Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Effects of an experimental infection with Actinobacillus pleuropneumoniae on the interferon-alpha and interleukin-6 producing capacity of porcine peripheral blood mononuclear cells stimulated with bacteria, virus or plasmid DNA

The effect of a bacterial infection on interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) production by porcine cells was studied in specific pathogen-free (SPF) pigs, infected intranasally with Actinobacillus pleuropneumoniae serotype 2. Three experimental groups of five pigs were used: infected non-treated pigs, infected pigs that were treated with enrofloxacin at disease onset, and non-infected, non-treated control pigs. Blood samples were collected from all pigs on the day of infection and on days 1, 4, 7, 13 and 17 post-infection. Sera were analysed for presence of antibodies to A. pleuropneumoniae and for the cytokines IL-6 and IFN-alpha. Ability to produce these cytokines was tested in vitro using whole blood cultures stimulated with inactivated virus (Aujeszky's disease virus infected porcine kidney cells (ADV/PK-15)), inactivated bacteria (A. pleuropneumoniae) or bacterial plasmid (pcDNA3). All cytokine inducers were used neat or pre-incubated with the transfectious agent lipofectin. IL-6 appeared in the serum of all infected non-treated animals but no IFN-alpha was found in the serum of any of the experimental pigs. Accordingly, the bacteria induced a substantial IL-6 but hardly any IFN-alpha production when tested in vitro. However, following incubation with lipofectin, the inactivated bacteria as well as pcDNA3 became efficient inducers of IFN-alpha in whole blood cultures. The increased IFN-alpha production, previously recorded in vitro during the acute phase of infection with A. pleuropneumoniae, was confirmed using lipofected plasmid DNA and it was indicated that leukocytes obtained from infected but apparently cured animals also exhibited an increased production of IFN-alpha. Thus, even mild/sub-clinical bacterial infections may affect cytokine production in pigs.     

光生物素cDNA探针对体内外蓝舌病病毒感染的检测

蓝舌病病毒(BTV)基因的第三片段(RMA_3)在不同型间有较高的同源性.用光生物素标记的其cDNA重组体pC7,检测2～22型BTV BHK细胞培养物全部为阳性,而相关病毒EHDV_1、EHDV_2和Ibraki病毒为阴性;同一探针检测17个型BTV攻毒羊的全血样品均为阳性,未感染的正常羊血细胞和BHK细胞培养物样品均为阴性.     

Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China

Babesia sp. Xinjiang was isolated from a splenectomised sheep infested by Rhipicephalus sanguineus and Hylomma anatolicum anatolicum, collected from sheep and cattle in Xinjiang province. It was considered to be a novel ovine Babesia species on the basis of its morphology, pathogenicity, vector tick species and alignments of 18S ribosomal RNA (18S rRNA) and internal transcribed spacers (ITS) gene sequences. Continuous in vitro cultures of the ovine parasite were established using infected sheep blood. In RPMI 1640 medium with 7.5% sheep red blood cells (RBCs) maintained in an incubator at 37 °C and 5% CO(2), the percentage of parasitized erythrocytes (PPE) peaked at 10% in 24- and 6-well plates. It increased to 20-50% with the same culture medium but with 2.5% RBC in 75 cm(2) flasks. Two clonal lines of Babesia sp. Xinjiang were screened using the limiting dilution method. Growth characteristics of these lines in vitro were measured by a microtiter-based spectrophotometric method and from the PPE. The generation time in sheep erythrocytes was between 15.20 h and 16.27 h. Furthermore, the host range of parasite was identified with in vitro culture and in vivo infection. Erythrocytes of sheep, cattle, sika deer and humans could be invaded into by lines in vitro, but the parasites could not propagate in human erythrocytes. The parasites could not enter erythrocytes from goats in vitro. However, in vivo, only sheep could be infected by lines. Finally, a Babesia sp. Xinjiang-like parasite (which shared 99.5% identity with the original strain of Babesia sp. Xinjiang) was isolated using this in vitro culture system from 1 of 19 sheep blood samples collected from western Gansu province, China.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Transmissible gastroenteritis virus antibody production in vitro by porcine peripheral blood leukocytes.

