Long-term culture of canine peripheral blood monocytes in vitro.

Various cultural conditions were assessed for their ability to maintain canine peripheral blood monocytes in vitro. Approximately ten days after incubation of peripheral blood leukocytes in Earle's minimum essential medium supplemented with homologous red cell lysates and normal horse serum, virtually a pure macrophage culture was obtained which could then be maintained for about two months. This culture was judged to be pure by surface marker analysis and their phagocytic activity. The number of monocytes could be increased by injecting the dogs with a chloroform extract from Listeria monocytogenes prior to collection of the blood.     

Activation of natural killer cells in newborn piglets by interferon induction

Natural killer (NK) cell activity in the peripheral blood lymphocytes (PBL) of newborn piglets, normally negligible, was stimulated by in vitro treatment with porcine type I interferon (IFN), and the NK activity of PBL from weaned piglets was augmented by the same treatment. Binding of the PBL to the PK-15 targets used in the single cell cytotoxicity assay for NK activity was not affected by age or by IFN treatment. When newborn piglets were treated with a single intravenous dose at 2 days of age of 0.5 mg/kg of polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), a synthetic IFN inducer, their IFN levels peaked at 6 h post-induction, and NK activity in their PBL peaked at 24 h post-induction at the level normally found in weaned piglets. The NK activity then declined until 7 days post-induction, when it increased again in a similar manner to that in untreated control piglets. Target-binding of the PBL was not affected by poly ICLC treatment of the piglets. Newborn piglets treated with poly ICLC and subsequently exposed to infection with transmissible gastroenteritis (TGE) virus showed a delay in onset of clinical signs of TGE compared with untreated control piglets. It was concluded that NK cells in newborn piglets can be activated by treatment of the piglets with poly ICLC, and that the presence of active NK cells is associated with some increase in resistance to challenge with TGE virus.     

Cell-mediated immunity in horses with sarcoid tumors against sarcoid cells in vitro

Cell-mediated immunity in horses with sarcoid tumor against sarcoid antigens was studied in vitro by means of mixed lymphocyte tumor cell culture assay and lymphocyte-mediated cytotoxicity of 52Cr-labeled target cells. When Mc-1 sarcoid cells were used as stimulatory cells for peripheral blood lymphocytes in the mixed lymphocyte tumor cell assay, a clear difference in the kinetics of the generated lymphocytic proliferative response could be detected between sarcoid and control horses. With sarcoid horses, their proliferative maximum was reached 3 days earlier than that of the control horses, and at this time their proliferative activity was significantly increased over that of control horses. When normal allogeneic fibroblasts were used as stimulatory cells, no such difference between sarcoid and control horses could be seen. The cellular cytotoxicity of peripheral blood lymphocytes from sarcoid and control horses against Mc-1 cells or normal allogeneic fibroblast targets was very low. However, the mean cytotoxicity against Mc-1 was slightly increased for sarcoid horses as compared with that of control horses. In contrast, the cytotoxicity against allogeneic fibroblasts was slightly lower for sarcoid than for control horses. In contrast, the cytotoxicity against allogeneic fibroblasts was slightly lower for sarcoid than for control horses. Furthermore, it was shown that sarcoid horses, but not control horses, had a slightly but consistently increased cytotoxicity against Mc-1 cells as compared with that against normal allogeneic fibroblasts.     

Tumoricidal effect of interleukin-2-activated killer cells in canines

We investigated the effect of both partially purified (TCGF) and recombinant interleukin-2 (rIL-2) on the tumor-directed cytotoxic activity of canine peripheral blood mononuclear cells (PBMC) using three normal canines. No cytotoxic activity was displayed by unstimulated effector cells at 3 h of incubation; however, the cytotoxic effect was observed in a 16-h assay. PBMC of all canines displayed significant levels of lytic activity after stimulation for 4 to 7 days with both types of IL-2 against a variety of allogeneic and xenogeneic neoplastic cells in 3-h 51Cr release assay. The cytotoxic activity of cultured cells increased proportionally in the 16-h assays. Morphological examination of the May-Grünwald and Giemsa stained cytocentrifuged slides of cultured cells on each day of assay showed an increase in large granular lymphocytes (LGLs) beginning on day 4 and reaching a peak on day 7 of culture.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Efficacy of atovaquone against Babesia gibsoni in vivo and in vitro

The therapeutic efficacy of atovaquone against Babesia gibsoni was examined in three dogs experimentally infected with B. gibsoni isolated from naturally infected dogs in Aomori Prefecture, Japan. Once parasitemia reached 10%, atovaquone was administered orally (30 mg/kg twice daily for 7 days). Within 2 days of atovaquone treatment, the parasite disappeared from blood smears without any clinical side effects. Anemia and thrombocytopenia were significantly improved in all the dogs. However, a polymerase chain reaction assay revealed that a B. gibsoni marker gene was intermittently present in peripheral blood after atovaquone therapy, indicating that the organism had not been eliminated, and parasites reappeared in blood smears 33 days after the last treatment. To investigate the change in sensitivity against atovaquone, an in vitro sensitivity test was performed using peripheral blood obtained from an untreated dog that was infected with the original parasite isolate, and from two of the experimentally infected and atovaquone-treated animals (blood was collected at the time of the post-treatment recurrence of the B. gibsoni infection). Atovaquone was added to the culture medium to final concentrations of 0.1, 1, 10, 100, and 1000 nM. For the untreated parasites, complete growth inhibition occurred at 1000 nM of atovaquone, whereas the recurrent parasites were inhibited by only 39.52 +/- 8.34% and 31.31 +/- 8.14% at this concentration after 48 h of incubation. Thus, the recurring parasites were less sensitive to atovaquone than the untreated originally isolated parasites.     

