Assessment of Antibody‐independent Cellular Cytotoxicity (AICC) of Porcine Neutrophilic Granulocytes by Quantitative Flow Cytometry. Lack of Modulation by Larval Products of Oesophagostomum dentatum

After infection of pigs by the larvae of Oesophagostomum dentatum, granulomas are formed around the third‐stage larvae in the submucosa of the gut which contain a considerable number of neutrophils. This has no obvious impact on the larvae, which develop to fourth‐stage larvae within these granulomas. We therefore asked, whether the products of O. dentatum larvae modulate the functional capacity of porcine neutrophils. The antibody‐independent cellular cytotoxicity (AICC) was chosen as a model system. This assay was developed for the pig and quantified using flow cytometry. Bovine lymphoblastoid cells (cell line Anna TA1) served as targets. The measurement of cytotoxicity was based on the determination of absolute numbers of vital target cells. This procedure proved to be reliable and required no additional labelling of target and/or effector cells. Porcine neutrophils, when stimulated with phorbol 12‐myristate 13‐acetate (PMA; 10 nmol/l), killed target cells at effector : target ratios between 1 : 1 and 9 : 1. AICC was not demonstrable after 4 h but could be observed between 16 h and 20 h after in vitro co‐culture. Killing of targets required close physical contact between effector and targets, since supernatants of PMA‐stimulated polymorphonuclear cells were not able to lyse the target cells. Homogenates of third‐ and fourth‐stage larvae of O. dentatum did not affect the vitality of porcine granulocytes or target cells in vitro, nor did they modulate the AICC capacity of porcine granulocytes.     

Cytotoxicity of bovine lymphocytes after treatment with lymphokines

Cytotoxic lymphocytes were generated from bovine peripheral blood mononuclear leukocytes after in vitro stimulation with lymphokines that contained interleukin-2. Lymphokine-stimulated cultures were cytotoxic to K562 cells (human natural killer [NK] targets) and YAC-1 cells (mouse NK targets), but not to HSB-2 cells (human NK targets) in a 4-hour, 51Cr-release assay. Cells generated after lymphokine activation also mediated antibody-dependent cellular cytotoxicity to HSB-2 cells. Appearance of effector cells as a function of time in culture, method of stimulation, and cold target competition experiments strongly indicated that direct cytotoxicity and antibody-dependent cellular cytotoxicity may have been mediated by the same cell. Cells generated by similar conditions were able to mediate cytotoxicity against infectious bovine rhinotracheitis virus-infected target cells, especially in an 18-hour assay.     

Natural killer (NK) activity of porcine blood lymphocytes against allogeneic melanoma target cells

Allogeneic PM/86 melanoma cells of Munich Troll miniature swine have been used for the demonstration of porcine peripheral blood NK cell activity. Compared with the specific lysis of xenogeneic K562-, U937- and Vero-target cells, NK cell-mediated cytotoxicity (NK-CMC) against PM/86 melanoma tumor cells was significantly lower in a 16 h chromium release assay. The target cell susceptibility to peripheral blood NK-CMC of both adult Troll miniature swine and German Landrace sows was very similar. Cold target inhibition assays revealed the allogeneic PM/86 melanoma cells to be the most powerful inhibitors of NK-CMC. Nylon wool non-adherent lymphocytes produced interferon (IFN)-alpha in different quantities upon contact with NK susceptible target cells. The NK effector cells could be stimulated to a higher lytic activity against all susceptible targets by a moderate dose of natural human interleukin-2 (nhuIL-2). The role of NK-CMC in melanoma tumor rejection and/or prevention of metastases is yet unknown in swine although porcine melanoma serves as a good model for the disease in man.     

The in vitro motility response to various anthelmintics of third-stage larvae of Oesophagostomum spp. from pigs

The in vitro activities of thiabendazole, levamisole, pyrantel, morantel and ivermectin against Oesophagostomum spp., the nodular worm of pigs, were determined and compared. The study was carried out using isolates of O. dentatum and O. quadrispinulatum, which had been defined in vivo. Infective larvae were exposed to the anthelmintics for 24 h and then placed in a micromotility meter. All the treatments significantly reduced the motility of the ensheathed L₃ larvae, but the micromotility meter was not able to differentiate between anthelmintic resistant and anthelmintic susceptible isolates.     

