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The primary objectives of this study were to describe the prevalence and risk factors for Cryptosporidium spp. and Giardia spp. infection in cows and calves during the calving season in western Canadian cow-calf herds. Through the calving season of 2002, fresh fecal samples were collected from 560 beef cows and 605 calves in western Canada. Feces were examined for the presence of Cryptosporidium spp. and Giardia spp. using a quantitative sucrose gradient immunoflourescent antibody test. Samples were collected from mature cows on 59 farms and from calves on 100 farms. Only 1.1% (5/560) of the cows were positive for Cryptosporidium spp. whereas 3.1% (19/605) of the calves were positive. Prevalence for Giardia spp. was much higher; Giardia spp. was detected in 17.0% (95/560) of the cow and 22.6% (137/605) of calf fecal samples. Data describing herd management practices, treatment and disease history, age, gender, breed and fecal consistency were gathered to assess potential risk factors associated with shedding. The association between the risk of shedding and average precipitation from December to June and ecological region were also evaluated. Risk factors for infection with Cryptosporidium spp. in either cows or calves could not be evaluated because the multilevel model would not converge due to the relatively low prevalence of the organism in this sample. The prevalence for Giardia spp. was sufficient to explore potential risk factors in both cows and calves. No risk factors were identified for Giardia spp. in beef cows following calving. After the construction of a multivariable model, the only significant predictors for Giardia spp. presence in beef calves was dam age and calf age. Calves born to 2-year-old heifers were 2.3 (95% CI, 1.09-5.06; P = 0.031) times more likely to be shedding Giardia spp. then calves born to cows that were 4-10 years of age. Calves that were 9-18 days of age and calves that were > 18 days of age were 22.4 (95% CI, 5.88-88.18; P < 0.001) and 150 (95% CI, 39.72-603.19; P < 0.001) times more likely, respectively, to be shedding Giardia spp. than calves < or = 4 days of age.

Neospora caninum, infectious bovine rhinotracheitis (IBR), and bovine viral diarrhea virus (BVDV) are important differentials for the diagnosis of infectious reproductive loss in beef herds. The objective of this study was to describe the serological status of both pregnant and non-pregnant beef cows from herds with varying levels of reproductive success. The study provided an opportunity to examine whether there were any associations between serological status for BVDV, IBR, and N. caninum and pregnancy status, as well as the subsequent risk of abortion, or stillbirth. Samples were collected from 2516 cows and heifers from 66 herds; 31 herds where the proportion pregnant was <90% and 35 randomly selected herds where the proportion pregnant was > or =90%. Of these samples 5.9% were positive for antibodies to N. caninum, 20.4% had titres >1:80 to IBR, 91.8% had titres > or =1:256 to BVDV type 1, and 23.9% had titres > or =1:256 to BVDV type 2. N. caninum antibody concentration was associated with an increased individual animal risk of non-pregnancy (OR(logS/P), 1.9; 95% CI, 1.2-2.9) and abortion (OR(pos/neg), 2.8; 95% CI, 1.1-7.5). The proportion of animals at pregnancy testing with antibodies to BVDV type 2 above 1:3000 (OR(10%changeinprevalence), 2.3; 95% CI, 1.5-3.5) was also associated with an increased risk of abortion. No other measures of antibody status were associated with reduced reproductive performance in this group of herds. Antibodies to Mycobacterium avium spp.paratuberculosis were also measured; 0.7% of samples were positive (sample to positive (S/P) >0.25) and 3.6% were suspicious (S/P, 0.10-0.25).

In transfection protocols, the expression levels of the transgene is important to define, still is difficult to obtain in certain cell lines such as those derived from T-lymphoma cells. In this study we evaluate transgene expression kinetics in the presence and absence of two well known transcription activators such as phorbol-12-myristate13-acetate (PMA) and Ionomicin (IO). Three murine T lymphoma cell lines (LBC, EL4 and BW5147) were transfected by electroporation using green fluorescent protein (GFP) as a reporter gene and analyzed by flow cytometry. Addition of PMA/IO resulted in a significant increase of the Mean Fluorescence Intensity but not in GFP-positive cell percentages, either in transient or stable transfected LBC and EL4 cells. Remarkable, BW5147 cells showed low GFP induction with a significant increment only in stable transfected cells. Our results demonstrated that CMV promoter activity can be enhanced in transfected lymphoma cells by PMA/IO suggesting that transgene expression levels can be optimized by means of the use of transcription activators.

Objective: To determine whether a combination viral vaccine containing modified-live bovine herpesvirus-1 (BHV-1) would protect calves from infection with a recent field isolate of BHV-1., Design: Randomized controlled trial., Animals: Sixty 4- to 6-month-old beef calves., Procedure: Calves were inoculated with a placebo 42 and 20 days prior to challenge (group 1; n = 10) or with the combination vaccine 42 and 20 days prior to challenge (group 2; 10), 146 and 126 days prior to challenge (group 3; 10), 117 and 96 days prior to challenge (group 4; 10), 86 and 65 days prior to challenge (group 5; 10), or 126 days prior to challenge (group 6; 10). All calves were challenged with BHV-1 via aerosol. Clinical signs, immune responses, and nasal shedding of virus were monitored for 14 days after challenge., Results: Vaccination elicited increases in BHV-1-specific IgG antibody titers. Challenge with BHV-1 resulted in mild respiratory tract disease in all groups, but vaccinated calves had less severe signs of clinical disease. Extent and duration of nasal BHV-1 shedding following challenge was significantly lower in vaccinated calves than in control calves. In calves that received 2 doses of the vaccine, the degree of protection varied with the interval between the last vaccination and challenge, as evidenced by increases in risk of clinical signs and extent and duration of viral shedding., Conclusions and Clinical Relevance: Results suggest that this combination vaccine provided protection from infection with virulent BHV-1 and significantly reduced nasal shedding of the virus for at least 126 days after vaccination.

