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1.
The staining characteristics of gamonts of Hepatozoon canis in peripheral blood smears were evaluated. Three stains, Diff-Quik stain, Giemsa stain and a modification of the naphthol-ASD-chloroacetate esterase stain, were compared. The staining characteristics of the infected cell and the parasite were different in each stain used. The modified naphthol-ASD-chloroacetate esterase stain showed the most difference between the infected and noninfected cells, as well as a difference in staining characteristics of the parasite nucleus from those of the host nucleus. Although the parasite was identified with all stains, a definitive diagnosis was more easily obtained with the naphthol-ASD-chloroacetate esterase stain.  相似文献   

2.
Radioimmunoassay for Anaplasma marginale antibodies in cattle   总被引：2，自引：0，他引：2  
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.  相似文献   

3.
OBJECTIVE: To assess the innate resistance of and transmission in naive Bos taurus cross Bos indicus and purebred Bos indicus cattle when placed in a paddock with cattle infected with Anaplasma marginale and carrying Boophilus microplus ticks. DESIGN: A group of 49 purebred B indicus, and 48 B indicus cross B taurus (50%, F1 generation) 24-month-old steers were kept in the same paddock with cattle artificially infected with a virulent isolate of A marginale and Boophilus microplus. The cattle were seronegative for A marginale at the start of the trial but had previously been exposed to Babesia bovis and B bigemina. PROCEDURE: Cattle were inspected twice weekly for 118 days. Whole blood, blood smears and serum samples were collected from the cattle on day 37 after exposure and then at regular intervals to day 83 after exposure to measure packed-cell volumes, parasitaemias and antibody titres to A marginale. Any animals that met preset criteria were treated for anaplasmosis. On day 83 all cattle were treated with an acaricide and cattle infected with A marginale were removed from the rest of the group. RESULTS: A marginale was detected in blood smears from 14 crossbred and 9 B indicus steers between days 56 and 72 after exposure. Five and two of the infected crossbred and B indicus steers required treatment, respectively. One of the Bos indicus cattle died as a result of the A marginale infection despite treatment. Antibodies to A marginale were detected in the 23 infected cattle. The mean packed-cell volume depression was 40 and 37% in the affected crossbred and Bos indicus groups, respectively. There was no significant difference detected in susceptibility between these two groups. CONCLUSIONS: Innate resistance of purebred B indicus and crossbred cattle was not significantly different. The results confirm that purebred B indicus and crossbred cattle are sufficiently susceptible to warrant the use of vaccination against Anaplasma infections.  相似文献   

4.
Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.  相似文献   

5.
Spherocytes were detected on blood smears of 2 Angus cows. The RBC from both cows had increased fragility in hypotonic saline solution, supporting the presence of spherocytes. Other laboratory tests revealed a macrocytic, regenerative anemia with anisocytosis, polychromasia, basophilic stippling, Howell-Jolly bodies, nucleated RBC, and Anaplasma marginale organisms. A complement-fixation test was positive for anaplasmosis in cow 1 and a direct Coombs' test was positive for immunoglobulin G in both cows.  相似文献   

6.
BACKGROUND: "Vacuolated" eosinophils (ie, eosinophils with empty, nonstaining granules) have been described previously in normal Greyhounds. However, to our knowledge, detailed studies of granules in vacuolated and normal eosinophils in this breed have not been performed. OBJECTIVE: The objective of this prospective study was to characterize some of the morphologic, ultrastructural, and cytochemical staining features of specific (primary) granules in both normal and vacuolated eosinophils in Greyhound blood. METHODS: Morphologic features of eosinophils in Wright's- and Diff-Quik-stained peripheral blood smears from 49 Greyhounds were compared with 200 blood smears from non-Greyhound dogs. Transmission electron microscopy was done on blood from 3 Greyhounds with vacuolated eosinophils and 3 with normal eosinophil granules. Blood smears from 4 of these dogs also were stained cytochemically with alkaline phosphatase (AP), chloracetate esterase (CAE), and alpha naphthyl butyrate esterase (ANBE). The morphologic features and tinctorial properties of vacuolated and normal eosinophils were compared. RESULTS: Twenty-six Greyhounds (53%) had vacuolated eosinophils and 23 (47%) had normal granulated eosinophils in smears stained with Wright's stain. Only 1% of eosinophils were vacuolated in non-Greyhound dogs. Twenty of the 23 (85%) Greyhounds with normal granulated eosinophils on Wright's-stained smears had vacuolated eosinophils in smears stained with Diff-Quik. Ultrastructurally, no morphologic differences were observed between granules of vacuolated and normal eosinophils. Both vacuolated and normal eosinophils in Greyhounds were positive for AP and negative for CAE and ANBE, as expected for normal dogs. CONCLUSION: Vacuolated eosinophils in Greyhounds likely reflect, at least in part, differential staining properties of the specific granules with different hematologic stains. Ultrastuctural and cytochemical features of eosinophil granules were similar in normal and vacuolated eosinophils from Greyhounds.  相似文献   

