Effect of progesterone on the vaccination and immune response against Chlamydia abortus in sheep.

Chlamydia abortus produces ovine enzootic abortion (OEA). Symptoms are not observed until the organism colonises the placenta, eventually causing abortion. Infected animals become carriers and will shed the organism in the following oestruses. This process suggests that sex hormones might play an important role in the physiopathology of OEA, affecting the success of chlamydial clearance and also jeopardising the effectiveness of vaccination. However, the mechanisms through which sex hormones are involved in chlamydial pathogenicity remain unclear. The aim of this study, therefore, was to determine the effect of progesterone on the immune response against C. abortus and on the protection conferred by an experimental inactivated vaccine in sheep. Eighteen sheep were ovariectomised and divided into four groups: vaccinated and progesterone-treated (V-PG), vaccinated and non-treated (V-NT), non-vaccinated and non-treated (NV-NT) and non-vaccinated and progesterone-treated sheep (NV-PG). Animals from both PG groups were treated with commercial medroxyprogesterone acetate impregnated intravaginal sponges before and during the vaccination (V-PG) or just before challenge (NV-PG). The animals from both V groups were subcutaneously immunised with an experimental inactivated vaccine, which was seen to confer high protection in previous studies. All sheep were challenged intratracheally with C. abortus strain AB7 and were sacrificed on day 8 post-infection. Morbidity was measured as the variation in rectal temperature and samples of sera were collected for antibody and cytokine (IFN-γ and IL-10) analysis by commercial ELISA. In addition, lung and lymph node samples were collected for chlamydial detection by qPCR and for histopathological and immunohistochemical analyses. Sheep from the V-PG group showed less severe or no lesions and lower morbidity than the other groups. They also had the highest abundance of regulatory T-cells. The sheep from V-NT also manifested high antibody levels against C. abortus and less severe lesions than those observed in non-vaccinated sheep, which showed high morbidity, low antibody levels and severe lesions, especially in NV-NT. These results confirm the effectiveness of the experimental vaccine employed and suggest that progesterone could enhance the effect.

Detection of staphylococcal enterotoxin and toxic shock syndrome toxin-1 (TSST-1) by immunoblot combined with a semiautomated electrophoresis system.

An immunoblot technique combined with a semi-automated electrophoresis system has been developed for the detection of staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1). The advantages of this method over other detection techniques include speed, sensitivity (10 ng/ml) and specificity. The use of semiautomated electrophoresis permits the routine detection of staphylococcal enterotoxins and TSST-1.

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells.

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.

Enterotoxin and toxic shock syndrome toxin-one production by staphylococci isolated from mastitis in sheep.

From 160 staphylococci isolated from ovine mastitis, 125 were identified as coagulase-positive staphylococci (CPS) and 35 as coagulase-negative staphylococci (CNS). Of these, 108 (87.8%) S. aureus produced at least one of the staphylococcal enterotoxins (SE) described. However, no CNS was found to be enterotoxigenic. Enterotoxin C (SEC) was the type most frequently produced. TSST-1 was shown to be produced by 91 (74.0%) of S. aureus, almost invariably in combination with SEC. Three CNS strains were also found to produce TSST-1 (two strains of S. xylosus and one strain of S. epidermidis).

Evaluation of virus excretion by cells persistently infected with the bovine leukaemia virus (BLV) using monoclonal antibodies.

BACKGROUND: bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukaemia. Studies in vitro usually require the use of infected cell lines, mostly to produce antigen. Two of the most widely used cell lines are FLK-BLV and BLV-bat2. OBJECTIVE: the dynamics of the excretion of BLV proteins and whole virus by the persistently BLV-infected cell lines mentioned above was studied using an indirect ELISA in combination with eight monoclonal antibodies (mAbs) and cow and rabbit serum. STUDY DESIGN: tissue culture flasks were seeded with different concentrations of cells (13000-67000 cells per cm2, corresponding to 1-5 million cells per 75 cm2 flask) and were studied for 20 days. Samples (1.5 ml) were removed every 24 h and the presence of BLV proteins was determined using an indirect ELISA assay in which the antigen reaction with the monoclonal antibodies was evidenced by peroxidase labeled anti-mouse immunoglobulins. RESULTS: cell line FLK-BLV produced a complete monolayer as early as 4 days after passage, 3 days earlier than BLV-bat2. Using mAbs, the amount of viral proteins in the supernatant showed a cyclic pattern, with two evident peaks at days ca. 8 and 16. These peaks occurred even in the absence of passage or medium change, which causes depletion of essential nutrients and acidity. In comparison to polyclonal serum, mAbs gave more clear and defined values and are useful for determining the dynamics of viral production. CONCLUSION: when aiming for high viral yield, BLV should be harvested between days 6 and 8 after passage, when viral shedding is at its maximum. These results are very useful for preparing antigen for monoclonal antibody production, or for techniques such as ELISA or Western blot.