Tissue sets from 36 snatch-farrowed colostrum-deprived (SF/CD) and 71 Caesarian-derived gnotobiotic swine infected with porcine circovirus type 2 (PCV-2) as neonates were examined and scored for the types and tissue distribution of histologic lesions associated with this viral infection. The occurrence and severity of these lesions were correlated with qualitative and quantitative determinations of viral burden in tissues by immunohistochemistry (IHC) and tissue titrations for infectious virus, respectively. These measures were, in turn, related to 1 of 3 categories of clinical disease expressed in PCV-2-infected swine as subclinical infection, preclinical postweaning multisystemic wasting syndrome (PMWS), and clinically evident PMWS, respectively. Statistically significant (P < 0.05 to 0.001) associations between both measures of viral burden, the severity of histologic lesions and the stage of disease were obtained. Discrimination between and among categories of disease was best accomplished by a combination of IHC and histopathology. The results of this study confirm that viral burden in PCV-2-infected tissues, specifically lymphoid tissues and liver, directly correlate with severity of clinical disease expression in PCV-2 infected swine.

The objective of this study was to describe a multiyear outbreak of calf diarrhea in an Alberta cow-calf herd and the impact of severe diarrhea on calf productivity. A retrospective analysis was performed through the use of detailed individual animal records and laboratory reports. The most significant laboratory finding was Salmonella Typhimurium isolated from the fecal samples of 2 ill members of the treatment crew and from 3 calves on postmortem examination. In 2002, at the peak of the outbreak, 90.3% (325/360) of the calves required treatment and 8.9% (32/360) of the calves died. In 2003, the severity of the problem had declined with only 20.9% (47/225) of the calves requiring treatment and 3.1% (7/225) of the calves dying. In both years, the weaning weights of treated calves were significantly reduced compared with those of nontreated calves. For calves weaned in 2002, after adjusting for the effect of calf sex, calves treated more than 6 times had 200-day adjusted weaning weights that were 15.2 kg (95% CI; 5.8 to 24.4 kg) lighter than of those calves treated only once or not at all (P = 0.0015, n = 321). For calves weaned in 2003, calves were 5.5 kg (95% CI; 0.23 to 10.8, P = 0.04, n = 207) lighter per treatment day with electrolytes, when calf sex and dam age were controlled for. Assuming that increased days of treatment or number of treatments is representative of disease severity, long-term calf performance is negatively affected by severe calfhood disease. The estimated lost revenue and treatment expenses, excluding the cost of labor, cow feed, and maintenance, was $22 800 in 2002 and $1589 in 2003.

The objective of this study was to determine whether a commercially available, saponin-adjuvanted, inactivated bovine respiratory syncytial virus (BRSV) vaccine would protect calves from experimental infection with virulent BRSV. This was a randomized controlled trial comprising 14, 8- to 9-week-old calves seronegative for BRSV Group 1 calves (n = 8) were not vaccinated and group 2 calves (n = 6) were vaccinated on days 0 and 19 with an inactivated BRSV vaccine. All calves were challenged with virulent BRSV on day 46. Clinical signs, arterial PO2, and immune responses were monitored after challenge. Calves were euthanatized on day 54 (8 d after challenge) and lungs were examined for lesions. Vaccination elicited increases in BRSV-specific immunoglobulin (Ig) G and virus neutralizing antibody titers. Challenge with BRSV resulted in severe respiratory tract disease and extensive pulmonary lesions in control calves, but no signs of clinical disease and minimal or no pulmonary lesions in vaccinated calves. Arterial blood oxygen values on day 53 (7 d after challenge) in control calves were significantly lower than those in vaccinated calves, which remained within normal limits. Control calves shed BRSV for several days after challenge, whereas BRSV was not detected on deep nasal swabs from vaccinated calves. In summary, the results indicated that this inactivated BRSV vaccine provided clinical protection from experimental infection with virulent virus 27 d after vaccination and significantly decreased the prevalence and severity of pulmonary lesions. Efficacy was similar to that reported for other commercial inactivated and modified-live BRSV vaccines.

Background: While cyclosporine A (CsA) is an effective therapy for nephrotic syndrome, it has nephrotoxic side effects. We compared the anti-proteinuric effects and nephrotoxicity in rats with passive Heymann nephritis (PHN) of CsA and mycophenolate mofetil (MMF)., Methods: PHN was induced in female Wistar rats. Two treatment groups consisting of 8 rats each received either 25 mg of CsA or 25 mg of MMF/kg body weight/day and were compared with untreated controls. Kidney function and proteinuria were monitored over 4 weeks. Western blots were used for densitometric analysis of renal cyclooxygenase-2 (COX-2) protein expression. Thromboxane B2 (TxB2) and 6-keto-PGF(1alpha) were determined by radioimmunoassays (RIAs) in renal tissue and urine., Results: Rats with PHN exhibited a marked proteinuria of 12.76 +/- 4.42 vs. 0.73 +/- 0.28 mg/24 h (p < 0.01) and showed increased glomerular concentrations of TxB2 and 6-keto-PGF(1alpha) (992.6 +/- 216.9 and 1,187.0 +/- 54.2 pg/mg protein, respectively) compared with healthy controls (595 +/- 196.17 and 729 +/- 297.84, respectively) and a strongly induced COX-2 protein expression. CsA and MMF treatment reduced PHN-related proteinuria to 2.10 +/- 1.47 and 1.47 +/- 7.2 mg/24 h, respectively. In rats with PHN, CsA induced a significant deterioration of renal function and enhanced urine excretion of thromboxane A2, paralleled by a significant, twofold increase in COX-2 protein expression and renal prostaglandins. By contrast, MMF treatment in rats with PHN was not nephrotoxic and had no effect on prostaglandin production. COX-2 protein expression under MMF was suppressed., Conclusion: While the antiproteinuric efficacy of MMF and CsA in PHN was comparable, the absence of nephrotoxicity might favor MMF in the treatment of nephrotic syndrome. The CsA-induced increase in COX-2 expression and COX-2-dependent prostacyclin may indicate a mechanism that compensates nephrotoxicity in the diseased and CsA-exposed kidney.

