Use of alpha-naphthyl acetate esterase staining to identify T lymphocytes in cattle

Peripheral blood lymphocytes (PBL), lymph nodes, and/or spleens from clinically normal cattle were examined for cytochemical staining of alpha-naphthyl acetate esterase (ANAE). Two types of positive-staining patterns in ANAE staining resulted. By a combination of ANAE staining and latex-ingesting test, diffuse ANAE-positive cells were considered as mononuclear phagocytic cells. Using erythrocyte rosettes, erythrocyte antibody complement rosettes, nylon-wool column technique, surface immunoglobulin (SIg) staining, and the ANAE staining technique, granular ANAE-positive lymphocytes were shown to be T lymphocytes. The frequency of T and B lymphocytes in PBL, spleens, and lymph nodes of clinically normal cattle was measured, using ANAE staining and SIg staining. In PBL, 47.7% were ANAE-positive and 26.9% were SIg-positive; in spleens, 22.4% were ANAE-positive and 53.7% were SIg-positive; and in lymph nodes, 38.5% were ANAE-positive and 28.3% were SIg-positive. The frequencies of T and B lymphocytes in PBL, spleens, and/or lymph nodes from cattle with enzootic bovine leukosis (EBL) and cattle with persistent lymphocytosis and in tumor cells from cattle with EBL were measured. When compared with those of clinically normal cattle, PBL, spleens, and lymph nodes of cattle with EBL and the PBL of cattle with persistent lymphocytosis contained numerous SIg-positive cells and few ANAE-positive cells. Tumor cells from cattle with EBL contained 7.3% ANAE-positive and 78.0% SIg-positive cells.     

Analysis of the bone morphogenetic protein 6 gene promoter region in young beef cattle affected by enzootic bovine leukosis

Enzootic bovine leukosis (EBL) is typically observed in cattle over 3 years old. However, some cases of EBL onset in young beef cattle have been reported in Japan. The mechanism for early EBL onset is unclear. In Japan, beef cattle are given large amounts of concentrated feed with low vitamin A. Bone morphogenetic proteins (BMPs) are regulators of cell proliferation, differentiation, and apoptosis, and thought to represent one of the key players in tumor malignancy. The purpose of this study was to evaluate the differences in BMP-6 methylation status between EBL beef cattle under 3 years old and other cattle. We investigated the methylation status of the BMP-6 promoter region in 32 EBL beef cattle under 3 years old. We also compared the methylation status of EBL dairy cattle to that of healthy cattle. Median methylation rate of the BMP-6 promoter region in EBL beef cattle under 3 years old was 8.9%, which was significantly higher than that of other groups. Hypermethylation of the BMP-6 promoter region might contribute to early onset of EBL in beef cattle under 3 years old, and animal feeding management practices specific to beef cattle may affect the methylation status of the BMP-6 promoter region.     

Tumor-associated antigen on bovine leukemia virus-induced bovine lymphosarcoma

Specific tumor-associated antigen (TAA) was detected on enzootic bovine leukosis (EBL) cells by monoclonal antibodies against TAA. One of the monoclonal antibodies, c143, reacted with all EBL tumor cells tested but not with bovine leukemia virus (BLV) antigens. c143 reacted slightly with bovine fetal thymus and mitogen-stimulated lymphocytes from BLV-free cows but not with normal bovine lymphoid cells. TAA may be a good tumor marker of EBL tumor cells. We sacrificed eight TAA-positive but clinically normal animals and examined them in order to elucidate whether or not they had gross or histological tumors. At necropsy, four animals had tumors macroscopically. Three animals had no tumors histologically but had initial lesions showing follicular hyperplasia and the TAA on affected lymph nodes. The one remaining showed medullary hyperplasia in the spleen but there were no findings of tumors. Thus, c143 is a useful tool not only for diagnosing EBL, but also for screening of BLV-infected cattle with potential to develop tumors in the future.     

Seroprevalence of bovine immunodeficiency-like virus and bovine leukemia virus in a dairy cattle herd.

To determine the prevalence of single vs. dual infection with bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV), sera (n = 95) from a dairy cattle herd were analyzed for anti-BIV and anti-BLV antibodies by an enzyme linked immunosorbent assay. Twenty-one percent (20/95) of samples were BIV-seropositive, while 52% (49/95) of the same samples were BLV-seropositive. A significantly greater percentage of BIV-seronegative samples were BLV-seropositive, 57% (43/75), than were BIV-seropositive samples, 30% (6/20). There was no significant correlation between data ranked from least to greatest amount of anti-viral antibody. Five cattle had persistent lymphocytosis (PL); all five were BLV-seropositive and two were BIV-positive. The mean anti-BLV titer was significantly greater in PL cattle, as compared at non-PL cattle, whereas there was no significant difference between the mean anti-BIV titer in PL cattle, as compared with non-PL cattle. These results provide additional information on the seroprevalence of naturally occurring BIV infection, and indicate that BIV can exist independent of other common infectious agents, such as BLV. Further, the results suggest that infection with BIV is not associated with an increased rate of infection with other infectious agents such as BLV.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

