The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation

The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1×10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1×10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Clinical relevance of monocyte procoagulant activity in horses with colic

Endotoxin-activated monocytes express a thromboplastin-like procoagulant activity on the cell surface that may serve as a focal point for formation of microvascular thrombi. Because coagulopathy is a common sequela to endotoxemia in the equine species, we investigated the ability of monocytes, isolated from horses with colic, to express procoagulant activity. On the day of admission, and on the third and fifth day of hospitalization, monocytes were isolated from 30 adult horses with colic. A coagulation profile, including prothrombin time, activated partial thromboplastin time, thrombin time, and plasma fibrinogen and serum fibrin degradation products concentrations, was determined at each sample collection. The concentration of endotoxin in the plasma was quantitated at the time of admission. Ten clinically normal adult horses served as controls. The procoagulant activity of monocytes isolated from horses with colic was significantly (P less than 0.05) greater than that of the monocytes isolated from clinically normal horses. On the first and third day of hospitalization, the mean prothrombin time was significantly (P less than 0.05) longer in horses with colic, compared with clinically normal horses, and was the most common abnormality in the coagulation profile on the day of admission (25/30; 83%). Mean fibrin degradation products concentration was significantly (P less than 0.05) greater in horses with colic on the day of admission and was the second most common abnormality in the coagulation profile on day 1 (23/30; 77%). In horses with colic, the mean prothrombin and activated partial thromboplastin times were significantly (P less than 0.05) longer in horses that did not survive, compared with horses that survived.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of immune-mediated hemolytic anemia in dogs with cyclophosphamide

A review of 60 cases of immune-mediated hemolytic anemia (IMHA) in the dog was performed in order to characterize the disease and to identify potential prognostic indicators. Dogs ranged in age from 1 to 13 years, with a mean age of 6.5 years. The 2 most commonly affected breeds were Cocker Spaniels and Labrador Retrievers. Fifty-two of the 60 dogs tested (87%) were autoagglutination positive and spherocytes were present in 45 (75%). Forty-one (89%) of 46 patients tested positive for the presence of immunoglobulin on the red blood cell surface (Coombs assay). The most common clinical signs at presentation were lethargy, weakness, pale mucous membranes, icterus, hemoglobinuria, and anorexia. PCV less than 25% was present in 59 (98%) dogs. At the time of presentation, 35 dogs (58%) had a nonregenerative anemia, whereas 25 patients (42%) had a regenerative response. Thrombocytopenia was seen in 41 (68%) dogs. Nine of 34 dogs (26%) had a prolonged prothrombin time, 19 of 34 (56%) had a prolonged activated partial thromboplastin clotting time, and 12 of 34 (35%) had abnormal fibrinogen concentrations. All dogs received prednisone at immunosuppressive doses (2.2-4.4 mg/kg PO as a single or divided dose every 24 hours) and cyclophosphamide as primary therapy. Forty-one dogs (63%) received cyclophosphamide at 50 mg/m2 q24h for 4 days, whereas 9 dogs (15%) received an initial high dose (200 mg/m2) followed by 3 days of a lower dose (50 mg/m2 q24h). No statistical difference in survival times was found for either protocol. Thirteen dogs were treated with azathioprine in addition to cyclophosphamide and prednisone. The median survival time of dogs that received all 3 drugs was 370 days as compared to 9 days for those dogs that were treated with cyclophosphamide and prednisone alone. Thirty-one (52%) dogs died from the disease, 13 (22%) dogs were alive, and 15 (25%) dogs were lost to follow-up. The median length of survival for all dogs was 21 days. Eight dogs that were discharged from the hospital suffered a relapse (PCV < 25%).     

The Role of Recreational Motivation in the Birding Participation–Environmental Concern Relationship

Previous research has generally not found a strong relationship between participation in outdoor recreation and environmental concern. Research has suggested, however, that the motivation underlying recreation can mediate this relationship for some nature-based recreation groups. We hypothesized that the motivation underlying participation in birding mediates the participation–environmental concern relationship. We collected data via mail-back surveys from 529 American Birding Association members. Using structural modeling, we compared a direct effects model with partial and full mediation models with motivation variables as the mediators. The partial mediation model was weakly supported for two out of three motivation variables. Fit indices showed all models to have less than adequate fit. The results suggested that participation may not lead to environmental concern and that other variables, including motivation, may be more influential.     

Iron deficiency and pseudo-iron deficiency in hospitalized horses

Values for serum ferritin, serum iron, and serum total-iron binding capacity were determined in 100 hospitalized horses. Six foals had iron deficiency, as indicated by low serum ferritin and iron concentrations. Twenty-eight horses had low serum iron content and normal or high serum ferritin content and were considered to have pseudo-iron deficiency, possibly in response to infection. Ten horses had serum ferritin concentrations that were quite high, probably indicating iron overload.     

Does Shrub Removal Increase Groundwater Recharge in Southwestern Texas Semiarid Rangelands?

Evapotranspiration (ET) is a key component limiting groundwater recharge past the root zone in semiarid regions. Vegetation management may alter groundwater recharge if ET is altered due to changes in vegetation type or cover. This study quantifies changes in groundwater recharge following vegetation cover change from native woodland to pasture in a semiarid region of southwest Texas. The Carrizo–Wilcox aquifer is a valuable groundwater resource in this area, where overuse by dependent farming practices has lowered aquifer levels significantly in the last 85 yr. Combining data from short-term (30 mo) monitoring of the changes in soil moisture and long-term (5–30 yr) changes in total soil chloride indicated deep drainage increased slightly where land had been cleared of vegetation. Annual recharge rates below rooting depths (standardized to 155 cm) averaged only 0.72 ±  mm · yr^-1 (mean ± SE) in areas not cleared of woody vegetation, as estimated by chloride mass balance. Upon clearing, 72% of the total chloride naturally occurring in the soil profile was flushed away within 30 yr, leading to an estimated 2.59 ±  mm · yr^-1 additional recharge. Deep soil moisture in recently cleared land increased by up to 17% during the growing season of wet years (double the average rainfall) but did not increase in dry or normal precipitation years, providing supporting evidence that more water penetrated below the roots under certain environmental conditions. These results demonstrate that brush management can increase recharge by modest, but measurable, amounts depending on site-specific soil characteristics and degree of reduction in vegetation.