Vertical fissures (sandcracks) affect approximately 20% of adult beef cattle in western Canada. The risk factors associated with the formation of these lesions are not well understood. This paper describes a case-control study that identifies the role of increased claw size (claw volume > 390 cm3, odds ratio 7.8) in the formation of the lesion. No connection was found between vertical fissures and horn hydration status. However, horn hydration was found to vary significantly between samples collected in the summer and winter, the latter samples having significantly reduced moisture content. The importance of these findings and the implications for the prevention of fissure formation are discussed.

During 1999, serum samples were collected from beef cows on pastures in western Canada. Some of the herds had a history of confirmed abortions associated with Neospora caninum infection. All these samples were initially analyzed using a single application of 1 common commercial enzyme-linked immunosorbent assay (ELISA) for antibodies to N. caninum. From these initial results, 239 positive and 250 negative samples were randomly selected for further testing. This group of samples was retested using the 3 commercially available ELISA tests for N. caninum as per the manufacturer's recommendations. The agreement between 2 of the ELISAs was good (K = 0.76); agreement of these 2 tests with the third test was much lower (K = 0.46 and 0.60). Quantitative agreement between tests measured by intraclass correlation coefficients was also acceptable between the first 2 tests but was almost zero when the first 2 tests were compared with the third. This information is necessary to understand the differences in seroprevalence reported in different regions from laboratories using different methods.

Background: In experimental glomerulonephritis, inhibition of cyclooxygenase 2 (COX-2) enhances the renocortical expression of pathogenic alpha(v) integrins., Aims: To study whether this effect is mediated by prostaglandin E(2) (PGE(2)) acting through its EP(3) receptor in cultured rat mesangial cells (MCs)., Methods: MCs were incubated with lipopolysaccharide (LPS), celecoxib, PGE(2), or the selective EP(3) agonist, MB28767. The expression of COX-2, EP(3), and alpha(v) integrin mRNA was measured by reverse transcriptase polymerase chain reaction., Results: LPS upregulated COX-2 expression 2.8-fold and alpha(v) integrin expression twofold. The COX-2 inhibitor celecoxib increased alpha(v) integrin mRNA expression twofold. Both exogenous PGE(2) and the specific EP(3) receptor agonist, MB28767, reduced constitutive alpha(v) integrin mRNA expression to half normal values. COX-2 dependent PGE(2) suppressed the expression of alpha(v) integrin mRNA mediated by the EP(3) receptor in MCs., Conclusions: These results suggest that COX-2 suppresses the expression of alpha(v) integrins by an increased production of PGE(2) activating its EP(3) receptor in glomerulonephritis.

A study was conducted in Alberta to determine the seroprevalence of Neospora caninum in feedlot calves purchased from various auction markets throughout western Canada. Four feedlots (1 feedlot from each of the Airdrie and High River areas and 2 feedlots from the Strathmore area) were selected for sampling. At each feedlot, a random 10% sample of feedlot steer and bull calves entering the feedlot from September 2001 to December 2001 were enrolled in the study until there were a maximum of 500 animals enrolled per feedlot. Blood samples were collected from 1976 male animals at the time of entry to the 4 study feedlots. The animals represented 375 groups purchased from 70 sale points throughout British Columbia, Alberta, Saskatchewan, and Manitoba. Of the 1976 animals tested, 128 animals tested positive for antibodies to N. caninum. The prevalence and adjusted 95% confidence limits for N. caninum in beef calves on entrance to the feedlot in western Canada were 6.5% (95% CI, 5.1 to 8.2). There were no significant (P > or = 0.05) associations between the risk of treatment, the risk of designation as "chronic," and the risk of death and antibodies to N. caninum either before or after adjusting for feedlot, entry weight, entry date, and clustering of disease within lots at each feedlot. In addition, there was no significant (P > or = 0.05) association between serological status and feedlot entry weight or average daily gain. Note that there was no information available on feed conversion because the calves were mixed within existing commercial feedlot pens and the actual feed intake of each animal could not be determined. Adjustment for the concentration of antibodies to bovine viral diarrhea virus on arrival did not change any of the examined associations between N. caninum status and calf health or performance. The results of this study demonstrated that the prevalence of N. caninum in feedlot calves in western Canada was less than the prevalence reported in the United States. Additional studies are required to determine whether the inferior rate of gain and feed efficiency observed in the southern United States with animals testing positive for antibodies to N. caninum also occurs under the management conditions used in western Canada.

Two experiments were conducted with 24 bulls in which semen collection was attempted by transrectal massage (RM) and electroejaculation (EE). In experiment 1, bulls received the following treatments on successive semen collection days: saline 10 min prior to electroejaculation (control); saline 10 min prior to 2 min of transrectal massage followed by electroejaculation; cloprostenol (CLO) 10 min prior to 2 min of transrectal massage followed by electroejaculation; oxytocin (OXY) 10 min prior to 2 min of transrectal massage followed by electroejaculation. Transrectal massage consisted of general, back and forth motion over the ampullae, prostate and urethra with a flattened hand. In experiment 2, bulls received saline (control), oxytocin, or cloprostenol 10 min before attempting semen collection by transrectal massage. Massage was applied specifically to the ampullae for a maximum of 5 min or until a semen sample was obtained. Electroejaculation was attempted in all bulls following transrectal massage. In experiment 1, semen was obtained in <1% of bulls by transrectal massage. However, by using an improved massage technique in experiment 2, semen was obtained in 97.2% of attempts. Semen was obtained in 96.9 and 98.9% of attempts by electroejaculation in experiments 1 and 2, respectively. Oxytocin treatment increased the time to penile protrusion during electroejaculation in experiment 1 and during massage in experiment 2. In experiment 1, oxytocin decreased the time to semen emission and tended to decrease the number of electroejaculation stimuli to semen emission. Cloprostenol treatment, in experiment 1, resulted in an increased number of electroejaculation stimuli to penile protrusion, but did not affect the number of stimuli required for semen emission. Massage of the ampullae prior to electroejaculation reduced both the time to semen emission and the number of electroejaculation stimuli required for semen emission. Transrectal massage of the ampullae was very effective in this experiment for producing semen emission, but quantity of semen samples was less than for electroejaculation. The usefulness of transrectal massage for semen collection in breeding soundness evaluations needs to be investigated further under field conditions.