Rapid detection of specific polyclonal and monoclonal antibodies against bovine leukemia virus.

ELISA and Western blot have been used for detecting specific antibodies or antigens for routine diagnostic laboratory tests and experimental protocols, as well as for screening hybridomas secreting antibodies. Although these techniques are sensitive, some slow growing hybridomas are identified as positive only when they are grown slowly long time. We standardized the dot-ELISA, a more sensitive technique, for the detection of antibodies against BLV. The main advantages of the dot-ELISA described in this study are (a) its sensitivity, detecting hybridomas which would otherwise be considered negative and discarded from the results of indirect ELISA and/or Western blot; and (b) the possibility of economizing reagents using as little as 1 microl of the antigen and 0.5 microl of antibody and conjugate. Different BLV-antigen preparations were bound to nitrocellulose membranes (NC), including cells lysed chemically (LYS) or by sonication (SOC), semi-purified virus (PV), and supernatant from infected cultures, either without treatment (SUP) or sonicated (SOS). The antigen preparations most adequate for detecting monoclonal antibodies against BLV and polyclonal antibodies in cattle sera were undiluted cell lysates (LYS) and semi-purified BLV (PV). When testing bovine sera, the supernatant (SUP) and sonicated supernatant (SOS) antigens gave a high background due to the presence of FCS which reacted with the anti-bovine labeled antibodies. In this study, 59 BLV specific antibody secreting hybridomas were identified using the dot-ELISA, compared to only 20 detected using iELISA, and doubtful reactions due to nonspecific binding to fetal calf serum (FCS) and cellular components were measured. The results of the improved dot-ELISA described may be stored at room temperature for future reference. Results were consistently reproducible in coated nitrocellulose membranes kept at different storage temperatures (-20 degrees C, 4 degrees C, and 25-30 degrees C) 48 h, 1 week and 5 months.

Comparison of four tests to evaluate the reactivity of rabbit sera against envelope or Gag-related proteins of bovine leukemia virus (BLV).

Bovine leukemia virus (BLV) has a long latency period during which animals are inapparently infected, may spread the disease, and are only detected by serological techniques or by the most cumbersome molecular biology techniques. We have compared techniques for detecting either total antibodies (ELISA), anti-p24 and Gag-related proteins (Western blot), or anti-gp51 (agar gel immunodiffusion, AGID, and syncytia inhibition, SI) in rabbits inoculated experimentally with inocula of variable immunogenicity. The two tests to detect antibodies to gp51 correlated well in sera clearly positive or clearly negative by either one, but correlation was poor in the intermediate groups. All sera positive by AGID were also positive by ELISA, but results did not agree in sera negative by AGID, ELISA proving to be more sensitive. Western blot was a good technique for detecting antibodies against Gag-related proteins. However, no band was identified to clearly correspond to anti-Env-related proteins. As for other retroviruses, testing of animals for infection with BLV should include the detection of antibodies anti-Gag and anti-Env proteins.

Comparison of methods for routine separation of coagulase-negative staphylococci from micrococci isolated from sheep.

A total of 176 Gram-positive, catalase positive cocci strains, isolated from sheep were studied by different routine tests for the differentiation of staphylococci and micrococci, comparing their validity and usefulness. By glucose fermentation and growth in the anaerobic portion of thioglycolate 85 and 73.6% respectively of coagulase negative staphylococci were misclassified as Micrococcus spp. Susceptibility to lysostaphin was an adequate test for the differentiation of the strains. Atypical results in the production of acid from glycerol/erythromycin were obtained in 11.8% of the coagulase negative strains and 16.7% of micrococci. The combined use of the selective media furazolidone agar and Schleifer and Krämer medium resulted in a fast and useful separation of ovine staphylococci and micrococci. The bacteriolytic activity misclassified 32.2% of the coagulase negative strains.

Macrophages infected with bovine leukaemia virus (BLV) induce humoral response in rabbits.

