Serum nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHBA) concentrations are used to evaluate energy status in peripartum dairy cows. Blood samples from 37 cows in the week before parturition and 47 cows in the first week after parturition from 3 dairy herds were taken 1h before the first feeding (-1h) as well as 4 and 10h after the first feeding. Nonesterified fatty acid concentrations were measured in samples from cows before calving and BHBA was measured in samples from lactating cows. Mean NEFA concentrations in the prepartum cows were significantly higher at -1h (0.20 mmol/L) than at 4h (0.14 mmol/L), but were not different between 4 and 10h (0.17 mmol/L). Using a cutpoint of NEFA > or = 0.4 mmol/L, 32% of cows had high concentrations at -1h compared with 16% of the same cows at 4 and 10h. There were no differences in mean BHBA between -1h (646 micromol/L) and 4h (596 micromol/L), but mean BHBA was higher at 10h (711 micromol/L) than at -1h. Using a cutpoint of BHBA > or = 1,400 micromol/L, there were no differences in the proportions of high BHBA, which were 9, 11, and 13% of cows at -1, 4, and 10h, respectively. Prandial effects on serum NEFA may affect interpretation of this analyte. In order not to misclassify cows when assessing energy status, samples for NEFA must at least be taken at a consistent time relative to feeding within a given herd. When sampling cows to monitor elements of energy metabolism in the prepartum period, there was twice the probability of detecting animals with NEFA values > or = 0.4 mmol/L if they were sampled 1h before the first feed delivery compared with sampling the same cows 4 or 10h after feeding.

The purpose of this study was to investigate the effects of isolates of noncytopathic type 2 Bovine viral diarrhea virus (ncpBVDV-2) of high and low virulence on the proliferation of bone marrow progenitor cells. Holstein calves 6 to 7 mo old and BVDV-naïve were inoculated intranasally with a BVDV isolate of high virulence (HV24515), a BVDV isolate of low virulence (LV11Q), or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 d, and the mean number of colony-forming unit-granulocyte-macrophage (CFU-GM) colonies was determined. Tritiated (3H)-thymidine uptake by BMMCs was determined to indicate overall proliferative capacity. Virus isolation was done on concurrent samples of BMMCs and peripheral blood. Virus was isolated from BMMCs and peripheral blood buffy-coat cells as early as day 2 or 3 after inoculation. Neutropenia developed in both groups inoculated with a BVDV isolate. However, in the calves given LV11Q, neutrophil counts rebounded earlier in response to increased proliferation of BMMCs, whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased (P = 0.0047) in BMMCs after inoculation compared with before inoculation in the calves given LV11Q but not in those given HV24515 or in the control calves. The median number of CFU-GM colonies was significantly decreased (P = 0.0164) after inoculation compared with before inoculation in the calves given HV24515, whereas there was no significant difference in the calves given LV11Q or in the control calves. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.

Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression, and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.

A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay. Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines. Cultures were prepared with and without 100 ng/mL of rbG-CSF. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF. Both culture media had the highest colony counts on day 5.

To investigate the hematologic abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), calves 6 to 8 mo old were inoculated with an isolate of either high virulence (HV24515) or low virulence (LV11Q); control animals received the same volume of uninfected cell-culture supernatant. Peripheral blood neutrophil, lymphocyte, and platelet counts decreased in all the virus-inoculated calves but were significantly lower and remained decreased longer in the calves given HV24515. For each isolate, a decrease in the number of mature myeloid cells in the bone marrow coincided with the development of neutropenia, but the depletion persisted significantly longer (4 to 6 d) in the calves given HV24515. In the bone marrow of calves given LV11Q, the number of proliferating myeloid cells increased in proportion to the decrease in the number of mature myeloid cells. In the calves inoculated with HV24515, BVDV antigen was observed in bone marrow cells when the peripheral blood counts were lowest. Megakaryocytes were the predominant cell type exhibiting positive BVDV staining; myeloid cells rarely stained positively. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that ncpBVDV-2 isolates of both high and low virulence caused decreased leukocyte and platelet counts, but only the high-virulence HV24515 isolate caused a delay in the production of myeloid proliferating cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.

