Tissue Impression Smears Research Articles

Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.

Entomological and rodent surveillance of suspected plague foci in agro-environmental and feral biotopes of a few districts in Maharashtra and Gujarat states of India.

Studies carried out on entomological and rodent surveillance in agroclimatic and feral biotopes of five districts of Maharashtra and two districts of Gujarat revealed that the terrain features of the seven districts surveyed were conducive to wild rodent species, Tatera indica, a natural reservoir of plague and vector flea species, Xenopsylla cheopis. A total of 214 Tatera indica and three Bandicoota bengalensis were collected from burrows by the digging method and 89 rat fleas were retrieved. The flea index calculated ranged from 0.26 to 1.0 in different districts. The examination of blood serum samples, contact tissue impression smears and tissue organs (heart, lung, liver and spleen) of these wild rodents did not reveal any evidence of plague activity. The natural harbourages of Tatera indica were found to be elevated land or raised margins of agricultural fields or barren land with thorny bushes/xerophytic plants and parthenium grass. Agricultural fields having wet soil were found to be the preferred harbourages of Bandicoota bengalensis. Movement of wild rodents toward human settlement was observed to be the common phenomenon during the harvesting season which may lead to the creation of favorable conditions for plague outbreaks.

Amplification of Pneumocystis carinii DNA on specimens scraped from slides

A method for the detection of Pneumocystis carinii by polymerase chain reaction using specimens obtained by scraping bronchoalveolar lavage or tissue impression smears is described. The smears were scraped into water and then absorbed onto a glass-fiber filter. After fixing with methanol, the specimen on the filter was digested with proteinase K. The digestion mixture was then clarified, and a portion of the clarified supernatant was used as a template for the amplification of a portion of the mitochondrial rRNA gene of P. carinii. Using this method of sample preparation, we were able to amplify P. carinii DNA from both unstained and Giemsa stained smears.

Studies on the cellular response in avian inflammation using a simple subcutaneous pouch model

A subcutaneous pouch model was developed and used for examination of cellular events in the local acute inflammatory response in the chicken. The model involved insertion of a pair of coverslips-dipped in the irritant-into the prepared subcutaneous pouch, and their replacement with plain coverslips at frequent intervals. The model was simple to operate, caused minimal distress to the experimental bird, and permitted multiple continual sampling of emigrating cells. The cytological features and migratory sequence of inflammatory cells produced by non-immunological and immunological stimuli were studied over a period of 48 h. The initial migration of leukocytes comprised heterophils and monocytes. This was soon followed by emigration of basophils. The basophilic reaction was pronounced in response to Escherichia coli endotoxin. Lymphocytes were prominent in the later stages. Also, in the later stages the monocytes apparently coalesced, formed syncytia and even what appeared to be distinct multinucleated giant cells. The results obtained by the present model corroborated the findings of earlier investigators recorded through the use of tissue sections and impression smears. It is suggested that the subcutaneous pouch model may offer an excellent system in avian inflammation research.

Diagnostic Parasitology

Diagnosis of parasitic infections in small animals is challenging, interesting, and fun, and involves the recognition of parasite stages based on size, morphology, color, and movement. Size and morphology are the major diagnostic parameters, and a calibrated microscope is the essential tool in the diagnostic laboratory. The fecal flotation technique is used routinely for the diagnosis of most parasites that reside within the gastrointestinal tract. The specific gravity and type of solution used often will influence the results obtained. A sugar flotation solution with a specific gravity of 1.27 is one that will float a majority of parasite stages with minimal distortion. Other techniques for diagnosis of specific gastrointestinal parasites include the direct smear and the merthiolate-iodine-formalin preservative method for diagnosis of Giardia sp, the Baermann technique for diagnosis of lungworm and other live larvae, the direct sedimentation technique for diagnosis of trematode eggs, the ether-formalin-sedimentation technique for diagnosis of trematode eggs and concentrating protozoan cysts from feces with high fat content, and the McMaster technique, a dilution modification of the fecal flotation technique. Techniques for evaluation of parasites in blood include the Knott's test, hematocrit method, and direct blood smear for diagnosis of microfilariae and thick and thin stained blood smears for diagnosis of Babesia sp, Haemobartonella sp, Cytauxzoon felis, Hepatozoon canis, and Trypanosoma cruzi. Tissue impression smears are used commonly for the diagnosis of Leishmania sp in dogs and biopsy specimens or aspirates are used for the diagnosis of Pneumocystis carinii. Diagnosis of acute toxoplasmosis can be accomplished with peritoneal and thoracic fluids, and organisms occasionally are detected in blood or spinal fluid. Serological tests for many parasitic diseases have been developed and often are used as supportive diagnostic tests.

A direct immunofluorescence test for the rapid detection of avian influenza virus antigen in tissue impression smears

A direct immunofluorescent test on tissue impression smears for the simple, rapid, cheap and sensitive detection of avian influenza virus antigen is described. Cross immunofluorescence tests carried out on four avian influenza conjugates demonstrated that a conjugated anti-serum prepared against one influenza virus subtype would stain heterologous subtypes. Direct immunofluorescence on tissue impression smears from broilers infected with either avirulent or highly pathogenic avian influenza viruses was compared with virus isolation in embryonated hens' eggs, chick embryo liver and chick embryo fibroblast cell cultures. Immunofluorescence was as sensitive as virus isolation in eggs. In some cases antigen was detected by immunofluorescence where virus isolation failed due to the presence of antibody or the onset of autolysis. Virus isolation in chick embryo liver was more successful than chick embryo fibroblasts but not as sensitive as embryonated eggs.

