Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

BACKGROUND: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. METHODS: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. RESULTS: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. CONCLUSIONS: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.

Comparative Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata.

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

Intra-observer repeatability of optical quality measures provided by a double-pass system.

PURPOSE: The aim was to evaluate the intra-observer repeatability of the ocular optical quality measurements provided by a double-pass system in healthy eyes. METHODS: A total of 24 healthy eyes of 24 patients with ages ranging from 20 to 60 years were included in the study. Three consecutive measurements were obtained by an experienced examiner with the Optical Quality Assessment System (Visiometrics) of the following parameters defined by the manufacturer as follows: cut-off spatial frequency for the modulation transfer function (COMTF), Strehl ratio, width of the point spread function (PSF) at 10 per cent of its maximal height (PSF10) and width of the PSF at 50 per cent of its maximal height (PSF50). Intra-observer repeatability for 3.0 mm pupil measurements was evaluated by the within-subject standard deviation (S(w) ) and intra-class correlation coefficient (ICC). RESULTS: The S(w) values for the different parameters evaluated were 4.34 cycles per degree for COMTF, 0.03 for the Strehl ratio, 1.14 arcmin for PSF10 and 0.36 arcmin for PSF50. The ICC values for these parameters were 0.746, 0.627, 0.783 and 0.814 for COMTF, Strehl ratio, PSF10 and PSF50, respectively. Statistically significant correlations were found between COMTF and the S(w) for PSF50 (r = -0.45, p = 0.03), and between the S(w) and the mean value for PSF50 (r = 0.42, p = 0.04). The significance of these correlations would vanish when considering the Bonferroni correction. CONCLUSIONS: Measurements provided by the Optical Quality Assessment System should be considered and interpreted with caution because their consistency seems to be limited, especially in eyes with poor optical quality. The limitation in the validity of measurements due to the use of infrared light instead of middle-wavelength light should also be considered.

Effects of cortisol on pregnancy rate and corpus luteum function in heifers: an in vivo study.

To determine whether glucocorticoids affect the function of the bovine corpus luteum (CL) during the estrous cycle and early pregnancy, we examined the effects of exogenous cortisol or reduced endogenous cortisol on the secretion of progesterone (P4) and on pregnancy rate. In preliminary experiments, doses of cortisol and metyrapone (an inhibitor of cortisol synthesis) were established (n=33). Cortisol in effective doses of 10 mg blocked tumor necrosis factor-induced prostaglandin F(2α) secretion as measured by its metabolite (PGFM) concentrations in the blood. Metyrapone in effective doses of 500 mg increased the P4 concentration. Thus, both reagents were then intravaginally applied in the chosen doses daily from Day 15 to 18 after estrus (Day 0) in noninseminated heifers (n=18) or after artificial insemination (n=36). Pregnancy was confirmed by transrectal ultrasonography between Days 28-30 after insemination. Plasma concentrations of P4 were lower in cortisol-treated heifers than in control heifers on Days 17 and 18 of the estrous cycle (P<0.05). However, the interestrus intervals were not different between control and cortisol-treated animals (P>0.05). Moreover, metyrapone increased P4 and prolonged the CL lifespan in comparison to control animals (P<0.05). Interestingly, in inseminated heifers, cortisol increased the pregnancy rate (75%) compared with control animals (58%), whereas metyrapone reduced the pregnancy rate to 16.7% (P<0.05). The overall results suggest that cortisol, depending on the physiological status of heifers (pregnant vs. nonpregnant), modulates CL function by influencing P4 secretion. Cortisol may have a positive influence on CL function during early pregnancy, leading to support of embryo implantation and resulting in higher rates of pregnancy in heifers.

Vector analysis of astigmatic changes after cataract surgery with implantation of a new toric multifocal intraocular lens.