Background: Renal cyclooxygenases (COX) produce the prostaglandins (PG) E2, I2 and thromboxane (TxA2), which interact with distinct G protein-coupled receptors. We investigated the expression of the three EP receptors EP2, EP3 and EP4 and the receptors for PGI2 (IP) and TxA2 (TP) in rats with passive Heymann nephritis (PHN). We studied their regulation by COX-2 inhibition with celecoxib., Materials and Methods: Four groups of Wistar rats were studied: healthy rats (group A), healthy rats treated with celecoxib (group B), rats with PHN (group C), and rats with PHN receiving celecoxib (group D). Expression of the mRNA for all receptors in the renal cortex and for the EP3 receptor in cultured mesangial cells (MCs) was determined by semiquantitative reverse transcriptase polymerase chain reaction. Stable prostaglandin metabolites were measured in the urine by radioimmunoassay., Results: Rats with PHN (group C) showed an 1.8-fold increase of cortical EP3 receptor mRNA expression as compared with controls (group A). In celecoxib-treated PHN rats (group D) the mRNA expression of the EP3 and EP4 receptors was significantly reduced to 1.0-fold and 0.7-fold induction, respectively. Furthermore, the excretion of bicyclo-prostaglandin E2 (PGE2) was inhibited by celecoxib. No changes were observed in the expression of the other PG-receptors. In cultured MC, PGE2 enhanced the EP3 mRNA expression., Conclusions: These data suggest a predominant role of the EP3 receptor in the transduction of PGE2-actions in PHN. It was concluded that COX-2-dependent PGE2 is able to potentiate its effects in the kidney by up-regulating its own receptors.

We studied the role of IL-2, IL-15, IL-10, TNF and IL-2 receptor complexes (IL-2R) produced constitutively by a T-cell lymphoma line (LBC) on their own proliferation. The constitutive expression of surface alpha, beta and gamma chains IL-2R was detected in tumor cells by flow cytometry. Using reverse-transcription PCR, mRNA for IL-2, IL-15, IL-10 and TNF were found to be present in LBC. In addition, tumor cells were found to constitutively express intracellular IL-2, IL-15, IL-10 and TNF. Despite the production of these cytokines by tumor cells, specific neutralising antibodies did not inhibit LBC proliferation; surprisingly, anti-IL-15 increased LBC cell growth. We also demonstrated that recombinant IL-2 or IL-15 enhanced LBC cell proliferation. Our data suggest that endogenous IL-2 and IL-15 may trigger the proliferation of lymphoma LBC cells, and so their growth could be regulated, at least partly, by IL-2/IL-15/IL-2R system. In addition, IL-10 and TNF, immunosuppressor and pro-metastatic cytokines, respectively, may promote the in vivo growth of the tumor. The fact that leukaemia-lymphoma cells produce simultaneously both IL-2 and IL-15 should be taken into consideration in the design of immunotherapy protocols directed to IL-2R.

Allelic variation (C to T transition that results in an Arg25Cys) in the leptin gene has been associated with increased fat deposition in beef cattle. We report that this same genetic variant is also present in dairy breeds. Body fat reserves play an important role in sustaining high milk production in early lactation, when energy intake is limited. To test for an association between the leptin single nucleotide polymorphism and milk productivity, we genotyped 416 Holstein cows and compared lactation performance data using a mixed model. Animals homozygous for the T allele produced more milk (1.5 kg/d vs. CC animals) and had higher somatic cell count linear scores, without significantly affecting milk fat or protein percent over the entire lactation. The increase in milk yield is most prominent in the first 100 d of lactation (2.44 kg/d), declining to 1.74 kg/d between 101 and 200 d in lactation. The milk yield advantage, observed in cows homozygous for the T allele, could represent a major economic advantage to dairy producers.

The analgesic efficacy of an epidural morphine/mepivacaine combination alone versus epidural morphine/mepivacaine in combination with meloxicam administered prior to the onset of anesthesia was assessed in 20 dogs undergoing cranial cruciate ligament repair. Numerical and visual analog pain scores were performed prior to anesthesia and at 6, 8, 12, 16, and 24 hours after epidural administration by a trained observer, blinded to treatment. An analgesiometer was used to determine the amount of pressure required to produce an avoidance response at the incision site. Animals that received meloxicam demonstrated a trend toward decreased pain scores over all time periods. Visual analog pain scores tended to be lower in dogs receiving meloxicam across all time periods, with a significant interaction between time and visual analog score at 6 and 8 hours (P < 0.05). No dogs receiving meloxicam required rescue analgesia, while 3 of 10 dogs in the epidural only group required rescue analgesia. Administration of meloxicam in addition to epidural morphine plus mepivacaine conveys improved analgesia as compared with epidural alone. Postoperative analgesia is reliably maintained for 24 hours following administration.

