Susan S. Ringler

In utero transmission of porcine torque teno viruses

Sera and selected tissue homogenates collected from gnotobiotic swine never exposed to the environment or other swine tissues were tested for the presence of porcine torque teno virus (TTV) DNAs by nested and non-nested polymerase chain reactions (PCR) using primers specific for the untranslated region of porcine genogroups (g) 1 and 2. Twenty-three of 105 (21.9%) gnotobiotic piglets were g1- and/or g2-TTV DNA positive. Twenty-three of 27 (85.2%) sow sera, collected at the time of Caesarian derivation of the litters contained either or both TTV genogroup DNAs. These data demonstrate that porcine TTV may be transmitted to piglets by the in utero route and that the incidence of fetal infection is high.

Vaccination of Gnotobiotic Piglets against Helicobacter pylori

To determine the effect of oral adjuvant-assisted and parenteral vaccination, germ-free piglets were vaccinated orally with and without labile toxin adjuvant or parenterally and challenged with viable Helicobacter pylori. All prechallenge vaccination regimens induced anti-H. pylori antibodies and suppressed bacterial colonization, but no vaccination regime completely prevented infection. Parenteral vaccination given after infection had no effect on bacterial colonization. Lymphocytic gastritis was present in all piglets challenged with live bacteria regardless of vaccination status. Neutrophilic gastritis was present in vaccinated challenged piglets but not in infected, unvaccinated piglets. Gastritis was not present in uninfected control piglets regardless of vaccination status. In gnotobiotic piglets, vaccination suppresses but does not prevent infection by H. pylori, and parenteral vaccination does not cure infected piglets. Vaccination does not ameliorate gastritis due to H. pylori in piglets but does induce neutrophilic gastric inflammation in some infected piglets.

Immunosuppression by canine distemper virus: modulation of in vitro immunoglobulin synthesis, interleukin release and prostaglandin E2 production.

In vitro or in vivo infection of canine mononuclear cells by canine distemper virus (CDV) in short-term microcultures resulted in suppression of lectin-induced 3H-thymidine incorporation. This suppressive effect was also evident in pokeweed mitogen-driven in vitro immunoglobulin synthesis and release. Lectin-induced interleukin-2 production by monocyte-depleted lymphocyte cultures was marginally affected by CDV, whereas interleukin-1 production by adherent mononuclear cells was significantly depressed. Monocyte cultures established from viremic dogs released prostaglandin (PG)E2. The results suggest that, in addition to a direct viral effect upon lectin responsive cellular population(s), CDV modulates monocyte functions by inhibition of interleukin-1 production and by enhancing PGE2 release.

Manifestations of the local gastric immune response in gnotobiotic piglets infected with Helicobacter pylori

Helicobacter pylori, a human gastric bacterial pathogen, was inoculated into gnotobiotic piglets and manifestations of the resultant gastric inflammation was analyzed by in situ immunochemistry and flow cytometric analysis of isolated lamina propria leukocytes (LPL) and peripheral blood leukocytes (PBL) recovered from infected and control piglets. Gastric mucosa tissue sections from uninfected control piglets were essentially negative for cluster differentiation- (CD-) positive leukocytes. Failure to isolate significant numbers of LPL from the gastric lamina propria confirmed this observation. A local and systemic immune response occurs in piglets after infection with H. pylori. This is manifest by the appearance of cells associated with a local immune response in gastric mucosa. In gastric tissue sections from H. pylori-infected piglets, CD4-positive leukocytes were sparse and closely associated with developing lymphoid follicles whereas the CD8-positive cellular phenotype was abundant. The latter formed a continuous band in the lamina propria just above the muscularis mucosa. Perivascular accumulations of lymphocytes in the outer muscular tunic(s) were strongly positive for expression of CD8 antigen. Class II-positive cells were prominent in CD8 lymphocytic infiltrates, developing follicles and vascular endothelia but were uniformly absent from gastric epithelia even in sites overlying areas of immunocyte proliferation and infiltration. Leukocytes possessing the monocyte and granulocyte markers were rare. Plasma cells containing IgA were common in the periphery of developing lymphoid follicles or distributed as discrete foci around individual gastric pits. Fewer numbers of IgG- and IgM-positive plasma cells were identified. When the LPL flow cytometry data were compared with the flow cytometry data obtained from PBL in these same H. pylori-infected piglets, leukocytes bearing the CD8 marker predominated in LPL whereas leukocytes bearing the CD4-reactive and MHC class II markers predominated in PBL. Finally, local ELISA antibody responses were measured in mucosal explant culture supernatants and compared with in vivo antibody levels in sera, bile, and gastric juice. Antibody activity, specific for H. pylori, was detected in supermatants and serum in all three isotypes in actively infected piglets whereas gastric juice lacked antibodies. Gastric explants prepared from piglets in which infection had been successfully eradicated failed to produce local antibody into supermatant fluids. These data support the concept that the gastric inflammation observed is mediated by local immunological events.

