Changes in peripheral blood leucocyte counts and subpopulations after experimental infection with BVDV and/or Mannheimia haemolytica

Leucocyte counts and subpopulations were studied in peripheral blood from calves experimentally infected in the respiratory tract with either bovine virus diarrhoea virus (BVDV) or Mannheimia haemolytica (Mh), or with a combination of both agents (BVDV/Mh). A non-inoculated control group was included. Peripheral blood samples were obtained for total leucocyte counts, and for neutrophil, lymphocyte and monocyte counts. The numbers of blood lymphocytes expressing the surface antigens CD4, CD8, WC1, B and IL-2R were analysed using flow cytometry. The results showed that BVDV inoculation induced a significant decrease in total leucocyte counts and in neutrophil and lymphocyte numbers, while Mh inoculation induced significant increases in total leucocyte counts and neutrophils, while the lymphocyte count decreased. In the BVDV/Mh group, the total leucocyte count and the lymphocyte numbers decreased significantly. In this group, the lymphocyte numbers remained on a very low level throughout the rest of the study. The numbers of CD4+, CD8+ and WC1+ lymphocytes decreased significantly compared with before inoculations mainly in the BVDV and BVDV/Mh groups. The drops were most pronounced in the BVDV/Mh group. The numbers of B+ lymphocytes and IL-2R+ cells did not change significantly.     

Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection.

Depletion in the number of lymphocytes and viral persistence are thought to be the most important outcomes of classical swine fever virus (CSFV) infection. To define the change in peripheral blood mononuclear cells (PBMC) and virus replication in leukocytes after CSFV infection, 8-week old pigs were infected with the LPC vaccine strain or virulent CSFV (HCV-YL strain). Changes in the relative number of PBMCs were analyzed by flow cytometry. The results showed a significant increase in the relative percentage of monocytes in PBMCs during acute CSFV infection of naive pigs (p < 0.05). Monocyte frequencies were not changed in LPC-vaccinated pigs and control pigs. There was also a significant decrease in the number of IgM+ cells (p < 0.05) and a slight decrease in the number of CD4+ lymphocytes after 5 days of infection. There was no change in the frequency of CD8+ lymphocytes in PBMCs after infection. To define which subpopulation of PBMCs was the target for CSFV infection, PBMC populations from CSFV infected pigs were separated and stained for virus antigen expression. Alveolar macrophages (AM) were also studied. The results showed that CSFV replicated in all PBMC subpopulations: CD4+, CD8+, and IgM+ lymphocytes, and monocytes as well as AMs. However, virus antigen expression was more intense in monocytes and AMs. The infection of lymphocytes may, therefore, contribute to the depletion in their numbers after infection and lead to defective antibody production during virulent CSFV infection.     

Changes in Distribution and Numbers of CD4+ and CD8+ T‐lymphocytes in Lymphoid Tissues and Intestinal Mucosa in the Early Phase of Experimentally Induced Early Onset Mucosal Disease in Cattle

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune‐mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post‐inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post‐inoculation was paralleled by a decrease of B‐lymphocytes and an increase of CD4+ T‐lymphocytes. An increased number of CD8+ T‐lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T‐lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T‐lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T‐lymphocytes in sites with lesions. This might indicate a cell‐mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T‐lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.     

Changes in distribution and numbers of CD4+ and CD8+ T-lymphocytes in lymphoid tissues and intestinal mucosa in the early phase of experimentally induced early onset mucosal disease in cattle

Mucosal disease (MD), one sequelae of bovine virus diarrhoea virus (BVDV) infection, causes severe lesions in lymphoid tissues and mucosal surfaces. Lesions are associated with the presence of cytopathogenic (cp) BVDV and initially characterized by apoptotic cell death. The objective of this investigation was to determine if this cell death is mediated only by the cp BVDV, which is known to induce apoptosis in cell culture or if immune-mediated host reactions might also contribute. Early onset MD was experimentally induced in calves by inoculation of persistently viremic calves with a closely related cp BVDV. Calves were euthanized in the early phase of infection between days 5 and 13 post-inoculation and tissues from tonsils, lymph nodes, Peyer's patches, jejunum and colon were collected. Presence of cp BVDV antigen was correlated with distribution of lymphocyte subpopulations in consecutive cryostat sections. In the lymphoid tissues, cp BVDV antigen was predominantly found in the lymphoid follicles. The increase of infected cells with time post-inoculation was paralleled by a decrease of B-lymphocytes and an increase of CD4+ T-lymphocytes. An increased number of CD8+ T-lymphocytes was seen in progressed lesions only. In the intestinal mucosa, initially multifocal, later diffuse infection with cp BVDV was accompanied by a multifocal or diffuse increase of CD4+ T-lymphocytes, respectively. Numbers of IgA+ plasma cells and CD8+ T-lymphocytes were decreased. The common change observed in lymphoid tissues and mucosa was the increase of CD4+ T-lymphocytes in sites with lesions. This might indicate a cell-mediated immune response to the cp BVDV. Besides their helper function to other cells of the immune system, activated CD4+ T-lymphocytes might also exert cytotoxic activity, induce apoptosis in target cells via Fas/Fas ligand binding and thus contribute to the severity of tissue lesions in MD.     

