Methyl Green-pyronin Research Articles

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.

Morphological quantitative changes in the number of lymphocytes, macrophages and plasma cells in the uterus and lymph nodes of rats exposed to the systemic administration of BCG.

This study was designed to investigate the effect of systemic administration of BCG on the morphological quantitative changes in the number of lymphocytes, macrophages and plasma cells in the uterus and lymph nodes of rats. Thirty female virgin Wistar Albino rats, aging 6 months and weighing between 200-250 g, were assigned to the two experimental groups; BCG treated and controls (n = 15). BCG group received 0.1 ml BCG in tail skin and control group received 0.1 ml saline at the same place. Two weeks after injections, rats in both groups were anesthesized with a high dose of ether and decapitated. Uterus and ileocecal lymph nodes were processed to determine a napthyl acid esterase (ANAE)-positive T lymphocytes and macrophages. The plasma cells were stained with the methyl green-pyronin method. It was found that the numbers of T lymphocytes, macrophages and plasma cells in the uterus and the ileocecal lymph nodes of BCG treated group significantly increased indicating the presence of an immune response to the systemic BCG administration. It was concluded that the systemic administration of BCG increases humoral and cellular immunity in endometrium, myometrium and regional lymph nodes. The immune deficiency system plays an important role in the pathogenesis of endometriosis. Therefore, the endometriosis might be prevented by using periodical administration of BCG. However, further experimental and clinical studies associated with these issue are required.

An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures

We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score ⩾1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the “rabbit skin irritation test”) and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.

Analytical and microscopical studies on the protective effect of ascorbic acid (vitamin C) and beta-carotene against the toxicity induced by fenitrothion on the liver of female albino rats

The organophosphate insecticide feneitrothion is a contact insecticide and selective acaricide. It is used as a fly, mosquito and cockroach, residual contact spray for farms and public health programs. The objectives of the present study were to evaluate the toxicity of fenitrothion on the female rate and the possible protective effects of ascorbic acid (vitamine C) and beta–carotene as antioxidant agents against the toxicity induced by fenitrothion. Sixty of adult female albino rats were randomly assigned to six equal groups including control group and groups treat
          
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  ! successive days. Ingestion of fenitrtothion caused a significant increase in ALT (alanine transferase), AST (aspartate transferase), and AP (alkaline phosphatase). It decreased signifiantly GL (glucose) level, AchE (acetyle cholinesterase) and GSH (glutathion reductase) activities, while, it had insignificant effects on TB (total bilirubine) and a slight decrease in TP (total protein). The histological study of female rat liver tissues by Hx & Eosin,P.A.S, and Methyl Green Pyronine revealed that, fenitrothion showed vascular and degenrative changes in the hepatic cells, Also, it caused a significant decrease in glycogen contents and depletion in of nucleic acids in hepatic cells. Treatments with ascorbic acid and beta–carotene plus fenitrothion hasn’t been caused any significant changes in all parameters in serum of female rats. Treatment with ascorbic acid plus fenitrothion resulted a significant improvement in all parameters tested regarding to the histological study, while, beta-carotene plus fenitrothion showed the same improvement except in glycogen content in hepatic cells .

Analytical and microscopical studies on the protective effect of ascorbic acid (vitamin C) and beta-carotene against the toxicity induced by fenitrothion on the liver of female albino rats

The organophosphate insecticide feneitrothion is a contact insecticide and selective acaricide. It is used as a fly, mosquito and cockroach, residual contact spray for farms and public health programs. The objectives of the present study were to evaluate the toxicity of fenitrothion on the female rate and the possible protective effects of ascorbic acid (vitamine C) and beta–carotene as antioxidant agents against the toxicity induced by fenitrothion. Sixty of adult female albino rats were randomly assigned to six equal groups including control group and groups treat
          
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  !" successive days. Ingestion of fenitrtothion caused a significant increase in ALT (alanine transferase), AST (aspartate transferase), and AP (alkaline phosphatase). It decreased signifiantly GL (glucose) level, AchE (acetyle cholinesterase) and GSH (glutathion reductase) activities, while, it had insignificant effects on TB (total bilirubine) and a slight decrease in TP (total protein). The histological study of female rat liver tissues by Hx & Eosin,P.A.S, and Methyl Green Pyronine revealed that, fenitrothion showed vascular and degenrative changes in the hepatic cells, Also, it caused a significant decrease in glycogen contents and depletion in of nucleic acids in hepatic cells. Treatments with ascorbic acid and beta–carotene plus fenitrothion hasn’t been caused any significant changes in all parameters in serum of female rats. Treatment with ascorbic acid plus fenitrothion resulted a significant improvement in all parameters tested regarding to the histological study, while, beta-carotene plus fenitrothion showed the same improvement except in glycogen content in hepatic cells .