BLV is a lymphotropic retrovirus which infects mainly B-cells. However, the possible infection of cells of the monocyte/macrophage lineage (M/M) might explain some aspects of the disease such as latency or disease progression. We infected sheep M/M with BLV either by culturing M/M with supernatant containing virus, or coculturing M/M with persistently infected cell lines. These BLV-infected M/M were inoculated into rabbits and the serological response was followed for two years. ELISA results using adsorbed sera showed a persistent production of specific antibodies from as early as the first week post inoculation. Two tests were used to detect the response against envelope glycoprotein gp51: Agar gel immunodiffusion (AGID) and a virus neutralization test read as syncytia inhibition (SI). Sera were positive by AGID after the second or third inoculation. Neutralizing titres (SI) were higher than those seen in control rabbits inoculated with persistently infected cell lines, suggesting that the virus may be expressed better in M/M. Gag-related proteins were analyzed by Western Blot (WB). Sera from rabbits inoculated with BLV-infected M/M recognized as many viral proteins as sera from BLV immunized control rabbits or infected cows, and this profile did not change with repeated inoculations. All these results suggest that BLV may infect M/M, where viral proteins are actively expressed to the point that they induce a humoral immune response in animals, and that animals get persistently infected.

Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot of nonspecific and specific viral proteins frequently detected in different antigen preparations of bovine leukemia virus.

Bovine leukemia virus (BLV) infection in cattle is seldom manifested clinically, and is routinely diagnosed by serologic tests such as enzyme-linked immunosorbent assay or Western blot (WB). Because of the difficulty in interpreting WB results, the aim of the present study was to determine which of the bands observed in WB were specifically produced by BLV and which corresponded to nonspecific proteins, either derived from medium components or of a cellular nature. Five different BLV antigen preparations from 2 cell lines (FLK-BLV and BLV-bat2) frequently used for the production of BLV antigen were compared. The protein profiles of these antigen preparations were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and WB. Fetal calf serum, required for cellular growth and important in induction of viral transcription in vitro, was identified as a source of irrelevant proteins. In this study, 15 nonspecific protein bands in the growth medium were observed. These bands interfered with the interpretation of results. A nonspecific protein (25 kD) that was highly reactive in cell lysate preparation from BLV-bat2 was also detected. The unequivocal identification of protein bands, both specific and nonspecific, seen in WB is important not for understanding the protein profile of antigen preparations but also for determining if an animal is BLV positive or negative.

Comparison of two PCR and one ELISA techniques for the detection of small ruminant lentiviruses (SRLVs) in milk of sheep and goats.

The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosis of small ruminant lentiviruses (SRLVs). Detection of the proviral genome by PCR, though sensitive, is difficult due to the heterogeneity of the SRLV genomes. One of the PCR techniques amplifies a fragment in the pol gene (pol-PCR) and the other PCR targets the LTR region of the proviral genome (LTR-PCR). Milk from 194 sheep and 163 goats from farms in the Central Spain was analyzed by both techniques and compared to results obtained by ELISA. When compared to the serologic assay, the agreement of both PCR techniques was very low (0.024 and 0.020 in sheep, and 0.124 and 0.114 in goats). In view of these results, it may be concluded that the efficacy of PCR for the diagnosis of SRLVs is low and a combination of PCR and ELISA should be used for diagnosis.

Altered plasma concentrations of sex hormones in cats infected by feline immunodeficiency virus or feline leukemia virus.

Gender differences may affect human immunodeficiency virus (HIV) infection in humans and may be related to fluctuations in sex hormone concentration. The different percentage of male and female cats observed to be infected by feline leukemia virus (FeLV) or feline immunodeficiency virus (FIV) has been traditionally explained through the transmission mechanisms of both viruses. However, sexual hormones may also play a role in this different distribution. To study this possibility, 17ß-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA) concentrations were analyzed using a competitive enzyme immunoassay in the plasma of 258 cats naturally infected by FIV (FIV(+)), FeLV (FeLV(+)), or FeLV and FIV (F(-)F(+)) or negative for both viruses, including both sick and clinically healthy animals. Results indicated that the concentrations of 17ß-estradiol and testosterone were significantly higher in animals infected with FIV or FeLV (P < 0.05) than in negative cats. Plasma concentrations of DHEA in cats infected by either retrovirus were lower than in negative animals (P < 0.05), and F(-)F(+) cats had significantly lower plasma values than monoinfected cats (P < 0.05). No significant differences were detected in the plasma concentration of progesterone of the four groups. No relevant differences were detected in the hormone concentrations between animal genders, except that FIV(+) females had higher DHEA concentrations than the corresponding males (P < 0.05). In addition, no differences were observed in the hormone concentrations between retrovirus-infected and noninfected animals with and without clinical signs. These results suggest that FIV and FeLV infections are associated with an important deregulation of steroids, possibly from early in the infection process, which might have decisive consequences for disease progression.

