Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.     

Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.     

Milk yield of primiparous beef cows from three calving systems and varied weaning ages

Primiparous beef cows produced in 3 calving systems were used in a 2-yr study with a completely random design to measure milk yield throughout a 190-d lactation (2002, n = 20; 2003, n = 24 per calving system). Calving occurred in late winter (average calving date = February 4 +/- 2 d), early spring (average calving date = March 30 +/- 2 d), and late spring (average calving date = May 26 +/- 1 d). Additionally, cows used in this study had been weaned at varied ages as calves, creating 6 dam treatments. Dam age at weaning was 140 (late spring), 190 (late winter, early spring, late spring), or 240 (late winter, early spring) d of age. Milk production was measured by using the weigh-suckle-weigh technique at an average of 20, 38, 55, 88, 125, 163, and 190 d in milk. Milk yield for the 190-d lactation period was calculated as area under the curve by trapezoidal summation. Data were analyzed with a model containing treatment, year, and their interaction. Orthogonal contrasts were used to separate effects when treatment was significant (P < 0.10). Total milk yield did not differ (P = 0.42) between cows in the late winter and early spring systems, but cows in the late spring system tended to differ (P = 0.09) from the average of the other 2 systems. Cows in the late spring calving system had increased milk yield in 2002 and lesser milk yield in 2003 compared with the other calving systems (treatment x year interaction, P < 0.001). Cows born in late spring that had been weaned at 140 d of age produced more (P = 0.05) total milk than those weaned at 190 d of age. Peak milk yield was affected (P < 0.001) by treatment and showed a treatment x year interaction (P = 0.006). Day of peak lactation differed among treatments (P = 0.002), with cows in the late winter system peaking later (P = 0.007) than early spring cows, and late spring cows peaking earlier (P = 0.004) than the average of late winter and early spring cows. The average date of peak lactation was May 4 for the late winter system, May 31 for the early spring system, and July 19 for the late spring system. Calf ADG differed (P < 0.001) for the late spring system compared with the average of the late winter and early spring systems, but the relationship interacted with year (P < 0.001). Cow BW and BW change differed among treatments (P < 0.004), with much of the difference associated with the amount of milk produced or the timing of peak lactation. Season of calving affects milk yield of primiparous cows grazing Northern Great Plains rangelands and ADG of their calves.     

Generation and purification of recombinant bovine pregnancy associated glycoprotein

Bovine pregnancy-associated glycoprotein-1 (bPAG-1) is predicted to play an essential role during pregnancy and is labelled as a potential biochemical marker of pregnancy in ungulates. We have compared the generation of the glycosylated form of recombinant bPAG-1 (rbPAG-1) by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells in attached cultures and evaluated the adaptation of the rbPAG-1 transfected cell line to suspension culture. The PAG cDNA was cloned from placental RNA obtained from a slaughtered cow on day 55 of pregnancy. The PAG-pRcRSV expression vector was transfected into HEK 293 and CHO cells. Western blot analysis showed that clonal HEK 293 cells expressed rbPAG-1 better than CHO cells in attached cultures. Transfected HEK 293 cells were adapted to suspension culture in spinner flasks and the rbPAG-1 purified to homogeneity using ion-exchange, pepstatin-sepharose affinity chromatographies and preparative SDS-PAGE. The expression of rbPAG-1 was immunocharacterised using a polyclonal antibody. Our findings indicated that 293 cells are suitable for production of glycosylated form of rbPAG-1 and that the availability of the recombinant glycoprotein will aid in further studies to elucidate the function and structure of the protein.     

Two techniques for supplementing interlocking nail repair of fractures of the humerus, femur, and tibia: results in 12 dogs and cats

OBJECTIVE: To describe 2 devices for improving stabilization of inadequately stabilized interlocking nail (ILN) repairs of the humerus, tibia, and femur in dogs and cats. STUDY DESIGN: Prospective study. ANIMALS: Twelve client-owned dogs and cats. METHODS: Two devices to further stabilize ILN repair of inadequately stabilized diaphyseal fractures were developed. Device 1 was an axial extension for the ILN that was connected to a conventional type I external skeletal fixator (ESF) with a short connecting bar. Device 2 had hybrid ILN bolt/ESF pins that were used to lock the ILN and serve as the pins for a type I ESF. Devices were used at the initial surgery when the stability of ILN repair was considered inadequate based on palpable fracture segment movement, insufficient medullary canal filling of the ILN at the fracture site, or when the ILN was used in a buttress mode. Outcome was obtained by recheck examinations, radiography, and telephone interview. RESULTS: Device 1 was applicable to fractures of the humerus and femur, but was not used for fractures of the tibia because the ILN extension would have interfered with the stifle. No gross loosening of the ILN/ESF extension connection to the ILN occurred. Device 2 was easily placed and used in the humerus, femur, and tibia. Device 2 allowed removal of the ILN interlock to one or both main fracture segments non-invasively. Clinically, both devices added stability compared with ILN repair alone. Both devices facilitated controlled destabilization of the fracture repair as healing progressed. Complications of pin tract infection, and premature hybrid bolt/ESF pin loosening resulting in premature ESF removal each occurred in 1 patient. Four of 28 hybrid ILN/ESF pins were grossly loose at 4- or 6-week postoperative recheck examinations. Outcomes were excellent (9), good (1), fair (1), and poor (1). CONCLUSIONS: Inadequately stabilized ILN repair of fractures can be stabilized by use of either device, both of which also permit controlled destabilization of the repair during healing. Device 2 can be used when non-invasive removal of the ILN interlock is desired during healing. CLINICAL RELEVANCE: These 2 devices should be considered as alternative methods for stabilization of inadequately stabilized ILN repairs in dogs and cats, or when controlled destabilization of an ILN fracture repair is desired.     

