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.

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.

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.

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.

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.

Comparative study of PCR as a direct assay and ELISA and AGID as indirect assays for the detection of bovine leukaemia virus.

The choice of a diagnostic method depends on the characteristics of the herd to be analysed. Two herds with different prevalences of enzootic bovine leukaemia were chosen to study the concordance between agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) methods. PCR, an increasingly used virological method, was performed with four sets of primers, amplifying different genomic regions (env, pol and tax), from DNA extracted either from peripheral blood monocytes (PBMCs) or milk leucocytes. The highest percentage of positive animals was obtained using PCR performed with DNA extracted from PBMCs using primers which amplified either env or pol, followed by PCR using PBMCs and primers which hybridized with tax, then ELISA using serum and finally AGID. The results of PCR were more consistent with PBMCs than when milk leucocytes were used.

Seroepidemiological survey of infection by feline leukemia virus and immunodeficiency virus in Madrid and correlation with some clinical aspects.

A study of 180 healthy cats found that 15.6% were feline leukemia virus (FeLV) positive, 8.3% were feline immunodeficiency virus (FIV) positive, and 1.1% were FIV and FeLV positive, which corresponded to 30.4, 13.8, and 2.6, of 115 cats with FIV- and FeLV-related symptoms, respectively. Differences were seen in the sexes and ages of the populations studied. Anemia, leukopenia, and lymphopenia were the most frequent hematological abnormalities in infected cats.

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.

Production and characterization of monoclonal antibodies against bovine leukaemia virus using various crude antigen preparations: a comparative study.

A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.

In vitro infection of cells of the monocytic/macrophage lineage with bovine leukaemia virus.

The oncogenic retrovirus bovine leukaemia virus (BLV) primarily infects B cells. Most infected animals remain asymptomatic for long periods of time before an increase in circulating B cells or localized tumours can be observed. This long clinical latency period may be explained by cells of the monocyte/macrophage lineage (M/M) becoming infected and acting as a reservoir for the virus, as shown for other retroviruses (human immunodeficiency virus-1, feline immunodeficiency virus). M/M cells in different stages of differentiation (HL-60, THP-1, U-937, J774, BGM, PM2, primary macrophages of sheep and cows) were cultured with BLV produced by permanently infected donor cells (FLKBLV and BLV-bat(2)). Donor cells were inhibited from multiplying by either irradiation or treatment with mitomycin C. In other experiments, supernatant from donor cells containing virus was used. In co-culture with the donor cells, the less differentiated monocytic cells showed severe cellular changes such as differentiation, vacuolization, cell lysis and membrane blebbing; apoptosis was a frequent phenomenon. Budding and extracellular viruses were also observed. The more differentiated macrophage cells, although they showed less signs of infection by microscopy, had a complete BLV protein profile, as seen by Western blotting; bands corresponding to p24CA (Gag) and its precursors were clearly seen. In addition, gp51SU was identified by syncytia formation assays. It is concluded that M/M cells may be infected by BLV, the consequences of the infection differing according to the type of cell.

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.

Origin and rapid evolution of a novel murine erythroleukemia virus of the spleen focus-forming virus family.

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent Mr of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an Mr of 60,000. During further in vivo passaging, a virus that encodes an Mr-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.

An array of novel murine spleen focus-forming viruses that activate the erythropoietin receptor.

The Friend spleen focus-forming virus (SFFV) env gene encodes a 409-amino-acid glycoprotein with an apparent Mr of 55,000 (gp55) that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. We reported previously the in vivo selection during serial passages in mice of several evolutionary intermediates that culminated in the formation of a novel SFFV (M. E. Hoatlin, E. Gomez-Lucia, F. Lilly, J. H. Beckstead, and D. Kabat, J. Virol. 72:3602-3609, 1998). A mouse injected with a retroviral vector in the presence of a nonpathogenic helper virus developed long-latency erythroblastosis, and subsequent viral passages resulted in more pathogenic isolates. The viruses taken from these mice converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives. Western blot analysis of cell extracts with an antiserum that broadly reacts with murine retroviral envelope glycoproteins suggested that the spleen from the initial mouse with mild erythoblastosis contained an array of viral components that were capable of activating EpoR. DNA sequence analysis of the viral genomes cloned from different factor-independent cell clones revealed env genes with open reading frames encoding 644, 449, and 187 amino acids. All three env genes contained 3' regions identical to that of SFFV, including a 6-bp duplication and a single-base insertion that have been shown previously to be critical for pathogenesis. However, the three env gene sequences did not contain any polytropic sequences and were divergent in their 5' regions, suggesting that they had originated by recombination and partial deletions of endogenously inherited MuLV env sequences. These results suggest that the requirements for EpoR activation by SFFV-related viruses are dependent on sequences at the 3' end of the env gene and not on the polytropic regions or on the 585-base deletions that are common among the classical strains of SFFV. Moreover, sequence analysis of the different recombinants and deletion mutants revealed that short direct and indirect repeat sequences frequently flanked the deletions that had occurred, suggesting a reverse transcriptase template jumping mechanism for this rapid retroviral diversification.

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.

Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization.

A group of 14 monoclonal antibodies (mAbs) to staphylococcal enterotoxin B (SEB) were obtained by fusion of Sp2/O myeloma cells with spleen cells from female BALB/c mice immunized with commercial SEB. The antibodies belonged to IgG1 and IgG2b subclasses. We evaluated the anti-SEB titres, competition assays and sensitivity of detection by indirect ELISA. Reactivity and cross-reactivity were also studied by indirect ELISA and confirmed by immunoblotting. All the mAbs reacted with SEB and with a second band which had a different electrophoretic mobility and probably represents an aggregate of SEB or SEB bound to membranes. Three mAbs reacted only with SEB and the rest showed cross-reactions with SEC1. No reactions were observed against any other serovar (SEA, SED and SEE) or other proteins.

Absence of HTLV-I and HTLV-II infection in prostitutes in the area of Seville, Spain.

Infection with human retroviruses other than HIV in Spain has only been reported in isolated cases in African immigrants and intravenous drug abusers infected with HTLV-I. The status of the prostitute population is unknown. The sera of 88 prostitutes in Seville were therefore tested for HIV, HTLV-I and HTLV-II, and relevant epidemiological data were collected on the health status, sexual practices etc. In the prostitute population studied 2.5% of the non-intravenous drug abusers and 20% of the intravenous drug abusers were positive for HIV. However, infection with HTLV-I/II could not be demonstrated in any of them.

Antibody profile of early HTLV-I infection.

To define the antibody profile of early seroconversion in infection with human T-cell lymphotropic virus type I (HTLV-I), consecutive serum samples from 10 subjects presumed to have seroconverted on the basis of the particle agglutination test were studied by three enzyme immunoassays and two confirmatory tests (radioimmunoprecipitation and western blot). 3 samples positive and 1 sample indeterminate in the confirmatory tests were reactive in one enzyme immunoassay, which used recombinant envelope antigen, but not in the other two enzyme immunoassays. 2 of 38 particle-agglutination-negative samples had a prozone effect. The confirmatory tests identified 8 seroconverters (7 women, 1 man); their serum samples were used to study the antibody reactivity by western blot assays to HTLV-I specific antigens (three recombinant proteins spanning the N-terminal, middle, and C-terminal env glycoprotein gp46; a recombinant transmembrane protein gp21; a recombinant tax protein; and three gag proteins [p28, p24, and p19]). All 8 seroconverters had antibody reactivities to the C-terminal region (aminoacid residues 229-308) of gp46 and to gag p19 and p24 when their seroconversion was detected.