Detection of antibody to Toxocara vitulorum perieneteric fluid antigens (Pe) in the colostrum and serum of buffalo calves and cows by Western blotting

Toxocara vitulorum, a nematode parasite in the small intestine of cattle and water buffaloes, causes high morbidity and mortality of 1-3 months old buffalo calves. This research evaluated the specific perieneteric antigens (Pe) reactivity of anti-T. vitulorum-Pe antibody (Tv-Pe-Ab) in both immune sera and colostrum from buffalo cows immediately post-partum from buffalo cows. The presence of Tv-Pe-Ab in sera of buffalo newborn calves was also examined at 1 day before and after suckling the colostrum as well as in sera from naturally infected calves at the beginning and peak of the maximum infection and then again during the period of rejection and post-rejection of the parasite. Pe antigens were characterized for Tv-Pe-Ab by SDS-PAGE and Western blot (WB). The SDS-PAGE showed that Pe contained nine protein bands (11, 14, 31, 38, 58, 76, 88, 112 and 165 kDa). All Pe bands were recognized by Tv-Pe-Ab in sera and colostrum of buffalo cows. Only the serum antibodies of buffalo calves at 1 day of age after suckling the colostrum and during the beginning of T. vitulorum infection recognized Pe antigen's nine bands. In contrast, serum antibodies from 1-day-old buffalo calves, taken before suckling colostrum, did not react with any protein band. In suckling calves, which reached peak egg output, rejection and post-rejection stages of the infection, serum Tv-Pe-Ab reactivity with lower molecular weight protein bands (11-76 kDa) was lost and only reactivity with the Pe protein bands of higher molecular weight (88, 112 and 165 kDa) remained.     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

Production of inhibin A and inhibin B in boars: changes in testicular and circulating levels of dimeric inhibins and characterization of inhibin forms during testis growth

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29–31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29–31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa monomer. Immunohistochemistry indicated that positive immunostaining for the subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.     

Relationship between the expression of key lipogenic enzymes,fatty acid composition,and intramuscular fat content of Limousin and Aberdeen Angus cattle

The amount and fatty acid composition of intramuscular fat (IMF) are major factors in determining the nutritional value of beef. Understanding the mechanisms regulating IMF formation is important for designing strategies for manipulation of IMF quantity and quality. The aim of this study was to investigate the contribution of key lipogenic enzymes towards intramuscular fat formation in Aberdeen Angus and Limousin crossbred steers. This study reports the presence of 150 kDa acetyl-CoA-carboxylase α (ACCα), 150 kDa fatty acid synthase (FAS), 37 kDa stearoyl-CoA desaturase (SCD), 50 kDa Δ6-desaturase (Δ6d), and 50 kDa Δ5-desaturase (Δ5d) immunoreactive bands in cattle muscle (as detected by Western blot analyses). A significant positive relationship was established between ACCα protein content and IMF content; FAS protein content and IMF, as well as between expression of these enzymes and the products of their reactions, saturated fatty acid. IMF level was also positively related to the expression of Δ6d and Δ5d proteins. However, no relationship was found between IMF and the amount of SCD protein. It is suggested that the rate of saturated fatty acids and PUFA biosynthesis might have a significant impact on IMF deposition in cattle.     

Expression of inhibin/activin subunits in the ovaries of fetal and neonatal mice

In the present study, the expression of inhibin/activin subunits in the mouse ovary from 13 days post-coitus (dpc) to 30 days postpartum (dpp) was investigated. Circulating FSH, LH, inhibin A, and inhibin B in neonatal to 30 dpp ovaries were measured. Inhibin/activin subunits (alpha, beta(A), beta(B) ) were weakly stained in 13 dpc ovarian stromal cells and increased with age. Inhibin alpha subunit was immunolocalized in follicular granulosa cells at each developmental stage. In 30 dpp ovaries, several large antral follicles were strongly stained for inhibin alpha subunit. Inhibin beta(A) subunit was weakly immunolocalized in granulosa cells until 20 dpp. Moreover, 2 to 3 antral follicles from 20 to 30 dpp were strongly stained for inhibin beta(A) subunit. There was relatively high immunoactivity for inhibin beta(B) subunit in neonatal to 30 dpp mouse ovaries. All three inhibin subunits were stained in theca-interstitial cells from 15 dpp onward. RIA data showed that a temporal increase in circulating FSH occurred around 10 dpp, while the plasma concentrations of LH were sustained at a relatively higher level from 8 to 15 dpp. Inhibin B was detectable in circulation early at 1 dpp (day of birth), and a clear increase in inhibin B occurred around 8 dpp. Circulating inhibin B gradually increased from 20 dpp to 30 dpp, indicating a negative correlation with FSH. Inhibin A levels were only measured on 25 and 30 dpp, and the levels were low. These results suggest that inhibins play an important role in early folliculogenesis in mice. In addition, inhibin B seems to be the main functional isoform from the neonatal to prepubertal stage in the mouse ovary.     

Changes in Ovarian Follistatin Levels During the Oestrous Cycle in Sheep may Serve as an Intraovarian Regulator

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day ?1 and 0) to mid‐luteal phase (days 11–13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.     

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 1 crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31/32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.     

Fertility in rats immunized with steroid-free bovine follicular fluid

Inhibin is a gonadal hormone that inhibits the release of follicle stimulating hormone (FSH) from the anterior pituitary gland. The objective of this study was to determine whether active immunization of male and female rats against inhibin rich, steroid-free bovine follicular fluid would increase inhibin antibody titre, onset of female puberty, pregnancy rate, litter size, testis weights, testosterone concentration and serum FSH. Immunization of rats with steroid free bovine follicular fluid stimulated production of anti-inhibin antibodies that immunoneutralized endogenous inhibins and increased levels of circulating FSH in immunized males. Inhibin immunoneutralization resulted in early vaginal opening in immunized females compared with controls and pregnancy rates were increased when immunized female rats were mated with immunized males. However, serum testosterone, testis weights and potential litter size remained unchanged. We conclude that methods to immunoneutralize inhibin may have merit as therapeutic procedures to enhance reproductive performance in domestic animals.     

The expression and localization of inhibin isotypes in mouse testis during postnatal development

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress follicle-stimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, α, β_A and β_B, in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin α, β_A and β_B expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin α, β_A and β_B were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin α was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin β_A and β_B immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.     

Purification, characterization and expression kinetics of heat shock protein 70 from Bubalus bubalis

The present work on Bubalus bubalis (buffalo) was designed to study heat shock protein 70 (HSP70) induction in lymphocytes, its purification and characterization. HSP70 induction and expression kinetics at different temperatures and time durations were also studied. HSP70 purification was carried out by immunoaffinity chromatography using adenosine di-phosphate (ADP-agarose column) and the characterization of the purified protein was done using western blotting by mouse monoclonal anti-HSP70. The molecular weight of HSP70 of buffalo lymphocytes was found to be approximately of 68 kDa and was less than that of bovine brain HSP70. The purified HSP70 was assessed using indirect inhibition enzyme-linked immunosorbent assay (ELISA). A good amount of HSP70 (1430 ηg HSP70/100 μl) was recovered after purification, out of the total 2040.40 ηg of HSP70/100 μl of cell supernatant. To assess the impact of temperature and time dependent variability in the induction and expression pattern of HSP70, buffalo lymphocytes were subjected to three different temperature treatments, viz.: (I) 38 °C for 48 h and further exposed the same cells at 45 °C for 3 h, (II) 42 °C for 3 h, and (III) 45 °C for 3 h, respectively. The respective cell viability was found to be 68%, 63%, and 51%. The HSP70 levels were 58.30 ± 4.37, 42.59 ± 9.04 and 21.95 ± 6.79 ng/million cells, respectively, at three temperature exposures. The results indicates that higher intensity and duration of temperature exposure cause higher HSP70 induction in buffalo lymphocytes to maintain cellular homeostasis with a threshold of thermal dose for maximum HSP70 expression. However immediate induction of HSP70 in the lymphocytes was dependent on magnitude of thermal exposure (stress level) and time of thermal exposure (stress duration). The present study on HSP70 and its induction will help likely to solve the problems related to the present scenario of thermo-adaptability in buffaloes.     