This study was designed to compare the analgesic effects of butorphanol with those of meloxicam following ovariohysterectomy. Fifteen dogs were premedicated with 0.05 mg/kg body weight (BW) of acepromazine by intramuscular (IM) injection, plus 0.2 mg/kg BW of meloxicam by subcutaneous (SC) injection. Fifteen dogs were premedicated with 0.05 mg/kg BW of Acepromazine, IM, plus 0.2 mg/kg BW of butorphanol, IM. Anesthesia was induced with thiopental, and dogs were maintained on halothane. All pain measurements were performed by 1 experienced individual, blinded to treatment. Pain scores and visual analogue scales (VAS) were performed at 2, 3, 4, 6, 8, 12, and 24 hours postpremedication. An analgesiometer was used to determine the pressure required to produce an active avoidance response to pressure applied at the incision line. Pain scores, VAS, and analgesiometer scores were analyzed by using a generalized estimating equations method. A significance level of P < 0.05 was considered significant. Animals that received meloxicam demonstrated significantly lower pain scores and VAS than did animals that received butorphanol in the first 12 hours after surgery. Results of this study suggest that meloxicam will produce better postoperative analgesia than will butorphanol. Mucosal bleeding times were performed on cooperative animals in the study group (11 butorphanol, 13 meloxicam). Bleeding times were performed prior to premedication, 6 hours following premedication, and 24 hours after premedication. The 6- and 24-hour readings were compared with baseline bleeding times by using a paired t-test with a Bonferroni correction (a significance level of P < 0.025). Bleeding times did not change significantly over time.

The TNF-family molecule, RANKL, is a key regulator of bone remodeling and essential for the development and activation of osteoclasis. Bone involvement signals diesease activity in non-Hodgkin's lymphoma and influences the progenesis. The molecular mechanism and soluble factors involved in osteoclastic activation in haematological malignancies remain unclear except for Multiple Myeloma and Adult T-cell Leukemia. The aim of this paper is to report the first case of Follicular Lymphoma with bone involvement displaying an aberrant expression of RANKL in malignant cells. The detection of RANKL in Follicullar Lymphoma may help to prevent bone lesion in patients by determining an appropriate treatment.

Background: Cyclooxygenase-2 (COX-2), the inducible isoform of the cyclooxygenases, is upregulated in various inflammatory renal diseases and responsible for prostaglandin formation. As prostaglandins are known to influence cell adhesion processes, we investigated the effect of COX-2 inhibition on the expression of alpha(v) integrins, which are also enhanced in renal diseases and control the adherence between the endothelium and the extracellular matrix (ECM) in the glomerulus., Methods: Healthy female Wistar rats and animals with previously induced passive Heymann nephritis (PHN) received either 5 mg/kg body weight/day celecoxib or a placebo. After 28 days, renal cortical mRNA expression of COX-2 and alpha(v) integrin subunits was determined., Results: Rats with PHN showed a significant 1.7-fold increase in renal cortical mRNA expression of alpha(v) integrin subunits. Treatment with celecoxib increased cortical alpha(v) integrin mRNA expression 2.2-fold (p < 0.05) in healthy animals and 4.0-fold (p < 0.05) in rats with PHN, but lowered COX-2 mRNA expression in rats with PHN to 0.8-fold (p < 0.05). An inverse correlation between the expression of COX-2 and alpha(v) integrins in rats with PHN was demonstrated., Conclusions: It is suggested that COX-2-derived prostaglandins suppress the expression of alpha(v) integrins. This implies a previously unknown role for COX-2 in chronic inflammation in the kidney., (Copyright 2003 S. Karger AG, Basel)

Lead poisoning is commonly diagnosed in cattle. In this study, 3 groups of cattle from different herds accidentally exposed to discarded lead batteries on pasture were intensively studied to determine the extent and severity of exposure. The losses from acute death due to lead toxicity were substantial in all the 3 study groups at 12%, 17%, and 4%. Blood samples were taken from all cattle around the time of the first diagnosis and then later in 2 of the 3 herds to monitor the change in lead concentrations over time. Asymptomatic lead toxicosis was observed in these herds. In these 3 groups, between 4% and 12% of asymptomatic cattle had blood lead concentrations consistent with acute lead poisoning (> 0.35 ppm), and between 7% and 40% of these asymptomatic animals were in the high-normal range (0.1-0.35 ppm). Because of the consistently high number of asymptomatic cattle with elevated lead levels, all cattle potentially exposed to a lead source should be tested before sale or slaughter to minimize the entry of lead into the food chain. The blood lead concentrations, which were monitored for months after the initial diagnosis, decreased slowly after the cattle were removed from the lead source. The prolonged retention of lead may be due to continued release and absorption of lead from metal particles in the reticulum or rumen. The mean reduction in the lead level was 0.046 ppm (95% CI, 0.017-0.075 ppm) every 30 days for these 2 herds. Using a single-component exponential model, the half-life of lead in the animals retested from Herds 1 and 2 was highly variable. The median half-life was 63 days (interquartile range, 34-107 days). One out of 8 pregnant heifers with high blood levels had a stillborn calf. There were no abortions or calf mortalities in this group. Blood samples were'collected from the calves around the time of birth. The concentrations of lead in the blood of the calves exposed in utero were low (0.010-0.095 ppm).

It is well documented that adjuvants improve the immune response generated by traditional viral vaccines, but less is known about the effects of adjuvants on the immune response elicited by DNA vaccines. In this study, we have investigated the use of RN-205 (immunomodulator containing a membrane rich in lipopolysaccharide from gram-negative bacteria) as an adjuvant and analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD). The comparison of the antibody response induced in mice by a mixture of the three different versions of DNA gD (membrane-anchored, secreted and cytosolic) formulated with or without RN-205 showed that the immunomodulator did not affect the total specific humoral response. The cellular immune response induced in mice immunized with vaccines plus RN-205 was higher than that obtained in mice vaccinated without RN-205, not only in the indexes of proliferation tests but in the number of IL-4 and gammaIFN secreting cells. When total spleen cells were marked with specific monoclonal antibodies against surface markers, a significant increase in the macrophage population of all the groups receiving RN-205 was observed. CD8 and CD4 positive cells were also increased but to a lesser extent. Our results indicate that the incorporation of RN-205 into DNA vaccines induces an increase of the cellular specific immune response in mice.