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.     

A dose-response investigation on the level of resistance to pyrantel citrate in nodular worms of pigs

This study was undertaken to determine the level of resistance against pyrantel citrate in strains of Oesophagostomum quadrispinulatum and Oesophagostomum dentatum which have previously been found resistant to this anthelmintic. Groups of pigs were artificially infected with batches of infective larvae which were previously found either susceptible or resistant to pyrantel citrate. After treatment with 1, 2 and 4 times the recommended dose (14 mg kg-1) of pyrantel citrate, the resistant O. quadrispinulatum population was reduced by 51.0, 76.2 and 86.1%, and O. dentatum by 41.2, 47.9 and 78.5%. The results indicated that O. dentatum was slightly more resistant (P less than 0.05) than O. quadrispinulatum to pyrantel citrate. Treatment of the susceptible worms with 1 and 2 times the recommended dose caused a reduction in worm numbers by 61.0 and 99.4%, respectively.     

Products of Fourth‐Stage Larvae of Oesophagostomum dentatum Induce Proliferation in Naïve Porcine Mononuclear Cells

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune‐modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum‐infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third‐ (L₃) and fourth‐stage larvae (L₄) of O. dentatum. Numbers of vital cells and blast‐transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L₄ (but not L₃) significantly increased the numbers of vital poMNC in vitro (up to 187 %). In addition, L₄ products (homogenates and supernates, 0.1–10 μg/ml) but not those of L₃ induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum‐infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen‐induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L₃ and L₄ indicate that the parasite alters its set of immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.     

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells.

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.     

Characterization of porcine xenoreactive cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatibility complex.     

Porcine Antimicrobial Peptide PR-39 Is Modulated by Lactoferrin in Marrow Cells

PR-39 is a porcine antimicrobial peptide that plays an important role in the innate defense mechanism. We produced monoclonal antibodies (MAbs) against PR-39 by fusing mouse myeloma cells with lymph node cells from BALB/c mice immunized with the reactive site sequence PR-11 of PR-39. Furthermore, we investigated the effect of lactoferrin on PR-39 gene levels and peptide concentrations in cultural supernatants of bone marrow granulocytes by RT-PCR and the competitive inhibition ELISA method established in this study. Two hybridomas 3H5 and 5H7 were selected for developing ascites and contained MAbs against PR-11, which were used for screening the specificity to PR-39 by competitive inhibition ELISA. We found that MAbs were successfully produced against PR-11 and the MAbs had a strong reaction with PR-39 excreted by porcine marrow granulocytes. The ascites had a relatively high titer and the purified MAbs were determined as IgG1. Incubation with 10 or 100 µg/mL lactoferrin significantly increased (P < 0.05) PR-39 mRNA levels in bone marrow granulocytes after 3 h and 6 h, but 1000 µg/mL lactoferrin reduced (P < 0.05) PR-39 mRNA expression after 3 h and 6 h compared with control (no lactoferrin added). The relative concentrations of PR-39 in supernatant secreted by marrow granulocytes were significantly increased by 1000 µg/mL lactoferrin after both 3 h and 6 h. These findings suggest a regulatory role of lactoferrin for the antimicrobial peptide PR-39 in marrow cells in vitro. It is possible that the regulation of antimicrobial peptide PR-39 expression may be one of the protective mechanisms of lactoferrin in pigs.     

Interactions between the predacious fungus Arthrobotrys oligospora and third-stage larvae of a series of animal-parasitic nematodes

Interactions between the predacious hyphomycete Arthrobotrys oligospora and third-stage larvae of nine animal-parasitic nematodes were tested in vitro. The trap-inducing capabilities of the ruminant trichostrongylus Cooperia oncophora, C. curticei, Haemonchus contortus and Ostertagia ostertagi and of equine cyathostomes were almost comparable to those of free-living soil nematodes, and significantly higher than those of the porcine Oesophagostomum dentatum and Oe. quadrispinulatum and of the murine Nematospiroides dubius. The trap-forming potential of Dictyocaulus viviparus was poor. All animal-parasitic nematodes were rapidly captured when fungal traps had been pre-induced in high numbers. The possible influence of predacious fungi on animal-parasitic nematode populations under natural conditions in the field is discussed.     

