An assessment of extraction and assay techniques for quantification of calpain and calpastatin from small tissue samples.

Our objective was to evaluate whether small (biopsy-sized) samples could be used to measure calpain and calpastatin activities in skeletal muscle. The accuracy of different separation and assay methods for the quantification of calpains and calpastatin from small (1.0 and 0.2 g) skeletal muscle samples was tested. In Exp. 1, the LM was removed from six lambs, and a 50-g subsample was processed using the reference method (DEAE-Sephacel chromatography and casein assay). Subsamples (1.0 and 0.2 g) also were processed using the two-step separation (1 mL DEAE-Sephacel and bulk elution using 200 and 400 mM NaCl) and heated calpastatin methods; in both cases, fractions were assayed with Bodipy-labeled and [14C]-labeled casein microassays. Finally, casein zymography was used to separate and quantify the calpain proteases from 1.0-and 0.2-g samples. The values obtained after processing the 50-g sample using the reference method were judged most accurate, and the alternative approaches were compared with these. For each extraction and assay approach, we considered: 1) the effect of the sample size on the mean activity; 2) increased or decreased variation of data; and 3) the correlation relative to the reference method. Where possible, we compared the ratio of calpain to calpastatin activities determined using the alternative approaches with the ratios found using the reference method. These methodologies were further investigated in Exp. 2, where single homogenates from different tissues (heart, spleen, lung, and muscle) were assayed using the alternative approaches. Experiment 1 established that most of the approaches suffered from poor correlations and/or unacceptable variation. By using a large, homogenous sample in Exp. 2, however, we determined that this error was not due to the methodologies themselves. Therefore, the unacceptable variation found in Exp. 1 resulted from the small sample size, and we recommend that large tissue samples (e.g., 50 g) should be used for calpain and calpastatin activity measurements in skeletal muscle instead of small tissue biopsies (e.g., 0.2 and 1.0 g).

Neospora caninum antigens defined by antigen-dependent bovine CD4+ T cells.

Neosporosis is an important cause of pregnancy loss in cattle worldwide. The objective of the present study was to identify Neospora caninum antigens as vaccine candidates using antigen-specific, short-term CD4+ T cells established from N. caninum-immunized and -challenged cows. Whole N. caninum tachyzoite lysate was separated into 6 fractions by DEAE anion-exchange chromatography using high-pressure liquid chromatography (HPLC). The CD4+ T-cell proliferation assay results indicated that antigenic activity was associated with proteins from HPLC fractions 4-6, with fraction 5 exhibiting the highest antigenic activity. Also, SDS-PAGE analysis revealed a 16-kDa protein in fractions 4-6 that was recognized by anti-N. caninum antibodies. This 16-kDa protein was absent in other fractions, and it may be a target of a T-cell response in cattle. Further identification of immunogenic proteins of N. caninum may facilitate development of subunit vaccines against neosporosis.

Trypanosoma vivax: a novel method for purification from experimentally infected sheep blood.

Trypanosoma vivax is the principal etiological agent of bovine trypanosomosis, a widely disseminated disease in tropical and subtropical regions. Here, we present a simple and reproducible method for the purification of T. vivax from experimentally infected and immunosuppressed sheep, using an isopycnic Percoll gradient, followed by DEAE-cellulose chromatography, with an estimated yield of 11-15%. This method could be used for the purification of T. vivax geographical isolates from various locations and from different natural hosts.

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax.

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.

High level activity of 2', 5'-oligoadenylate synthetase in dog serum.

Most animal cells that are exposed to interferon (IFN) experience an increase in the activity of 2', 5'-oligoadenylate synthetase (OAS), which is an important effector of IFN's antiviral action. OAS activity has been widely used in clinical chemistry as an indicator of IFN activity. In this study, we found that OAS activity in canine serum is 46.0 +/- 40.4 nmol/dl/hr, which is 10- to 100-fold higher than in other animals such as the cat (1.9 +/- 2.1), rabbit (4.0 +/- 1.1), and guinea pig (0.3 +/- 0.6). The canine OAS protein was detected by Western blotting using a 68M-10 monoclonal anti-murine OAS antibody, and was found to be composed of at least three distinct molecular species of p40 class OAS. Among these, the 40 and 42 kDa components were determined to be the major species in serum and fibroblast cell lines, respectively.

Isolation of ovulation-inducing factors in the seminal plasma of Bactrian camel (Camelus bactrianus) by DEAE-cellulose chromatography.