A retrospective case-control study was conducted based on the records of 80 dogs with visceral haemangiosarcoma (HSA) and 200 dogs with various diseases that had clinical features similar to HSA. All dogs were more than 1 year old, had histologically confirmed disease, and had a complete blood count performed prior to the final diagnosis. A standard protocol was used to count acanthocytes on one blood film from each dog. Acanthocyte count had a maximum diagnostic sensitivity of 53.8% (and specificity of 61.5%) at a cutpoint of ≥1 acanthocyte/2,000 red blood cells. A diagnostic specificity of 100% (and sensitivity of 7.5%) was achieved at a cutpoint of >71 acanthocytes/2,000 red blood cells. The precision of acanthocyte count, within and between raters, varied from poor (unweighted kappa = 0.26) to good (weighted kappa = 0.71) due to the subjective nature of the identification of acanthocytes. Although dogs with acanthocytes were more likely to have HSA (P=0.02), and dogs with HSA had higher acanthocyte counts than controls (P=0.003), acanthocyte count had limited ability to distinguish between dogs with HSA and unaffected dogs with similar signs, as indicated by the receiver operator characteristic plot, which lay approximately along the diagonal. There was no level of acanthocytosis at which HSA could be ruled out, and although HSA could be ruled in at counts >71 acanthocytes/2,000 red blood cells, only six of the 80 dogs with HSA in the study could be identified by this cutpoint.

Bovine leukemia virus (BLV) is a common retroviral infection of cattle. Infection is accompanied by integration of BLV into the host cell genome and is persistent for the life of the individual as is the presence of anti-BLV antibodies. Lymphosarcoma occurs in a small fraction of infected adult individuals but otherwise there is little or no associated disease. Viremia is undetectable, however, BLV is expressed readily once infected cells are cultured in vitro. A sandwich enzyme-linked immunosorbent assay (sELISA) was optimized, using murine monoclonal antibodies, to quantify the major internal structural protein (p24) produced in short-term cultures of peripheral blood mononuclear cells (PBMCs). Optimal production of BLV p24 was achieved utilizing RPMI supplemented with 10% fetal bovine serum (FBS), pH 7, and 5 x 10(6) cells per ml. Cultures were terminated at 24 h. The sELISA was linear between 30 and 900 ng/ml and the limit of detection was 1.2 ng/ml. At three concentrations of p24, intra- and inter-assay coefficients of variation (CV) varied between 9.2 and 13.3 and 5.1 and 12.9%, respectively.

Cancer chemotherapy agents cause damage in the bone marrow, resulting in leukopenia during treatment and secondary cancers after recovery from the original disease. We created an experimental model of alkylation-based chemotherapy using ethylnitrosourea (ENU) to investigate the effect of niacin status on cancer induction. For 4 wk, nontumor-bearing weanling Long-Evans rats were fed niacin-deficient (ND) diets or were pair-fed (PF) identical quantities of a niacin-adequate diet. One week after the initiation of niacin feeding protocols, ENU treatment began (12 doses, 30 mg/kg by gavage, every other day). At the end of dietary modulation and ENU treatment, all rats were fed a high quality control diet and monitored for weight loss (>5%) and palpable tumors (>1cm), at which point they were necropsied for the presence of disease. The morbidity curves were significantly different; ND rats reached 20% morbidity 10 wk earlier than PF rats. In the first 20 wk after ENU treatment, ND rats developed 17 malignancies, including 11 leukemias, whereas PF rats developed 3 malignancies with 2 leukemias. In the end, there was a 47% greater average number of malignancies in ND vs. PF rats, despite a more rapid onset of morbidity. In short-term studies, niacin deficiency caused an 80% decrease in bone marrow NAD(+). Basal poly(ADP-ribose) levels were dramatically reduced by niacin deficiency. A single dose of ENU increased poly(ADP-ribose) levels fivefold in PF rats, whereas levels in ND rats remained 90% lower. Niacin deficiency did not alter the initial accumulation of DNA damage, indicating that drug metabolism is not an underlying factor in the diet-induced changes. These data show that niacin deficiency alters poly(ADP-ribose) metabolism in the bone marrow and increases the risk of nitrosourea-induced leukemias.

Many chemotherapeutic agents function by damaging the DNA of rapidly dividing cells, leading to side effects in the bone marrow, including anemia and leukopenia during chemotherapy and the development of secondary leukemias in the years following recovery from the original disease. We have created an animal model of alkylation-based chemotherapy, in nontumor-bearing rats, to investigate the effect of niacin deficiency on the side effects of chemotherapy [2 x 2 design, niacin-deficient (ND) vs. pair-fed (PF) control, and ethylnitrosourea (ENU) vs. vehicle control (C)]. Weanling Long-Evans rats were fed ND diet or PF niacin replete diet for 4 wk. ENU or C treatment started after 1 wk of feeding and consisted of 12 doses delivered by gavage, every other day. At 4 wk postweaning, niacin deficiency and ENU treatment ended, the rats were fed a high-quality control diet (AIN-93M) and the recovery of blood variables was monitored. ND alone decreased growth rate and caused anemia and neutrophilia. ENU treatment alone caused anemia, lymphopenia, neutropenia and an increase in circulating reticulocytes. In combination, ND and ENU treatment synergistically decreased hematocrit. ND prevented the ENU-induced increase in reticulocyte numbers observed in control rats. ND also increased the severity of ENU-induced lymphopenia. A combination of ND and ENU abolished the neutrophilia caused by ND alone. In summary, ND significantly increased the susceptibility of young Long-Evans rats to ENU-induced bone marrow suppression, suggesting that niacin-deficient cancer patients may benefit from supplementation.

