Local immune response to larvae of Rhipicephalus microplus in Santa Gertrudis cattle.

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick-resistant phenotypes. There was a tendency for more CD3+ , CD4+ , CD8+ , CD25+ , γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4+ cells. Most of the cattle developed intra-epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.

Global differential gene expression in the pituitary gland and the ovaries of pre- and postpubertal Brahman heifers.

To understand genes, pathways, and networks related to puberty, we characterized the transcriptome of two tissues: the pituitary gland and ovaries. Samples were harvested from pre- and postpubertal Brahman heifers (same age group). Brahman heifers () are older at puberty compared with , a productivity issue. With RNA sequencing, we identified differentially expressed (DEx) genes and important transcription factors (TF) and predicted coexpression networks. The number of DEx genes detected in the pituitary gland was 284 ( < 0.05), and was the most DEx gene (fold change = 4.12, = 0.01). The gene promotes bone mineralization through transforming growth factor-ß (TGFß) signaling. Further studies of the link between bone mineralization and puberty could target . In ovaries, 3,871 genes were DEx ( < 0.05). Four highly DEx genes were noteworthy for their function: (a γ-aminobutyric acid [GABA] transporter), (), and () and its receptor . These genes had higher ovarian expression in postpubertal heifers. The GABA and its receptors and transporters were expressed in the ovaries of many mammals, suggesting a role for this pathway beyond the brain. The pathway has been known to influence the timing of puberty in rats, via modulation of GnRH. The effects of at the hypothalamus, pituitary gland, and ovaries have been documented. and its receptors are known factors in the release of GnRH, similar to and GABA, although their roles in ovarian tissue are less clear. Pathways previously related to puberty such as TGFß signaling ( = 6.71 × 10), Wnt signaling ( = 4.1 × 10), and peroxisome proliferator-activated receptor (PPAR) signaling ( = 4.84 × 10) were enriched in our data set. Seven genes were identified as key TF in both tissues: , , , , , , and a novel gene. An ovarian subnetwork created with TF and significant ovarian DEx genes revealed five zinc fingers as regulators: , , , , and . Recent work of hypothalamic gene expression also pointed to zinc fingers as TF for bovine puberty. Although some zinc fingers may be ubiquitously expressed, the identification of DEx genes in common across tissues points to key regulators of puberty. The hypothalamus and pituitary gland had eight DEx genes in common. The hypothalamus and ovaries had 89 DEx genes in common. The pituitary gland and ovaries had 48 DEx genes in common. Our study confirmed the complexity of puberty and suggested further investigation on genes that code zinc fingers.

SNP detection using RNA-sequences of candidate genes associated with puberty in cattle.

Fertility traits, such as heifer pregnancy, are economically important in cattle production systems, and are therefore, used in genetic selection programs. The aim of this study was to identify single nucleotide polymorphisms (SNPs) using RNA-sequencing (RNA-Seq) data from ovary, uterus, endometrium, pituitary gland, hypothalamus, liver, longissimus dorsi muscle, and adipose tissue in 62 candidate genes associated with heifer puberty in cattle. RNA-Seq reads were assembled to the bovine reference genome (UMD 3.1.1) and analyzed in five cattle breeds; Brangus, Brahman, Nellore, Angus, and Holstein. Two approaches used the Brangus data for SNP discovery 1) pooling all samples, and 2) within each individual sample. These approaches revealed 1157 SNPs. These were compared with those identified in the pooled samples of the other breeds. Overall, 172 SNPs within 13 genes (CPNE5, FAM19A4, FOXN4, KLF1, LOC777593, MGC157266, NEBL, NRXN3, PEPT-1, PPP3CA, SCG5, TSG101, and TSHR) were concordant in the five breeds. Using Ensembl's Variant Effector Predictor, we determined that 12% of SNPs were in exons (71% synonymous, 29% nonsynonymous), 1% were in untranslated regions (UTRs), 86% were in introns, and 1% were in intergenic regions. Since these SNPs were discovered in RNA, the variants were predicted to be within exons or UTRs. Overall, 160 novel transcripts in 42 candidate genes and five novel genes overlapping five candidate genes were observed. In conclusion, 1157 SNPs were identified in 62 candidate genes associated with puberty in Brangus cattle, of which, 172 were concordant in the five cattle breeds. Novel transcripts and genes were also identified.

