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Anaplasma marginale: effect of the treatment of cattle with an interferon gamma-neutralizing monoclonal antibody or the nitric oxide synthetase inhibitor aminoguanidine on the course of infection.

Cattle undergoing initial infection with the rickettsia Anaplasma marginale were treated with either a monoclonal antibody (MoAb) with neutralizing activity for bovine interferon gamma (IFN-gamma) or aminoguanidine (AG), a specific inhibitor of the inducible form of nitric oxide synthetase (iNOS). Plasma levels of MoAb and AG were measured over the time of administration. The course of A. marginale infection was not altered in the MoAb-treated cattle relative to untreated controls. In cattle treated with AG however, A. marginale infection was significantly ameliorated, as judged by lower parasite levels and decreased anaemia in these cattle relative to the controls. The implications of these findings in relation to the basis for immunity against this economically important haemoparasite are discussed.

Anaplasmosis↗

Human interferon alpha fails to inhibit the development of Babesia bigemina and Anaplasma marginale infections in cattle.

Studies were undertaken to determine whether human interferon alpha (HuIFN-alpha) administered orally could inhibit the development of clinical disease caused by the intraerythrocytic protozoan Babesia bigemina and the intraerythrocytic rickettsia Anaplasma marginale in cattle. HuIFN-alpha did not inhibit intraerythrocytic multiplication of either of the two parasites, suggesting that there is no role for HuIFN-alpha administered orally in the control of these pathogens.

Anaplasmosis↗

Attempts to transmit Anaplasma marginale with Hippobosca rufipes and Stomoxys calcitrans.

Three attempts to transmit anaplasmosis with field collections of Hippobosca rufipes were unsuccessful, despite the fact that the flies had been fed initially on splenectomized cattle acutely infected with Anaplasma marginale. However, 1 out of the 3 attempts, made concurrently with the others, to transmit this organism with Stomoxys calcitrans was successful. The prepatent period was 27 days.

Anaplasmosis↗

Detection of colonies of Anaplasma marginale in salivary glands of three Dermacentor spp infected as nymphs or adults.

Salivary glands from males of 3 Dermacentor species (D andersoni, D variabilis and D occidentalis) that were infected with either the Virginia or Idaho isolate of Anaplasma marginale as nymphs or adults were examined for colonies of A marginale by use of light and electron microscopy. Prior to dissection of salivary glands, exposed ticks were held at 25 C for 15 to 18 days, followed by a 3-day incubation at 37 C. Ticks of 2 species transmitted A marginale to calves; the third tick species was confirmed infected by demonstration of typical colonies in tick gut cells, but transmission was not attempted; Colonies of A marginale were seen with light microscopy in salivary glands of all 3 species of ticks; they were located in acinar cells that contained simple granules. Colonies varied morphologically from small, compact ones to larger structures that contained distinct organisms and often were adjacent to the host cell nucleus. Electron microscopy confirmed that the colonies were rickettsial organisms. Morphologic features of A marginale varied and included reticulated forms, forms with electron-dense centers, and small particles; these various forms were similar to those described previously in midgut epithelial cells of ticks. We believe that the organism seen within tick salivary glands may replicate in the glands before its transmission to the vertebrate host.

Anaplasma↗

Detection of Anaplasma marginale rickettsemia prior to onset of clinical signs by using an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay was developed by using monoclonal antibodies to conserved epitopes on the Anaplasma marginale MSP1a surface protein. The assay sensitivity was 1.1 (+/- 0.5)% parasitized erythrocytes, and all infected cattle were detected prior to development of 2.0%-parasitized erythrocytes. Positive tests preceded the onset of anemia by a mean of 2 days. The assay was specific for anaplasmosis, as demonstrated by nonreactivity with another common hemoparasitic pathogens.

Anaplasmosis↗

Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission.

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.

Amino Acid Sequence↗

Molecular basis for surface antigen size polymorphisms and conservation of a neutralization-sensitive epitope in Anaplasma marginale.

Anaplasmosis is one of several tick-borne diseases severely constraining cattle production and usage in many parts of the world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginale carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates of A. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the mspl alpha gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.

Amino Acid Sequence↗

Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5.

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.

Anaplasmosis↗

Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood.