Flow cytometric analysis of canine umbilical cord blood lymphocytes

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.     

Phenotypic analysis and effects of sequential administration of activated canine lymphocytes on healthy beagles

We attempted to accumulate the basic data for evaluation of activated lymphocyte therapy for small animal medicine. The peripheral blood mononuclear cells (PBMCs) collected from healthy dogs were activated using anti-CD3 antibody and human recombinant (hr) interleukin (IL)-2 and reactivated using hr interferon (IFN)-alpha and hr IL-2. The property of obtained cells was compared with PBMCs. The number of cells was shown to have increased approximately>50 -fold by cultivation. The proportion of CD8+ cells was significantly increased, the cytotoxicity of the cultured cells was revealed to have been reinforced. Additionally, CD56 mRNA levels tended to have increased. The cells obtained by this method were confirmed to be activated lymphocytes. Furthermore, we investigated the effects of sequential administration of the obtained cells to healthy dogs. By sequential administration of the activated lymphocytes, the cell proliferative activity, proportion of CD4+ cells and CD8+ cells, and serum IFN-gamma concentration were shown to have increased, and no severe adverse effects were observed. Consequently, activated lymphocytes could be induced using anti-CD3 antibody and IL-2 in healthy dogs, and sequential administration of activated lymphocytes reinforced the recipient's immunity.     

The effect of different isolation procedures on canine leucocyte populations and on lectin-induced lymphocyte proliferation

Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.     

Effect of radical scavengers on canine peripheral blood mononuclear lymphocyte-mediated cytotoxicity and production of active oxygen

Production of active oxygen by canine peripheral blood mononuclear lymphocytes (PBLs) from beagle dog was examined by luminol-dependent chemiluminescence production. The canine PBLs rapidly produced the active oxygen in parallel with the number of cells when PBLs were cocultured with canine leukemia-derived CL-1 cells as target cells. Cytolysis of the target cells and active oxygen production were inhibited linearly by the addition of benzoic acid and n-propyl gallate as hydroxyl radical scavenger. However, superoxide dismutase and tiron which are scavengers of superoxide anion did not inhibit the cytotoxicity so much at low concentrations that inhibited the induction of luminol-dependent chemiluminescence. These results suggest that hydroxyl radical production by stimulated PBLs might be playing a major role of cytotoxic action in the case of canine system.     

Intraepithelial but not lamina propria lymphocytes in the porcine gut are affected by dexamethasone treatment

It is well established that glucocorticoids are key regulators of the immune system and act as immunosuppressive agents in high concentrations. In the pig, effects on the gut immune system and trafficking of lymphocytes between tissues and blood plasma were not investigated so far. Twelve pigs of 70 kg were fed 0.4 mg portions of dexamethasone (Dexa) twice daily for 9 days or remained untreated (controls) and were sacrificed for tissue collection at the end of Dexa treatment. Another six pigs with jugular vein catheters were left untreated for 7 days (control period) and then received Dexa for 9 days. Blood was drawn twice during the control period and at days 3, 6 and 9 of the Dexa period for characterization of peripheral blood leukocytes. Cells were obtained from thymus, mesenteric lymph nodes, jejunal mucosa and Peyer's patches. Lymphoid cells from gut tissue were isolated from two fractions: the EDTA-fraction, containing the intraepithelial lymphocytes (IEL), and the Collagenase-fraction, containing the lamina propria lymphocytes (LPL). In all samples, cell counts and phenotypic characterization of cells by flow cytometry (FCM) were performed. In thymus, Dexa led to a more than 90% reduction of the absolute cell number, which was mainly found in the CD4+CD8+ subpopulation. Dexa effects on lymphocytes from mesenteric lymph nodes were less severe (50%) and led mainly to a decrease (71%) of B-lymphocytes. The number of lymphocytes in the EDTA-fraction (IEL) of the jejunal mucosa decreased significantly by 56% in the Dexa-treated animals compared to the controls, whereas the number of lymphocytes in the Collagenase-fraction (LPL) decreased only moderately. In the Peyer's patches, a decreasing tendency in the number of lymphocytes in the EDTA-fraction was observed which, however, was not significant. In blood, monocytes and granulocytes were significantly increased in an order of 60%. The data show that supraphysiological amounts of Dexa remarkably reduce cell numbers in thymus and also in the intraepithelial compartment of the jejunal mucosa and ileal Peyer's patches. In blood, a notable homeostasis was observed for several leukocyte populations whereas both monocytes and granulocytes increased.     