Pathological evaluation of rat endometriosis model

Objective: To clarify a pathogenesis and evaluate a therapeutic effect in a disease, it is important that experimental animal model is established and that the lesions in the model are examined pathologically. Experimental rat endometriosis has been developed by uterine autotransplantation already, and has been used in various investigations of endometriosis. However, almost all previous reports are only focused on implanted uterine tissues, therefore, little is known about morphology of the lesions in detail, specifically in peritoneum attached uterine transplants. Design: In this experiment, we induced rat endometriosis by surgical autotransplantation of uterine tissue and examined morphological alterations of uterus-attached peritoneum with light and electron microscope following the time lapse after the implantation. Materials/Methods: SLC-Sprague-Dawley Rats (8-week old) were maintained on a 12-hour (light) to 12-hour (dark) for 2 weeks, and these rats were used for induction of endometriosis models by the surgical autotransplantation technique of uterine square (5 × 5 mm). The mesenteries were autotransplanted as a comparative control. The implanted areas of peritoneum including abdominal muscle were obtained from anesthetized rats at 4, 7, 14 days after uterine autotransplantation and were examined under a light and electron microscope. To detect DNA fragmentation, these samples were analyzed by TUNEL method. In order to examine localization of mast cells and plasma cells in each specimens, these were performed with Toluidine blue stain (for mast cells) and Methylgreen pyronin stain (for plasma cells). Results: On our light and electron microscopic observations in rat endometriosis models, stromal tissues of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophil, plasma cells, lymphocytes and macrophages. These lesions increased following time lapse after implantation, however, these infiltrated cells disappeared and the proliferation declined finally. Our findings suggest that uterine autotransplantation induced infiltration of allergic inflammatory-related cells and proliferative lesion in peritoneal stroma attached endometrial epithelium, these lesions in rat models are similar to those in endometriosis patients. Conclusions: Our findings suggest that uterine autotransplantation induced immune response, which may be hypersensitivity reaction, in peritoneal stroma attached endometrial epithelium. We believe that these morphological data of rat endometriosis are very important to clarify pathogenesis and progress of human endometriosis. Supported By: No support.

Large quantity of ribosomal RNA exists extracellularly in mouse spleen.

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen 'supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green-Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that 'thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.

Pyronin Y as a fluorescent stain for paraffin sections.

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green-pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogen granules.

Immunohistochemical Study of Interstitial Keratitis in an Animal Model

Purpose: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA). Methods: A mixture of OA (5 μg/mL) and Freund's complete adjuvant was initially injected subcutaneously (0.4 mL) into the rabbit's back and footpad (0.2 mL). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 mL) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 mL) was injected into the corneal stroma. We observed the cornea on days 1–3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done. Results: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils into the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10. Conclusions: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells.

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23. In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.

Response of viral hepatitis B and C to interferon alpha treatment: Possible role of MGP-Positive lymphoid cells

Fifty-six needle liver biopsies from 28 patients with viral hepatitis B (15 cases) and hepatitis C (13 cases) treated with Interferon alpha for a minimum of six months were studied with the aim of determining the role of MGP-positive lymphoid cell infiltrates in the response to treatment with interferon. Besides assessing the MGP-positive cells, the following parameters were assessed and graded: (i) Inflammation in and around the portal tracts, (ii) fibrosis, nodularity and cirrhosis, (iii) piece-meal necrosis (inter-face hepatitis), (iv) lobular activity, (v) stainable iron. Sec-tions were stained with H&amp;E, PAS, PASD, Perl, Masson Trichrome and MGP (Methyl Green Pyronin). All the parameters were graded 1 to 3 except stainable iron which was noted as present or absent.Fourteen patients (50%) demonstrated histological evidence of a response to treatment. Seven of these showed a very pronounced response with the post treatment liver bi-opsies returning almost to normal. Five of the seven, 27.8% of all MGP stained cases and 71% of all brisk responses, contained ten or more MGP-positive lymphoid cells in the portal tracts.In contrast, the non-responsive and worsened cases con-tained less than five MGP-positive cells as well as a pre-dominantly small mature lymphocytic infiltrate in the por-tal tracts.Severity of piece-meal necrosis, lobular activity, portal tract inflammation, the presence or absence of stainable iron, were not of predictive value in the response of viral hepatitis to treatment with interferon alpha.It appears that MGP-positive lymphoid cells, most prob-ably immune competent activated lymphoid cells, have a positive influence on, and are of predictive value for, the response to treatment of viral hepatitis B and C with inter-feron alpha.

