Esperanza Gomez-Lucia

Evaluation of a novel nested PCR for the routine diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV)

Laboratory diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) usually involves both viruses, as the clinical signs are similar and coinfection may occur. Serological methods may not represent an accurate diagnosis: maternal antibodies or cross-reactions may give false positive results to FIV, and false negative results may occur in latent FeLV status, or in certain FIV infection stages. A nested polymerase chain reaction (PCR) technique was designed to detect FeLV, FIV and feline endogenous retrovirus simultaneously. The detection of endogenous sequences was considered indicative of successful DNA extraction. The technique was used to diagnose FIV and FeLV in the blood cells of 179 cats. The κ value with the serological data was 0.69 for FeLV and 0.87 for FIV. The joint detection of FeLV and FIV by this novel nested PCR is sensitive, specific, fast and convenient, and its applicability for clinical diagnosis is promising, as the direct evidence of the presence of the virus is more realistic than the indirect data provided by the serological detection.

Use of recombinant interferon omega in feline retrovirosis: from theory to practice.

Abstract Type-I interferons (IFNs) are cytokines that have non-specific antiviral activity, participating mostly in innate defense mechanisms. Their administration has been proposed to treat several viral and immunomediated diseases as an immunomodulatory therapy. Due to its availability, recombinant human interferon-alpha (rHuIFN-α) has been studied in relation to feline retrovirosis, both in vitro and in vivo. However, IFNs are species-specific and antibodies have been shown to develop in response to the high rHuIFN-α doses necessary for an effective therapy. A recombinant feline IFN has been developed, which has been characterized as interferon-omega (rFeIFN-ω), designed to overcome these problems. Nonetheless, very few studies have been undertaken to evaluate its efficacy in cats naturally infected with FIV or FeLV. In an initial study, we here demonstrated that rFeIFN-ω can dramatically improve the clinical condition of infected cats, and induce improvement of hematologic parameters. Minor changes or no change was observed for hypergammaglobulinemia, CD4/CD8 ratio, proviral load, viremia and RT activity, suggesting that the overall effect of IFN was on innate immunity. More studies are needed in order to better understand its in vivo mechanisms.

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.

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.

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.

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).;

Plasma Electrophoretogram in Feline Immunodeficiency Virus (FIV) and/or Feline Leukaemia Virus (FeLV) Infections

Summary 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 α2‐globulin fraction were observed in FeLV+ group (A/G mean, 0.88 ± 0.08; α2‐globulin, mean, 0.84 ± 0.07 g/dl) when compared with non‐infected group (A/G mean, 1.06 ± 0.08; α2‐globulin mean, 0.68 ± 0.04 g/dl). The α1‐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.

Viability of Listeria monocytogenes in Milk Treated with Hydrogen Peroxide

The susceptibility of Listeria monocytogenes to hydrogen peroxide in sterilized and raw milk was studied. In raw milk, L. monocytogenes was less susceptible to H2O2 than milk microflora. The ratio of L. monocytogenes to total milk micro-organisms (natural microflora plus added L. monocytogenes ) increased when raw milk was stored at refrigeration temperature (4°C), due to a selective enrichment of Listeria present in milk. In sterilized milk, a concentration of 0.0495% H2O2 and 9 h were required to produce complete destruction of L. monocytogenes when this microorganism was in pure culture, although a reduction in listeria counts was observed at 1.5 h. When sterilized milk was simultaneously contaminated with L. monocytogenes , Staphylococcus aureus and Streptococcus faecalis , a decrease in L. monocytogenes count during the first 24 h was observed at 0.0495% H2O2. From this time L. monocytogenes recovered and multiplied reaching levels similar to the initial counts at the end of the experiment.

Enterotoxin production by strains of Staphylococcus intermedius and Staphylococcus aureus isolated from dog infections.

Sixty-six strains of S. intermedius and 10 of S. aureus isolated from infected dogs were examined for enterotoxin production. 39.5% of the strain (37.9% of S. intermedius and 50% of S. aureus) produced one or more enterotoxins. The predominant types produced by S. intermedius were C1 and C2, and only two of the strains synthesized enterotoxin A. One of the S. aureus strains produced the toxic shock syndrome toxin 1.

Growth of Staphylococcus aureus and Enterotoxin Production in Homemade Mayonnaise Prepared with Different pH Values

The ability of Staphylococcus aureus to grow and produce enterotoxins in homemade mayonnaise prepared at different pH values was studied. Ten enterotoxigenic strains, producing one or two enterotoxin types (A, B, C, or D) were inoculated into mayonnaise samples with pH adjusted to values ranging between 4.0 and 5.8, and incubated at 37°C for 7 d. Counts were made on days 1, 3, 5, and 7 and extracts were prepared on day 7 to detect enterotoxin by ELISA. An important difference was seen between those samples prepared with pH below or equal to 4.9 and those over or equal to 5.0; in the range of pH between 4.0 and 4.9 the average of staphylococcal population was 100 CFU/g; at pH 5.0 it was 1.6 × 105, and at pH 5.15 and above it was at least 8 × 106 CFU/g. Enterotoxin was detected only at initial pH over 5.15 and when final pH was not less than 4.7. The highest amount of enterotoxin corresponded to 157.8 ng of SEB/100 g of mayonnaise.