Effects of dietary lysine and energy density on performance and carcass characteristics of finishing pigs fed ractopamine

Two hundred sixteen crossbred barrows and gilts (84.3 kg BW) were used to test the effects of dietary energy density and lysine:energy ratio (Lys:ME) on the performance, carcass characteristics, and pork quality of finishing pigs fed 10 ppm ractopamine. Pigs were blocked by BW and gender, allotted to 36 pens (six pigs per pen), and pens were assigned randomly within blocks to dietary treatments (as-fed basis) arranged in a 2 x 3 factorial design, with two levels of energy (3.30 or 3.48 Mcal/kg) and three Lys:ME (1.7, 2.4, or 3.1 g lysine/Mcal) levels. Pigs were fed experimental diets for 28 d, and weights and feed disappearance were recorded weekly to calculate ADG, ADFI, and G:F. Upon completion of the feeding trial, pigs were slaughtered and carcass data were collected before fabrication. During carcass fabrication, hams were analyzed for lean composition using a ham electrical conductivity (TOBEC) unit, and loins were collected, vacuum-packaged, and boxed for pork quality data collection. Energy density had no (P > 0.22) effect on ADG or ADFI across the entire 28-d feeding trial; however, pigs fed 3.48 Mcal of ME were more (P < 0.02) efficient than pigs fed 3.30 Mcal of ME. In addition, ADG and G:F increased linearly (P < 0.01) as Lys:ME increased from 1.7 to 3.1 g/Mcal. Carcasses of pigs fed 3.48 Mcal of ME were fatter at the last lumbar vertebrae (P < 0.08) and 10th rib (P < 0.04), resulting in a lower (P < 0.03) predicted fat-free lean yield (FFLY). Conversely, 10th-rib fat thickness decreased linearly (P = 0.02), and LM depth (P < 0.01) and area (P < 0.01) increased linearly, with increasing Lys:ME. Moreover, FFLY (P < 0.01) and actual ham lean yield (P < 0.01) increased as Lys:ME increased in the diet. Dietary energy density had no (P > 0.19) effect on pork quality, and Lys:ME did not (P > 0.20) affect muscle pH, drip loss, color, and firmness scores. Marbling scores, as well as LM lipid content, decreased linearly (P < 0.01) as Lys:ME increased from 1.7 to 3.1 g/Mcal. There was a linear (P < 0.01) increase in shear force of cooked LM chops as Lys:ME increased in the finishing diet. Results indicate that 3.30 Mcal of ME/kg (as-fed basis) is sufficient for optimal performance and carcass leanness in pigs fed ractopamine. The Lys:ME for optimal performance and carcass composition seems higher than that currently used in the swine industry; however, feeding very high Lys:ME (> 3.0 g/Mcal, as-fed basis) to ractopamine-fed pigs may result in decreased marbling and cooked pork tenderness.     

Effects of supplemental protein type on intake, nitrogen balance, and site, and extent of digestion in whiteface wethers consuming low-quality grass hay

Two experiments were conducted to determine the effects of supplementing ruminally degradable intake protein (DIP) or ruminally undegradable intake protein (UIP) on N balance (Exp. 1; n = 6 wethers; initial BW = 48.7 +/- 4.6 kg) and site and extent of digestion (Exp. 2; n = 5 wethers; initial BW = 36.9 +/- 3.1 kg) in whiteface wethers consuming (as-fed basis) 69% blue grama and 31% love grass hay (mixture = 7.5% CP, 73.0% NDF, 36.0% ADF [DM basis]). Treatments were 1) no supplement (Control), 2) a supplement (219 g/d, as-fed basis) low in UIP (70 g/d of CP; 24.8 g/d of UIP), and 3) a supplement (219 g/d, as-fed basis) high in UIP (70 g/d of CP; 37.1 g/d of UIP). Both experiments were replicated 3 x 3 Latin square designs, with identical feeding and supplementation. Wethers had ad libitum access to the forage mixture and fresh water, and received supplement once daily. In Exp.1, forage intake (percentage of BW) was greatest (P = 0.04) for control, but total DMI (g/d) was greatest (P = 0.05) for lambs consuming supplement. Apparent total-tract OM digestibility was numerically greater (P = 0.11) for supplemented wethers than for controls, whereas total-tract ADF digestibility tended (P = 0.08) to be greater for control wethers. Lambs fed supplements consumed and retained more (P < or = 0.01) N (% of N intake) compared with controls, but no difference (P = 0.22) was observed between low and high UIP treatments. Similar to Exp. 1, forage intake (percentage of BW) tended (P = 0.06) to be greater for control than for supplemented wethers in Exp. 2. Ruminal NDF digestibility was 16.3% greater (P = 0.02) for supplemented wethers than for controls. Postruminal NDF and N digestibilities were greatest (P < or = 0.03) for controls, but apparent OM digestibility did not differ among treatments at all sites. Duodenal N flow was greatest (P = 0.05) for high UIP and least for control wethers. Nonmicrobial N flow was greater (P = 0.02) for high UIP compared with low UIP or controls. Control wethers had greater (P = 0.05) microbial efficiency. Ruminal ammonia concentration tended (P = 0.08) to be greatest for wethers fed low UIP and least for controls, with high-UIP wethers having intermediate ammonia concentrations. Results from these experiments suggest that in lambs fed low-quality forage there was no difference in apparent total-tract digestion or N balance (percentage of N intake) between lambs fed supplements that had the same CP but differed in the proportion of UIP and DIP; however, supplementing protein (regardless of UIP:DIP ratio) to wethers consuming low-quality forage increased N balance.