Interactions Between Geminiviruses in a Naturally Occurring Mixture: Pepper huasteco virus and Pepper golden mosaic virus

ABSTRACT Pepper huasteco virus (PHV) and Pepper golden mosaic virus (PepGMV) are found in mixtures in many horticultural crops in Mexico. This combination constitutes an interesting, naturally occurring model system to study several aspects of virus-virus interactions. Possible interactions between PHV and PepGMV were studied at four levels: symptom expression, gene expression, replication, and movement. In terms of symptom expression, the interaction was shown to be host-dependent because antagonism was observed in pepper, whereas synergism was detected in tobacco and Nicotiana benthamiana. PHV and PepGMV did not generate viable pseudorecombinant viruses; however, their replication is increased during mixed infections. An asymmetric complementation in movement was observed because PHV was able to support the systemic movement of PepGMV A whereas PepGMV did not support the systemic distribution of PHV A. Heterologous transactivation of both coat protein promoters also was detected. Several conclusions can be drawn from these experiments. First, viruses coinfecting the same plant can interact at several levels (replication, movement) and in different manners (synergism, antagonism); some interactions might be host dependent; and natural mixed infections could be a potential source of geminivirus variability by generating viable tripartite combinations that could facilitate recombination events.     

γ-Amino Butyric Acid Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims to ascertain the influence of gamma-amino butyric acid (GABA)(A or B) receptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates GABA-AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)-alpha at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to a specific GABA(A or B) receptor agonist or antagonist at various doses (0.1-10 mm). GABA(A) (muscimol; no E(2), n = 7 perifusion chambers, with E(2), n = 11) or GABA(B) (baclofen; no E(2), n = 8, with E(2), n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20-30 min after exposure to 10 mm GABA(A or B) antagonists (bicuculline, no E(2), n = 7: from 4.6 +/- 0.7 to 33.0 +/- 0.4, with E(2), n = 17: from 11.9 +/- 1.4 to 32.8 +/- 6.0; CGP52432, with E(2), n = 14: from 14.0 +/- 2.6 to 28.8 +/- 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABA(B) but not GABA(A) antagonist-stimulated AVP release was enhanced in the presence of E(2). In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E(2) through GABA(B) receptors in vitro.     

Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and Epididymis

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak‐to‐moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.     

Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

Screening wild plants of Capsicum annuum for resistance to pepper huasteco virus (PHV): Presence of viral DNA and differentiation among populations

Plants collected in thirteen wild populations of Capsicum annuum from Northwest Mexico were tested for resistance to the pepper huasteco begomovirus (formerly subgroup III) (PHV) that is transmitted by the white fly Bemisia tabaci Genadius. Plants were inoculated using both grafting and biolistic methods. Presence of viral DNA was detected by dot blot hybridization and densitometry. Populations varied in their resistance to PHV. Plants of only two of the populations either did not develop disease symptoms or showed very light symptoms after inoculation. In some cases, symptoms appeared several days after inoculation. In plants of these populations viral DNA was detected by dot-blot hybridization assays but they appear to be a good source of resistance (symptomless) for use in breeding programmes. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Effect of a Chronic Stressor, Lameness, on Detailed Sexual Behaviour and Hormonal Profiles in Milk and Plasma of Dairy Cattle

The objectives of the present study were to quantify the effects of a biological chronic stressor (lameness) on the duration and frequency of different oestrous behaviours in parallel with milk hormone profiles. Dairy cows 51.8 ± 1.4 days postpartum (n = 59), including 18 non‐lame control cows, were scored for lameness and closely observed for signs of oestrus having had their follicular phases synchronized by administration of gonadotrophin‐releasing‐hormone (GnRH) followed by prostaglandin F_2α (PG) 7 days later. Lameness shortened the period when herd‐mates attempted to mount the lame cows (1.83 ± 0.69 h vs 5.20 ± 1.53 h; p = 0.042) but did not affect the overall duration of total behaviours (lame 12.3 ± 1.3 h vs non‐lame 15.2 ± 1.3 h). Lameness also lowered the intensity of oestrus [1417 ± 206 points (n = 18) vs 2260 ± 307 points (n = 15); p = 0.029]. Throughout the synchronized oestrous period, lame cows mounted the rear of herd‐mates less frequently (p = 0.020) and tended to chin rest less (p = 0.075). Around the period of maximum oestrous intensity, lameness also diminished the proportion of cows mounting the rear of another cow and chin resting (p = 0.048, p = 0.037, respectively). Furthermore, lame cows had lower progesterone values during the 6 days before oestrous (p ≤ 0.05). Fewer lame cows were observed in oestrus following PG (non‐lame 83%, lame 53%; p = 0.030); however, if prior progesterone concentrations were elevated, lame cows were just as likely to be observed in oestrus. In conclusion, following endogenous progesterone exposure, lameness shortens the period when herd‐mates attempt to mount lame cows but does not affect the incidence of oestrous. However, lame cows are mounted less frequently and express oestrus of lower intensity. This is associated with lower progesterone prior to oestrus but not with abnormal oestradiol or cortisol profiles in daily milk samples.     

Respiratory mechanics of horses during stepwise treadmill exercise tests, and the effect of clenbuterol pretreatment on them

Normal Standardbred horses were given an incremental exercise test on a horizontal treadmill to evaluate the influence of exercise on gas exchange, resistance, dynamic compliance and inertance of the respiratory system. The exercise test consisted of 2 min exercise steps at each of the following speeds: 2.4 m/sec (walk), 4.5 m/sec (slow trot), 7.0 m/sec (fast trot) and 10 m/sec (gallop). At rest and after 1 min of exercise at each step, airflow, tidal volume, respiratory frequency, pharyngeal, mid-oesophageal and transdiaphragmatic pressures and arterial blood gas tensions were measured. The same horses were subsequently treated intravenously with clenbuterol (0.8 microgram/kg) and an identical exercise test and measurement performed 10 min after clenbuterol injection. In response to exercise, there were large increases in tidal volume, respiratory frequency, airflow and pressures. Exercise was associated with a decrease in upper airway resistance but total pulmonary resistance was unchanged. Exercise did not alter inertance or dynamic compliance, horses became hypoxaemic, and at 10 m/sec (galloping) also developed hypercarbia. Treatment with clenbuterol did not alter any of these measurements in response to exercise. These data suggest that dilation of upper airways occurs during exercise, and that inertial forces are important in strenuously exercising horses and may influence the accuracy of dynamic compliance determinations at high exercise intensities.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum

An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumoniae in porcine serum is described. The results are presented as an ELISA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied to the ELISA plate was diluted 1 in 20 in PBS - Tween. Mean ELISA ratios ranged from 1.2 +/- 0.3 for pigs without porcine enzootic pneumonia (PEP) lesions to 5.5 +/- 1.5 for pigs observed with a PEP lesion reacting positively with immunofluorescent histopathology. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofluorescent histopathology had a mean ELISA ratio of 4.9 +/- 1.7. The ELISA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known M hyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed M hyorhinis showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3) for M hyopneumoniae.