Partial purification and characterization of Gastrothylax crumenifer somatic antigens

The soluble extracts of Gastrothylax crumenifer isolated from the rumen of buffalo (Bubalus bubalis) were fractionated on a Sephadex G-200 column. A total of eight major fractions (F1, F2, F3, F4, F5, F6, F7, and F8) were separated from the whole homogenate of G. crumenifer, and each of these fractions was tested for their antigenicity by ELISA against rabbit hyperimmune sera. It was observed that F1, F2, F3 and F4 were highly antigenic, F6 and F7 were moderately antigenic and F5 and F8 were poorly antigenic. The individual fractions analysed after SDS-PAGE and Western blotting indicated that the antigenic fractions of G. crumenifer are of low molecular weight, in the range of <14-50kDa, and predominant antigenic components which were evident in most of the Sephadex profiles were of Mr 15, 18, 19, 23-24 and 28-32kDa.     

Plasma concentrations of inhibin A in cattle with follicular cysts: relationships with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones

We investigated the profiles of circulating levels of inhibin A and total inhibin in beef cows with follicular cysts in relation to the patterns of follicular development and circulating gonadotropins and steroid hormones. Turnover of follicular waves was monitored in five cows every 2 days for 70 days from 10 days after detection of estrus without ovulation. The mean interwave intervals were 19.6 ± 1.0 days (n = 18 waves with cysts from the five cows). Circulating levels of inhibin A were approximately 170 pg/ml before emergence of follicular waves with cysts and increased (P < 0.05) concomitantly with follicle emergence. High concentrations of inhibin A (greater than 300 pg/ml) were noted for 7 days during the growth phase of cystic follicles, but inhibin A levels decreased gradually when development of the cysts reached a plateau. This profile of inhibin A was similar to those of total inhibin and estradiol, but was inversely related to the changes in plasma FSH concentrations. LH pulse frequency and mean concentrations of LH in cows with cysts were higher than those observed in the luteal phase of normal cyclic cows. These results indicate that the capacity to secrete inhibin, as well as estradiol, is maintained in cystic follicles, the growth of which is extended by LH secretion at levels greater than those seen in the normal luteal phase. Inhibin A plays an important role in the extension of interwave intervals by suppressing recruitment of a new cohort of follicles.     

Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts

A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90 kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230 kDa. LC-nanoESI-MS/MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence "ILVGLIGTNR", that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081).     

Follicular and hormonal dynamics during the estrous cycle in goats

Transrectal ultrasonography of ovaries was performed daily in 6 goats for 3 consecutive estrous cycles. Blood samples collected daily were measured for concentrations of FSH, inhibin A, and estradiol-17beta. Follicular and hormonal data were analyzed for associations between the follicular waves and hormonal concentrations. During the interovulatory intervals, follicular growth and regression occurred in a wave like pattern (2-5 waves), and the predominant patterns were three and four follicular waves. In addition, there was no significant difference among the diameters of dominant follicles during the growth phase of the follicular waves. The number of 3 mm follicles peaked on days 0, 7, and 11 in interovulatory intervals that had three follicular waves and on days -1, 5, 11, and 15 in those that had four follicular waves. Plasma concentrations of FSH increased around the day of follicular wave emergence and declined with the growth of follicles. Circulating FSH increased again concomitant with regression of dominant follicles in the anovulatory wave, whereas FSH levels remained low in the ovulatory wave. Inhibin A was negatively correlated with FSH, while it was positively correlated with estradiol-17beta, suggesting that inhibin A is a product of healthy growing follicles and that it contributes to the suppression of FSH secretion. In conclusion, the growth of ovarian follicles in goats exhibits a wave-like pattern, and follicular dominance is less apparent in goats. Moreover, inhibin A may be a key hormone for regulation of the follicular wave through suppression of FSH secretion in goats.     