Demonstration of cytotoxic lymphocytes to virus-infected target cells in pigs inoculated with transmissible gastroenteritis virus.

Lymphocytes, cytotoxic to virus-infected target cells, were induced in pigs orally exposed to transmissible gastroenteritis virus. They were studied and experiments were carried out by using autochthonous testicle cells as target cells to avoid genetic incompatibility of effector lymphocytes and target cells. Cytotoxic lymphocytes were demonstrated in Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood on postinoculation day (PID) 7. Cytotoxic activity of lymphocytes increased thereafter and reached the maximal amount at PID 21. Lymphocyte cytotoxicity was somewhat greater in lymphocytes of peripheral blood and spleen than in those of Peyer's patches and mesenteric lymph nodes after PID 14. On the contrary, lymphocyte reactivity to the viral antigen measured by lymphocyte proliferative assay was higher in Peyer's patch and mesenteric lymph node cells than in peripheral blood and splenic cells. Lymphocyte cytotoxicity was depressed by treating effector cells with anti-porcine thymocyte serum and complement. However, lymphocyte suspensions treated with anti-porcine thymocyte serum and complement were still cytotoxic to some extent against virus-infected target cells, although T lymphocytes were completely excluded by the treatment. This suggests that cytotoxic mechanism other than the direct action of cytotoxic T lymphocytes may be involved in the cytotoxicity assay systems used in the present studies. In experiments in which allogenic cells (testicle cells of siblings) were used together with autochthonous cells as targets, lymphocyte cytotoxicity was equally expressed against both autochthonous and allogenic target cells in 2 of 3 experiments. However, lymphocyte cytotoxicity was greater against autochthonous cells than against allogenic target cells in 1 of 3 experiments.     

Porcine Antimicrobial Peptide PR-39 Is Modulated by Lactoferrin in Marrow Cells

PR-39 is a porcine antimicrobial peptide that plays an important role in the innate defense mechanism.We produced monoclonal antibodies(MAbs)against PR-39 by fusing mouse myeloma cells with lymph node cells from BALB/c mice immunized with the reactive site sequence PR-11 of PR-39.Furthermore,we investigated the effect of lactoferrin on PR-39 gene levels and peptide concentrations in cultural supernatants of bone marrow granulocytes by RT-PCR and the competitive inhibition ELISA method established in this study.Two hybridomas 3H5 and 5H7 were selected for developing ascites and contained MAbs against PR-11,which were used for screening the specificity to PR-39 by competitive inhibition ELISA.We found that MAbs were successfully produced against PR-11 and the MAbs had a strong reaction with PR-39 excreted by porcine marrow granulocytes.The ascites had a relatively high titer and the purified MAbs were determined as IgG1.Incubation with 10 or 100 μg/mL lactoferrin significantly increased(P0.05)PR-39 mRNA levels in bone marrow granulocytes after 3 h and 6 h,but 1000 μg/mL lactoferrin reduced(P0.05)PR-39 mRNA expression after 3 h and 6 h compared with control(no lactoferrin added).The relative concentrations of PR-39 in supernatant secreted by marrow granulocytes were significantly increased by 1000 μg/mL lactoferrin after both 3 h and 6 h.These findings suggest a regulatory role of lactoferrin for the antimicrobial peptide PR-39 in marrow cells in vitro.It is possible that the regulation of antimicrobial peptide PR-39 expression may be one of the protective mechanisms of lactoferrin in pigs.     

Antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against cells infected with porcine transmissible gastroenteritis virus.

