BOVINE BABESIOSIS: COMPARISON OF FLUORESCENT ANTIBODY AND GIEMSA STAINING IN POST-MORTEM DIAGNOSIS OF INFECTION

SUMMARY Following experimental infections of cattle, Babesia argentina was detected in smears of heart, lung and kidney, immediately, and up to 8 hours post-mortem (PM), using Giemsa staining, while smears of brain were positive for up to 28 hours after death. Direct fluorescent antibody (DFA) staining detected B. argentina in heart and lung smears for 12 hours, in kidney smears for 16 hours, and in brain smears for 28 hours after death. Following experimental infections with B. bigemina, this organism could be recognised morphologically in smears of heart, lung and kidney immediately after death, but by one hour and up to 8 hours PM, its morphology resembled B. argentina in Giemsa stained films. A few red blood cells inside and outside brain capillaries were infected with organisms resembling B. argentina up to 16 hours after death. DFA staining detected B. bigemina in heart and lung smears for 12, and in kidney smears for 16 hours after death. A few infected red blood cells inside and outside capillaries were also seen in brain smears 16 hours after death. Organ smears could be held at 22°C for 5 days and both species of Babesia detected by either Giemsa or DFA staining.B. argentina was diagnosed by Giemsa staining of organ smears from 27 field cases of babesiosis. DFA confirmed B. argentina in 22, identified B. bigemina in 3 and failed to detect parasites in the other 2 cases. B. argentha was diagnosed by Giemsa staining in 10 and B. bigemina in 1 blood film from field cases. DFA confirmed B. argentina in 4 cases and B. bigemina in the 1 case. Serum from 5 of 12 animals which died in the field from natural B. argenfina infec- tions, had fluorescent antibody titres 2 64. After an experimental primary blood infection with B. argentina and while parasites were still detectable, 18 of 72 cattle had titres 2 64. Because there was no significant statistical difference between these results, it was con- cluded that the 12 field cases were due to primary infections, not recrudescences or hetero- logous strains or variants.     

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen.     

Granules of blood eosinophils are stained directly by anti-immunoglobulin fluorescein isothiocyanate conjugates

Direct staining of the granules of blood eosinophils by anti-immunoglobulin fluorescein isothiocyanate (FITC) conjugates was observed when feline blood smears were tested for presence of feline leukemia virus (FeLV) antigen by immunofluorescent antibody. When blood smears of other species including swine, horses, cattle, dogs, sheep, birds, and human beings were examined, direct staining of eosinophils by FITC conjugates was also detected. This FITC staining was restricted to eosinophils and was not observed in neutrophils, lymphocytes, and platelets. Direct FITC staining of eosinophils does not represent a problem in immunofluorescent test for the detection of FeLV infection in cats, as long as the eosinophils, which can easily be recognized as such, are excluded from the spectrum of interpreted cells.     

Morphologic characterization of specific granules in Greyhound eosinophils

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.     

A COMPARISON OF DIRECT FLUORESCENT ANTIBODY AND GIEMSA STAINING FOR THE POST-MORTEM DIAGNOSIS OF ANAPLASMOSIS

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.     

Modifications of the immunofluorescence assay for feline leukemia virus group-specific antigens.

Observations and minor modifications are presented concerning the immunofluorescence assay for feline leukemia virus (FeLV) group-specific antigens (GSA) in blood cells of cats. Data are given regarding absorption of goat FeLV GSA antiserum in vivo in cats, absorption of the antiserum in vitro with feline blood cells, and the comparative efficacy of various chemical fixatives in preservation of FeLV GSA for immunofluorescent staining. The best results were obtained with in vitro absorption of antiserum and methanol fixation of FeLV GSA in blood smears.     

Haemobartonella detection in cat blood smears]

Two useful staining methods for the diagnosis of haemobartonellosis on blood smears and distribution of Haemobartonellae on erythrocyte surfaces are shown.     

What is your diagnosis? Blood smear from an injured red‐tailed hawk

Abstract: An injured juvenile red‐tailed hawk (Buteo jamaicensis) was evaluated at the Veterinary Medical Teaching Hospital at the University of California, Davis. The hawk was quiet, alert, and emaciated, and had a closed comminuted, mid‐diaphyseal ulnar fracture. CBC results included heterophilia with a left shift, monocytosis, and increased plasma fibrinogen concentration. The blood smear included rare heterophils containing small, dark blue inclusions approximately 1–2 μm in diameter that ranged from round to coccobacillary in shape and formed variably shaped aggregates; the morphology of the inclusions was suspicious for Chlamydophila or Ehrlichia spp. pathogens. The hawk died, and histopathologic examination of tissues obtained at necropsy found severe multifocal histiocytic and heterophilic splenitis in addition to chronic hepatitis, myocarditis and epicarditis, meningoencephalitis, and airsacculitis. Using immunohistochemistry the presence of Chlamydia/Chlamydophila spp. antigen within multiple tissues was confirmed. Chlamydophila psittaci DNA was demonstrated in whole blood and fresh splenic tissue via real‐time PCR. Direct fluorescent antibody staining of air‐dried blood smears was positive in rare leukocytes for Chlamydia/Chlamydophila spp. antigen, and immunocytochemical staining of blood smears for Chlamydia/Chlamydophila spp. antigen was focally positive in rare heterophils. These findings may represent the first reported diagnosis of natural avian C. psittaci infection by visualization of organisms in peripheral blood heterophils. Immunocytochemical evaluation of blood smears was valuable in confirming the diagnosis and may be a useful antemortem test to discriminate between bacteria and other inclusions within heterophils.     

