Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

Differential effects of -mannose and 2-deoxy- -glucose on attempted powdery mildew fungal infection of inappropriate and appropriate ramineae

Barley, oat and wheat were used as both inappropriate hosts (IH) and appropriate hosts (AH) for three formae speciales of the fungus Blumeria graminis, the causal agent of powdery mildew disease. Treatment with either the glucose analog 2-deoxy- -glucose (DDG) or with -mannose dramatically suppressed penetration resistance in IH and to a much lesser extent in AH combinations. Other effects of DDG and -mannose were strikingly dissimilar. DDG greatly reduced localized autofluorescence at fungal attack sites on epidermal cells, and prevented hypersensitive epidermal cell death (HR). -mannose had little effect on autofluorescence or HR. DDG arrested the development of fungal haustoria and apparently prohibited biotrophy leading to secondary hyphae. -mannose allowed haustorial development and functional biotrophy leading to the production of elongating secondary hyphae. This suggests that B. graminis is in some way capable of utilizing -mannose as a carbon substrate. Results with IH combinations paralleled those of known mlo -barley responses to DDG and -mannose. Results are discussed in relation to specific physiological processes known to be influenced by either DDG or by -mannose, or by both compounds.     

Do public attitudes affect conservation effort? Using a questionnaire-based survey to assess perceptions,beliefs and superstitions associated with frogs in South Africa

Globally, amphibians are among the least appreciated vertebrates and are often negatively perceived by the general public. Such attitudes are particularly prevalent in South Africa, where fear, superstitions and myths associated with frogs are pervasive in some cultures. These attitudes could have harmful consequences both for the animals concerned and conservation efforts. This study aimed to investigate attitudes to frogs throughout South Africa across multiple age and ethnic groups. We used a multi-language survey in both hard-copy and online versions. Respondents (n = 2 295) comprised both genders across six age groups and five ethnic groups. A functionalist approach using structural equation modelling was used to assess how liking of frogs and cultural beliefs varied with socio-demographic factors. Attitudes varied significantly between groups, with males more likely to like frogs than females and age and level of education also significantly linked to liking. The influence of cultural beliefs and lack of knowledge also directly influenced negative attitudes towards frogs. More than 60% of respondents expressed an interest in learning more about frogs, indicating an opportunity for improving attitudes through educational outreach. The findings of this study can be used to inform current efforts to protect amphibians in South Africa through social interventions.     

Genetic characterization of 2 novel feline caliciviruses isolated from cats with idiopathic lower urinary tract disease

Feline caliciviruses (FCVs) are potential etiologic agents in feline idiopathic lower urinary tract disease (I-LUTD). By means of a modified virus isolation method, we examined urine obtained from 28 male and female cats with nonobstructive I-LUTD, 12 male cats with obstructive I-LUTD, and 18 clinically healthy male and female cats. All cats had been routinely vaccinated for FCV. Two FCVs were isolated; I (FCV-U1) from a female cat with nonobstructive I-LUTD, and another (FCV-U2) from a male cat with obstructive I-LUTD. To determine the genetic relationship of FCV-U1 and FCV-U2 to other FCVs. capsid protein gene RNA was reverse transcribed into cDNA, amplified, and sequenced. Multiple amino acid sequence alignments and phylogenetic trees were constructed for the entire capsid protein, hypervariable region E, and the more conserved (nonhypervariable) regions A, B, D, and F. When compared to 23 other FCV isolates with known biotypes, the overall amino acid sequence identity of the capsid protein of FCV-U1 and FCV-U2 ranged from 83 to 96%; identity of hypervariable regions C and E ranged from 58 to 85%. Phylogenetically, FCV-U1 clearly separated from other FCV strains in phenograms based on nonhypervariable regions. In contrast, FCV-U2 consistently segregated with the Urbana strain in all phenograms. Clustering of isolates by geographic origin was most apparent in phenograms based on nonhypervariable regions. No clustering of isolates by biotype was apparent in any phenograms. Our results indicate that FCV-UI and FCV-U2 are genetically distinct from other known vaccine and field strains of FCV.     

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.