Detection of bovine leukaemia virus (BLV) in tissue samples of naturally and experimentally infected cattle

Enzootic bovine leukaemia (EBL) which is caused by the bovine leukaemia virus (BLV) still plays a remarkable role despite a significant success in sanitation programmes. In the Federal Republic of Germany it was not possible to eradicate the disease until today. Sporadically during slaughter or necropsy of cattle neoplastic lesions of the lymphatic tissues are observed that need to be clarified with regard to BLV as etiological agent. Due to the fact that in most instances no serological data are available from the respective animals and blood drawings from the original holdings are not easy to obtain the polymerase chain reaction (PCR) opens new avenues as supplementary diagnostic tool to test unfixed lymphatic tissues for the presence of BLV proviral DNA. Lymph node tissues from 10 naturally or experimentally BLV-infected cattle, which have been monitored virologically and serologically, and tissues from 4 negative animals were processed, DNA was extracted and subjected to PCR to amplify BLV env gene specific sequences. The results show that in cattle with BLV-induced leukosis as well as in cattle, which were clinically healthy and unsuspicious at slaughter or at post-mortem, either with persistent lymphocytosis (PL) or without, BLV proviral DNA could be detected easily in samples of lymphatic tissues and in high concordance with serological data. In this article data from the National and OIE reference laboratory for EBL at the Friedrich-Loeffler-Institut (FLI, Germany) are presented. Elaborated laboratory protocols for processing of tissue samples and performing of BLV-PCR are recommended.     

Thymoma‐associated lymphocytosis in a dog

A 9‐year‐old female spayed English Springer Spaniel was evaluated for a cranial mediastinal mass and lymphocytosis. Flow cytometric immunophenotyping of peripheral blood lymphocytes revealed 97% as CD3 positive, confirming a T‐cell lineage. Additionally, T‐cell subset assessment showed 53.2% to be double‐negative T‐lymphocytes, expressing neither CD4 nor CD8 surface markers. The number of double‐negative lymphocytes in circulation coincided with the number of T‐cell receptor (TCR) γδ‐expressing T‐cells in circulation. Molecular T‐cell clonality analysis of TCR Gamma (TCRG) gene rearrangement showed a polyclonal expansion of T‐lymphocytes. Histopathology confirmed the mass to be a thymoma, supporting the diagnosis of thymoma‐associated T‐cell lymphocytosis. Resolution of the lymphocytosis after removal of the thymoma provided further evidence for this diagnosis. To the authors' knowledge, this case is only the second report of thymoma‐associated peripheral lymphocytosis in the veterinary literature, and is the first to report a confirmed thymoma‐associated peripheral γδ T‐cell lymphocytosis in a dog.     

Study on Proliferation and Apoptosis of EBL Cells Induced by P48 Protein of Mycoplasma bovis

The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung (EBL) cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10 μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation (P<0.01),while 0.1 and 0.5 μg/mL protein concentration had no inhibitory effect (P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.     

牛支原体P48蛋白诱导EBL细胞增殖和凋亡的研究

试验旨在研究牛支原体P48蛋白对胎牛肺（embryonic bovine lung,EBL）细胞增殖和凋亡的影响。收集不同时间段、不同浓度P48蛋白与EBL细胞共孵育的样品,通过MTT法检测细胞增殖率;DAPI染核法观察EBL细胞核形态变化;流式细胞技术检测该蛋白诱导EBL细胞的凋亡率;实时荧光定量PCR检测凋亡标志物的mRNA相对表达变化;Western blotting方法检测Bax和Beclin-1蛋白表达水平。结果显示：当作用时间为72 h,P48蛋白浓度在10 μg/mL时,对EBL细胞增殖有极显著的抑制作用（P<0.01）,在0.1和0.5 μg/mL时对EBL细胞的增殖的抑制作用不显著（P>0.05）;经蛋白诱导12 h细胞核形态未有明显变化,24 h细胞核形态发生皱缩和凝聚,48和72 h细胞核发生碎裂;凋亡标志基因mRNA表达在2和12 h没有明显提高,在24、48、72 h有显著提高,与作用时间呈正相关,同时凋亡相关蛋白Bax和Beclin-1的表达随之显著提高。流式细胞技术结果显示P48蛋白诱导EBL细胞凋亡率为48.44%。综上表明,牛支原体P48重组蛋白能够抑制EBL细胞的增殖,促进细胞凋亡,为进一步揭示牛支原体的致病机制提供参考依据。     

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.     

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.     

