Mesenteric Lymph Node Sections Research Articles

Comparative diagnosis of Mycobacterium avium subspecies paratuberculosis in the tissues of clinical and subclinical sheep of paratuberculosis endemic farm

Paratuberculosis (Johne’s disease) is the chronic infectious granulomatous enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis. Due to the lack of definite diagnostic tests, the early detection of the disease is difficult. The present study was undertaken to assess comparative efficacy of diagnostic methods routinely used in the identification and confirmation of Mycobacterium avium subspecies paratuberculosis (MAP) in the tissue samples of naturally infected clinical and subclinical sheep. The ileum and mesenteric lymph node (MLN) tissues were collected from 20 paratuberculous sheep from organized farm of Rajasthan. These animals were further classified as paucibacillary (PB) (n = 8) or multibacillary (MB) (n = 12) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN sections from PB and MB and uninfected control groups were tested by indirect immunoperoxidase technique (IPT), Ziehl Neelsen’s (ZN) method, bacterial culture, IS900 PCR and 251 gene PCR. In PB sheep, IPT and ZN were positive in 87.5 and 50% of intestinal sections and 75 and 37.5% of MLN sections, respectively. In MB sheep, IPT and ZN had equal sensitivity. The overall sensitivity of IPT was found to be superior (95%) than ZN method (80%) in demonstration of acid-fast bacteria in intestinal tissues. On the bacterial cultural examination of the intestinal and MLN tissues using Herrold’s egg yolk medium, MAP was isolated in eight (66.6%) MB and in four (50%) PB sheep. Among 20 sheep, IS900 PCR detected 13 (65.0%) and 251 gene PCR detected 16 (80%) sheep as positive for MAP. In the PB sheep, the culture, ZN test and PCR sensitivity was found low in comparison to MB sheep. The sensitivity of IPT in PB sheep was better than other tests in detecting the MAP infection, which underlines its utility in confirmation of subclinical and clinical cases of paratuberculosis in sheep. The sensitivity of 251 gene PCR assay was found better than IS900 gene PCR and has potential to be used as screening test for clinical and subclinical paratuberculosis in sheep flocks.

Longitudinal Study of Clinicopathological Features of Johne’s Disease in Sheep Naturally Exposed toMycobacterium aviumSubspecies Paratuberculosis

The objective of this study was to describe chronological changes in infection status and enteric lesions of sheep naturally exposed to Mycobacterium avium subspecies paratubercuolosis. Samples of terminal ileum (TI) and mesenteric lymph node (MLN) were collected from 77 Merino sheep via surgical biopsy at 12, 18, and 24 months of age and necropsy at 36 months of age. Infection status at each sampling period was determined by fecal, TI, and MLN culture. Quantitative grading schemes were used to gauge the severity of granulomatous inflammation and degree of mycobacterial colonization affecting TI and MLN sections. Incidences of infection and disease were steady throughout the study; 46 of the 77 (59.7%) sheep became infected, and 30 of the 77 (39.0%) developed Johne's disease. Infection was first detected after 18 months of age in many sheep, and age when infection was first detected was not associated with clinical outcome. Culture of MLN detected 44 of the 46 (95.6%) infected sheep and initial lesions always involved MLN. Sheep typically developed lesions within 6-12 months following detection of infection by culture. The severity of enteritis and mycobacterial colonization progressed at variable rates among sheep. Severe multibacillary enteritis never regressed, and affected sheep expressed clinical signs within the following 12 months. Lymphocyte-rich paucibacillary enteritis was observed in 3 sheep, causing clinical signs in one and progressing to severe multibacillary enteritis in another. Six of the 46 (8.7%) biopsy-culture-positive sheep later had negative cultures at necropsy, suggesting recovery from infection. Further study is needed to identify factors associated with clearance of infection or progression of disease.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats ( Suricata suricatta) with fatal toxoplasmosis in Argentina

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats ( Suricata suricatta) in the zoo of La Plata, Argentina and the in vitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1–5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3′-SAG2, 5′-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Development of a new monoclonal antibody to ovine chimeric IgE and its detection of systemic and local IgE antibody responses to the intestinal nematode Trichostrongylus colubriformis.