PURPOSE: To analyze the astigmatic changes after cataract surgery with the implantation of a new toric multifocal intraocular lens (IOL) using the Alpins vector method. SETTING: Vissum-Instituto de Oftalmológico de Alicante, Alicante, Spain. DESIGN: Prospective case series. METHODS: After cataract surgery with implantation of an AT Lisa 909M toric multifocal IOL, the visual, refractive, corneal topographic, internal astigmatism, defocus curves, and contrast sensitivity outcomes were evaluated over 6 months. Refractive astigmatic changes (target astigmatism [TIA]; surgically induced astigmatism [SIA]; difference vector; magnitude of error; flattening effect; torque) were analyzed using the Alpins vectorial method. RESULTS: The study enrolled 23 eyes (12 patients). Postoperatively, there was a significant reduction in refractive cylinder (P < .01) with an associated visual improvement for near (P = .03) and distance (P=.01). The magnitude of SIA vector was significantly larger than the TIA 3 months postoperatively (P = .03). The mean magnitude of the difference vector was 0.67 diopter (D) ± 0.51 (SD) at 6 months. The mean magnitude of error remained positive and close to zero over the entire follow-up (P ≥ .19). The mean magnitude of torque vector was 0.49 ± 0.54 D at 6 months. Significant positive correlation was found between the magnitude of torque and the difference vector (r ≥ 0.70, P<.01). CONCLUSION: The new toric IOL restored distance and near visual function with excellent predictability in eyes with moderate to high corneal astigmatism having cataract surgery.

Vector analysis of astigmatic changes after cataract surgery with toric intraocular lens implantation.

PURPOSE: To analyze the astigmatic changes after cataract surgery with toric intraocular lens (IOL) implantation and determine the relationship between these changes and the final visual outcomes. SETTING: Vissum-Instituto de Oftalmológico de Alicante, Alicante, Spain. DESIGN: Prospective case series. METHODS: This study comprised eyes with visually significant cataract that had surgery with Acrysof toric IOL implantation. Refractive, corneal topography, internal astigmatism, and contrast sensitivity outcomes were evaluated over 6 months. The Alpins vectorial method was used to analyze the target (TIA) and surgically (SIA) induced astigmatism, difference vector, magnitude of error, flattening effect, and torque. RESULTS: The study enrolled 27 eyes (21 patients). Postoperatively, there was a significant reduction in refractive cylinder (P < .01) with an associated visual improvement (P < .01). The magnitude of the SIA vector was significantly lower than the TIA 1 month and 6 months after surgery (P < .01). The mean magnitude of the difference vector was 0.91 diopter (D) ± 0.45 (SD) at 6 months, with no significant differences between postoperative visits (p > 0.19). The mean magnitude of torque vector was 0.59 ± 0.42 D at 6 months. There was a significant negative correlation between refractive cylinder and the magnitude of torque (r ≤ -0.54, P < .01) and the difference vector (r = -0.98, P < .01). CONCLUSION: The toric IOL restored distance visual function in eyes with cataract and significant corneal astigmatism, although there was a small trend toward undercorrection related to misalignment of treatment. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Comparative analysis of clinical outcomes obtained with a new diffractive multifocal toric intraocular lens implanted through two types of corneal incision.

PURPOSE: To analyze and compare the clinical outcomes obtained after cataract surgery with the implantation of a new multifocal toric intraocular lens (IOL) using two different types of corneal incision. METHODS: Retrospective study including 64 eyes of 35 patients who underwent surgery with implantation of the AT LISA 909M multifocal toric IOL (Carl Zeiss Meditec) in 2 different ophthalmologic centers using different criteria for corneal incision size: sub-1.8 mm (micro-incision surgery [MICS] group) and 2.2 mm (mini-incision group). Visual, refractive, and corneal topographic outcomes were evaluated during 6-month follow-up. Additionally, refractive astigmatic changes were analyzed using the Alpins vectorial method. RESULTS: Significant reductions of refractive sphere and cylinder were observed postoperatively (P<.03), with associated visual improvements for near and distance (P<.01) in both groups. Mean postoperative magnitudes of difference vector, torque, and magnitude of error in the overall sample were 0.52±0.36 diopters (D), 0.36 ± 0.36 D, and 0.08 ± 0.38 D, respectively. A mean overcorrection of 4% in refractive astigmatism was found. Mean angle of error was 0.37± 5.50° and -4.51±13.16° for the MICS and mini-incision groups, respectively (P=.09). Significant positive correlations were found between the magnitudes of torque and difference vector (r=0.78, P<.01) as well as between the magnitude of torque and absolute angle of error (r=0.76, P<.01). CONCLUSIONS: Implantation of the AT LISA toric IOL using corneal incisions <2.2 mm provides excellent predictability for astigmatic correction.

Vector analysis of evolutive corneal astigmatic changes in keratoconus.