The purpose of this study was to demonstrate the in vitro production of transmissible gastroenteritis virus (TGEV)-specific antibodies by peripheral blood leukocytes (PBL) harvested from piglets infected with TGEV. Piglets were infected with the virulent Purdue strain of TGEV and at intervals postinfection their PBL were cultivated in the presence of TGEV antigen, control antigen or pokeweed mitogen (PWM). The culture supernatants were tested for TGEV antibodies by a fixed cell enzyme immunoassay. Antibodies were never found in the supernatants of unprimed PBL cultures from control piglets, nor in cultures stimulated with control antigen, and antibodies were produced more frequently in response to stimulation of primed PBL with viral antigen than with PWM. In PBL cultures stimulated with viral antigen, TGEV antibodies of the IgG class were produced more frequently than IgA class antibodies. Optimal antibody responses were produced by PBL harvested two weeks after infection and cultivated at a concentration of 10(7) cells/mL for five days.     

Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus.

The in vitro cell tropism of non-cytopathogenic (ncp) and cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) was studied in primary dissociated brain cell cultures derived from ovine fetuses of different gestational ages. The cell types infected were identified by double immunofluorescence using antibodies against BVDV and cell type-specific markers. In cultures infected with ncp BVDV viral antigen was present in neurofilament (NF 200 kDa)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes and fibronectin-expressing cells. Estimation of the percentages of individual cell types infected with ncp BVDV indicated a tropism for NF 200-positive neurons. In cultures infected with cp BVD virus cytopathic changes were observed beginning at 40 hours post infection. Viral antigen was present in vacuolated NF 200-, GFAP- and fibronectin-positive cells. In comparison with non-infected control cultures a considerable reduction of the number of the different cell types was seen.     

The use of interleukin-2 receptor expression as a marker of cell-mediated immunity in goats experimentally infected with Mycobacterium avium ssp. paratuberculosis

The purpose of the present work was to demonstrate cell-mediated immune response to paratuberculosis in experimentally infected animals, using quantification of interleukin-2 receptor (IL-2R) expression on activated lymphocytes by means of in vitro stimulation with Mycobacterium avium ssp. paratuberculosis-derived purified protein derivative (PPDp). A whole-blood technique was developed, and optimal conditions for quantification of IL-2R expression on caprine lymphocytes, using monoclonal antibodies (anti-bovine IL-2R-alpha) and low cytometrical analysis, were determined. Different PPDp-antigen concentrations and incubation times were compared. The whole-blood method was also compared to the more traditional IL-2R assay using peripheral blood mononuclear cultures (Hesketh et al., 1993). Cross-reactivity to Mycobacterium avium was studied at different mycobacteria-PPD concentrations. An immune response could be demonstrated in animals infected with Mycobacterium avium ssp. paratuberculosis. We found that a PPDp concentration of 10microgml(-1) together with an incubation time of 72h, gave the best results using the whole-blood method. The whole-blood method eliminates many laborious steps involved in lymphocyte separation, and the effects of all the constituents of blood are expressed in a way which corresponds more to in vivo conditions. The risk of selecting subpopulations of lymphocytes during cell separation is avoided.     

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

In vitro cultivation of a south African isolate of an Anaplasma sp. in tick cell cultures

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08' E, 25 degrees 38' S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatienne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Limitations of in situ hybridization for the detection of bluetongue virus in blood mononuclear cells

In situ nucleic acid hybridization was tested for the ability to detect bluetongue virus (BTV) nucleic acids in blood mononuclear cells. A standard protocol was devised and applied to the demonstration of BTV genetic sequences in cultured bovine mononuclear cells that had been infected in vitro. In situ hybridization using biotinylated single-stranded RNA probes, in the presence of 50% formamide at 50 C, demonstrated an intense, positive signal in 0.001-0.01% of the BTV-infected cultured mononuclear cells. The protocol was applied to isolated mononuclear cells from an experimentally infected heifer. No infected cells were observed by this method, although the blood specimens were obtained during peak viremia.     