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals. These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.

In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo.

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID(50) of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0.05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.

Production and characterisation of monoclonal antibodies recognising ovine IgE

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86 000 Daltons) and immunoglobulin light chain (28 000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.

Identification of Aleutian mink disease parvovirus transcripts in macrophages of infected adult mink.

We examined Aleutian mink disease parvovirus (ADV) mRNA expression in lymph nodes of adult mink infected with ADV by Northern (RNA) blot and in situ hybridization. In Northern blot analysis, ADV transcripts were detected in the poly(A) RNA fraction extracted from mesenteric lymph nodes of two of five mink 10 days after intraperitoneal inoculation with the virulent Utah I strain of ADV. In strand-specific in situ hybridization, ADV DNA and mRNA were detected in some macrophagelike cells located in the medullary sinus in mesenteric lymph node sections from two of six infected mink by using biotinylated probes. In suspensions of lymph node cells, about 30% of the cells phagocytic for latex particles contained ADV DNA and about 1% of these cells contained ADV mRNA. In peritoneal exudate cells, about 20% of the macrophagelike cells contained ADV DNA and about 2% of these cells contained ADV mRNA. These results indicated that some macrophages in ADV-infected mink contained ADV mRNA and were target cells in ADV infection.

Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone.

Using quantitative techniques we have shown elsewhere that dendritic cells (DC) migrate from blood into the spleen, under the control of T cells. Here we traced the localization of DC within the spleen and sought to explain the means by which they entered. DC were labeled with a fluorochrome, Hoescht 33342, and injected intravenously. Spleens were removed 3 or 24 h later and DC were visualized within particular areas that were defined by mAbs and FITC anti-Igs. At 3 h most DC were in the red pulp, whereas by 24 h the majority had homed to T-dependent areas of the white pulp and may have become interdigitating cells. Lymphoid DC, isolated from spleen and perhaps normally present in blood, may thus be a migratory stage distinct from the relatively fixed interdigitating cells. We also developed a frozen section assay to investigate the interaction of DC with various lymphoid elements. When DC were incubated on sections of spleen, at 37 degrees C but not at 4 degrees C they attached specifically within the marginal zone and did not bind to T areas; in contrast, macrophages attached only to red pulp and T cells did not bind specifically. However, DC did not bind to sections of mesenteric lymph node, whereas T cells localized in particular regions at 4 degrees C but not at 37 degrees C, probably the high endothelial venules. DC may thus express "homing receptors," similar to those of T cells, for certain endothelia. We propose that T cells can modify the vascular endothelium in certain areas to allow egress of DC from the bloodstream.

Generation and Characterization of Murine Monoclonal Antibodies Specific for Bovine Immunoglobulin E

Monoclonal antibodies were produced against serum-derived bovine immunoglobulin E (IgE). Culture supernatants of hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA). Supernatant-derived antibodies were concentrated and further characterized using ELISA, reverse cutaneous anaphylaxis, immunohistochemical staining, and immunoblotting of IgE-containing samples separated by SDS-polyacrylamide gel electrophoresis (PAGE). Eight monoclonal antibodies showed specificity for bovine epsilon immunoglobulin heavy chain. Two antibodies (E2 and E32) reacted in immunoblots of SDS-PAGE of serum IgE under reducing conditions. Additionally, E2, E5, and E32 detected epsilon chain in serum separated by SDS-PAGE and then renatured. Antigen-specific IgE was detected in Western blots by E5 and E32. Immunoperoxidase staining of IgE-containing cells in mesenteric lymph node sections was detected with E5, E21 and E32. All eight antibodies produced positive reverse cutaneous anaphylaxis reactions in calf skin. All functioned well in ELISA as a plate-sensitizing reagent for quantitation of total IgE; E5 and E32 worked well as a primary antibody in antigen-specific IgE assays. These antibodies will be useful in research applications and in diagnostic assays.