Characterizing “Blue Blobs”

To investigate the nature and origin of "blue blobs" (Bbs) in atrophic Pap smears in postmenopausal women and to study their clinical significance. A retrospective study of 412 atrophic Pap smears from postmenopausal women was done to detect the presence of Bbs. The smears from 24 cases showing Bbs were further studied to evaluate the nature of the Bbs with special stains, immunohistochemistry and electron microscopy. Bbs showed a heterogeneous morphology, with variable numbers and staining intensity. The diameter of Bbs was approximately equivalent to that of a parabasal/intermediate squamous cell. Special stains showed Bbs to be positive for periodic acid-Schiff and methyl green pyronin and negative for mucicarmine and calcium. Immunohistochemistry revealed Bbs to be positive for cytokeratin, epithelial membrane antigen and carcinoembryonic antigen and negative for vimentin and muscle-specific actin. Some Bbs had residual ghost nuclear shadows. Electron microscopy revealed cellular skeletons with residual tonofilaments enmeshed within a loose cytoskeleton matrix and nuclei with variable degrees of degeneration. Special stains, immunohistochemistry and electron microscopy indicated that Bbs represent parabasal/intermediate squamous cells exhibiting various degree of degeneration. In general, Bbs appear to be of no clinical significance except as a source of potential diagnostic error.

Methyl green-pyronine staining of porcine organotypic skin explant cultures: an alternative model for screening for skin irritants.

We describe a new alternative method for screening for skin irritants by using fresh intact porcine skin biopsies. Test chemicals were applied to the epidermis of the biopsies, which were then incubated for different times in tissue culture medium at 37°C and with 5% carbon dioxide. A decrease in epidermal keratinocyte RNA staining, visualised in frozen sections by using a modified methyl-green pyronine (MGP) staining procedure, was employed as a marker of irritancy. If a decrease in epidermal RNA was observed after incubation for 4 hours (strong irritant), the chemical had an MGP score of 3; if after incubation for 24 hours (moderate irritant), the MGP score was 2; and if after incubation for 48 hours (weak irritant), the MGP score was 1. If no keratinocyte cytotoxicity was observed after incubation for 48 hours, the chemical was classified as non-irritant (MGP score = 0). At least three ears were used per chemical. The average MGP score was used to classify the chemical. Based on the MGP score for 20% sodium dodecyl sulphate, chemicals classified as strong or moderate irritants by using the MGP test were grouped together as category R38 chemicals. Weak irritants or non-irritants were not classified (NC). The MGP staining correctly identified 23 of 25 skin irritants for which reference data were available.

Apoptosis in renal proximal tubules of rats treated with low doses of aminoglycosides.

Kidney cortex apoptosis was studied with female Wistar rats treated for 10 days with gentamicin and netilmicin at daily doses of 10 or 20 mg/kg of body weight and amikacin or isepamicin at daily doses of 40 mg/kg. Apoptosis was detected and quantitated using cytological (methyl green-pyronine) and immunohistochemical (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining, in parallel with a measurement of drug-induced phospholipidosis (cortical phospholipids and phospholipiduria), cortical proliferative response ((3)H incorporation in DNA and histoautoradiography after in vivo pulse-labeling with [(3)H]thymidine), and kidney dysfunction (blood urea nitrogen and creatinine). Gentamicin induced in proximal tubules a marked apoptotic reaction which (i) was detectable after 4 days of treatment but was most conspicuous after 10 days, (ii) was dose dependent, (iii) occurred in the absence of necrosis, and (iv) was nonlinearly correlated with the proliferative response (tubular and peritubular cells). Comparative studies revealed a parallelism among the extents of phospholipidosis, apoptosis, and proliferative response for three aminoglycosides (gentamicin >> amikacin congruent with isepamicin). By contrast, netilmicin induced a marked phospholipidosis but a moderate apoptosis and proliferative response. We conclude that rats treated with gentamicin develop an apoptotic process as part of the various cortical alterations induced by this antibiotic at low doses. Netilmicin, and still more amikacin and isepamicin, appears safer in this respect. Whereas a relation between aminoglycoside-induced tubular apoptosis and cortical proliferative response seems to be established, no simple correlation with phospholipidosis can be drawn.

Histochemical characterization of the lead intranuclear inclusion bodies.