The objective of this study was to determine whether porcine peripheral blood leukocytes and intestinal intraepithelial leukocytes can mediate antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. Peripheral blood leukocytes collected from six young adult pigs and intraepithelial leukocytes from a further five pigs were used as effector cells in chromium release assays against PK-15 cells persistently infected with transmissible gastroenteritis virus. Both peripheral blood leukocytes and intraepithelial leukocytes were capable of mediating antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against PK-15 transmissible gastroenteritis cells. While the peripheral blood leukocytes mediated lower levels of specific 51Cr release in spontaneous cell-mediated cytotoxicity than in antibody-dependent cell-mediated cytotoxicity, the intraepithelial leukocytes were more effective in spontaneous cell-mediated cytotoxicity than antibody-dependent cell-mediated cytotoxicity.     

Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs.

The aim of this study was to examine interactions between Ascaris suum and Oesophagostomum dentatum infections in pigs with regard to population dynamics of the worms such as recovery, location and length; and host reactions such as weight gain, pathological changes in the liver and immune response. Seventy-two helminth-na?ve pigs were allocated into four groups. Group A was inoculated twice weekly with 10000 O. dentatum larvae for 8 weeks and subsequently challenge-infected with 1000 A. suum eggs, while Group B was infected with only 1000 A. suum eggs; Group C was inoculated twice weekly with 500 A. suum eggs for 8 weeks and subsequently challenge-infected with 5000 O. dentatum larvae, whereas Group D was given only 5000 O. dentatum larvae. All trickle infections continued until slaughter. Twelve pigs from Group A and B were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 12 pigs from the each of the four groups were slaughtered 28 days p.c.i.. No clinical signs of parasitism were observed. The total worm burdens and the distributions of the challenge infection species were not influenced by previous primary trickle-infections with the heterologous species. Until day 10 p.c.i. the ELISA response between A. suum antigen and sera from the O. dentatum trickle infected pigs (Group A) pigs were significantly higher compared to the uninfected Group B. This was correlated with a significantly higher number of white spots on the liver surface both on Day 10 and 28 p.c.i. in Group A compared to Group B. The mean length of the adult O. dentatum worms was significantly reduced in the A. suum trickle infected group compared to the control group. These results indicate low level of interaction between the two parasite species investigated.     

Natural cell-mediated cytotoxicity to cells infected with infectious bovine rhinotracheitis virus

Cell-mediated cytotoxicity against viral-infected cells was demonstrated in a 6-hr 51Cr release assay. Peripheral blood mononuclear leukocytes from both infectious bovine rhinotracheitis virus (IBRV)-infected and noninfected cattle exhibited preferential lysis against IBRV-infected primary bovine embryonic kidney (BEK) cells compared to cells infected with pseudorabies virus and noninfected BEK cells. Addition of specific antibody to the assay did not enhance cytotoxicity. The effector cell was a nonadherent cell which was either spontaneously enriched or generated during in vitro cultivation. Maximal cytotoxic activity was detected in peripheral blood mononuclear cells cultured for 3 to 5 days. Several factors affected the magnitude of cytotoxicity during the assay: target cell type, concentration of viral inoculum, duration of effector and target cell contact. It is suggested that target cell lysis was a form of natural cell-mediated cytotoxicity mediated by a cell which has different characteristics from the typical human and murine NK cell.     

Characteristics of a flubendazole resistant isolate of Oesophagostomum dentatum from Germany.

Two controlled tests were performed to investigate the benzimidazole resistance of a nodular worm isolate "GIBZ" from a pig breeding farm in Germany. In Trial I, groups of five pigs, artificially infected with Oesophagostomum larvae isolated from that farm were treated with flubendazole at a single dose of 5 mg kg(-1) bodyweight (BW) or remained untreated. In Trial II, three groups of three pigs each infected with larvae after a further laboratory passage of this isolate were treated with flubendazole either at a single dose of 5 mg kg(-1) BW or at a divided dose of 1.5 mg kg(-1) BW daily for 5 consecutive days, or with fenbendazole at a single dose of 5 mg kg(-1) BW, the fourth infected group remained untreated. The respective doses of anthelmintics were mixed with a small amount of feed and administered to individual pigs in both trials. Fecal egg counts before and after treatment and post-mortem worm burdens 7 days after (last) treatment were examined to assess the anthelmintic efficacies. Only infections with Oesophagostomum dentatum were found in both trials. In Trial I, the mean worm count reduction by flubendazole was 30% as compared to the untreated controls. In Trial II, flubendazole administered at a single or divided dose reduced the mean worm burden by 0 and 85%, respectively, whereas fenbendazole was 100% effective. These results establish resistance to flubendazole in the isolate "GIBZ" of O. dentatum. The failure to reveal side resistance to fenbendazole may be explained by that the currently recommended dose rate of this compound is supra-optimal for porcine nodular worms.     

