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.

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.

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.