Seminal plasma proteins and their relationship with percentage of morphologically normal sperm in 2-year-old Brahman (Bos indicus) bulls.

The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (p<0.05). A multiple regression analyses of these SPPs, using three models; non-parametric Tree Model, Generalized Additive Model, and a step-wise selection method were conducted, and 6 SPPs could be used to predict PNS24; four SPPs had positive and two had negative association with PNS24. Together these spots explained 35% of the phenotypic variation in PNS24. Using mass spectrometry (MALDI-ToF and TripleToF-MS) the SPPs with positive relationship contained mainly apolipoprotein A-I (1310), protein DJ-1 and glutathione peroxidase 3 (2308), phosphoglycerate kinase 1 (6402) and apolipoprotein A-I and secretoglobin family 1D member (8008). The SPPs inversely associated with PNS24 were clusterin/seminal plasma protein A3 (1411) and epididymal secretory protein E1 (8108). This is the first comprehensive report on the association between seminal plasma protein composition in Bos indicus Brahman bulls and sperm morphology.

Evaluation and histological examination of a Campylobacter fetus subsp. venerealis small animal infection model.

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0% to 100% and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion.

Seminal plasma proteome of electroejaculated Bos indicus bulls.

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4 ± 2.3 and 64 ± 3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70 kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 d-isomerase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase 1. In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.

The integrity of sperm chromatin in young tropical composite bulls.

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r=0.34, P=0.0076), Mot (r=0.36, P=0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r=0.31, P=0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r=-0.28, P=0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r=-0.53, P<0.0001), the percentage of sperm with head abnormalities (r=0.68, P<0.0001) and the percentage of intact sperm (Int) with SBH (r=-0.26, P=0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3(+), CD4(+), CD8(+) and gammadelta T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for gammadelta T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Evaluation of formulas for optimal positioning of central venous catheters.

OBJECTIVE: To evaluate the accuracy of formulas designed to estimate the optimum intravenous length of central venous catheters. DESIGN: A prospective study of catheter insertion sites to evaluate the accuracy of predetermined formulas that predict the intravascular insertion length required to avoid intracardiac catheter tip placement. SETTING: A 320-bed tertiary hospital. PATIENTS: Critically ill patients requiring central venous access for therapy or monitoring. MAIN RESULTS: The formulas accurately predicted required intravascular length of the central venous catheter in 217 of 228 (95%) cases. The formula for predicting catheter length was most accurate when the subclavian vein was used. It was least accurate when the right internal jugular vein was selected. CONCLUSIONS: The formulas can accurately predict the required length of catheters and thereby reduce the possibility of complications and save time and expense.

Does continuous positive airway pressure compensate for loss of glottic function during tracheal intubation?

Adult patients with acute lung injury (ALI) exhibit increased PaO2 when receiving continuous positive airway pressure (CPAP). Some have increased PaO2 after extubation. To determine the role a competent glottis played in improving gas exchange, we anesthetized seven rabbits and inserted central venous and carotid artery catheters. After recovery from anesthesia, ALI was induced with oleic acid (0.08 ml/kg). Twenty-four hours later, the animals were sedated and placed in a sling. The pHa and blood gas tensions were measured. The animals were placed supine and were given inhalation anesthesia to facilitate tracheal intubation. A polyethylene catheter was placed slightly distal to the tracheal tube outlet to measure tracheal pressure (PT). Intubated rabbits were repositioned in the sling and were given either zero end-expiratory pressure (ZEEP) or 5 cm H2O CPAP, alternately. After the animals had breathed room air for 60 min, pHa and blood gas tensions were again measured, and PT was recorded. Animals were extubated, but the PT catheter was left in place. Data were collected again 60 min later, the catheter was removed, and the animals were returned to their cages. Forty-eight hours after onset of ALI, the protocol was repeated.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of glottic mechanism on pulmonary function after acute lung injury.

We measured arterial gas tensions, respiratory timing, and intratracheal pressure in 12 rabbits to investigate the consequences of translaryngeal intubation with normal and subsequently injured lungs. Data were collected before, during, and after intubation. Intubation in normal rabbits precipitated no untoward effects on gas exchange or respiratory phase timing. However, there was significant elevation of subglottic pressure during expiration following extubation. Central venous injection of oleic acid (0.08 ml/kg) induced an acute lung injury that after 24 h was characterized by reduced PaO2 and dynamic lung-thorax compliance and tachypnea. Intubation in animals with acute lung injury was associated with a significant decline in arterial oxygenation, tachypnea, and increased PCO2. Expiratory tracheal pressure and expiratory time were greater and PaCO2 and respiratory rate were lower following extubation. We conclude that translaryngeal intubation following acute lung injury exacerbates already compromised pulmonary function by preventing a compensatory expiratory braking maneuver by the glottic apparatus.