Rapid staining techniques in cytopathology: a review and comparison of modified protocols for hematoxylin and eosin,Papanicolaou and Romanowsky stains

The objective of this study was to review and compare rapid protocols for fixation and staining of cytologic smears. We used fresh surgical specimens from dogs and horses to evaluate and modify, if necessary, previously described rapid staining protocols. Slides were wet-fixed, rehydrated or air-dried. Rapid Papanicolaou, hematoxylin and eosin (H&E), and Romanowsky stains were applied, including modification of Diff-Quick stain. The modified rapid staining protocols were simple to use and gave results within 5 minutes that were comparable to those obtained with traditional methods. Advantages of rehydrated vs wet-fixed smears included consistent preparations, a clean background, and equally good or superior nuclear detail.     

Comparison of Various Staining Procedures in the Identification of Hepatozoon canis Gamonts

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.     

Scanning electron microscopy of Heinz bodies in feline erythrocytes.

Whole blood and partially lysed blood films from 5 cats having 20 to 91% of the erythrocytes containing Heinz bodies were examined, using the light microscope and the scanning electron microscope. Heinz bodies were detected in the intact erythrocytes from 3 of the cats as abruptly elevated and distinctly demarcated protuberances in various shapes, sizes, and locations. The Heinz bodies were located just beneath the cell membrane either centrally or near the cell margin and varied in their projectional magnitude. Brilliant cresyl blue staining of blood of these 3 cats revealed prominent Heinz bodies within, and projecting from, the erythrocytes. In contrast, Heinz bodies were not identified on scanning electron microscopy of intact erythrocytes of the remaining 2 cats even though Heinz bodies were found on their blood films stained with brilliant cresyl blue. Scanning electron microscopy of partially lysed blood smears of all 5 cats revealed Heinz bodies of various sizes in the erythrocyte ghosts. Furthermore, blood smears from the 3 cats having distinct Heinz bodies in intact erythrocytes revealed small dense intracellular granules distributed singly or coalesced in small clumps. Further aggregation of these clumps was assumed to result in the formation of a single large Heinz body. The 3-dimensional nature of Heinz bodies was clearly apparent in lysed blood smears.     

Improved mouse blood smears using the DiffSpin slide spinner

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.     

Frequency and severity of mastocytemia in dogs with and without mast cell tumors: 120 cases (1995-1997).

OBJECTIVE: To elucidate frequency of detection on blood smears and severity on quantitative buffy coat evaluation of mastocytemia between dogs without mast cell tumors (MCT) and dogs that had MCT, and to expand the list of diseases associated with mastocytemia in dogs without MCT. DESIGN: Retrospective study. ANIMALS: 94 dogs without MCT and 26 dogs with MCT. PROCEDURE: Medical records of all dogs with mast cells detected on blood or buffy coat smears during a 2-year period were reviewed. Dogs with mastocytemia were grouped by disease into dogs with MCT and dogs without MCT. Twenty-five of the dogs without MCT that had mast cells detected on blood smears also had evaluations of buffy coat smears. Quantitative buffy coat results of the 25 dogs without MCT were compared with those of the 26 dogs with MCT. RESULTS: 95.5% of blood smears with mast cells detected during CBC determination were from dogs without MCT. For these dogs, diagnoses included inflammatory disease (28.2%), regenerative anemia (27%), neoplasia other than MCT (25.9%), and trauma (11.8%). Dogs with MCT had a mean of 71.4 mast cells/buffy coat smear, whereas dogs without MCT had a mean of 276.2 mast cells/buffy coat smear. The 2 highest counts of mast cells/buffy coat smear were for dogs without MCT. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of results of quantitative buffy coat evaluations, severity of mastocytemia in dogs without MCT often exceeds that detected during tumor staging in dogs with MCT. Random detection of mast cells in blood smears during CBC determination in dogs is usually not secondary to MCT.     

Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.

For the diagnosis of chlamydiosis in dead and live birds different methods were compared for their sensitivity and specificity. The specificity of the modified Giménez staining and the direct immunofluorescence (DIF) test for direct demonstration of Chlamydia psittaci in organ, cloacal and/or conjunctival smears was examined. The sensitivity of the isolation of Chlamydia psittaci in 6 days embryonated specific pathogen free (SPF) chicken eggs, Buffalo Green Monkey (BGM) cell line, McCoy cell line and Vero cell line was compared. On smears, the direct immunofluorescence test was more specific than the modified Giménez staining. The concordance between the results of both detection methods was 80%. The BGM cell culture was the most sensitive artificial host for isolation of Chlamydia psittaci, followed by the embryonated eggs, the Vero cell line and the McCoy cell line. The concordance between the results of isolation in BGM cell culture and eggs was 96.5%, while it was 86% between the results of isolation in BGM cell culture and Vero cell culture and only 65.5% between the results of isolation in BGM cell culture and McCoy cell culture. For dead bird species, chlamydiosis could be diagnosed more often using DIF on smears than with isolation. The concordance between the results of the DIF on smears and isolation followed by DIF was 91%.     