ABSTRACTA 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.

A rapid method for the isolation of endothelial cells from canine adrenal glands is described. The technique is based on collagenase digestion of adrenal glands following mechanical tissue disruption. The endothelial cells attach and spread onto plastic petri dishes coated with a commercial attachment factor approximately one hour after seeding. Duration to first passage was approximately one week. Doubling times were 2-3 days. Cells were positively identified with immunoperoxidase staining for von Willebrand factor antigen.

The National Cancer Institute Working Formulation (NCI-WF) for the subjective classification of human non-Hodgkin's lymphoma is readily applicable to the classification of bovine lymphomas. Forty-nine cases of bovine lymphoma were analysed morphometrically to see if nuclear size and cleavage were distributed continuously or discretely between different NCI-WF tumour cell types. The mean nuclear area (±standard error of the mean, SE) was significantly greater (P<10−6) in cell from the different types of diffuse largecell lymphoma than in cells from the different types of small-cell lymphoma (42·91±1·21 μm2 vs 19·33±1·08 μm2, respectively); there was no overlap between the two groups. The mean nuclear area (±SE) of cells from diffuse large-cell lymphomas was significantly greater (P<10−4) than that of cells from small non-cleaved lymphomas (42:74±1·72 μm2 vs 27·54±1·08 μm2, respectively), and there was again no overlap between the two groups; these two cell types are difficult to distinguish by any criteria other than size. Additionally, the cell-to-cell variability in nuclear area within a given tumour was significantly greater (P<0·001) for the diffuse large-cell type than for the small non-cleaved cell type. The mean nuclear form factor (±SE) and mean nuclear contour indices (±SE) of the diffuse large cleaved cell type (0·53±0·02 and 5·08±0·11, respectively) were significantly different (P<0·01 to 10−6) from the same parameters in the diffuse large-cell type (0·82±0·01 and 3·94±0·04, respectively). Some of the major criteria of the NCI-WF used subjectively to discriminate between bovine lymphoma cell types were supported by morphometric measurements. The magnitude of the differences in nuclear morphological characteristics between bovine lymphoma cell types was such that there was no overlap.

We found the rate of bovine immunodeficiency-like virus (BIV) infection among bulls to be 9.6% using serology and 12.6% when tested by the polymerase chain reaction (PCR) for the presence of BIV provirus in peripheral blood leukocytes. Previously, we determined the frequency of BIV infection among the general dairy cow population in Ontario to be 5.5% based on serological analysis. Apparently, serological testing detects only 77% of BIV-infected bulls. Since almost a quarter of BIV-infected bulls may be seronegative, it is recommended that the PCR test be used to identify BIV-infected individuals. It is clear from the data presented here and supported by experimental studies that the latent carrier state is a relatively common outcome in ruminants infected with BIV.

A prospective analysis of 38 cases of bovine lymphoma was performed. Thirty-five of the lymphomas were considered enzootic because of serologic or molecular biologic association with the bovine leukemia virus (BLV). The three remaining cases were in cattle <1 year of age and were not associated with BLV and hence were considered sporadic lymphomas. Utilizing the National Cancer Institute Working Formulation for the morphologic classification of human non-Hodgkin's lymphoma, 54.3% (19/35) of the enzootic lymphomas were classified as the diffuse large cleaved cell type. The cells of the 35 enzootic lymphomas expressed a consistent immunophenotype represented by MHCII+, γ-heavy chain+, and λ-light chain+; this constellation of markers indicated a mature B-cell phenotype. The three sporadic lymphomas failed to express MHCII antigen despite the presence of other B-cell antigens. There was no correlation between morphologic cell type and immunophenotype in these bovine lymphomas.

