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.

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.

Avian pox infection in Spanish Imperial eagles (Aquila adalberti).

A cutaneous lesion, previously known as "warts", affecting the featherless parts of face and legs has long been recognized in juvenile Spanish Imperial eagles (Aquila adalberti). This paper describes the presentation, microbiological, histopathological, and electron microscopic findings of lesions and diagnosis of poxvirus infection in nine juveniles. Lesions consisted of single or multiple nodules with a crust and surrounded by skin swelling. Seventy-eight percent of the swabs taken from lesions yielded bacterial growth, with Escherichia coli being the most common bacterium isolated. Histopathology revealed typical pox lesions in all cases. Histopathological changes found consisted of proliferative epithelium, with ballooning degeneration of keratinocytes and lymphocyte infiltrates extending into underlying dermis. Avianpox virus was confirmed by the presence of eosinophilic intracytoplasmatic inclusion bodies in the affected cells on light microscopy, and diagnosis confirmation was performed by electron microscopy of biopsies from all nine eagles.

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.

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.

Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples.

DNA-based methods have emerged as an additional tool for Brucella infection-confirmation at a herd level. However, their implementation may require the use of specialized equipment. In this context the recently developed loop-mediated isothermal amplification (LAMP) technique may constitute an additional and cost-effective tool for rapid and specific DNA detection, especially in low income areas. In the present study the usefulness of a newly developed LAMP assay aiming at the multicopy-IS711 sequence was assessed on a variety of clinical samples (n=81 from abortions and ewes; cattle, n=3; swine, n=4) that were analyzed in parallel using real-time PCR and bacteriology. Although overall sensitivities obtained with the three methods were comparable (p>0.05), our results highlighted the complementarity between bacteriology and molecular-based methods for increased sensitivity. Significant differences (p<0.05) were observed with all techniques depending on the nature of the sample. Our results demonstrate the potential of the IS711-LAMP technique for direct Brucella detection.

Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain.

Two hundred and five isolates of Pasteurella multocida from pigs were phenotypically and genetically characterised by determining their biovar, capsular type, virulence-associated genes and pulsed-field gel electrophoresis (PFGE) profiles. All isolates were identified as P multocida subspecies multocida and most were assigned to biovar 3 (58 per cent) and biovar 2 (39.5 per cent). Biovar 1 represented 2.4 per cent of the isolates. According to the capsular type, the great majority of the isolates (79.0 per cent) belonged to capsular type A, 18.5 per cent belonged to capsular type D and 2.4 per cent were of capsular type F. All isolates harboured ompH, psl, oma87, ptfA, nanB, nanH, tonB, hgbA, sodA and sodC genes, while none of them possessed the transferrin-binding protein gene tbpA. The prevalence of toxA, pfhaA and hgbB genes was variable (7.8, 40.5 and 60.5 per cent of the isolates, respectively). After PFGE typing, isolates of biovar 2 and 3 were grouped in two different clusters (A and B) at a level of 45 per cent similarity. In addition, isolates of biovar 2 and 3 exhibited statistically significant differences (P<0.05) in the virulence-associated hgbB and pfhA genes (biovar 3 was hgbB(+) pfhA(-), while biovar 2 was hgbB- pfhA(+)).

Lactobacillus ceti sp. nov., isolated from beaked whales (Ziphius cavirostris).

Biochemical and molecular genetic studies were performed on three isolates of an unknown Gram-positive, catalase-negative and rod-shaped organism isolated from the lungs and liver of two beaked whales. The organisms were tentatively identified as Lactobacillus spp. based on cellular morphology and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the novel isolates as members of the genus Lactobacillus, but the isolates did not correspond to any recognized species of this genus. The novel strains shared the same phenotypic characteristics and exhibited 100 % 16S rRNA gene sequence similarity. The nearest phylogenetic relatives of the novel isolates were Lactobacillus satsumensis DSM 16230T (94.2 % 16S rRNA gene sequence similarity), Lactobacillus salivarius JCM 1047 (94.0 %), Lactobacillus nagelii ATCC 700692T (94.0 %) and Lactobacillus saerimneri DSM 16049T (93.8 %). The novel isolates could be distinguished from these species and other related species of the genus Lactobacillus by physiological and biochemical tests. On the basis of these phenotypic, physiological and phylogenetic findings, it is proposed that the new isolates from whales be classified as a novel species of the genus Lactobacillus, Lactobacillus ceti sp. nov. The type strain is 142-2T (=CECT 7185T=CCUG 53626T).

Characterization of Aerococcus viridans isolates from swine clinical specimens.

We present here the biochemical and genetic characterization and antimicrobial susceptibility of 58 isolates of Aerococcus viridans isolated in pure culture from different clinical specimens of normally sterile body sites of pigs. A. viridans isolates were commonly susceptible to beta-lactam antimicrobials and exhibited a great genetic heterogeneity as determined by pulsed-field gel electrophoresis typing. The results indicate that A. viridans might be included in the list of possible etiological agents causing disease in pigs.

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.

Applicability of an immunoblot technique combined with a semiautomated electrophoresis system for detection of staphylococcal enterotoxins in food extracts.

We studied the usefulness of an immunoblot technique for the detection of staphylococcal enterotoxins (SEs) in strains and food extracts. Food samples (milk, yogurt, hot dog sausage, cheese, and mayonnaise) were artificially contaminated with SEA through SEE. Protein A did not interfere with the results; it appeared on electrophoresis gels as bands with molecular weights higher than those of the SEs. Other food proteins were not revealed by the technique. The immunoblot technique proved to be fast, specific, and sensitive for the detection of SEs in foods.

Enterotoxin production by staphylococci isolated from healthy goats.

The ability of 342 staphylococcal isolates from different anatomical sites in healthy goats to produce staphylococcal enterotoxins (SE) was investigated. SE were produced by 74.3% of the 70 coagulase-positive strains and by 22% of the coagulase-negative strains studied. Most enterotoxigenic strains were isolated from the skin of udders and teats and from milk. SEC was the SE type most frequently produced, either alone (67.9%) or in combination with others. Five coagulase-negative species not previously reported as SE producers were identified (Staphylococcus chromogenes, S. warneri, S. sciuri, S. saprophyticus, and S. lentus). SEA, SEB, and SEC were detected in the milk of 17 of the 133 healthy goats studied. These results suggest that the goat is an important reservoir of enterotoxigenic staphylococci, most of which produce SEC.

Production of staphylococcal enterotoxins and TSST-1 by coagulase negative staphylococci isolated from ruminant mastitis.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.

Detection of enterotoxins and TSST-1 secreted by Staphylococcus aureus isolated from ruminant mastitis. Comparison of ELISA and immunoblot.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1) was studied in 81 strains of Staphylococcus aureus isolated from cases of mastitis in cattle, goats and sheep. SE and TSST-1 were detected by two techniques: ELISA double antibody sandwich, and an immunoblot technique combined with a semiautomated electrophoresis system. More Staph. aureus strains isolated from sheep produced enterotoxins than those from goats and cattle. SEC was the predominant type in all isolates from these animal species. The highest proportion of strains producing TSST-1 were obtained from sheep, twice as many as those from goats or cows. The two techniques gave similar results, as all the strains positive by immunoblot were also positive by ELISA, and only three were positive by ELISA but negative by immunoblot.