Early events in the immunopathogenesis of feline retrovirus infections.

Feline leukemia virus and feline immunodeficiency virus (FIV) are lymphotropic retroviruses that cause a wide range of diseases in domestic cats. Although it is known that both viruses are capable of infecting T lymphocytes and that infected cats are lymphopenic, it was not known how infection with either virus might alter specific lymphocyte subpopulations. Using a panel of monoclonal antibodies to feline lymphocyte subpopulations, we examined, by use of flow cytometric analysis, lymphocyte changes in cats naturally infected with FeLV or FIV and explored the early stages in the immunopathogenesis of experimentally induced infection with these viruses. Both groups of naturally infected cats had T-cell lymphopenia. In the FIV-infected cats, the T-cell decrease was principally attributable to loss of CD4+ cells, whereas CD8+ and B-cell numbers remained normal. This led to inversion of the CD4+ to CD8+ ratio in these cats. In contrast, the T-cell lymphopenia in FeLV-infected cats resulted from decrease in CD4+ and CD8+ cells, which led to a CD4+ to CD8+ ratio within normal limits. Experimentally induced infection with these 2 viruses supported these findings. Infection with FIV induced early (10 weeks after infection), chronic inversion of the CD4+ to CD8+ ratio. In contrast, infection with FeLV did not alter CD4+ to CD8+ ratio in the first 20 weeks after infection.     

Role of bovine viral diarrhea virus biotype in the establishment of fetal infections

OBJECTIVE: To examine the role of bovine viral diarrhea virus (BVDV) biotype on the establishment of fetal infection in cattle. ANIMALS: 30 mixed-breed pregnant cows. PROCEDURE: Pregnant cows were inoculated oronasally with either i-WNADL, originating from an infectious BVDV cDNA clone of the National Animal Disease Laboratory (NADL) isolate, or the parental virus stock, termed NADL-A. RESULTS: All cows developed neutralizing antibodies to BVDV, and virus was commonly isolated from peripheral blood mononuclear cells or nasal swab specimens of NADL-A inoculated cows; however, virus was rarely isolated from specimens of i-WNADL inoculated cows. i-WNADL did not cause fetal infection, whereas all fetuses harvested from NADL-A inoculated cows at 6 weeks after inoculation had evidence of infection. Immunoblot analysis of fetal virus isolates revealed the absence of NS3, confirming a noncytopathic (NCP) biotype BVDV in the NADL-A stock. The sequence of the NCP contaminant (termed NADL-1102) and the i-WNADL genome were virtually identical, with the exception of a 270 nucleotide-long insert in the i-WNADL genome. Phylogenetic analyses revealed that NADL-1102 forms a monophyletic group with 6 other NADL genomes. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the contaminating NCP virus in the NADL-A stock was the ancestral NADL virus, which originally infected a bovine fetus and recombined to produce a cytopathic (CP) variant. Following oronasal infection of pregnant cows, viremia and transplacental transmission of CP BVDV to the fetus is rare, compared with the high occurrence of maternal viremia and fetal infection observed with NCP BVDV.     