X-linked hereditary nephritis (HN) in Samoyed dogs is a model for human HN (Alport's syndrome). Angiotensin converting enzyme (ACE) inhibitors have been shown to slow the progression of renal disease in animal models and human patients. To determine the effect of ACE inhibitor treatment on X-linked HN in Samoyed dogs, a group of affected and a group of normal males were each randomly divided into two subgroups, which were either treated with an ACE inhibitor or left untreated. ACE inhibitor treatment caused significant increases (P < 0.05) in plasma renin activity in normal and affected dogs, confirming its effectiveness, but did not lower systemic blood pressure. Three of four affected treated dogs had improved weight gains and, overall, treated dogs survived 1.36 times longer than affected untreated dogs (P < 0.05). ACE inhibitor treatment of affected dogs significantly delayed (P < 0.05) the onset of an increase in serum creatinine concentration, tended to delay the decline of glomerular filtration rate and effective renal plasma flow (ERPF), significantly improved (P < 0.05) the ERPF at 110-154 days of age, and significantly slowed (P < 0.01) the rate of increase of proteinuria. Affected treated dogs showed a significant (P < 0.05) transient reduction in glomerular basement membrane splitting. Thus, ACE inhibitor treatment of Samoyed dogs with X-linked HN produced beneficial effects with respect to renal function, renal structure, and survival.

Six yearling sheep were inoculated intraperitoneally with peripheral blood from two sheep infected with the bovine immunodeficiency-like virus (BIV) strain R29. An additional five sheep served as sham-inoculated controls. Of the six sheep given BIV, five seroconverted, one of them remaining seropositive for the duration of the study. The polymerase chain reaction demonstrated BIV provirus in three of the five serologically positive animals. At necropsy, 1 year after inoculation, histological changes were found only in the lymphoid tissues. In sheep exposed to BIV, mild though significant increases were seen in the (1) number of splenic periarteriolar lymphoid sheaths, (2) number of secondary follicles in hilar and prescapular or popliteal lymph nodes, and (3) medullary sinus cellularity in prescapular and popliteal lymph nodes.

Biopsy and necropsy specimens, comprising 107 primary carcinomas and three mesenchymal tumours, were reviewed from 110 dogs with cancer of the bladder, urethra, or both. Histological classifications developed for the assessment of human bladder cancer were found to be readily applicable to the dog. These classifications are based on histological features, including the pattern of growth, the cell type, the grade of transitional tumour and the depth of invasion of the bladder wall. Features associated with localized disease in canine transitional cell carcinoma included papillary architecture, ''in-situ'' tumour, low tumour grade and a strong peritumoral lymphoid cell reaction.Features of tumours with metastasis included infiltrating and non-papillary architecture, increasing tumour grade, depth of invasion, vascular invasion and presence of peritumoral fibrosing reaction. Wide variability was found within single tissue samples, indicating that multiple sample sites are necessary for the adequate characterization of a given lesion.Statistically significant correlations were found between: tumour grade and depth of invasion (P<0.0001); tumour grade and presence of metastases (P<0.029); and peritumoral desmoplasia and metastases (P<0.029).It was concluded that canine bladder cancer could be classified for the purpose of clinical management with a modified World Health Organization system as developed for human tumours. (C) 1995 Academic Press Limited

Of the 920 cows tested, 56.7% showed antiretroviral serological reactivity. Prevalence rates (95% confidence interval) of antiretroviral antibodies among individual dairy cows in Ontario were: BIV 5.5% (4.0-7.0), BLV 25.7% (22.9-28.6), and BSV 39.6% (36.4-42.8). The following percentages of cows showed serological reactivity against the specified retroviruses: BIV 2.3%, BLV 14.0%, BSV 27.5%, BIV and BSV 1.3%, BIV and BLV 0.9%, BLV and BSV 9.9%, BIV and BLV and BSV 0.9%. These rates of sero-positivity are similar to those found in other countries. Serological test results were not adjusted for sensitivity and specificity. The prevalence rates of antibodies to the three retroviruses (BIV, BLV, and BSV) were significantly different, but no associations were observed between specific retroviral serological test results among individual cows. The prevalence rates of BIV and BSV seropositivity were constant across Ontario, whereas, there was a significant trend for the prevalence rate of BLV seropositivity to decrease going from southwestern to eastern Ontario; cows in eastern Ontario had approximately half the prevalence rate of those in southwestern Ontario. Cows that were seropositive for BSV were significantly older than BSV seronegative cows. There was no association between culling rate and BSV serology. Significant negative associations were found with winter or summer housing of calves separate from adults and summer outdoor exercise for dry cows. The use of calf hutches in the summer had a significant positive association with BSV seropositivity. Regression analyses were done to assess the association of retroviral (BIV, BLV, and BSV) seropositivity on calving interval, milk somatic cell count, and milk production. Serological test results for BIV, BLV, and BSV were entered into all models and all models were adjusted for intra-cluster (intraherd) correlation. Herd size and age were found to be important confounding variables. BIV seropositivity was not associated with any changes in production using this approach, however when considered in isolation BIV seropositivity remained associated with decreased milk production. BLV seropositivity was significantly associated with longer calving intervals and higher somatic cell counts in older cows. As well, in older cows, BSV seropositivity was significantly associated with higher milk production.