestion of the spleen, epicardial and endocardial petechiae were present in three fetuses and one of them had lungs with some hemorrhagic lobules. Cardiac blood films of all the fetuses stained by the Pappenheim's panoptic method showed Anaplasma marginale in two to 20% of red corpuscles. When stained with acridine orange and immunofluorescent methods blood films of the first fetus specifically showed A. marginale.     

Co-feeding studies of ticks infected with Anaplasma marginale

Ticks often cluster at preferred feeding sites on hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. While the major route of infection of ticks with pathogens is via the bloodmeal during feeding on a parasitemic host, non-systemic transmission of viruses and spirochetes has been shown to occur from infected to uninfected ticks at common feeding sites on uninfected hosts. In this research, two separate studies were done using the tick-borne rickettsial pathogen of cattle, Anaplasma marginale. In one study we tested whether A. marginale could be transmitted non-systemically from infected to uninfected Dermacentor variabilis males while co-feeding on rabbits. Infection of ticks was determined by allowing them to transmission feed on susceptible cattle and by DNA probe and microscopy studies on salivary glands. In the second study, we tested whether the co-feeding of male and female ticks on parasitemic cattle would increase the acquisition and development of A. marginale in males. A. marginale infections in salivary glands were determined by quantitative PCR after the ticks were allowed to transmission feed on susceptible cattle. Non-systemic transmission of A. marginale did not occur from infected and uninfected ticks that fed at the same site on rabbits and, therefore, does not appear to be a means of A. marginale transmission. A. marginale infections in male ticks were not increased while co-feeding with females. Thus, co-feeding of adult Dermacentor spp. does not appear to influence the dynamics of A. marginale transmission.     

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.     

The natural history of Anaplasma marginale

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1α, provided information about the biogeography and evolution of A. marginale, and msp1α genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.     

Anaplasma marginale in tick cell culture

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Percutaneous infection of nymphal Dermacentor andersoni with Anaplasma marginale

Newly replete nymphal Dermacentor andersoni (principals) were percutaneously exposed to Anaplasma marginale by injection of either intact or lysed infected bovine erythrocytes. Control nymphs were fed on calves with anaplasmosis. The subsequently molted adults were examined for infection by light microscopy, and companion ticks were tested for infectivity by allowing them to feed on susceptible calves. When they fed as adults, both control ticks and percutaneously inoculated principals transmitted A marginale to susceptible calves. Prepatent periods in calves varied according to the method by which nymphs were infected. Colonies of A marginale were found in all ticks that acquired infection by feeding, but colonies were not observed in any ticks exposed percutaneously. The possible developmental cycle of A marginale in artificially infected ticks is discussed.     

Acute phase response in cattle infected with Anaplasma marginale

This study was undertaken to evaluate the acute phase responses via the assessment of the concentration of serum sialic acids (total, lipid bound and protein bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins (Hp and SAA) in 20 adult crossbred cattle naturally infected by Anaplasma marginale. The infected animals were divided into 2 subgroups on the basis of parasitemia rate (<20% and >20%). Also, as a control group, 10 clinically healthy cattle from the same farms were sampled. Our data revealed significant decreases in red blood cell count (RBC), hematocrite (PCV) and hemoglobine (Hb) in infected cattle compared to healthy ones. Conversely, the concentrations of Hp, SAA, ceruloplasmin, fibrinogen, serum sialic acids and the circulatory IFN-γ and TNF-α were increased in the diseased cattle (P<0.05). In addition, it was evident that the progression of parasitemia in infected cattle did not induce any significant alterations in the hematological indices (RBCs, PCV and Hb) and the concentrations of Hp, SAA, ceruloplasmin and fibrinogen. SAA was the most sensitive factor to change in the diseased cattle. Therefore, increase in SAA concentration may be a good indicator of inflammatory process in cattle naturally infected with Anaplasma marginale.     

Differential extraction of antigens of Anaplasma marginale

Strain-, group-, and genus-specific antigens of the Florida isolate of Anaplasma marginale had different solubilities in zwitterionic, nonionic, and anionic detergents. On the basis of their solubility in nonionic detergent, antigens were grouped into 3 classes: (i) a 108-kilodalton (kD) group-specific antigen and a series of poorly defined antigens (70 to 100 kD) were completely soluble in nonionic detergent; (ii) 96 kD and 75 kD group-specific antigens, and a 91-kD strain-specific antigen were completely insoluble in nonionic detergent; and (iii) a 47-kD group-specific antigen and a 41-kD genus-specific antigen were partially soluble in nonionic detergent. A mercaptoethanol-sensitive antigen of 200 kD also was present in the insoluble fraction. Differences in solubility of taxonomic antigens of Anaplasma may reflect differences in function. Antigens totally insoluble in nonionic detergent were possibly integral membrane proteins, and those completely soluble in nonionic detergent were more likely peripheral membrane proteins that were less tightly bound to hydrophobic components of the membrane. Seemingly, all antigens were associated with membranes. Bovine preimmune and immune sera used did not react with any proteins of noninfected RBC.     

DNA probes for the detection of Anaplasma centrale and Anaplasma marginale

Anaplasmosis can be diagnosed either by immunological techniques or by direct microscopic examination of blood smears. Both methods are time-consuming and labour intensive. The use of DNA probes in an hybridization assay may simplify the diagnosis of anaplasmosis in cattle and sheep. A genomic DNA library of Anaplasma centrale was constructed in an expression vector and screened to detect clones containing A. centrale DNA. Four probes which hybridized to A. centrale and Anaplasma marginale DNA were isolated. One of these (AC-1) hybridized only to A. centrale DNA, whereas AC-2, AC-3 and AC-4 could detect DNA from both A. centrale and A. marginale. Probes AC-1 and AC-2 could detect 127 ng and 8 ng DNA respectively, while AC-3 and AC-4 detected 64 ng A. centrale DNA.