Effects of hyperoxia in the presence of acute lung injury.

We employed a multiple-dose, oleic acid (OA) model to evaluate the susceptibility to oxygen toxicity of rabbits with acute lung injury (ALI). The rabbits were partitioned into four groups: ALI group (n = 8) received OA (0.04 ml/kg iv) and again at two consecutive 24-h intervals and breathed room air (RA); hyperoxic O2/ALI group (n = 8) underwent similar OA injection protocol and breathed an FIO2 greater than or equal to 0.96; oxygen group (n = 8) received identical injection protocol with normal saline (NS) and breathed an FIO2 greater than or equal to 0.96; and control (CTR) group (n = 5) received isovolume NS injections and breathed RA. Arterial blood pH and gas tensions were measured before and 24, 48, and 60 h after ALI. Surviving animals were killed at 72 h and body weight (BW) was determined at autopsy; then the lungs were removed and weighed (LW). The mortality for animals exposed to hyperoxia was significantly (p less than or equal to .02) greater than those breathing RA, regardless of the presence or absence of ALI. Blood pH was lower (p less than or equal to .05) in all animals but the CTR group. However, acidemia was significantly greater in both hyperoxic groups compared to animals in the CTR and ALI/RA. Inflation and deflation lung-thorax compliances were lower (p less than or equal to .05) and percent lung weight of terminal body weight (LW/BW) was higher (p less than or equal to .05) after hyperoxia with or without ALI compared to CTR animals regardless of FIO2.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic and gas exchange alterations during Intralipid infusion in patients with adult respiratory distress syndrome.

Hemodynamic and pulmonary gas exchange consequences of 20 percent intravenous fat emulsion infusion (3.0 +/- .3 mg/kg/min) were evaluated in 19 patients who demonstrated ARDS. Lipid infusion precipitated a significant reduction in PaO2/FIO2 from 241 +/- 50 to 184 +/- 41 (mean +/- SD) and increased MPAP from 26.0 +/- 5.1 to 31.8 +/- 4.8 mm Hg, pulmonary vascular resistance from 149 +/- 78 to 179 +/- 61 dyne.s/cm5 and pulmonary venous admixture (Qva/Qt) from 20.7 +/- 15.2 to 30.6 +/- 8.6 percent. Further analysis revealed that the magnitude of increased Qva/Qt was greater in patients who manifested septicemia (N = 10) compared to those who did not (N = 9): 12.3 vs 7.3 percent, respectively. We conclude that intravenous lipid administration was associated with increased MPAP and Qva/Qt in patients with ARDS, particularly when accompanied by septicemia. Although these alterations resolved after the lipid infusion was terminated, we suggest that prudent measures should be taken to guarantee adequate oxygenation during intravenous fat emulsion therapy in patients suffering from ARDS.

Cardiopulmonary effect of various inspiratory flow profiles during controlled mechanical ventilation in a porcine lung model.

We studied the influence of inspiratory flow (VI) patterns on gas exchange and hemodynamics after metacholine inhalation challenge (MIC) in seven swine mechanically ventilated with a constant, sinusoidal, accelerating, and decelerating flow pattern. Blood gas and expired CO2 tensions, pulmonary mechanics, and hemodynamics were measured during each pattern. Flow pattern sequence was randomized and MIC was repeated at 30 to 45-min intervals. MIC reliably reduced PaO2, and increased PaCO2 and peak tracheal pressure (PT). There was no significant difference in gas exchange or hemodynamics between various VI curves. Insufflation with an accelerated VI manifested a significantly higher PT than any other pattern. We conclude that no VI contour studied offers a unique advantage for gas exchange after MIC in swine. However, since accelerated gas flow generates significantly higher PT values, it is not recommended in the presence of significant airway resistance.

Hemodynamics and intrathoracic pressure transmission during controlled mechanical ventilation and positive end-expiratory pressure in normal and low compliant lungs.