Responses of european green lizards Lacerta viridis following administration of Leishmania agamae promastigotes

European green lizards ( Lacerta viridis) were injected intraperitoneally, subcutaneously or orally with viable Leishmania agamae promastigotes. Neither promastigotes nor amastigotes were later found in blood and tissue impression smears, or in blood and selected organ cultures. However, by the use of an immunoperoxidase technique, parasite antigens were detected in the liver, stomach, small intestine, kidney, gonad, heart, lung and skin but not in the bone marrow, brain or spleen. Non-precipitating antibodies with β 2-electrophoretic mobility were induced against L. agamae. They were detected in the sera by enzyme-linked immunosorbent assay 3–7 days post-infection. The titres increased significantly above background levels ( P < 0.001) and reached maxima after 6–7 weeks, with 27 out of 29 lizards producing antibodies. The mean serum protein concentration significantly increased after infection ( P < 0.005) with no significant differences in mean values between male and female animals. Lizard sera separated into 7 components on cellulose acetate membranes with migration rates comparable to albumin, α- and β-globulins of human serum; γ-globulins were absent. Significant decreases occurred ( P < 0.05) in the albumin fraction, with significant increases in the β-globulin region of anti- L. agamae sera. C-reactive protein was not detected in either normal or immune lizard sera.

Antigen distribution and humoral response in the lizard, [formula omitted], after injection with [formula omitted

The response of the spiny-tailed agamid lizard, Agama caudospinosum , to administration of Leishmania agamae promastigotes was investigated. Lizards given a single injection of promastigotes showed no signs of clinical infection. Neither promastigotes nor amastigotes were found in blood and tissue impression smears, nor in blood and selected body organ cultures. However, parasite antigens were demonstrated by an immunoenzyme method only in the liver, small intestine, stomach, spleen and kidney. Non-precipitating serum antibodies with γ-electrophoretic mobility were detected by enzyme-lined immunosorbent assay 1 week post-injection and a maximum titre was reached after 6 weeks. The mean immune serum protein concentration increased significantly ( P<0.005) about two-fold over the controls after injection. Decreases occurred in the β-globulin region of anti- L.agamae sera ( P<0.001) whilst the γ-globulin fraction was increased ( P<0.005) following electrophoresis on cellulose acetate membranes. C-reactive protein was not detected in any of the sera. These data show that although A.caudospinosum failed to become infected by L.agamae promastigotes, which had been isolated from agamids, it did exhibit antigen distribution and a humoral response similar to other reptiles.

Inactivation of rabies virus in reagents used for the fluorescent rabies antibody test.

Procedures for inactivating rabies virus in reagents used for the fluorescent rabies antibody test are described. Mouse brain adsorbing suspensions containing greater than or equal to 10(9) 50% lethal doses of virus per ml were rendered noninfectious by treatment with 0.1% beta-propiolactone or by heating at 56 degrees for greater than or equal to 30 min. Viable virus in tissue impression smears was inactivated by acetone fixation at 50 degrees C for greater than or equal to 30 min or by immersion in 0.1% beta-propiolactone at 37 degrees C for 2 h. Inactivated reagents gave specific and sensitive reactions in the fluorescent rabies antibody test.

Comparison of two methods used to prepare smears of mouse lung tissue for detection of Pneumocystis carinii

The laboratory diagnosis of Pneumocystis carinii pneumonia in humans includes the identification of cysts in stained lung tissue impression smears. By using a mouse model, we compared the number of cysts in lung tissue impression smears with those contained in a concentrate of homogenized lung tissue. Eleven C3H/HEN mice developed P. carinii infection after corticosteroid injections, a low protein (8%) diet, and tetracycline administered in drinking water. Impression smears were prepared with freshly bisected lung tissue. Smears of concentrates were prepared with sediment from centrifuged lung tissue homogenates. All smears were made in duplicate, stained with toluidine blue O or methenamine silver, coded, randomized, and examined. The concentrate preparations contained more cysts per microscopic field than the impression preparations (P less than 0.01). Concentrates prepared by grinding with a mortar and pestle contained more cysts than concentrates prepared by blending with a Stomacher (P less than 0.05). Cysts were detected equally well with either the toluidine blue O or silver stain (not significant). Lung tissue concentrates were superior to lung tissue impressions for detecting P. carinii cysts in mice. Use of lung tissue concentrates should be considered for the diagnosis of human P. carinii infection.

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: Tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm 3H/mg tissue or as the percentage change in [ 3H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena. All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.

A CASE OF HUMAN MELIODOSIS: CLINICAL, EPIDEMIOLOGICAL AND LABORATORY FINDINGS.

Summary The clinical, epidemiological and laboratory aspects of the first confirmed case of human melioidosis reported in Ecuador are presented. The results of conventional bacteriological and serological testing and of animal inoculation confirmed the presumptive diagnosis of infection with Pseudomonas pseudomallei made by the fluorescent antibody test. Specifically stained fluorescent organisms were demonstrated in tissue impression smears prepared from the organs of guinea pigs experimentally infected with the specimen cultures. The value of immunofluorescence for screening of specimens was emphasized by the rapidity with which Pasteurella pestis and P. tularensis were excluded from consideration as probable etiologic agents of this infection and the short period of time required to make a presumptive identification of Ps. pseudomallei. The role of fluorescent antibody procedures in bacteriological and histopathological laboratories is discussed. A procedure for minimizing erroneous diagnoses of melioidosis is proposed.