Expression of conserved mucin domains by epithelial tissues in various mammalian species

Mucins are related to infectious and non-infectious diseases in Veterinary and Human Medicine. MUC1 mucin is a transmembrane glycoprotein expressed on the apical surface of human epithelia while MUC5AC is the predominant secreted mucin expressed in human gastric epithelium and goblet cells of lung and eyes. MUC5AC C-terminus cysteine rich regions and the cytoplasmic tail of MUC1 domains are conserved among several mammalian species. Objective: to compare the expression of MUC1 and MUC5AC mucins in mammalian epithelia. CT33 anti-MUC1 cytoplasmic tail (MUC1CT) polyclonal antibody and 45M1 anti-MUC5AC monoclonal antibody were employed. By immunohistochemistry, MUC1CT was expressed in most tissues while MUC5AC was restricted to gastric surface epithelium and goblet cells from trachea and lung. By western blot, MUC1CT showed a band at approximately 35 kDa in most tissues; MUC5AC revealed bands at >180 kDa in stomach and lung secretions from rat, cat, pig and cow. When rat MUC5AC was immunoprecipitated, a band at about 180 kDa was obtained.     

野猪甘露聚糖结合凝集素的分离纯化及其多克隆抗体的制备

对野猪血清样品用溴化氰活化的甘露聚糖结合凝集素-Sepharose4B亲和柱亲和层析,获得纯化的野猪MBL样品,再进行SDS-PAGE电泳,显示有1条32kDa主带,2条36kDa和56kDa副带。选取32kDa的蛋白带制备免疫原,免疫家兔制备抗血清,Western blot方法鉴定该抗体可以特异性识别野猪血清中的MBL分子。本研究表明采用亲和层析法可以获得野猪血清中MBL-A分子,制备的多克隆抗体可以用于检测天然MBL分子,将为野猪MBL的生物学特性及与抗病力相关性的研究提供基础。     

Seasonal Variations in Seminal Plasma Proteins of Buffalo

The study was designed to evaluate the influence of season on semen characteristics and seminal plasma protein profile of buffalo bull semen. Thirty‐six ejaculates were collected in three seasons (winter, summer and rainy) from six adult Bhadawari bulls, and semen characteristics were evaluated immediately after collection. The seminal plasma was harvested by centrifugation and protein profiling, and percentage protein fractions were analysed by SDS‐PAGE. The significant effect of season was observed on ejaculate volume, sperm concentration, progressive motility, percentage live spermatozoa, hypo‐osmotic swelling test (HOST) and acrosomal integrity. The electrophoretogram of seminal plasma proteins revealed 20 protein bands in winter, 23 bands in rainy and 25 bands in summer seasons, illustrating the significant effect of seasons on seminal plasma proteins. Among these protein bands, 18 bands were observed common in semen samples of all three seasons while protein bands of 46, 55, 58, 144 and 160 kDa were found in rainy and summer seasons. The protein bands of 48 and 60 kDa were observed only in winter season, whereas 184 and 200 kDa were reported in summer season only. The protein fractions (protein%) of common protein bands observed in three seasons revealed a significant effect of season on protein bands of 24.5, 66, 70, 72, 84 and 86 kDa. From the study, it was pertinent that bull seminal plasma contains specific proteins in particular season, which may be associated with some of the semen characteristics, and these proteins could be used as markers of the semen quality of buffalo bulls.     

