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.

Corynebacterium sphenisci sp. nov., isolated from wild penguins.

Six unidentified gram-positive, rod-shaped organisms recovered from the cloacae of apparently healthy wild penguins were characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of a cell wall based on meso-diaminopimelic acid and long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types, consistent with the genus Corynebacterium. Corynomycolic acids, which are characteristic of the genus, were also detected, albeit in small amounts. Comparative 16S rRNA gene sequencing studies showed that the unidentified organisms were phylogenetically related to corynebacteria and represent a novel subline associated with a small subcluster of species that includes Corynebacterium xerosis, Corynebacterium amycolatum and Corynebacterium freneyi. The unknown isolates were readily distinguished from their closest phylogenetic relatives and all other Corynebacterium species with validly published names by using a combination of biochemical and chemotaxonomic criteria. Based on both phenotypic and 16S rRNA gene sequence considerations, it is proposed that the unknown isolates recovered from penguins be classified as a novel species in the genus Corynebacterium, Corynebacterium sphenisci sp. nov. The type strain is CECT 5990T (= CCUG 46398T).

Isolation of Corynebacterium falsenii and description of Corynebacterium aquilae sp. nov., from eagles.

Biochemical, molecular chemical and molecular genetic studies were performed on seven unidentified gram-positive, rod-shaped organisms recovered from eagles. The strains were provisionally identified as Corynebacterium jeikeium with the commercial API Coryne system, but they were able to grow under anaerobic conditions and were non-lipophilic. Comparative 16S rRNA gene sequencing studies demonstrated that the isolates belonged phylogenetically to the genus Corynebacterium. Three strains were identified genotypically as Corynebacterium falsenii; the remaining four strains corresponded to a hitherto unknown lineage within the genus Corynebacterium, associated with a small subcluster of species that included Corynebacterium diphtheriae and its close relatives. The unknown bacterial strains were readily distinguished from these and other species of the genus by biochemical tests. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown bacterial strains from eagles should be classified as Corynebacterium aquilae sp. nov. (type strain is S-613T = CECT 5993T = CCUG 46511T).

Corynebacterium spheniscorum sp. nov., isolated from the cloacae of wild penguins.

Twenty unidentified Gram-positive, rod-shaped organisms were recovered from the cloacae of apparently healthy wild penguins (Spheniscus magellanicus) and subjected to a polyphasic taxonomic analysis. On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Corynebacterium, although the organisms did not appear to correspond to any recognized species. Lipid studies confirmed this generic placement, and comparative 16S rRNA gene sequencing showed that the unidentified organisms represent a hitherto unknown subline, associated with a small subcluster of species that includes Corynebacterium diphtheriae and its close relatives. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from penguins be classified in the genus Corynebacterium, as Corynebacterium spheniscorum sp. nov. The type strain is strain PG 39T (=CCUG 45512T =CECT 5986T).

Lactococcus lactis subsp. lactis infection in waterfowl: first confirmation in animals.

We report the first description, confirmed by bacteriologic and molecular (polymerase chain reaction and pulsed-field gel electrophoresis) analysis, of an infection in animals caused by Lactococcus lactis subsp. lactis, affecting waterfowl.

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.

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.

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.

Bovine tuberculosis and the endangered Iberian lynx.

We report the first case of bovine tuberculosis in a free-living Iberian lynx (Lynx pardina), an extremely endangered feline, from Doñana National Park in Spain. The isolate (Mycobacterium bovis) correlates by molecular characterization with other isolates from wild ungulates in the park, strongly suggesting an epidemiologic link. Mycobacterium bovis infects many animal species, with wild and free-ranging domestic ungulates being the main reservoirs in nature (1).

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.

Haemorrhagic septicaemia by Aeromonas salmonicida subsp. salmonicida in a black-tip reef shark (Carcharhinus melanopterus).

One black-tip reef shark (Carcharhinus melanopterus) was found dead without previous signs of disease. Major lesions consisted in cutaneous erythema, mainly at the base of the fins, focal to diffuse inflammatory lesions in gills and intestinal wall, and discrete haemorrhages in the same organs, liver and kidneys. Microcolonies of Gram-negative rods were observed in the lamina propia of gills, underneath the intestinal mucosa and randomly distributed in the renal and hepatic parenchymas. Also, emboli containing Gram-negative rods were observed in capillaries of these organs. Aeromonas salmonicida subsp. salmonicida was isolated in pure culture from gills, liver and intestine. Specific immunostaining confirmed the relationship between the isolate and lesion-associated bacteria. No previous reports on this infection in sharks have been found in the literature.

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.