猪瘟病毒感染对猪外周血T淋巴细胞亚群及TNF-α和IFN-γ的影响

为探讨猪瘟病毒(CSFV)弱毒株T株和野毒株G株感染对猪外周血T淋巴细胞亚群、TNF-α和IFN-γ的影响,本研究应用流式细胞术和ELISA等方法检测CSFV感染猪与未感染猪的白细胞凋亡、CD4+与CD8+T淋巴细胞亚群数量的动态变化以及TNF-α和IFN-γ的动态变化。结果表明,猪感染CSFV T株和G株第4 d和第7 d后CD4+T淋巴细胞比例分别为28.6%、26%和26%、20%,未感染前分别为33.4%和36.8%。猪感染CSFV T株和G株第4 d、第7 d后CD8+T淋巴细胞比例分别为41%、32%和38%、25%,感染前分别为43.8%和48.8%。外周血白细胞凋亡的检测结果显示,猪感染CSFV T株和G株第7 d后,白细胞凋亡比例分别为8.35%和9.89%,未感染的猪为1.63%。ELISA检测结果表明,猪感染CSFV T株和G株第7 d后,TNF-α的产生量分别为553.4 pg/mL和594.2 pg/mL;IFN-γ的产生量分别为8.2 pg/mL和9.8 pg/mL,未感染猪分别为498 pg/mL、12.5 pg/mL。以上结果提示,CSFV感染会引起机体免疫相关细胞及免疫分子发...     

Effect of infection of turkeys with haemorrhagic enteritis adenovirus isolate on the selected parameters of cellular immunity and the course of colibacillosis

The aim of the study was to determine the effect of a Polish low-virulence isolate of haemorrhagic enteritis adenovirus (HEV) on the immune system in turkeys and on the course of colibacillosis in birds infected under laboratory conditions. Turkeys were infected per os with HEV at the dose of 10(4.3)EID50/mL and with E. coli (APEC) (serotypes 078:K80:H9) at the dose of 4x10(9)CFU/mL by injection to the thoracic air sac. The birds infected with the HEV were infected with the APEC either simultaneously or after 5 days. Five days after HEV infection, the percentages of subpopulations of the CD3+CD4+ and CD3+CD8alpha+ T cells and the IgM+ B cells were determined in blood and spleens of the HEV-infected turkeys and in the control (uninfected) birds. The course of colibacillosis was more severe in turkeys infected with the APEC 5 days after infection with the HEV than in those infected with the HEV and APEC simultaneously and than in those infected only with APEC. Five turkeys out of the 18 infected with the APEC 5 days after infection with HEV, died. Their body weights were statistically significantly lower with higher FCR values 41 days after the infection in comparison to turkeys in the other groups. A considerable decrease in the percentage of the T and B cells subpopulations in the blood were found in turkeys infected with the HEV and while the percentage of CD3+CD4+ T cells subpopulation in the spleen increased significantly, the contribution of the CD3+CD8alpha+ T cells and IgM+ B cells subpopulations were decreased. These changes in the immune system of turkeys, occurring 5 days after infection with the HEV, made them more susceptible to infection with the APEC.     

Investigation of T-cell responses and viral mRNA persistence in lymph nodes of pigs infected with porcine rubulavirus

Selected lymphocyte subpopulations were studied and the distribution of viral mRNA were investigated during acute and persistent porcine rubulavirus (PoRV-LPMV) infection in Vietnamese pot-bellied pigs. Six pigs infected with PoRV-LPMV at 17 days of age exhibited clinical signs 7-10 days post-inoculation (pi). One infected piglet died 11 days pi while the other five recovered around day 13 pi and survived until euthanasia on day 277 pi. Increased numbers of CD8+, CD4+ and CD2+ T cells were detected during the acute phase of infection while CD8+ cells were elevated throughout the infection, including during the persistent stage. Specific antibodies against the haemagglutinin-neuraminidase protein of PoRV-LPMV were detected during persistent infection. Although infectious virus could not be recovered from tissues from any of the infected pigs at necropsy 277 days pi, PoRV-LPMV mRNA was detected in lymph nodes, pancreas and central nervous system using a nested polymerase chain reaction technique. Continued lymphocyte interaction with viral RNA may be an important factor in promoting cellular and humoral responses during persistent PoRV-LPMV infection.     

CSFV免疫状态下外周血T淋巴细胞亚群变化及对PRRS的免疫保护作用

建立CSFV疫苗免疫状态下猪繁殖与呼吸综合征病毒（PRRSV）感染动物模型。测定CSFV抗体阻断率和PRRSV抗体S/P值,根据CSFV抗体阻断率和PRRSV感染情况分析猪外周血中T淋巴细胞亚群变化和对PRRS的保护促进作用。采用流式细胞术（FAC）检测猪外周血T淋巴细胞CD3、CD4、CD8亚群的变化。结果,CSFV...     

Changes in macrophage and lymphocyte subpopulations of lymphoid tissues from pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV)

We used an immunohistochemical method to investigate changes in macrophage and lymphocyte subpopulations in various lymphoid tissues of pigs in the acute phase of porcine reproductive and respiratory syndrome virus (PRRSV) infection. The numbers of CD8+ cells and B-cells varied among lymphoid tissues after PRRSV infection. In the infected pigs, numbers of CD8+ cells increased in systemic lymphoid tissues whereas numbers of B-cells increased in mucosa-associated lymphoid tissues. There was no difference in the distribution of virus-infected cells and macrophages between lymphoid tissues of the infected pigs. These changes may be associated with the establishment of virus persistence or the emergence of concurrent infection in mucosal organs.     

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.     

Bronchoalveolar immune defense in cattle exposed to primary and secondary challenge with bovine viral diarrhea virus

To evaluate immune defense mechanisms against bovine viral diarrhea virus (BVDV), four calves received primary and secondary intrabronchial infections with the cytopathic, type I Singer strain of BVDV. The cellular and humoral responses to these site-specific infections with BVDV were monitored by sequential bronchoalveolar lavages (BAL) conducted prior to infection (day 0, non-infected controls) and on days 4, 7, 10, 17 (day 31, secondary infection), 35, 38, 41, 48 and 62 post-infection. Peak quantities of BVDV were recovered from BAL on day 4. BVDV clearance from the lung was complete between days 17 and 31. Immune clearance of BVDV from the lower airways upon secondary infection was swift, within 4 days, and sustained throughout a 1-month period. Total numbers of BAL CD4(+) and CD8(+) T-lymphocytes increased >200-fold by day 10, and increased to levels >70-fold higher than background by 4 days after a secondary BVDV infection. gammadelta(+) T-lymphocytes increased 100-fold by day 7 and remained at levels at least 10-fold higher than pre-infection throughout the study. B-lymphocytes increased to levels 30-fold greater than pre-infection levels by day 10, and further increased to levels 100-fold higher following secondary BVDV infection. Activation (defined by the phenotype CD25(+)/CD62L(-)) and memory (defined by the phenotype CD45RO(+)/CD45R(-)) profiles of lymphocytes in the lower airways were characterized. Activated subpopulations of CD4(+) and CD8(+) cells increased nearly 300- and 150-fold, respectively, by day 10, and to levels 100- and 50-fold 4 days after the secondary infection. Memory subpopulations of CD4(+) and CD8(+) cells increased to levels 170- and 120-fold, respectively, by day 10, and to levels approximately 400- and 300-fold, respectively, 7 days after the secondary infection. The primary antibody response consisted of increased titers of anti-BVDV-specific IgA in bronchoalveolar lavage fluid (BALF). A strong secondary antibody response with high levels of anti-BVDV-specific IgA and IgG in BALF before day 4 post-secondary BVDV infection, likely contributed, along with cellular defenses, to the rapid clearance of virus from the lung upon secondary exposure. These results demonstrate that primary infection of the bovine lung with BVDV initiates cell-mediated immune responses capable of clearing the virus within 2-3 weeks. Furthermore, populations of immune-activated and memory T-lymphocytes, combined with BVDV-specific antibody production, contribute to rapid BVDV clearance upon secondary exposure to the virus.     

Antiviral effect of dietary germanium biotite supplementation in pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Germanium biotite (GB) is an aluminosilicate mineral containing 36 ppm germanium. The present study was conducted to better understand the effects of GB on immune responses in a mouse model, and to demonstrate the clearance effects of this mineral against Porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected pigs as an initial step towards the development of a feed supplement that would promote immune activity and help prevent diseases. In the mouse model, dietary supplementation with GB enhanced concanavalin A (ConA)-induced lymphocyte proliferation and increased the percentage of CD3+CD8+ T lymphocytes. In pigs experimentally infected with PRRSV, viral titers in lungs and lymphoid tissues from the GB-fed group were significantly decreased compared to those of the control group 12 days post-infection. Corresponding histopathological analyses demonstrated that GB-fed pigs displayed less severe pathological changes associated with PRRSV infection compared to the control group, indicating that GB promotes PRRSV clearance. These antiviral effects in pigs may be related to the ability of GB to increase CD3+CD8+ T lymphocyte production observed in the mice. Hence, this mineral may be an effective feed supplement for increasing immune activity and preventing disease.     

Immunophenotypical characterization of lymphocyte subpopulations of the uterus of non-pregnant and pregnant goats

The increased susceptibility during pregnancy to certain pathogens that cause abortions may be related to changes in the distribution and phenotype of lymphocyte subpopulations in the uterus. Histological, electron microscopic and immunocytochemical techniques were used in this study to examine whether such variations occur in different stages of the reproductive cycle of goats. The study of non-pregnant goats showed that most uterine lymphocytes were T cells and displayed both an intraepithelial and stromal distribution. CD8+ T lymphocytes were more numerous than CD4+ T lymphocytes. In the endometrial epithelium two lymphocyte subpopulations were observed: non-granulated CD2+ CD8+ T lymphocytes and granulated CD2+ CD8- T lymphocytes. During gestation, no lymphocytes were observed in the placentomal area, while a decreased number of T lymphocyte subpopulations were found in the inter-placentomal area. In the inter-caruncular epithelium, non-granulated CD2+ CD8+ T lymphocytes disappeared, whereas the granulated CD2+ CD8- T lymphocyte subpopulations increased their number and changed their morphology.     

Bovine viral diarrhea virus type 1- and type 2-specific bovine T lymphocyte-subset responses following modified-live virus vaccination

Expression of CD25 (interleukin-2 receptor alpha chain) was used to monitor antigen-specific activation of T lymphocyte subsets (CD4+, CD8+, and gamma delta T cells) from cattle immunized with modified-live virus (MLV) bovine viral diarrhea virus (BVDV) vaccines. Two groups of 15 animals each were vaccinated with one dose of either BVDV genotype 1 (BVDV-1) or BVDV-1 and BVDV genotype 2 (BVDV-1/2). Six animals negative for both BVDV antibody and BVDV virus were used as negative controls. Three animals vaccinated 7 and 5 weeks before the start of the experiment with MLV BVDV-1 vaccine served as positive controls. Blood samples were taken from the negative control group, the positive control group, and the BVDV-1/2 group 0, 21, 35, 60, and 90 days after vaccination. Blood samples were taken from the BVDV-1 group 0, 21, and 90 days after vaccination. Isolated peripheral blood lymphocytes from immunized and control animals were incubated for 5 days with and without BVDV-1 or BVDV-2. Compared with nonvaccinated animals, a significant (P <.05) increase in expression of CD25 by CD4+ (60 days), CD8+, and gammadelta T (35 to 90 days) lymphocytes from the group given BVDV-1/2 was detected following in vitro exposure to BVDV-1 or BVDV-2 after vaccination. The CD8+ and gammadelta T cells from the group vaccinated with BVDV-1 had significantly (P <.05) increased expression of CD25 compared with nonvaccinates following postvaccination exposure to in vitro BVDV-1 but not to BVDV-2. There was no significant difference between the two vaccinated groups in CD25 expression on any of the T cell subsets in response to BVDV-1 or BVDV-2 exposure. A single administration of MLV BVDV vaccine may be more effective at stimulating CD8+ and gammadelta T cell-specific immune responses to the homologous genotype than to the heterologous genotype.     

Experimental inoculation of calves with laboratory strains of bovine viral diarrhea virus

Diarrhea, erosions and ulcers of the oral mucosa, with conjunctival and nasal discharges, were observed in six calves inoculated with a mixture of two laboratory cytopathic reference strains of bovine viral diarrhea virus (BVDV)-Oregon C24 V and NADL. The clinical picture was accompanied by biphasic body temperature elevation, transient leukopenia and a decrease in the number of lymphocytes. High dose of viruses and multiple routes of inoculation promoted the development of clinical and hematological changes typical for BVDV infection although laboratory strains were used.     

Influence of chronic caprine arthritis-encephalitis virus infection on the population of peripheral blood leukocytes

The influence of caprine arthritis-encephalitis (CAE) virus infection on the population of peripheral blood leukocytes in goats was evaluated. For this purpose two groups of adult dairy female goats were formed. The experimental group consisted of 17 goats, which had been naturally infected for many years. The control group comprised 29 non-infected goats, which originated from CAE-free herd. All goats were clinically healthy. Whole blood was collected and tested in hematological analyzer and light microscope to assess the total number of leukocytes and the percentage of four leukocyte populations--neutrophils, eosinophils, monocytes and lymphocytes. Then, flow cytometry with monoclonal antibodies against several surface antigens (namely CD14, CD2, B-B2, CD4, CD8h, TCR-N6, WC1-N2 and WC1-N3) was performed to assess the proportion of lymphocyte subpopulations. Statistically significant differences (alpha < or = 0.01) were observed only in the subpopulations of T lymphocytes--percentage of all subpopulations were significantly higher in the group of seropositive goats. No statistically significant differences were revealed with respect to the total number of blood leukocytes, the average percentage of blood leukocyte populations and proportions of both T and B lymphocytes.