Purification and Characterization of Liver Ferritins from Different Animal Species

The ferritins were purified from liver homogenates of buffalo, camel, cattle, sheep and shark by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and iron content of affinity-purified liver ferritins ranged from 0.008 to 0.052 mg/g and 3.17% to 11.4% respectively. As they are glycoproteins, the ferritins contained variable amounts of neutral carbohydrates. Except for shark ferritin, the ferritins all exhibited immunological cross-reactivity with anti-buffalo liver ferritin and anti-horse spleen ferritin by immunodiffusion and immunoelectrophoresis. Gel electrophoresis, gel filtration and ultracentrifugal analysis indicated the presence of a monomeric ferritin in all cases. SDS-gel electrophoresis of shark ferritin gave a protein band of 18 kDa. Ovine, buffalo and bovine ferritin comprised two protein subunits, the H (20 and 21 kDa) and the L types (18 and 19 kDa). Oligomeric ferritin subunits with molecular weights of 27, 37 and 55 kDa were also found for bovine and buffalo ferritin. SDS-PAGE of camel ferritin revealed a complex pattern with four prominent bands of 61, 51, 44 and 39 kDa. Two fast-migrating components of 15 and 16 kDa were also found in the purified liver ferritins, including reference preparations. The PO₄ ^3–/Fe ratios of purified shark (0.10) and bovine ferritin (0.12) were similar to that of standard equine spleen ferritin (0.11). However, the ratio was higher in ovine (0.17), camel (0.22) and bovine (0.26) ferritins. The amino acid compositions, molecular weights and sedimentation coefficients of the different liver ferritins were similar.     

Comparison of Sarcocystis neurona isolates derived from horse neural tissue

Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan.Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed that Sarcocystis spp. from the eight horses appeared the same, but different from the grackle isolate. One Michigan horse isolate (MIH6) had two bands at 72 and 25kDa that were more prominent than the UCD1 isolate and other Michigan horse isolates. Western blot analysis showed that merozoites of eight of eight equine-derived isolates, and the UCD1 S. neurona isolate had similar bands when developed with serum or CSF of an infected horse. Major bands were seen at 60, 44, 30, and 16kDa. In the grackle (Cornell) isolate, bands were seen at 60, 44, 29, and 16kDa. DNA from merozoites of each of the eight equine-derived isolates and the grackle-derived isolate produced a 334bp PCR product (Tanhauser et al., 1999). Restriction fragment length polymorphism (RFLP) analysis of these horse isolates showed banding patterns characteristic for S. neurona. The grackle (Cornell) isolate had an RFLP banding pattern characteristic of other S. falcatula species. Finally, electron microscopy examining multiple merozoites of each of these eight horse isolates showed similar morphology, which differed from the grackle (Cornell) isolate. We conclude that the eight Michigan horse isolates are S. neurona species and the grackle isolate is an S. falcatula species.     

Purification and Characterization of IgM-like Immunoglobulin from Turbot (Scophthalmus maximus L.)

A total of 40 turbot (Scophthalmus maximus) were immunized 3 times during a 3 months period using DNP-HSA whereafter serum samples were collected and pooled. Specific immunoglobulins (Ig) were affinity purified on an agarose column with immobilized DNP-BSA and further purified by gel filtration whereafter monospecific rabbit anti Ig serum was generated. Size exclusion chromatography and non-reduced SDS-PAGE indicated a MW of 8-900 kDa of the dominant antigen binding proteins from turbot serum. Reduced SDS-PAGE showed this fraction to be composed of disulphide linked heavy and light chains with MWs of 79 and 27–29 kDa, respectively, indicating a tetrameric structure. Isoelectric focusing of the 800–900 kDa Ig showed several bands between pH 5.5 and pH 5.8. Mean Ig concentration in serum of 10 turbot was measured to 6.48 mg/ml (SD 5.4) using rocket immunoelectrophoresis. Low molecular weight antigen binding molecules were copurified with the dominating immunoglobulins with an estimated MW of 500 kDa. Reducing SDS-PAGE of this fraction revealed molecules with MWs of 97,79,57,29,and 27 kDa.