Interpretation of canine and feline blood smears by emergency room personnel

Background: Interpretation of blood smears is commonly used to provide rapid laboratory evaluation of animals in veterinary emergency practice, but the accuracy of results of blood smear interpretation by emergency room personnel (ERP) compared with evaluation by trained veterinary clinical pathology personnel is unknown. Objective: The goal of this study was to compare blood smear evaluation by ERP with that of clinical pathology personnel. Methods: All animals that had a CBC determined by a diagnostic laboratory and had blood smears evaluated by personnel at the Foster Hospital for Small Animals Emergency Room between September 2008 and July 2009 were eligible for study inclusion. ERP who evaluated blood smears completed standardized forms with estimates of the WBC and platelet counts and evaluation of RBC and WBC morphology. Results from point‐of‐care assessment were compared with automated or manual results reported by the veterinary diagnostic laboratory. Results: One hundred and fifty‐five blood smears were evaluated. There was moderate agreement (κ value, 0.63; 95% confidence interval [CI]: 0.52, 0.74) between estimated platelet counts by ERP and automated counts. Poor agreement was found between estimated WBC counts by ERP and automated counts (κ value, 0.48; 95% CI: 0.37, 0.60). Specific abnormalities with a high likelihood of clinical significance, eg, toxic change, nucleated RBCs, spherocytes, hemoparasites, and lymphoblasts, were not predictably identified by ERP. Conclusions: ERP interpretation of canine and feline blood smears should be used cautiously and should not replace evaluation by a veterinary diagnostic laboratory.     

An immunoperoxidase monoclonal antibody stain for rapid diagnosis of infectious bursal disease

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.     

Prevalence of bovine trypanosomosis in Central Mozambique from 2002 to 2005

The study is the result of analyzing 16895 blood smears of cattle collected at 180 sites in the provinces of Manica, Sofala, Zambézia and Tete in Mozambique. Of the blood smears 73.9% were from Manica, 11.8% from Tete, 8.5% from Sofala and 5.8% from Zambézia; 75.6% of these were collected from smallholder cattle. Infections with trypanosomes were highest in smallholder cattle from Sofala Province with 36.8% of the 872 blood smears examined positive for trypanosomes, and lowest in cattle of commercial farmers in Manica Province with only 6.2% of 2252 blood smears being positive. Trypanosoma congolense was the predominant species, followed by Trypanosoma vivax and Trypanosoma brucei sensu lato. Trypanosoma brucei, which also infects humans, was more frequent in the districts of Buzi, Mutarara and Morrumbala with 15.1%, 10.5% and 9.8% of all examined cattle in 2005 being infected with it, respectively. The results show a significant increase in the infection rate with trypanosomes compared with results obtained in previous years by the Regional Veterinary Laboratory in Manica Province and by the Regional Tsetse and Trypanosomiasis Control Programme in Zambézia, Tete and Sofala provinces.     

A comparison of polychromasia and reticulocyte counts in assessing erythrocytic regenerative response in the cat.

Three staining techniques were evaluated for use in assessing the erythrocytic regenerative responses in the cat. Using new methylene blue as a vital stain, the aggregate reticulocyte count closely corresponded to the reticulocyte count on air-dried smears stained with new methylene blue and to the polychromatophilic erythrocyte count on Wright's stained smears.     

Use of a polymerase chain reaction assay to detect infection with Eperythrozoon wenyoni in cattle.

OBJECTIVE: To determine whether a polymerase chain reaction (PCR) assay could be used to detect Eperythrozoon wenyoni in the blood of cattle. DESIGN: Prospective study. ANIMALS: 95 cattle from various herds in Alabama and Georgia and 96 bulls enrolled in Auburn University's Alabama Beef Cattle Improvement Association Bull Test program. PROCEDURE: Blood samples were collected by means of venipuncture of the median caudal vein and submitted for a CBC and PCR assay. Blood smears were made immediately after blood collection and examined by means of light microscopy. RESULTS: Three of 95 cattle from herds in Alabama and Georgia and 5 of 96 bulls enrolled in the Bull Test program had positive PCR assay results. Organisms were seen in blood smears from only 5 of these 8 animals. Organisms were not seen in blood smears from any animals for which results of the PCR assay were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a PCR assay may be an effective method for detecting E wenyoni infection in cattle and that the PCR assay may be a more sensitive test than evaluation of blood smears.     

Development of a technique for quantification of reticulocytes and assessment of erythrocyte regenerative capacity in birds

OBJECTIVE: To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. SAMPLE POPULATION: Blood samples from a red-tailed hawk and 31 ill birds. PROCEDURES: A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22 degrees C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa-stained blood smears, a polychromatophil percentage was similarly determined. RESULTS: 4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [rho] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.