7.
Irregularly speculated red blood cells (IS-RBC) were quantified on fresh blood fixed in glutaraldehyde and were compared to RBC shape changes observed on Wright's-stained blood smears, RBC histograms, and RBC distribution widths (RDW). IS-RBC were infrequently found in healthy control dogs. Twenty dogs with increased IS-RBC were evaluated. The most common clinical diagnoses were lymphosarcoma (seven cases), glomerulonephritis (two cases), hemangiosarcoma (two cases), and chronic liver disease (two cases). Five cases had evidence of disseminated intravascular coagulopathy. In 12 of the 20 cases, keratocytes, schizocytes, and/or acanthocytes were detected in the monolayer area of blood smears. In the other seven cases, keratocytes, schizocytes, and/or acanthocytes were found only in thick areas of the smears. Acanthocytes were the most frequent cell type seen, while schizocytes were absent or present only in low numbers. RBC histograms had a shoulder on the left side of the tracing in six of the 20 cases, suggesting the presence of RBC fragments; however, cases with evidence of platelet aggregation had similar shoulders in RBC histograms. Red cell distribution widths were increased in 12 of the 20 cases with IS-RBC; however, the increase in RDW did not correlate with the presence of schizocytes and was most likely the result of reticulocytosis. This study suggests that quantitative evaluation of RBC shape is a more sensitive method for detection of mild RBC fragmentation when compared to blood smear evaluation, RBC histograms, or RDW. Additionally, acanthocyte-type cells were the most frequent shape change seen in dogs with evidence of RBC fragmentation.  相似文献   

8.
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.  相似文献   

9.
OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.  相似文献   

10.
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.  相似文献   

11.
Push smears of mouse blood prepared for differential white blood cell (WBC) determination often have many lysed WBCs, numerous RBC "ghosts", and poor morphology of intact RBCs. The purpose of this study was to compare the quality of peripheral blood smears prepared by 3 different methods and to optimize a technique for mouse blood differential WBC determination. Peripheral blood smears were prepared from blood obtained from clinically normal adult mice and human adults. Differential WBC counts, numbers of lysed WBCs/100 intact WBCs, and RBC morphology were compared in blood smears made using the standard push method with undiluted blood, the push method with blood diluted 1:5 with bovine serum albumin, and in centrifugally-prepared smears made with the DiffSpin Slide Spinner (StatSpin, Norwood, Mass, USA). The number of damaged WBCs in mouse versus human samples using the push method was compared using an unpaired Student's t test. ANOVA was used to compare differences in WBC differential counts and numbers of damaged WBCs among the 3 methods for each species. In addition, unpaired Student's t tests were used to compare each method against the other methods, within species. The number of damaged WBCs/100 intact WBCs was approximately 3 times higher in mouse than in human push smears (P=0.002). There was no significant difference in WBC differential cell counts among the 3 methods in either species. However, compared with both push techniques, a significantly (P <.01) greater number of intact cells was observed with the DiffSpin technique for mouse blood samples (damaged WBC/100 intact cells = 4.4 +/- 2.6 for DiffSpin smears, 9.5 +/- 3.9 for push smears with added albumin, and 31.3 +/- 10.2 for standard push smears). DiffSpin mouse blood smears consistently had better RBC morphology when compared with standard push smears. In conclusion, the DiffSpin Slide Spinner produced optimal smears of mouse blood for WBC differential determination and analysis of RBC morphology.  相似文献   

12.
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.  相似文献   

13.
Direct fluorescent antibody (DFA) and Giemsa staining of Anaplasma marginale were compared in smears collected serially at post-mortem (PM) from 11 experimentally Infected calves. Once smears had been prepared and air-dried they could be held for at least 5 days before staining with either technique with no noticeable change in staining quality. DFA staining was more sensitive in detecting anaplasms in smears than Giemsa staining. Anaplasma spp could be differentiated from Babesia bovis and B. bigemina by DFA staining but there were cross reactions between A. marginale and A. centrale. Blood smears prepared from subcutaneous vessels in the legs provided better diagnostic material than kidney, heart and lung smears. Brain smears were not suitable for PM diagnosis using either staining technique.  相似文献   