Domestic livestock have occasionally been used as sentinels of environmental health. Historically, these studies focused on measuring blood or tissue levels of a specific environmental contaminant. For many environmental exposures, however, there is no appropriate biomarker for exposure. The objective of this study was to examine productivity and health information from domestic livestock as one indicator of the potential long-term environmental impact of natural gas developments in a rural area. Intensive biological accounting methods were developed to measure the health and productivity of cow-calf herds surrounding a new sour natural gas processing plant. From the fall of 1991 through calving 1997, cow production records from 7040 recorded bull contacts were examined from seven area cow-calf herds. Detailed information was also collected on other risk factors known to influence beef herd productivity. The median risks for nonpregnancy, abortion, calving late, stillbirth, and calf mortality for local herds did not differ from those of other published reports. There was no significant change in the risk of nonpregnancy, abortion, calving late, stillbirth, or calf mortality., (Copyright 2001 Academic Press.)

This study examines the long-term impact of a Neospora caninum-associated abortion outbreak in a large cow-calf herd in northern Alberta. Blood samples were collected 4 times from all bred females and heifer calves born during the spring before the outbreak: (1) at the time of the outbreak, (2) the following spring, (3) the subsequent fall, and, finally, (4) the second spring after the outbreak. The samples were analyzed using a commercially available enzyme-linked immunosorbent assay for N. caninum. Calves born immediately following the outbreak were also monitored. Individual calving or abortion records were available from all cows for 2 calving seasons. All cows and heifers were pregnancy tested after the 2 subsequent breeding seasons. At the time of the abortion outbreak in 1997, 81% of all bred females and 87% of the heifer calves were serologically positive. In spring 1998, 49% of the cows and 47% of the heifer calves remained positive. In fall 1998, 48% of the remaining cows and heifers were serologically positive. After the first breeding season following the outbreak (1998), 13.5% of the heifers and 22.2% of the cows were open (not pregnant). Animals that were serologically positive in the spring were more likely to be open in the fall (odds ratio, 2.0; 95% confidence interval, 1.1 to 3.7). No subsequent associations with increased risk of abortion, stillbirth, or nonpregnancy were identified.

The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

Blood samples were collected from 1806 pregnancy-tested cows from 174 herds at a northern Alberta auction mart in the fall of 1998. One hundred sixty-two (9.0%) of these samples were positive for antibodies to N. caninum. Thirty-five of 260 samples (13.5%) collected from the same region in the 1980s were also serologically positive for N. caninum.

Two experiments were conducted to determine whether caudal epidural lidocaine anesthesia reduces a stress response to electroejaculation. In the 1st experiment, changes in cortisol and progesterone concentrations in serial blood samples were used to assess the stress response to restraint (control), transrectal massage, caudal epidural injection of saline, electroejaculation after caudal epidural injection of lidocaine, and electroejaculation without epidural lidocaine. In the 2nd experiment, behavioral responses were subjectively scored in bulls that were electroejaculated with or without caudal epidural lidocaine anesthesia. Cortisol and progesterone concentrations were significantly elevated after electroejaculation, whether or not bulls received caudal epidural anesthesia. Elevations in cortisol and progesterone were lower and fewer bulls vocalized during electroejaculation when given caudal epidural anesthesia; however, the differences were not significant.

Abortion outbreaks caused by infection with Neospora caninum in beef cattle have not been well documented. Neospora caninum infection was confirmed in 4 fetuses that were aborted by cattle in a 350-head beef herd; an additional 58 cattle aborted during the next 2.5 months. Overall, 44.4% (157/354) of the cattle in the herd did not become pregnant or experienced fetal loss during this period. Initially, 81.3% (282/347) of the herd was seropositive for antibodies to N caninum, and seropositive cows were 6.2 times as likely to not be pregnant as were seronegative cows. Other potential causes of abortion were not identified in this herd. Following the outbreak, few cattle in the herd or in a second exposed herd seroconverted, but high antibody titers persisted in most seropositive cattle through the end of the calving season. Evidence of cow-to-fetus transmission of the organism was detected in > 82% of seropositive cows.

CD44 is a transmembrane glycoprotein involved in cell-cell and cell-substrate interactions. As a cell surface molecule, CD44 may be shed or released into the circulation by proteolytic enzymatic mechanisms. Therefore, soluble CD44 can be found in cell culture supernatants as well as in plasma. In this study we evaluated the levels of soluble total CD44 (sCD44) in serum samples of patients with breast and colorectal carcinoma as well as non-Hodgkin's lymphoma in order to correlate prognosis with sCD44 expression. Besides, we evaluated other clinical tumour markers routinely used, Cancer Antigen (CA) 15.3 and CA 19.9. We investigated 132 serological samples from breast cancer patients, 48 sera from colorectal tumours, 48 samples from stage IV non-Hodgkin's lymphoma and sera from 80 individuals without evidence of cancer or autoimmune disease. Breast cancer patients were divided into three groups: a) patients with no clinical evidence of positive nodules and no metastatic disease; b) patients with positive nodules; and c) patients with metastasis. sCD44 mean serum levels in these groups were 198+/-54 ng/ml, 221+/-78 ng/ml and 242+/-119 ng/ml, respectively, while the marker CA 15.3 values were 15.6+/-6.6 U/ml, 14.0+/-5.8 U/ml and 211.5+/-358.9 U/ml, respectively. sCD44 levels for colorectal tumour were 243+/-72 ng/ml, while CA 19.9 serum levels were 230+/-270 U/ml. Stage IV non-Hodgkin's lymphoma sCD44 levels were 398+/-160 ng/ml. sCD44, CA 15.3 and CA 19.9 values for healthy individuals without evidence of any cancer pathology were 223+/-58 ng/ml, 16.4+/-6.2 U/ml and 33+/-14 U/ml, respectively. From these results we conclude that sCD44 might be used as a reliable marker for patients with non-Hodgkin's lymphoma. However, sCD44 levels failed to correlate with prognosis, tumour burden or metastasis in breast and colorectal cancer patients. Neither was any correlation found between high CA 15.3 or CA 19.9 levels and soluble CD44 serum level.