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain.

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.

Plasma electrophoretogram in feline immunodeficiency virus (FIV) and/or feline leukaemia virus (FeLV) infections.

The electrophoretogram of 89 cats, including those infected by feline immunodeficiency virus (FIV+), feline leukaemia virus (FeLV+) and non-infected, showed statistically significant differences in several of the fractions. FIV+ cats had very high protein values (mean, 8.10 g/dl), mostly because of hypergammaglobulinemia (mean, 2.81 g/dl) as compared with non-infected animals and FeLV+. In addition, in these FIV+ animals, the albumin/globulins ratio (A/G) was very low (mean, 0.72). Statistically significant differences in A/G and alpha2-globulin fraction were observed in FeLV+ group (A/G mean, 0.88 +/- 0.08; alpha2-globulin, mean, 0.84 +/- 0.07 g/dl) when compared with non-infected group (A/G mean, 1.06 +/- 0.08; alpha2-globulin mean, 0.68 +/- 0.04 g/dl). The alpha1-globulin fraction was higher in double infected animals (FIV and FeLV positive, F-F) (3.55 g/dl), than in FeLV+ or FIV+ cats (3.10 and 3.07 g/dl respectively), but no statistical conclusions may be drawn from this fact because of the low number of F-F animals. This technique may help to assess the initial clinical status of retrovirus-infected cats, and the clinical course of these chronic diseases, specifically during and after suitable therapy.

Toxic shock syndrome toxin 1 (TSST-1) production by staphylococci isolated from goats and presence of specific antibodies to TSST-1 in serum and milk.

The ability of staphylococcal strains isolated from different anatomical sites in 133 healthy goats to produce toxic shock syndrome toxin 1 (TSST-1) and the presence of antibodies to this toxin in serum and milk were studied. The enzyme-linked immunosorbent assay method was used to detect both the toxin and the presence of antibodies. Of a total of 342 staphylococcal strains studied, 86 (25.2%) were found to produce TSST-1. Specific antibodies to TSST-1 were found in the serum of 57 (42.9%) of the animals studied and the milk of 63 (47.4%) of the animals. These results suggest that goats are frequently in contact with staphylococci that produce TSST-1, a toxin usually associated with Staphylococcus aureus strains isolated from cases of toxic shock syndrome in humans.

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 (TSST-1) Production During Induced Goat Mastitis.

Six Staphylococcus aureus enterotoxigenic type strains were inoculated into the udders of healthy goats in order to check the "in vivo" production of staphylococcal enterotoxins and toxic shock syndrome toxin-one (TSST-1). Staphylococcal enterotoxins and TSST-1 produced by toxigenic strains in milk samples were detected by the enzyme-linked immunosorbent assay method. S. aureus strains FRI-100, FRI-1173, and CC-92 produced high levels of SEA, SEB, and TSST-1, respectively, in the inoculated goats' udders.

Detection of antibodies to staphylococcal enterotoxins in the serum and milk of healthy goats.

A study was made of the presence of antibodies (Ab) to staphylococcal enterotoxins A to E (SEA-SEE) in the serum and milk of 133 healthy goats, using a competitive ELISA method. Antibodies to some enterotoxins were detected in 83 sera (62.4%) and in 41 (30.8%) milk samples. In serum, antibodies to all SE types were detected, the most frequent being antibodies to SEA (24.8%). Milk contained antibodies to SEA, SEB and SEC, the latter being the most frequent (24.8%). A statistical study was performed to correlate the number of animals harbouring antibodies to a given enterotoxin with the presence in these animals of staphylococci producing that enterotoxin.

Incidence of antibodies reactive with toxic shock syndrome toxin 1 in bovine milk.

Fourteen samples of bovine milk, purchased in six states, were found to contain significant antibody titers to toxic shock syndrome toxin 1. This reactivity seems to have been due primarily to antibodies of the immunoglobulin A class. Some of these antibodies survived the cheese-making process.