PURPOSE: To evaluate by vector analysis the corneal astigmatic changes occurring in keratoconic corneas during a 3-year follow-up and to determine the relationship between these changes and other clinical changes. METHODS: Keratoconic eyes (n = 114) of 75 patients ranging in age from 14 to 70 years were retrospectively reviewed in four different centers. In all cases, a 3-year follow-up was completed after the diagnosis of keratoconus. Visual, refractive, keratometric, aberrometric, internal astigmatism (IA), and pachymetric changes were evaluated during the follow-up. In addition, corneal astigmatic changes were evaluated by examining the following parameters, using a modification of the Alpins vectorial method: evolutive astigmatism (EA) and angle of error (AE). RESULTS: An increase in the magnitude of refractive (P = 0.02) and corneal astigmatism (P = 0.05) was found. The mean magnitude of EA was 1.21 ± 0.97 D at 3 years, with no significant changes at each annual visit (P ≥ 0.52). Mean absolute AE increased significantly by the end of the follow-up (P < 0.01). Absolute AE and the increase in corneal astigmatism were found to correlate at 2 years (r = 0.675, P < 0.01). This correlation became poorer at 3 years (r = 0.352, P = 0.02). The magnitude of the EA was also found to be significantly correlated with central corneal thinning (r = -0.441, P = 0.02). Multiple regression analysis revealed that the magnitude of EA at 3 years correlated significantly with the baseline sphere and IA (R² = 0.86, P < 0.01). CONCLUSIONS: Corneal vector astigmatic changes are related to some signs of keratoconus progression and are therefore predictive.

Immunomodulatory and anti-fibrotic effects of ganglioside therapy on the cardiac chronic form of experimental Trypanosoma cruzi infection.

Heart failure and sudden death are the most common causes of death in patients with Chagas' disease. The main drug available for Chagas treatment is benznidazole, which eradicates Trypanosoma cruzi parasites during the acute stage of infection. However, its effectiveness during the chronic phase remains unclear. Ganglioside GM1 administration in chronically infected patients resulted to be an effective treatment for the cardiac manifestations of Chagas' disease. However, the precise mechanisms of GM1-induced improvement during chronic T. cruzi infection still remain unknown. The aim of the present study was to evaluate the potential benefits of ganglioside GM1 treatment during the chronic stage of murine chagasic infection, analyzing its influence on myocardial pathology as well as its immunomodulatory effects. The results obtained showed that GM1 therapy diminished the extent of myocardial fibrosis induced by T. cruzi in chronically infected mice. In addition, GM1 treatment resulted in a significant reduction in the myocardial expression of the fibrogenic cytokine TGF-ß as well as the proinflammatory cytokines and chemokines IFN-γ, TNF-α and CCL5/RANTES. Our experimental data indicate that GM1 could be a promising immunomodulatory agent with capacity to limit the inflammatory process leading to myocardial tissue damage in chronic chagasic patients.

Acute changes in circulating concentrations of progesterone and nitric oxide and partial pressure of oxygen during prostaglandin F2alpha-induced luteolysis in cattle.

To examine whether oxygen (O(2)) and nitric oxide (NO) are temporally associated with the acute changes in luteal function during luteolysis, we determined the real-time changes in the circulating concentrations of progesterone (P4) and nitrite/nitrate (the stable metabolites of NO) and the partial pressure of oxygen (pO(2)) during prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in cattle. Catheters for frequent blood sample collection were inserted into the ovarian vein (OV), jugular vein (JV) and aorta abdominalis (AA) in 12 cows on Day 9 of the oestrous cycle (oestrus=Day 0). On Day 10, the cows were randomly divided into two groups and treated with a luteolytic dose of a PGF(2alpha) analogue or saline solution (control). Blood samples were collected at -2, -1, 0, 0.25, 0.5, 0.75, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). Injection of a PGF(2alpha) induced a significant decrease in the concentrations of P4 in OV plasma within 2 h. The decrease in P4 concentrations was preceded by an increase in the NO concentrations in the blood collected from OV, JV and AA. Basal pO(2) was significantly higher in OV blood than in JV blood (P<0.05). PGF(2alpha) injection increased pO(2) in OV blood between 0.5 and 2 h. These results demonstrate that PGF(2alpha) induced an acute increase in pO(2) and NO in the ovarian circulation and suggest that O(2) and NO are involved in the early events of CL regression, including inhibition of P4 secretion and output, in cattle.

Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines.

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.

Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.