E.tenella初次感染雏鸡外周血T淋巴细胞亚群及增殖能力变化的动态研究

应用流式细胞术（ FCM）和MTS比色法检测雏鸡初次感染柔嫩艾美尔球虫（ E． tenella）后外周血中CD3＋CD4＋、CD3＋CD8＋T淋巴细胞及其增殖能力的动态变化。数据显示,E． tenella初次感染雏鸡后7～20 d CD3＋CD4＋、CD3＋CD8＋T 淋巴细胞比例明显高于对照组雏鸡（ P＜0．05和P＜0．01）,并且均于12 d时达到峰值。初次感染后7～20 d雏鸡外周血中CD4＋／CD8＋T淋巴细胞亚群的比值明显升高,于感染后16 d时达到1．78。 E． tenella初次感染雏鸡后12～20 d外周血淋巴细胞增殖能力明显高于对照组雏鸡（ P＜0．05）,并于16 d达到峰值。试验表明E． tenella初次感染雏鸡能激活淋巴细胞产生增殖反应,CD3＋CD4＋、CD3＋CD8＋T淋巴细胞在抵抗E． tenella初次感染过程中有重要作用。     

Culture conditions for blastogenic responses of bovine mammary mononuclear cells

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.     

Influence of different tumour types on natural cytotoxicity (NK cell activity) and mitogen-induced lymphocyte proliferation in isolated blood lymphocytes from 110 dogs with tumours

The cell-mediated immune response of blood lymphocytes from 110 untreated dogs with different tumours was evaluated. The influence of different tumour types on the cellular immune system was examined by assessing the percentage of isolated large granular lymphocytes (LGL), in vitro natural cytotoxicity and mitogen-induced lymphocyte proliferation. Although the overall natural cytotoxicity of dogs with different tumours was decreased, the overall difference from control values was not statistically significant. However, mitogen-induced lymphocyte proliferation was significantly depressed in dogs with tumours in comparison with the controls. Dogs with mammary carcinomas showed significantly lower natural cytotoxicity than controls and dogs with myeloid neoplasms showed significantly lower mitogen-induced lymphocyte proliferation. Abnormalities exist not only in natural cytotoxicity but also in mitogen-induced lymphocyte proliferation. For the dog, this is the first study to assess the influence of different tumours using a combined evaluation of natural cytotoxicity and mitogen-induced lymphocyte proliferation in such a large number of animals.     

Lymphocyte-associated immune responses to canine distemper and measles viruses in distemper-infected gnotobiotic dogs.

Cellular immune responses of peripheral blood lymphocytes to canine distemper virus and measles virus were determined in vaccinated or infected gnotobiotic dogs, using the technique of syncytia inhibition. Cross-reactivity between viruses was detected in both groups of dogs. Peak responses in vaccinated dogs occurred 11 days after vaccination and declined to base-line levels by 3 weeks, whereas responses in infected dogs were present 30 days after inoculation. Fractionation experiments with peripheral blood lymphocytes indicated that synctia inhibition is probably mediated by T lymphocytes.     

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.     

Canine dendritic cells from peripheral blood and lymph nodes

Canine dendritic cells were prepared from peripheral blood or lymph nodes using a series of steps including fractionation on bovine plasma albumin (BPA), irradiation with 4000 R, incubation for 16–18 hours, and refractionation on BPA. Dendritic cells were recovered in the low density (LD) fraction containing approximately 0.6% of the unfractionated cells. Measured by the incorporation of ³H-thymidine, the response of the high density (HD) cells to neuraminidase-galactose oxidase (NGO) was lower than that of the unfractionated lymph node cells (LNC) but increased in a concentration dependent manner after the addition of a population of cells enriched for dendritic cells (30–70% by morphologic criteria). Cooperation between HD- and LD- cells was not restricted to identity of the major histocompatibility complex. Canine dendritic cells also displayed stimulatory activity higher than unfractionated peripheral blood mononuclear cells (PBMC) in a one way mixed leukocyte culture (MLC). Canine dendritic cells were nonadherent to plastic, were of low density, and remained viable and functional after irradiation. For the first time, canine dendritic cells have been identified in peripheral blood and lymph nodes and have been shown to act as accessory cells in the response of lymphocytes to NGO and as stimulator cells in a MLC.     

Relative quantification of canine CD56 mRNA expression by real-time polymerase chain reaction method in normal tissues and activated lymphocytes

Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Dog lymphocyte cultures facilitate the isolation and growth of virulent canine distemper virus.

Optimal conditions for the isolation and growth of virulent canine distemper virus (CDV) in canine thymic and peripheral blood lymphocyte cultures were determined. Peak virus titers were seen from 3 to 6 days postinoculation of lymphocytes and depended on the multiplicity of infection. Dog lymphocytes were at least as susceptible as canine macrophages to infection with virulent CDV. Virus replication in lymphocytes resulted in higher virus titers than in dog lung macrophages. Peripheral blood lymphocytes (PBL) from CDV-immune dogs were as susceptible to CDV as were PBL from susceptible dogs.