Activation specificity of commonly employed mitogens for canine B- and T-lymphocytes.

Six day in vitro cultures of canine mononuclear leukocytes with or without prior carbonyl iron depletion were established with 5 commonly employed mitogens. Cultures were assessed for B-cell differentiation by induction of cytoplasmic and supernatant released IgG, M and A and T-cell differentiation by expression of Thy-1 antigen on the cell surface of lymphoblasts. The mitogen concanavalin A was shown to be a restricted T-cell mitogen, whereas pokeweed mitogen and protein A from Staphylococcus aureus stimulated maximal B-cell differentiation. These data establish the mitogenic specificity for canine lymphocytes in unfractionated peripheral blood leukocyte cultures and will permit an in vitro evaluation of various substances upon T- and B-lymphocyte functions.

A Novel Method for the Detection of Porcine Circovirus Type 2 Replicative Double Stranded Viral DNA and Nonreplicative Single Stranded Viral DNA in Tissue Sections

Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.

Cell surface markers of the canine natural killer (NK) cell

Studies were performed to determine which of several cell surface markers are expressed on canine peripheral blood leukocyte (PBL) natural killer (NK) cells. Chromium-51 release assays showed a decrease in NK activity after depletion of PBL by carbonyl iron ingestion and adherence to IgG-antibody-coated ovine erythrocytes (EA gamma) and to IgM-antibody-complement-coated ovine erythrocytes (EA mu C). Effector cell adherence to and subsequent lysis of canine thyroid adenocarcinoma (CTAC) target cell monolayers provided direct visual identification of the putative canine NK cell. These surface immunoglobulin-negative cells, individually identified by their physical adherence to dead CTAC target cells, failed to form nonimmune rosettes with guinea pig erythrocytes or rosettes with EA mu or EA mu C. However, 39.0 +/- 4.2% of these adherent cells formed rosettes with EA gamma and 73.3 +/- 0.8% expressed the canine T-lymphocyte marker, Thy-1.

The effects of human interferon-alpha upon in vitro canine immune responses

The effects of varying amounts (100.0-0.01 units/ml) of human interferon (IFN) alpha upon in vitro canine immune responses were studied. Exogenous heterologous species IFN-alpha suppressed B-cell differentiation in a dose-dependent fashion and enhanced interleukin-2 production (P less than 0.05) by activated T-lymphocytes. Interferon enhanced natural killer (NK) cytotoxicity when tested against NK-resistant target cells (less than 0.05). One hundred units IFN/ml increased interleukin-1 production by canine monocytes, but this effect was not statistically significant. Exogenous IFN had no discernible effect upon lectin-induced lymphocyte blastogenesis. The results of this study demonstrate that human IFN-alpha does affect various canine lymphocyte functions and these effects depend upon the in vitro assay system employed.

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs

OBJECTIVE To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV). SAMPLE POPULATION 22 commercially available M hyopneumoniae bacterins. PROCEDURES Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe. RESULTS Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays. CONCLUSIONS AND CLINICAL RELEVANCE Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Isolation of Pure Populations of Helicobacter heilmannii-like Bacteria

Large spiral bacteria similar in morphology to Helicobacter felis are common in the stomachs of many animal species including cats, dogs, pigs, non-human primates, and a variety of wild carnivores.1, 3–5, 8 These bacteria, first referred to as spirilla,5, 10 and later as Gastrospirillum sp,6 are most likely members of the helicobacter genus. Several isolates have been given the provisional name, H. heilmannii. 9 They are not culturable in vitro, however, and speciation has been limited to PCR amplification and sequencing of bacterial 16S rRNA genes in infected gastric scrapings.9 Thus, study of these bacteria is hindered by the lack of pure populations free from contaminating bacteria and other debris.