Oral infection with a Shiga toxin-negative Escherichia coli O157:H7 strain elicits humoral and cellular responses but does not protect sheep from colonisation with the homologous strain

We have previously shown that rectally inoculated sheep excrete Escherichia coli O157:H7 during weeks to months without developing a clear antibody response. However, antibodies against this bacterium were observed in naturally infected sheep, which most likely became orally infected. To understand this difference, sheep were orally inoculated with the same Shiga toxin-negative E. coli O157:H7 strain that was used for the rectal inoculation. A primary oral inoculation resulted in shedding of E. coli O157:H7 in the faeces and detection of antibody responses against intimin, EspA and EspB. The antibody titres waned as shedding decreased. A secondary inoculation resulted in longer shedding, even though a booster antibody response occurred. Cellular responses followed a similar pattern as the antibody levels, albeit with a lower secondary response. The presence of antigen-specific antibody-secreting cells indicates involvement of both a systemic response in the spleen and a local immune response in the terminal rectum. These results suggest that E. coli O157:H7 has to pass the small intestine to evoke antibody responses.     

Pharmacokinetics of topically applied ciprofloxacin in tears of mesocephalic and brachycephalic dogs

OBJECTIVE: To evaluate the pharmacokinetics of ophthalmic ciprofloxacin in the tear film of normal mesocephalic and brachycephalic dogs. ANIMALS STUDIED: Twenty mesocephalic dogs and 15 brachycephalic dogs. PROCEDURES: Thirty-five microliters of ciprofloxacin were placed on the cornea of both eyes of each dog. Five brachycephalic dogs were used twice. A tear-test strip placed in the ventral cul de sac for 30 s was used to obtain samples. The tear film of each eye was sampled once at eight time-points post administration, resulting in five samples at each time-point. Samples were evaluated using high performance liquid chromatography. Data from the two skull types were compared using the unpaired two-tailed t-test. RESULTS: The mean concentration of ciprofloxacin in the tears of mesocephalic dogs was 192.8 +/- 269.97, 140.6 +/- 91.06, 56.60 +/- 28.47, 13.6 +/- 6.3, 43.25 +/- 59.71, 16.6 +/- 10.62, 15.6 +/- 13.16 and 6.25 +/- 9.84 microg/g at 5, 10, 15, 30 min and 1, 2, 4 and 6 h, respectively. The mean concentration of ciprofloxacin in the tears of brachycephalic dogs was 272.6 +/- 106.21, 144.4 +/- 142.32, 131.2 +/- 147.07, 75 +/- 80.07, 40.8 +/- 30.35, 35 +/- 21.98, 52.75 +/- 51.87 and 8.6 +/- 12.10 microg/g at 5, 10, 15, 30 min and 1, 2, 4 and 6 h, respectively. There was no statistical difference in tear concentration at any time-point between skull types. CONCLUSIONS: Topical application of ciprofloxacin resulted in a mean tear concentration of ciprofloxacin that remained above the MIC(90) levels for most pathogenic bacteria for 6 h in normal mesocephalic and brachycephalic dogs.     

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non‐reproductive tissues

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.     

Effects of low‐density lipoproteins as additive on quality parameters and oxidative stress following cryopreservation of mithun (Bos frontalis) spermatozoa

Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p < .05) improvement in viability, sperm and nuclear abnormality, acrosome integrity, motility (progressive and in cervical mucus), cholesterol content, and reduction in the leakage of intracellular enzymes in Group II. Moreover, intactness of acrosome and biochemical membranes was protected significantly (p < .05) in addition to significant (p < .05) improvement in binding per cent and binding index in IVF assay in extender containing 8% LDL. These results demonstrate that although cryopreservation of mithun's spermatozoa in EY was comparable with other species, addition of 8% LDL holds a clear advantage over EY or 10% LDL.     

Receptor-specific binding of purified F4 to isolated villi.

Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT+Sta+STb+) were performed and the purity and yield of F4ac were compared. Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10(11) bacteria/ml). The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10(12) bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10(12) bacteria, i.e. 26.2%) and the relative contamination higher (1.0 mg per 10(12) bacteria, i.e. 73.8%). A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels. The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained. The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay. Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb.