Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5×10¹⁰ (group I), 3×10¹⁰ (group II) or 6×10¹⁰ (group III) infected erythrocytes mixed with STDCM® adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1×10⁸ infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses (P0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.     

Efficacy of attenuated Anaplasma marginale vaccine under laboratory and field conditions in Colombia

Four-month-old Holstein-Friesian calves were inoculated with 3 different doses (1, 2, and 3 ml) of attenuated Anaplasma marginale vaccine. Vaccinated calves showed mild anaplasma parasitemia, slight decrease in packed cell volume, low serologic conversion, and no clinical illness. An artificial challenge exposure of vaccinated and unvaccinated calves with virulent Colombian A marginale showed that the vaccine provided protection against clinical signs of the disease, including parasitemia and anemia. The volume of the vaccinal dose did not alter the degree of protection provided. A 2nd group of 8- to 9-month-old Holstein-Friesian calves was then inoculated with 3 ml of anaplasma vaccine and premunized with both Babesia bigemina and Babesia argentina while being housed in an area free of these diseases. Calves were moved to an enzootic region heavily infested with various arthropods, including ticks, for natural field challenge exposure. Control calves, which were not given anaplasma vaccine, suffered clinical illness manifested by severe anemia and an average weight loss of 50.6 kg due to anaplasma field challenge exposure. In contrast, vaccinated calves did not show anemia and their weight loss was 3.9 kg.     

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.     

Infectivity virulence and immunogenicity of Anaplasma centrale live blood vaccine

Cross-bred Bos taurus calves, aged between 6 and 8 months, were inoculated with the Onderstepoort Anaplasma centrale live blood vaccine. One group of 15 calves were inoculated once only, while a 2nd group of 15 were revaccinated 6 months later. All the animals were challenged with approximately 1 X 10(10) Anaplasma marginale parasites of a known virulent strain 8 months after the first vaccination. The results of blood smear examination and the card agglutination test indicated that only 20 out of 30 animals vaccinated contracted A. centrale infections after the first attempt, and 3 out of 5 after the second. The vaccine conferred only partial immunity to challenge with a virulent A. marginale strain.     

Development of a recombinant Anaplasma marginale DNA probe

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.     

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.     

Comparison of three oxytetracycline regimes for the treatment of persistent Anaplasma marginale infections in beef cattle

Anaplasmosis, caused by the tick-borne rickettsia, Anaplasma marginale, is an economically important disease of cattle in the United States and worldwide. Cattle that recover from acute infection become carriers in which low or microscopically undetectable A. marginale rickettsemia persists. Tetracycline antimicrobials are currently the only drug used in the US for treatment of acute anaplasmosis. There are currently no drugs specifically licensed for elimination of persistent infections. This study tested the efficacy of three oxytetracycline treatment regimens to clear A. marginale from cattle that were persistently infected. Forty Angus x Simmental steers, aged 6-12 months were experimentally infected with A. marginale. After the steers recovered from acute infection, seroconverted, and were confirmed infected using nested PCR followed by DNA hybridization, the carrier status of each animal was ascertained by sub-inoculation of blood into a separate, splenectomized Holstein calf. The steers were then blocked by bodyweight and randomly assigned as follows to four treatment groups: Treatment A, 300 mg/ml solution of oxytetracycline (Tetradure LA-300, Merial Canada Inc.) administered at 30 mg/kg, by intramuscular (i.m.) injection on day 0; Treatment B, the same 300 mg/ml solution of oxytetracycline administered at 30 mg/kg, i.m. on day 0 and again on day 5; Treatment C, a 200 mg/ml solution of oxytetracycline (Liquamycin LA-200, Pfizer Animal Health) administered at 22 mg/kg, intravenously (i.v.), q 24 h for 5 days (a treatment dose that corresponds with current Office International des Epizooties (OIE) recommendations for treatment prior to export). The fourth group consisted of untreated infected control cattle. All steers were still nested PCR and cELISA positive at 60 days after treatment. Infection was confirmed by subinoculation of blood into a splenectomized Holstein calf. These results demonstrated that the treatment regimens tested failed to clear A. marginale infections in carrier cattle.     

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.     

Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale

High titered antibody from rabbits immunized with Anaplasma centrale or from cattle recovered from A. centrale infection bound predominantly to several 33-36 kDa polypeptides present in both A. centrale and the Israel-NT isolate of Anaplasma marginale. High titered bovine antibody against the Israel-NT isolate of A. marginale also reacted predominantly with A. centrale polypeptides in this size range. The immunodominance of the 33-36 kDa polypeptides and their cross-reactivity indicate that these shared epitopes may be primarily responsible for the cross-protective immunity between A. centrale and A. marginale.     

人工感染边缘无浆体对犊牛血清生化及血液流变学的影响

为通过人工感染的方法建立牛边缘无浆体病动物模型,研究边缘无浆体(A.marginale)感染对犊牛血液主要生化指标和血液流变学指标的影响,本实验采用地塞米松注射液长期抑制一月龄犊牛的免疫力,经颈静脉接种含有A.marginale的血液,定期采血,测定其血液主要生化指标和血液流变学指标。结果表明,感染A.marginale后,血液主要生化指标与对照组相比,谷丙转氨酶(GPT)含量极显著升高(p0.01),谷草转氨酶(GOT)含量显著升高(p0.05),血糖(GLU)含量显著降低(p0.05),总胆固醇(T-CHO)含量极显著降低(p0.01),而碱性磷酸酶(ALP)、血铁(Fe)、尿素(BUN)、乳酸脱氢酶(LDH)、α-羟丁酸脱氢酶(α-HBD)和肌苷(Cr)含量均无显著性变化;血液流变学主要指标与对照组相比,全血还原高切比粘度和红细胞刚性指数(IR)极显著降低(p0.01),全血中切比粘度、全血低切比粘度和全血还原低切比粘度显著降低(p0.05),全血高切比粘度、血浆比黏度(ηp)、红细胞压积(PCV)和红细胞聚集指数(AI)均无显著性变化。     

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.     

Effects of long-term estrogen implants in beef heifers

Crossbred beef heifers (n = 78) were assigned randomly to one of three treatments. Heifers received either no implant, one estradiol-releasing implant (Compudose), or two estradiol-releasing implants. Heifers were implanted at birth and then reimplanted every 150 d. Calves were maintained with the cows until weaning at approximately 200 d of age. Heifers were placed in the feedlot as one group and fed a growing diet for 56 d. Following the growing phase, heifers were segregated into their respective treatment groups and fed until selected by industry buyers for harvest. Beginning at 1 yr of age and continuing every 14 d until puberty or harvest, heifers were palpated per rectum, and blood samples were collected for determination of ovarian activity and attainment of puberty. Serum progesterone of > or =1 ng/mL and(or) palpation of a detectable corpus luteum were criteria of puberty. At weaning and again at harvest, an x-ray was taken of the left front leg of six heifers selected randomly from each group. Metacarpal bones III and IV from the same animals were collected at harvest and transected for determination of epiphyseal plate closure. The x-ray scores and the actual measurements had a correlation of .94. Epiphyseal plate closure occurred in a dose-related manner, with heifers on the higher dose of estrogen having earlier plate closure than heifers on the lower dose. At harvest, reproductive tissues and carcass data were collected for all heifers. Eighteen of 25 untreated control heifers (P < .05), 6 of 26 heifers treated with one implant, and 2 of 26 heifers treated with two implants attained puberty by the end of the experiment. No differences (P > .10) were detected among treatment groups for carcass traits. These data suggest that early and continuous treatment of heifers with estradiol implants can retard reproductive function.     

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.