CD44 is a widely expressed cell-surface transmembrane glycoprotein involved in diverse adhesive processes. Its isoforms have been implicated in tumor progression and are considered a promising marker for evaluation of the metastatic potential of various tumors. Several methods have been described for the analysis of CD44 isoforms in tumor cells, including immuno-histochemistry, RT-PCR followed by hybridization and nested RT-PCR. We describe an alternative nested PCR for the analysis of CD44 isoform expression in various malignancies. Total RNA was isolated from various shock-frozen tissues from human tumors, reverse-transcribed and PCR-amplified using CD44-specific primers. Reverse-transcription was performed by two different methods, either using Tth-polymerase or MMLV-RT. Exon-specific amplification was then carried out using specific primers for each variable exon. Amplification products were assayed by agarose gel electrophoresis. Comparison of the patterns obtained from the first amplification and from the exon-specific amplification allowed to identify exons expressed by tumor tissues, as well as the genomic organization of CD44 isoforms. The method developed proved to be sensitive, reliable and inexpensive in comparison with other methods. It can be performed even in solid tumors and for numerous samples, and is suitable for laboratories with limited resources.

Objective: To determine seroprevalence of Neospora caninum infection in beef herds and the association between serologic status and rate of abortion, stillbirth, calf mortality, and reproductive failure., Design: Longitudinal study., Animals: 419 cows from 8 beef herds in central Alberta., Procedure: 1,391 serum samples from a serum bank were analyzed, using ELISA, and results were compared, using logistic regression, with productivity data on individual cows obtained from a database established during a 4-year period., Results: 30% of cows were seropositive at some point during the 4-year period. Risks of abortion (odds ratio [OR], 5.7) and stillbirth (OR, 2.8) in seropositive cows were significantly greater than in seronegative cows. Risks of being culled for any reason (OR, 1.9) or for reproductive failure (OR, 2.5) in seropositive cows were also significantly greater than in seronegative cows. Changes in titer with time in individual cows and a lack of association between serologic status of dam versus daughter suggest that postnatal transmission was possible in these herds. However, horizontal transmission did not appear to play a substantial role in abortions that occurred in these herds., Clinical Implications: Neosporosis should be investigated as a potential source of economic loss to the beef industry.

Three tests are routinely done to assess blood status of selenium in cattle: serum selenium, whole blood selenium, and glutathione peroxidase. The objective of this study was to compare the various analytical methods for determining blood selenium status in groups of mature cows and beef calves. Twenty to 30 blood samples per herd were collected from 8 beef herds in central Alberta and 1 dairy in Alberta herd twice a year from the spring of 1992 through the fall of 1995, and once from 185 spring calves in 2 beef herds in Saskatchewan. Serum and whole blood samples were submitted to 1 laboratory and whole blood samples were submitted to a 2nd laboratory. Samples for glutathione peroxidase determinations were submitted to a 3rd laboratory. Pearson's correlation coefficients and Cohen's kappa were calculated for each possible comparison among the different measures. The best agreement was observed between serum and whole blood analysis within Laboratory A. The remaining comparisons reflected poor agreement. Comparison of herd-level assessment resulted in better agreement than comparison of individual sample results among laboratories and procedures for all combinations tested. Serum selenium analysis was the only laboratory procedure for which external reference material was utilized. Serum selenium, whole blood selenium, and glutathione peroxidase measure different compartments of the blood selenium pool. The time frame of interest, supplementation practices, and the stability of recent dietary intake determine the optimum assessment method for individual animals or herds. Determination of the serum status or of blood selenium is more consistently measured at the herd-level than for individual samples.

Objective: To determine the association between a leak of sour natural gas (more than 30% hydrogen sulfide) from a pipeline in a river valley and the health of beef cattle in the intensively ranched surrounding area., Design: Prospective cohort study., Sample Population: 13 herds of cattle within 4 km (2.5 miles) of the leak and 10 herds outside the 4-km zone., Procedure: Distance of herds from the leak site was determined, using geographic information system technology. Information about speed and direction of winds was obtained from a local meteorologic station and an ambient air-quality monitoring trailer. Health and productivity data for surrounding beef herds, as well as exposure information, were collected and analyzed., Results: An association was not found between total herd calf mortality and herd distance from the leak, wind-aided exposure, location in the river valley, signs of irritation consistent with exposure to the gas, or reports of odors of gas at the time of the leak. Management changes reported in response to the gas leak were identified as risk factors for total herd calf mortality. Other herd-level risk factors associated with increased calf mortality ratio included a median calving date in February and percentage of twin births for a herd., Clinical Implications: In this example, we did not detect an association between productivity of cattle and exposure to sour natural gas. Several methods can be used for ranking potential exposure after discovery of a leak.

As it has been suggested that an autocrine mechanism may control tumour cell growth, in this work cells from a spontaneous murine T lymphocyte leukaemia (LB) expressing the interleukin-2 receptor (IL-2R) (CD25) were evaluated in vitro for IL-2-mediated autocrine growth. Cells grew readily in culture and proliferation was enhanced by the addition of recombinant IL-2 but inhibited by monoclonal antibodies against either IL-2 or IL-2 receptor, in the absence of exogenous IL-2. Cyclosporin A also inhibited LB cell growth. However, when exogenous IL-2 was added together with cyclosporin A, cell proliferation proved similar to controls. Using reverse transcription polymerase chain reaction (PCR), mRNA for IL-2 was found to be present in tumour cells. Our findings support the hypothesis that LB tumour cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture.

An improved two-chamber diffusion cell was developed for the study of drug transport across cultured cell monolayers. The cell monolayer was grown on a horizontal support membrane of polycarbonate, which could be rotated providing theoretically predictable thicknesses of the diffusion boundary layer in the donor and the receiver solution as a function of rotation rate. Permeation measurements were performed using the support membrane with and without a cell monolayer. The Madin-Darby-Bovine-Kidney (MDBK) cell line was employed, and permeability coefficients of model solutes (salicylic acid, mannitol, testosterone) across the three distinct mass transport barriers (i.e., the cell monolayer, the support membrane, and the diffusion boundary layer) were determined. The permeability of the diffusion boundary layer followed the theoretical dependence on the rotation rate; absolute values, however, deviated from predictions. Permeability coefficients for all three transport barriers varied substantially between solutes. This variation was the strongest for the permeability coefficient for the cell monolayer and resulted in a varying relative significance of these three barriers in controlling permeation kinetics. This improved diffusion apparatus permits the measurement of unbiased permeability values of solutes across the cell monolayer, notably when the cell monolayer is not absolutely the rate-determining barrier.