Ovulation in the Bactrian camel (Camelus bactrianus) depends upon the ovulation-inducing factor in the seminal plasma; however, little research has been conducted to isolate and identify the factor. The current study attempts to isolate and identify the bioactive fractions from the seminal plasma of Bactrian camel. The seminal plasma was fractionated by diethylamino-ethylcellulose (DEAE)-cellulose chromatography and five protein fractions were obtained. The bioactive of each fraction was estimated by rat pituitary tissue culture in vitro and by the intramuscular injection of the bioactive fraction to the female camels in vivo. The concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary culture media before and 6 h after the addition of each fraction and in the peripheral blood plasma collected from the camel immediately before and hourly after the injection of the active fraction were measured by radioimmunoassay. The results demonstrated that the third fraction (L3) had the bioactive potential to stimulate the release of LH in vitro from 11.82 +/- 1.77 to 25.63 +/- 3.84 mIU/ml after the addition of L3 to the culture media. The in vivo concentrations of LH in the blood plasma of the camel increased from 6.43 +/- 0.14 before to 15.50 +/- 2.64 ng/ml 6 h after injection of L3. However, the concentrations of FSH did not show any significant changes either in vitro or in vivo. The results clearly demonstrated the existence of LH-releasing associated fractions in the seminal plasma that appears to be separated by DEAE-cellulose matrix and the isolated L3 fraction might be the ovulation-inducing factor or one of its components.

The calpain system in three muscles of normal and callipyge sheep.

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibition of the calpains in postmortem muscle is modulated in some as yet unknown manner.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

Detection of annexins I and IV in bronchoalveolar lavage fluids from calves inoculated with bovine herpes virus-1.

Annexins are phospholipid-binding proteins and are abundant in the lung. Annexins I and IV, but not II and VI, have been detected in bronchoalveolar lavage (BAL) fluids from calves inoculated with Pasteurella haemolytica, the pathogen for calf pneumonia. In this study, BAL fluids from calves with experimental pneumonia induced by inoculation to right lung lobes of bovine herpes virus-1 (BHV-1), the major viral pathogen for pneumonia, were examined for detection of annexins I and IV. Of 6 calves inoculated with BHV-1, annexins I and IV were coincidentally detected in BAL fluids from right lung lobes of 4 calves, but not in BAL fluids from left lung lobes of 6 inoculated calves or those from left and right lung lobes of 3 control calves. Annexin II and VI were not found in any BAL fluids examined. These results, together with previous findings on calves inoculated with Pasteurella haemolytica, suggest that the release of annexins I and IV onto the alveolar surface is an essential event occurring in response to pulmonary infections of BHIV-1 and Pasteurella haemolytica.

Cross-bred cattle protected against Hyalomma anatolicum anatolicum by larval antigens purified by immunoaffinity chromatography.

Antigens from larvae of Hyalomma anatolicum anatolicum were extracted and purified by immunoaffinity chromatography using immunoglobulin ligands from cross-bred animals immunized with soluble larval antigen. Affinity-purified antigen (Aff-TLE) and a total larval extract (TLE) were used to immunize cross-bred (Bos indicus x Bos taurus) cattle. The group immunized with Aff-TLE rejected 71.6% of larvae and 77.3% of nymphs. However, the rejection percentages were lower in the TLE-immunized group. No significant changes in the feeding period, moulting percentages or moulting period of engorged larvae and nymphs were recorded. There was, however, a significant decrease in the number of resultant nymphs p < 0.01) and adults (p < 0.01) in the ticks fed on the Aff-TLE-immunized group. The Aff-TLE antigen was 93.3% purified. SDS-PAGE analysis identified a 39 kDa protein, reported for the first time, as the antigen responsible for the induction of resistance in the host.

Differential complement activation by bovine IgG2 allotypes.

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.

Identification of bovine decorin in the fetal bovine rumen.

Immunohistochemical localization of bovine decorin was examined with its biological analysis in the fetal bovine rumen. By immunohistochemical staining, monoclonal antibody (mAb) 2B6, which recognizes chondroitin 4-sulfate and/or dermatan sulfate (DS), reacted specifically to the lower mesenchymal region in the developing ruminal wall. Biochemical analysis of the extract from the developing rumen revealed that molecule detected immunohistochemically by mAb 2B6 was small DS proteoglycan, bovine decorin. These results support the view that bovine decorin is involved in organization of the fetal bovine ruminal mesenchyme as a collagenous tissue.

Antibody development in pigs inoculated with live or killed cultures of Brucella abortus.

As shown by density gradient ultracentrifugation and column chromatography, pigs formed IgM antibodies during the first week following vaccination with Brucella abortus, strain 19. At this time their sera reacted in both plate and tube agglutination but not in complement-fixation tests. A few days later, when IgG antibodies had developed, agglutination titers were still high and some activity was recorded in hemolytic complement-fixation tests. A similar sequence was observed in pigs repeatedly inoculated with phenol-killed suspensions of B. abortus. As the proportion of IgM to IgG antibodies decreased, agglutinin titers fell in relation to complement-fixing titers. In some animals the conglutinating complement absorption test became positive earlier than the plate agglutination.