Bovine natural cell mediated cytotoxicity (NCMC): activation by cytokines

Incubation of bovine peripheral blood mononuclear leukocytes (PBML) with the cytokines (CK) IL-2, alpha-IFN, gamma-IFN or IL-4 resulted in significant increases in natural cell mediated cytotoxicity (NCMC) over endogenous levels, as determined in an 18 h 51Cr-release assay using the human K562 or mouse Yac-1 target cell lines. Endogenous cytotoxic activity of bovine natural effector cells (NEC) using K562 or Yac-1 target cells was minimal (killing less than 8%). After 18 h of incubation with the CK hurIL-2, alpha-bovrIFN, gamma-bovrIFN or hurIL-4, NEC had significant increases in cytotoxic activity for both K562 and Yac-1 target cells. Significant increases in cytotoxic activity were not found after incubation of NEC with IL-1 or beta-IFN. Specific killing varied with CK concentration in a dose dependent manner and was proportional to effector:target cell ratio. Activation of the bovine NEC by CK was rapid, occurring within 6-12 h of incubation with alpha-IFN or gamma-IFN and within 12-18 h of incubation with IL-2. Incubation of bovine PBML with IL-2 and alpha- or gamma-IFN or with alpha-IFN and gamma-IFN showed that these CK do not act in a synergistic manner to increase NCMC in the bovine NEC.     

Characteristics of Yorkshire swine natural killer cells

The present study examined the properties of NK activity in Yorkshire swine. The results support other porcine studies which indicate the swine NK system has both similarities and differences to this system in other species. Profiles of NK activity indicated swine NK cells are highly reactive against the YAC-1 lymphoma, the K-562 myeloid leukemia, the P-815 mastocytoma, and the TU-5 virally transformed fibroblast. In contrast, the MOLT-4 and SB leukemias are NK resistant. Kinetic studies indicated that in Yorkshire swine, NK lysis begins 6 h after mixing effectors and targets. The kinetics of the lytic reaction differ both from other breeds of swine and from other species, where cytotoxicity is readily measured in 4-h assays. The delayed lysis was not due to delayed target cell recognition, because Yorkshire swine NK cells are rapidly bound to tumor targets. The delayed lysis seems to be due to a refractoriness in the NK lytic mechanism. This delay may relate to the morphologic finding that the target-binding cell in Yorkshire swine appeared quite different from the large granular lymphocyte (LGL) reported as the NK effector in humans and rodents. Indeed, in light microscopic studies the typical tumor-binding cell in Yorkshire swine is a small, apparently nongranular, lymphocyte. Analysis of NK activity at the single cell level was performed with single effector-tumor conjugates immobilized in agarose. Generally, lysis by target binders paralleled sensitivity to lysis in 51Cr release tests, indicating lysis in agarose may be used as an NK index in swine. Like other species, swine NK cells were found to be nonadherent lymphocytes with a characteristic tissue distribution. Peripheral blood and spleen had the highest levels of NK activity. Lymph node cells displayed a small amount of NK activity which was limited to the YAC-1 target, while thymocytes showed no appreciable NK activity against any of the cell lines tested.     

Development and distribution of Oesophagostomum columbianum in young lambs after oral or intraruminal infection

There was no difference in the establishment or rate of development of Oesophagostomum columbianum in young lambs after infective larvae were administered either orally or by injection directly into the rumen. However, the linear distributions in the intestine of encysted third-stage larvae differed according to the route of infection. The distributions of fourth-stage larvae, after migration to the large intestine, did not differ. It is suggested that the distribution of parasitic third-stage O. columbianum is host-dominated, depending on the site of exsheathment and the rate of passage of ingesta, but that of fourth-stage and adult worms involves active site selection by the parasite.