Cytopathic effect of lentogenic strains of Newcastle disease virus in cultured chicken muscle cells

The ability of lentogenic strains of Newcastle disease virus (NDV) to inhibit the development of embryonic chicken muscle cells in vitro is described. In contrast to the uninfected control, which displayed a thick network of branched multinucleated muscle fibers, cultures infected with NDV exhibited a severely reduced potential to differentiate. The few differentiated muscle fibers that formed in the presence of NDV were poorly developed, displayed an attenuated morphology, and detached from the substrate 2 to 3 days before uninfected cultures showed signs of cell aging. When NDV-infected cultures were stained before muscle fiber detachment with a fluorescent-labeled NDV-specific antibody, the infected muscle fibers reacted strongly relative to neighboring myoblasts and fibroblasts. All six strains of NDV induced a similar inhibitory effect, and no strain-specific differences were detectable. These results indicate that differentiating muscle fibers are highly susceptible to infection by NDV in vitro and that this tissue exhibits an early NDV-induced cytopathic effect.     

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela

Background: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. Objective: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. Methods: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. Results: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. Conclusions: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi

OBJECTIVE: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi. SAMPLE POPULATION: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi. PROCEDURES: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled. RESULTS: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%. CONCLUSIONS AND CLINICAL RELEVANCE: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.     

Failure of antigen-stimulated gammadelta T cells and CD4+ T cells from sensitized cattle to upregulate nitric oxide and mycobactericidal activity of autologous Mycobacterium avium subsp. paratuberculosis-infected macrophages

The function of gammadelta T cells during ruminant paratuberculosis (Johne's disease) is presently unknown. An ex vivo system was used to test the hypothesis that gammadelta T cells are capable of activating Mycobacterium avium subsp. paratuberculosis-(M. paratuberculosis)-infected macrophages. Peripheral blood-derived macrophages were infected in vitro with live M. paratuberculosis, and autologous LN-derived gammadelta T cells or CD4+ T cells were co-cultured with infected macrophages for 48h, at which time bacterial survival as well as production of nitrites and IFN-gamma was evaluated. Incubation of M. paratuberculosis-infected macrophages with autologous gammadelta T cells did not result in reduced intracellular bacterial viability compared to infected macrophage cultures without added T cells. IFN-gamma production by-infected cultures containing added gammadelta T cells was not enhanced compared to that of infected macrophages alone. Although infection of macrophage cultures caused increased production of nitrites at both post-infection day (PID) 0 and PID 60, the addition of gammadelta T cells did not further increase nitrite production. In contrast, addition of PPD-stimulated CD4+ T cells obtained at PID 60 to M. paratuberculosis-infected macrophages resulted in significantly increased IFN-gamma production compared to cultures without added T cells or cultures containing unstimulated CD4+ T cells or unstimulated or antigen-stimulated gammadelta T cells. However, the increased production of IFN-gamma by co-cultures containing PPD-stimulated CD4+ T cells did not result in increased bacterial killing or increased production of nitrites compared to cultures without added T cells. In additional in vitro experiments, M. paratuberculosis-infected macrophages, but not uninfected macrophages, were unable to increase nitrite production when stimulated with recombinant IFN-gamma. Taken together, the data suggest that (1) gammadelta T cells do not produce significant IFN-gamma and do not significantly increase NO production from M. paratuberculosis-infected macrophages in vitro, (2) the production of significant IFN-gamma by antigen-stimulated CD4+ T cells from infected calves is insufficient to enhance mycobacterial killing or nitrite production by infected macrophages, and (3) macrophages may have an impaired NO response following intracellular M. paratuberculosis infection, even in the presence of significant concentrations of IFN-gamma.     

Attempts to cultivate Ehrlichia phagocytophila in vitro

Whole blood, buffy coat and plasma from sheep infected with tick-borne fever (TBF) were inoculated into embryonated hen eggs, primary chicken embryo fibroblasts, primary foetal lamb kidney cells, mouse L cells, and ovine pulmonary macrophage cultures. Infected buffy coat cells were fused with primary chicken embryo fibroblasts, mouse L cells, ovine pulmonary macrophages and BHK-21 cells, using UV irradiated Sendai virus. Evidence of multiplication was not obtained.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.