A battery of histological and histochemical techniques was applied on the lead intranuclear bodies that have resuted in the kidneys of adult Wistar male rats receiving lead acetate in their diet to determine their nature. The intranuclear inclusion bodies have stained strongly with xanthene, anthraquinone, and trisulfonated basophilic dyes and weakly with dyes containing both positive and negative radicals, and they have responded negatively to acidophilic cationic dyes. They have also reacted positively to proteins and lead stains, but weakly to lipid stains, and negatively to Feulgen and methyl green pyronin techniques. The intranuclear bodies proved to be lead lipoprotein complexes containing sulfyhydryl groups and are basic in nature with orthochromatic, eosinophilic, argyrophilic, osmophilic, fuchsinophilic, and sudanophilic characteristics.

Focal Necrotizing Endometritis

From routine sign-out of endometrial biopsy specimens, a group of 15 endometria were identified that have a characteristic histologic pattern of inflammation that is not included in present classifications of endometritis. All but one of the women were premenopausal, and all presented with abnormal vaginal bleeding. The lesion is characterized by a patchy, focal inflammation, usually composed of lymphocytes with a variable number of neutrophils and rare macrophages centered around endometrial glands and extending into the glandular lumen with disruption and partial or subtotal necrosis of the endometrial glandular epithelium. These foci were widely dispersed, never confluent, and could be overlooked easily. Plasma cells were not found in any of the endometrial specimens despite methyl green pyronine staining of the samples. Based on the histologic characteristics of this process we have designated it focal necrotizing endometritis. The clinical significance, if any, of focal necrotizing endometritis is currently unknown.

In situ localization of heat-shock and histone proteins in honey-bee (apis mellifera l.) larvae infected with paenibacillus larvae

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%.After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells.

Theileria lestoquardi – maturation and quantification in Hyalomma anatolicum anatolicum ticks

The maturation and quantification of Theileria lestoquardi (T. hirci) parasites in unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was studied using (1) methyl green pyronin (MGP) staining of salivary glands, (2) in vitro infection of peripheral blood mononuclear cells (PBM) with parasites harvested from infected ticks and (3) a semi-quantitative polymerase chain reaction (PCR). With MGP staining the greatest infection rate was seen in unfed ticks. Feeding resulted in a gradual reduction in the number of infected acini with a concomitant increase in the maturity of the parasites. In vitro infection of sheep PBM with titrated group-up tick supernate (GUTS) demonstrated that infectivity peaked between 2 and 4 days of tick feeding whereas GUTS prepared from unfed ticks was not infective. The polymerase chain reaction (PCR) was both sensitive and specific, detecting T. lestoquardi DNA in unfed and partially fed ticks, with a maximum sensitivity of 0.022 infected acinus/tick in 2-day fed ticks, though it gave no indication of the infectivity of the parasite.

Evaluation of B and T lymphocytes and plasma cells in colonic mucosa from healthy dogs and from dogs with inflammatory bowel disease

The aim of this study was to investigate the subpopulations of lymphocytes in the colonic mucosa of healthy dogs and dogs with inflammatory bowel disease (IBD). Fourteen normal dogs and 13 dogs with IBD were examined. Endoscopic biopsy specimens of colonic mucosa from each dog were stained specifically for pan T lymphocytes (CD3) and pan B lymphocytes (CD79a), and for plasma cells with methyl green pyronin (MGP) stain. Cells were counted by means of a grid and statistical analysis was performed on the data collected. B and T lymphocytes were also counted in the glandular epithelium of normal dogs and dogs with IBD and the normal and abnormal groups compared statistically. Healthy dogs had significantly lower numbers of T cells in the lamina propria and glandular epithelium and significantly lower numbers of B cells in the lamina propria. Significant group differences for plasma cells were not evident. Our results indicate that in IBD a chronic cellular immune reaction is present in the diseased gut involving increased numbers of B and T lymphocytes.

Detection of Theileria lestoquardi (hirci) in ticks, sheep, and goats using the polymerase chain reaction.

Theileria lestoquardi (= T. hirci) is a protozoan parasite of sheep and goats that is morphologically and biologically similar to T. annulata, the causative agent of bovine tropical theileriosis. Both parasites are transmitted by ixodid ticks of the genus Hyalomma. However, because of their morphological similarity, they cannot be distinguished in the salivary glands of infected ticks by traditional staining methods such as Feulgen or Methyl green-pyronin. Thus a need has arisen for sensitive and specific diagnostic tests that will distinguish between the two species in the vector tick, allowing the epidemiology of both diseases to be clearly defined. A contribution to this has been the development of a polymerase chain reaction using specific primers which amplify, only in T. lestoquardi-infected ticks, a 785 bp fragment of the gene that codes for a 30 kD merozoite surface protein. The sensitivity of this test and its application to the detection of T. lestoquardi in infected H. anatolicum anatolicum ticks, in the blood of three species of domestic ruminants and in cell cultures established in mononuclear cells of sheep and goats is also discussed.