PEEP can significantly reduce cardiac output. This reduction in cardiac output is frequently attributed to transmission of airway pressure to intrathoracic vascular structures. We designed an acute lung injury (ALI) model in swine (n = 7) characterized by low lung thorax compliance (CLT) and compared the fractional transmission of airway pressure to pleura (PPL) and pericardium (PPC) and hemodynamics to normal animals (n = 5) during controlled mechanical ventilation (CMV) and PEEP. Fractional transmission of PEEP to PPL and PPC was reduced significantly from 62 +/- 8% and 54 +/- 19 (SD)% to 34 +/- 7% and 36 +/- 9% in normal and ALI animals, respectively. End-inspiration tracheal pressure was significantly higher in the low compliant group; thus, cardiac output was equally depressed in both groups despite reduction in fractional airway pressure transmission in ALI animals, possibly because absolute inspiratory PPL and PPC were similar due to increased pressure required to inflate injured lungs. The results of this investigation do not support the presumption that low CLT blunts hemodynamic consequences of CMV and PEEP.

Cardiopulmonary effects of Intralipid infusion in critically ill patients.

Cardiopulmonary effects of 500 ml of 20% iv fat emulsion (Intralipid) infusion in two groups of patients who required mechanical ventilation were evaluated in our Critical Care Center. Group 1 included ten patients void of signs or symptoms of sepsis. Group 2 consisted of ten patients exhibiting clinical and laboratory signs and symptoms of sepsis. Data were measured before and immediately after Intralipid infusion and when serum lipemia cleared. Intralipid infusion precipitated a significant increase in venous admixture (Qsp/Qt) from 13.7 +/- 3.6 (SEM) to 18.0 +/- 6.5% and from 22.0 +/- 4.8 to 25.8 +/- 7.0% in groups 1 and 2, respectively. Mean pulmonary artery pressure (MPAP) increased from 22.7 +/- 4.2 to 29.2 +/- 8.1 mm Hg and 26.4 +/- 6.8 to 28.0 +/- 4.0 mm Hg in groups 1 and 2, respectively. When serum lipemia cleared, Qsp/Qt and MPAP returned to preinfusion levels. We conclude that Intralipid infusion increases pulmonary artery pressure and venous admixture in critically ill patients. These changes are temporary and coincidental with serum lipemia rather than presence or absence of sepsis. Adequate levels of oxygenation should be confirmed during Intralipid infusion in patients with borderline oxygenation.

National survey of methods and criteria used for weaning from mechanical ventilation.

A national survey of hospital-based respiratory care departments was conducted to quantitate the use of various techniques and criteria employed to facilitate weaning of patients from mechanical ventilatory support. Responses were partitioned into private, nonprivate and university/university-affiliated institutions with further subdivision into bed-size groups of not more than 50 beds, 51 to 150 beds, 151 to 300 beds, and not less than 301 beds. Intermittent mandatory ventilation (IMV) was the most frequently used weaning technique in 90.2% of the responding hospitals. IMV was also listed as the primary mode of mechanical ventilatory support (71.6%). IMV to a T-tube system was the most common weaning protocol in nonuniversity hospitals. IMV to 3 to 5 cm H2O continuous positive airway pressure and IMV to T-tube were equally utilized in university/university-affiliated centers. PaCO2 was identified the most often (20.7%) and physiologic deadspace ratio the least often (2.5%) as a weaning criterion. We conclude that IMV is probably the most widely practiced weaning technique and that a variety of weaning criteria were employed.

Use of sodium nitroprusside in post-coronary bypass surgery. A plea for conservatism.

In 292 patients who underwent coronary artery bypass graft (CABG) surgery, seven patients developed sodium nitroprusside (SNP) toxicity in the postoperative period. Duration of infusion varied between 26 to 160 hrs and total SNP dose ranged from 1.8 to 12 mg/kg body weight. All patients were critically ill and required ventilatory support in the postoperative period. Tachyphylaxis to SNP requiring increase of SNP dose for control of hypertension, and loss of consciousness were the major signs of toxicity. Other commonly described signs of SNP toxicity were absent in those patients. Discontinuation of SNP therapy and treatment with sodium thiosulfate was followed by improvement in four patients. Three patients who failed to regain consciousness later died because of hemodynamic, pulmonary and/or renal complications. Our observation suggests that recommended doses of SNP may be toxic in unstable post-CABG patients. We recommend that the dose and duration of SNP infusion be minimized in critically ill patients.