Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

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.     

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.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.     

Cultivation of Anaplasma marginale from cattle in a Dermacentor cell line

A tick cell line derived from Dermacentor variabilis (RML-15) was inoculated with bovine RBC infected with Anaplasma marginale. Two hours after inoculation, numerous RBC were phagocytized by the tick cells. After one passage of the cell culture, numerous groups of Anaplasma-like particles were seen in the tick cell cytoplasm. Increased numbers of Anaplasma-like particles also were present. Seemingly, Anaplasma can multiply in tick cells.     

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.     

Prevalence and genetic diversity of Anaplasma marginale strains in cattle in South Africa

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in South Africa and results in considerable economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle raised in communal and commercial farms in the north-eastern and south-western regions of the Free State Province, South Africa, that varied in rainfall and vegetation. Seroprevalence to A. marginale was determined in 755 cattle by an Anaplasma spp. competitive enzyme-linked immunosorbent assay and ranged from 44% to 98% and was similar in both regions. While Anaplasma centrale was not targeted in this study, A. marginale infections were identified by species-specific msp1alpha polymerase chain reaction in 129 of 215 of the samples studied. Similar genetic diversity of A. marginale strains was found in both the north-eastern and south-western regions. The sequences of 29 A. marginalemsp1alpha amplicons from South African strains revealed considerable genetic diversity providing 14 new repeat sequences. However, 42% of MSP1a repeat sequences were not unique to this region. These results indicated the presence of common genotypes between South African, American and European strains of A. marginale. Cattle movement between different parts of South Africa was suggested by the presence of identical A. marginale MSP1a genotypes in north-eastern and south-western regions of the Free State Province. Control strategies for anaplasmosis in South Africa should therefore be designed to be protective against genetically heterogeneous strains of A. marginale.     

Molecular and serological prevalence of Anaplasma marginale in cattle of North Central Morocco

A cross sectional study was conducted to investigate the epidemiological distribution of Anaplasma marginale in North Central Morocco. Blood samples from five provinces of Morocco were collected from apparently healthy cattle (n=668) and simultaneously analyzed by a nested polymerase chain reaction (nPCR) assay and competitive enzyme-linked immunosorbent assay (cELISA). The overall prevalence of A. marginale was 21.9% by nPCR and 16.5% by cELISA. The Kappa coefficient between nPCR and cELISA indicated a modest level of agreement (0.54). The prevalence of A. marginale varied significantly according to the province and the month of sampling. However age, gender and breed did not have a significant effect on the prevalence of this pathogen. The highest prevalence of A. marginale was found in the Gharb, a sub-humid area while the lowest was reported in the Saiss, a semi-arid area. These results indicate that an A. marginale infection are widespread in the country and suggests that either or both techniques are excellent tools for epidemiological studies and control programs.     

Immunization of cattle with Anaplasma marginale derived from tick cell culture.

Anaplasmosis is a hemolytic disease of cattle caused by the ehrlichial tick-borne pathogen Anaplasma marginale. Killed vaccines used for control of anaplasmosis in the US used antigen harvested from infected bovine erythrocytes which was often contaminated with bovine cells and other pathogens. In this study, we performed an initial cattle trial to test A. marginale harvested from tick cell culture as an immunogen for cattle. Eleven yearling Holstein cattle were immunized with the cell culture-derived A. marginale and 11 cattle were non-immunized contact controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale in an oil-based adjuvant. Two immunizations were administered subcutaneously 4 weeks apart and the cattle were challenge-exposed 10 weeks after the second immunization with A. marginale infected blood. Maximum antibody levels as determined by an A. marginale specific competitive ELISA were observed 2 weeks after the last immunization. Antibody responses against major surface proteins (MSPs) 1a and 1beta1 were also characterized and immunized cattle demonstrated a preferential recognition for MSP1beta1. Cattle immunized with the cell culture-derived A. marginale had a significantly lower percent reduction in the packed cell volume (P<0.05) after challenge exposure as compared with the controls and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

Attempted transmission to cattle of Anaplasma marginale from overwintered Dermacentor andersoni ticks.

Since the 1983 summer outbreak of anaplasmosis in southern Saskatchewan, the role of the tick, Dermacentor andersoni as an overwintering reservoir for Anaplasma marginale has been questioned. The purpose of this study was to determine if spring-collected ticks carried virulent A. marginale. Sixteen splenectomized calves were assigned randomly to two groups of 14 principals and two controls. Adult D. andersoni, collected in April from areas having high transmission rates of A. marginale, were confined to the ears of the principals by special bags and allowed to feed for eight days. The two control calves were subsequently challenged intravenously with blood from a calf infected with the Virginia strain of A. marginale. Principals and controls were monitored for 60 and 50 days postexposure respectively for signs of infection by clinical, hematological and serological procedures. None of the principals developed anaplasmosis but both control calves developed signs of disease.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle

A modified indirect fluorescence antibody technique was used for the serodiagnosis of Anaplasma marginale infections in cattle. Nonspecific antibodies adherent to infected erythrocytes were removed, using acidic glycine buffer. Evans blue was used as a counterstain.