A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9/10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.0001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test.

Anaplasma↗

Selective in vivo depletion of CD4(+) T lymphocytes with anti-CD4 monoclonal antibody during acute infection of calves with Anaplasma marginale.

To investigate the in vivo role of CD4(+) T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4(+) T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginale in two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3(+) CD4(+) and CD4(+) CD45R(+) (naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginale antibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginale major surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4(+)-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4(+) T lymphocytes would abrogate resistance to acute infection.

Acute Disease↗

Transmission of Anaplasma marginale with adult Boophilus microplus ticks fed as nymphs on calves with different levels of rickettsaemia.

Nine splenectomised calves were infested with dissimilar numbers of adult Boophilus microplus ticks 72 h after collection as engorged nymphs from three non splenectomised calves with different levels of Anaplasma marginale rickettsaemia. Successful transmission of A. marginale appeared to be more dependent on the level of rickettsaemia of the donor calves than on the number of ticks attaching to the splenectomised calves, since infection was transmitted only when the rickettsaemia was 0.3% or greater. Field transmission would thus depend on the rate of tick migration amongst susceptible hosts and the rickettsaemia level of cattle on which the ticks fed previously.

Anaplasma↗

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.

Anaplasma↗

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

Amino Acid Sequence↗

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.

Anaplasma↗

Transmission of Anaplasma marginale by Boophilus microplus: retention of vector competence in the absence of vector-pathogen interaction.

Whether arthropod vectors retain competence for transmission of infectious agents in the long-term absence of vector-pathogen interaction is unknown. We addressed this question by quantifying the vector competence of two tick vectors, with mutually exclusive tropical- versus temperate-region distributions, for genetically distinct tropical- and temperate-region strains of the cattle pathogen Anaplasma marginale. The tropical cattle tick Boophilus microplus, which has been eradicated from the continental United States for over 60 years, was able to acquire and transmit the temperate St. Maries (Idaho) strain of A. marginale. Similarly, the temperate-region tick Dermacentor andersoni efficiently acquired and transmitted the Puerto Rico strain of A. marginale. There were no significant quantitative differences in infection rate or number of organisms per tick following feeding on cattle with persistent infections of either A. marginale strain. In contrast, the significantly enhanced replication of the Puerto Rico strain in the salivary gland of B. microplus at the time of transmission feeding is consistent with adaptation of a pathogen strain to its available vector. However, the transmission of both strains by B. microplus demonstrates that adaptation or continual interaction between the pathogen and vector is not required for retention of vector competence. Importantly, the results clearly show that reestablishment of acaricide-resistant B. microplus in the United States would be associated with A. marginale transmission.

Anaplasma↗

Transmission of Anaplasma marginale Theiler by males of Dermacentor andersoni Stiles fed on an Idaho field-infected, chronic carrier cow.

The role of ticks and carrier cattle in epizootics of bovine anaplasmosis was further clarified by demonstrating unequivocally, for the first time, that male ticks fed on a chronic carrier cow naturally infected with Anaplasma marginale can transmit this parasite intrastadially and biologically when subsequently fed on susceptible cattle. These data indicate that field epizootics of acute anaplasmosis may be initiated by males of tick vector species that feed on carrier cattle and subsequently transfer to susceptible cattle.

Anaplasma↗

The oxidant defence system in water-buffaloes (Bubalus bubalis) experimentally infected with Anaplasma marginale.

The glutathione (GSH) -oxidant defence system protects the erythrocytes and leucocytes from oxidative damage. Leucocyte -superoxide dismutase (SOD), GSH-peroxidase (GSH-px), GSH-reductase (GR), GSH-S-transferase (GSH-S-t) and arginase were examined in samples from buffaloes infected with Anaplasma marginale. All the enzymes, except arginase, were also studied in the red cell haemolysates from these animals. GSH-S-t, GSH- and glutathione-reductase (GR) levels in leucocytes decreased in infected animals suggesting a decline in the efficiency of the GSH-oxidant defence system. SOD levels increased but there was no change in leucocyte-arginase activity due to infection. Infection caused no significant changes in red cell SOD, GSH-px, GR and GSH. However, GSH-S-t significantly decreased (P less than 0.05).

Anaplasmosis↗