The relationship between tumorigenicity and expression of MHC class II molecules in a class II-negative murine leukaemia cell line (LBC) was studied. Analysis of structural DNA sequences encoding MHC class II proteins was performed by Southern blot with DNA isolated from both the original LB tumour and LBC cell line, digested with EcoRI, BamHI and HindIII and hybridised with specific probes for I-A alpha d and I-A beta d chains. Similar patterns were obtained for LB, LBC and normal BALB/c lymphocytes. In vitro treatment with IFN-gamma (20 - 1000 IU ml-1) failed to induce the expression of MHC class II antigens in LBC cell line. LBC cells were tri-transfected by a liposome-mediated protocol with I-A alpha d, I-A beta d genes and pSV2neo. Cells were selected for growth in medium containing Geneticin (G418). Surviving transfectants were cloned and three I-A+ clones were obtained after 20 days (LBCT cells). Syngeneic mice inoculated with 1.0 x 10(3) LBCT (I-A+) cells failed to develop a tumour, whereas the DT50 of mice injected with 1.0 x 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). Furthermore, specific CTL response against tumour cells was significantly enhanced upon priming with irradiated LBC-transfected cells (27 +/- 2%) compared with irradiated LBC cells (15 +/- 1.5%) in a 4 h 51Cr-release assay. It is suggested that neoexpression of MHC class II molecules enhances anti-tumour response by transforming tumour cells into professional antigen-presenting cells (APCs), which may be used to improve tumour-specific immunity in the autologous host.

LB leukemia is a nonimmunogenic T cell tumor which spontaneously arose in a BALB/c mouse; efforts to induce immunological rejection of the leukemic cells have always failed. The leukemic cells grow rapidly and progressively in the syngeneic host invading spleen, lymph nodes and liver. A cell line (LBC) was developed from the original tumor. Both the original tumor and the cell line have been characterized as expressing the Thy 1+, CD3-, CD25+, MHC class I+, class II-, CD4- (original tumor), CD4+ (cell line), CD8+, gp70-, J11d.2+ phenotypes. Immunization of syngeneic mice with irradiated LBC cells induced cytotoxic T lymphocytes as well as anti-LBC antibodies which reacted with components of 14, 16 and 27 kDa present on LB tumor cells, LBC cell line and normal thymocytes but not on normal lymph node cells. Immunization of syngeneic mice with LBC cells partially protected them against subsequent challenge with the original tumor cells. The effect of sera from tumor-bearing mice and the super-natants from short term cultures were studied on cell proliferation. An inhibitory activity was demonstrated in these fluids, which was abrogated by addition of exogenous IL-2. ELISA showed the presence of soluble IL-2R alpha chain both in the conditioned medium as well as in the serum, which was demonstrated to be responsible for the inhibitory activity. The soluble IL-2R was produced by LB leukemic cells and exerted the inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2. Using reverse-transcription PCR, mRNA for IL-2 was found to be present in tumor cells. Our findings indicate that LB cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. The relationship between tumorigenicity and expression of MHC class II was also investigated. In vitro treatment with IFN-gamma failed to induce the expression of class II antigens in LBC cell line. Therefore these cells were tri-transfected by a liposome-mediated protocol with 1-A alpha d, I-A beta d genes and pSV2neo. Cells were selected to grow in medium containing Genetecin (G418) and surviving transfectants were cloned. Three I-A+ clones were obtained (LBCT) and were used to induce a specific CTL response against tumor cells. Syngeneic mice inoculated with 10(3) LBCT cells failed to develop a tumor while the DT50 of mice injected with 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). It is suggested that neoexpression of MHC class II molecules enhances anti-tumor response by transforming tumor cells into professional antigen-presenting cells, which may be used to improve tumor-specific immunity in the autologous host.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24-48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non-specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 10(5)-1.3 x 10(5) M(r) serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsin or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing-thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL-2 abrogated inhibitory activity. ELISA showed the presence of soluble IL-2R both in LB conditioned medium and in above serum fraction. It is demonstrated that the inhibitory factor, soluble IL-2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL-2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2.

A methodology for the preparation of a polymerized antigen from house dust mites Dermatophagoides Pteronyssinus is described. Polymerization was carried out by glutaraldehyde treatment in PBS and Sephacryl S300 column fractionation. A polymer of 135 Kd was obtained. Rabbits immunized with the soluble polymer (HDMP) elaborated specific antibodies as measured by a Dot-immunobinding assay. The reaction was inhibited by previous incubation with the HDMP antigen. It is suggested that the polymerized antigen may be employed in the treatment of patients with house dust mite anaphylaxis.

This paper presents a model of spontaneous BALB/c lymphoma (LB) useful for the study of the relationship between the tumor and the immune system. Characterization of this tumor was done by FACS and it was determined that the tumor cells are T lymphocytes which express both CD4 and CD8 membrane markers but not the viral protein gp 70. In addition, tumor cells express IL-2 receptors and class I, but not class II, H-2 antigens. Antibodies were not detected in sera or ascitic fluid of syngeneic mice bearing a tumor transplant while both sera and ascites contained a suppressor factor that inhibited in vitro the proliferation of tumor cells and blastogenesis of normal BALB/c splenocytes after Concanavalin A stimulation. The results indicate that this tumor may be used as an experimental model for adult T cell leukemia as well as for the isolation and characterization of cytokines which may be responsible for the immunological unresponsiveness found in certain cancer patients.