14.
The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was compared with that of RBC from control and water-deprived poults. At different hypotonic NaCl concentrations, lysis of RBC from infected poults was 10 to 35% less on day 4 postinoculation (PI) and 50 to 65% less on day 7 PI than that of controls. Red blood cells of poults deprived of water for 3 days were also resistant to lysis; the percent lysis was roughly the same as that of RBC from infected poults at day 7 PI. Incubating control RBC in plasma from infected poults, in extracts of infected ceca, or at different pH levels did not increase their resistance to lysis, suggesting that neither a stabilizing factor in the plasma that had rapid effect on the RBC nor a transient shift in blood pH was involved. Mean RBC size differed little among infected, water-deprived, and control poults (14.0-14.2 X 8.0-8.1 X 3.8 microns). However, although 3.5% of RBC population of control and water-deprived poults were immature (mid to late polychromatic erythrocytes), only 0.4% of the RBC of infected poults were immature. The data suggest that reduced water intake as well as other factors may be involved in the decreased osmotic fragility of RBC from poults infected with E. adenoeides.  相似文献   

15.
Recurring and spontaneously curing spring haemoglobinuria was recently reported in a small sheep flock in a selenium deficient area of northern Hungary. In blood smears of two animals showing clinical signs, Anaplasma-like inclusion bodies were seen in erythrocytes. To extend the scope of the study, 156 sheep from 5 flocks and 26 cattle from 9 farms in the region were examined serologically with a competitive ELISA to detect antibodies to Anaplasma marginale, A. centrale and A. ovis. The seropositivity in sheep was 99.4%, and in cattle 80.8%. A. ovis and A. marginale were identified by PCR and sequence analysis of the major surface protein (msp) 4 gene in sheep and cattle, respectively. Haemoglobinuria, an unusual clinical sign for anaplasmosis might have been a consequence of transient intravascular haemolysis facilitated by selenium deficiency in recently infected sheep, as indicated by the reduction of mean corpuscular haemoglobin concentration (MCHC). Membrane damage was also demonstrated for parenchymal cells, since their enzymes showed pronounced elevation in the plasma. Ticks collected from animals in the affected as well as in neighbouring flocks revealed the presence of Dermacentor marginatus, Ixodes ricinus and D. reticulatus, with the dominance of the first. The present data extend the northern latitude in the geographical occurrence of ovine anaplasmosis in Europe and reveal the endemicity of A. ovis and A. marginale in Hungary.  相似文献   

16.
A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.  相似文献   

17.
The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.  相似文献   

18.
The incidence of blood parasites in trade cattle was surveyed with emphasis on tick-borne parasites, using blood smears and immunofluorescent antibody (IFA) techniques. With the blood smear method, about 9 and 8.9% of cattle examined were found positive for Babesia bigemina and Anaplasma marginale, respectively. Percentage infections with other parasites were 3.33, 1.92, 0.75, 0.75 and 0.58, respectively, for Babesia bovis, Trypanosoma brucei, Anaplasma centrale, Eperythrozoon and Theileria species as well as Trypanosoma congolense. The incidence of A. marginale infection was at its peak during the rainy season while B. bigemina was most prevalent during the dry season. There were mixed infections of Anaplasma and Babesia (1.42%); Babesia and trypanosomes (1.00%); Babesia and Eperythrozoon (0.75%) and Babesia and Theileria (0.75%). Using the indirect fluorescent antibody test, 93, 55 and 68% of cattle sera examined were found to be positive for B. bigemina, B. bovis and A. marginale, respectively. Forty-nine percent of the positive sera of B. bigemina had highest titres. The importance of using serological means for determining the endemic levels of tick-borne diseases in cattle in Nigeria is discussed.  相似文献   

19.
The apparent suppression of Anaplasma marginale infection by Eperythrozoon teganodes in a splenectomized calf has been reported. A splenectomized calf, inoculated with 500 ml of blood having 23% erythrocytes infected with A. marginale, developed eperythrozoonosis on the fourth day post inoculation. A. marginale parasitaemia remained very low during the patent eperythrozoonosis. A. marginale parasites started to increase in number only after E. teganodes infection had been controlled with neoarsphenamine. A splenectomized calf treated identically, but not showing E. teganodes parasites in the peripheral blood, developed clinical anaplasmosis and fulminant parasitaemia within 3-4 days post inoculation.  相似文献   

20.
Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.  相似文献   

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