HAEMATOLOGICAL INVESTIGATION OF A MULTIPLE CASE LEUCOSIS HERD

Adult cattle in a Queensland dairy herd with a history of deaths from lymphosarcoma were sampled regularly over a 4 year period for the identification of animals with persistent lymphocytosis (PL). Twenty-one of 94 animals that were sampled at least 6 times had PL. At the initial sampling 27% of the animals had lymphocytosis. Culling of haematologically positive animals in the first 18 months of the investigation reduced this to 5.3%, but cessation of the culling programme resulted in a gradual increase in the percentage of animals with lymphocytosis. Four deaths from lymphosarcoma occurred in adult animals, but only in the first 18 months of the investigation. Two of these animals had lymphocytosis and two lymphoblastic leukaemia. The calf of one of the latter cows developed lymphoblastic leukaemia and lymphosarcoma by the time it was 6 months of age. Although histological evidence of lymphosarcoma was lacking in a number of clinically normal animals with lymphocytosis, haematological investigation identified a group of animals within the herd that may develop lymphosarcoma.     

Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle

Lectins, polyclonal antibodies and monoclonal antibodies (MAbs) were evaluated as markers for bovine lymphocytes obtained from healthy animals and from cattle infected with bovine leukemia virus (BLV). In the blood from healthy cattle the proportion of cells identified as T lymphocytes with the lectin Helix pomatia (HP) (67.8 +/- 6.2%) using the indirect immunofluorescence technique was similar to the proportion of cells identified by the MAbs P5 (66.1 +/- 3.8%) and BLT-1 (59.8 +/- 7.1%). The proportion of B cells in blood from healthy animals identified with a polyclonal antibody to bovine IgM (18.0%) was similar to that identified with a MAb to bovine IgM (16.2%). However, greater variation between individual values was detected with the MAb (SD = 8.2) than with the polyclonal antibody (SD = 4.0). In the blood from BLV-infected cattle with persistent lymphocytosis, both the polyclonal and the MAb revealed a threefold increase of B cells. A proportion of the B cells had an increased amount of immunoglobulin molecules in their plasma membrane as indicated by flow cytometry. The proportion of T lymphocytes, identified by the MAb P5, was reduced to one-third of that in non-infected cattle. The indirect HP labelling gave inconsistent results and seems not to detect solely T lymphocytes among blood lymphocytes from BLV-infected cattle.     

Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Spontaneous and mitogen-induced RNA synthesis by blood lymphocytes from bovine leukemia virus-infected and normal cows

Peripheral blood lymphocytes (PBL) from normal and bovine leukemia virus (BLV)-infected cattle were prepared by density gradient technique and incubated with and without phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). RNA synthesis was determined at different periods of incubation by 3H-uridine incorporation. PBL from BLV-infected cows with persistent lymphocytosis (PL) showed the highest spontaneous RNA synthesis. PBL from BLV-infected cows with normal lymphocyte counts synthesized more RNA than cells from normal animals. Decreased mitogen responses were observed in PBL from infected cows with PL in comparison to normal and BLV-infected cattle without PL. PHA and PWM did not show significant differences in their degree of stimulation of RNA synthesis.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

The eradication experience of enzootic bovine leukosis from Lithuania

Before 1985 the situation regarding enzootic bovine leukosis (EBL) in Lithuanian cattle was described only haphazardly. In 1986 serological investigations were initiated together with an eradication programme. The EBL bovine leukosis virus (BLV) situation was monitored by the Institute of Immunology Vilnius University, national and regional veterinary laboratories. Starting in 1986 all EBL-positive cattle were separated from negative cattle into BLV-infected and BLV-free herds. To create the latter, calves were fed pasteurized milk. The seroprevalence in 1990 was 7.29%, but it steadily declined to 0.32% in 2006.     

Seroprevalence of enzootic bovine leukosis in Trakya district (Marmara region) in Turkey

In this study, 481 cattle belonging to 77 farms from nine localities in Trakya district in the Marmara region of Turkey were blood sampled and serologically tested for enzootic bovine leukosis (EBL). Antibodies to bovine leukosis virus (BLV) were detected in 51 cattle sera (11%) belonging to nine farms in five localities. Cattle tested were mostly female Holstein or Brown Swiss of ages ranging between 18 months and 10 years. Analysis of the relationships between age, breed or sex and seropositivity to EBL in seropositive herds indicated no significant associations (p>0.05). The relationship between seropositivity and haematological changes was also studied, and seropositive cattle had higher lymphocyte percentage and lower neutrophil percentage than seronegative cattle (p<0.001).     

A three-year study of enterohemorrhagic Escherichia coli O157 on a farm in Japan

A long-term study was performed on the prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 in bovine faeces. The present study was conducted on heifers raised on a farm showing a high isolation rate of EHEC O157 in previous years. The prevalence of EHEC O157 isolated from faecal samples was 10.6% (222/2104), 5.6% (181/3225), and 5.6% (153/2744) from 1998 to 2000, respectively. The numbers of EHEC O157-positive heifers for the same 3 years were 46.3% (185/400), 36.8% (147/399), and 31.7% (130/410), respectively. The seasonal prevalence of EHEC O157 varied according to the year. Most positive heifers excreted the EHEC O157 only once during the survey, though it was excreted 2 or 3 times by some heifers. The results obtained in the present study showed that the farm examined was heavily contaminated with EHEC O157. It is assumed that EHEC O157 does not remain in individual cattle long-term, but does exist long-term on farms due to repeated infection.