A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus.

Experimental infection of rabbits with a recombinant bovine herpesvirus type 5 (BoHV-5) gI, gE and US9-negative

Bovine herpesvirus type 5 (BoHV-5) is a major cause of viral meningoencephalitis in cattle. The expression of different viral proteins has been associated with BoHV-5 neuropathogenesis. Among these, gI, gE and US9 have been considered essential for the production of neurological disease in infected animals. To evaluate the role of gI, gE and US9 in neurovirulence, a recombinant from which the respective genes were deleted (BoHV-5 gI-/gE-/US9-) was constructed and inoculated in rabbits of two age groups (four and eight weeks-old). When the recombinant virus was inoculated through the paranasal sinuses of four weeks-old rabbits, neurological disease was observed and death was the outcome in 4 out of 13 (30.7 %) animals, whereas clinical signs and death were observed in 11/13 (84.6%) of rabbits infected with the parental virus. In eight weeks-old rabbits, the BoHV-5 gI-/gE-/US9- did not induce clinically apparent disease and could not be reactivated after dexamethasone administration, whereas wild type BoHV-5 caused disease in 55.5% of the animals and was reactivated. These findings reveal that the simultaneous deletion of gI, gE and US9 genes did reduce but did not completely abolish the neurovirulence of BoHV-5 in rabbits, indicating that other viral genes may also play a role in the induction of neurological disease.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade the brain and to establish latent infection. Additional studies are underway to determine the biological and molecular mechanisms underlying the inability of BoHV-5TKΔ to reactivate from latency.

Metabolic evaluation of dairy cows submitted to three different strategies to decrease the effects of negative energy balance in early postpartum

In early lactation dairy cattle suffer metabolic alterations caused by negative energy balance, which predisposes to fatty liver and ketosis. The aim of this study was to evaluate the metabolic condition of high yielding dairy cows subjected to three treatments for preventing severe lipomobilization and ketosis in early lactation. Fifty four multiparous Holstein cows yielding >30 L/day were divided into four groups: control (CN= no treatment), glucose precursor (PG= propylene-glycol), hepatic protector (Mp= Mercepton®), and energy supplement with salts of linolenic and linoleic faty acids (Mg-E= Megalac-E®). Treatments were administrated randomly at moment of calving until 8 weeks postpartum. Blood samples were collected on days 1, 7, 14, 21, 28, 35, 42 and 49 postpartum. Body condition score (BCS) was evaluated at the same periods and milk yield was recorded at 2nd, 4th, 5th, 6th, 7th, and 8th weeks of lactation. Concentrations of non-esterified fatty acids (NEFA), albumin, AST, ß-hydroxybutyrate (BHBA), cholesterol, glucose, total protein, urea and triglycerides were analyzed in blood samples. Cut-off points for subclinical ketosis were defined when BHBA >1.4 mmol/L and NEFA >0.7 mmol/L. General occurrence of subclinical ketosis was 24% during the period. An ascendant curve of cholesterol and glucose was observed from the 1st to the 8th week of lactation, while any tendency was observed with BHBA and NEFA, although differences among treatments were detected (p<0.05). BCS decreased from a mean of 3.85 at 1st week to 2.53 at 8th week of lactation (p=0.001). Milk yield was higher in the Mg-E group compared with the other treatment groups (p<0.05) Compared with the CN group, the treatments with Mp and PG did not show significant differences in blood biochemistry and milk yield. Cows receiving PG and Mg-E showed higher values of BHBA and NEFA (P<0.05), indicating accentuated lipomobilization. Supplementation with Mg-E also resulted in significant higher concentrations of cholesterol, BHBA, urea, AST and lower values of glycemia. This performance may be explained by the highest milk yield observed with this treatment. Treatments with PG and Mp did not improve milk yield, compared with control cows, but did not show metabolic evidence of ketosis, fat mobilization or fatty liver. These results suggest that treatment with Mg-E improves milk production but induces a higher negative energy balance leading to moderated lipomobilization and ketone bodies production, increasing the risk of fatty liver.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Signaled two-way avoidance learning using electrical stimulation of the inferior colliculus as negative reinforcement: effects of visual and auditory cues as warning stimuli

The inferior colliculus is a primary relay for the processing of auditory information in the brainstem. The inferior colliculus is also part of the so-called brain aversion system as animals learn to switch off the electrical stimulation of this structure. The purpose of the present study was to determine whether associative learning occurs between aversion induced by electrical stimulation of the inferior colliculus and visual and auditory warning stimuli. Rats implanted with electrodes into the central nucleus of the inferior colliculus were placed inside an open-field and thresholds for the escape response to electrical stimulation of the inferior colliculus were determined. The rats were then placed inside a shuttle-box and submitted to a two-way avoidance paradigm. Electrical stimulation of the inferior colliculus at the escape threshold (98.12 ± 6.15 (A, peak-to-peak) was used as negative reinforcement and light or tone as the warning stimulus. Each session consisted of 50 trials and was divided into two segments of 25 trials in order to determine the learning rate of the animals during the sessions. The rats learned to avoid the inferior colliculus stimulation when light was used as the warning stimulus (13.25 ± 0.60 s and 8.63 ± 0.93 s for latencies and 12.5 ± 2.04 and 19.62 ± 1.65 for frequencies in the first and second halves of the sessions, respectively, P<0.01 in both cases). No significant changes in latencies (14.75 ± 1.63 and 12.75 ± 1.44 s) or frequencies of responses (8.75 ± 1.20 and 11.25 ± 1.13) were seen when tone was used as the warning stimulus (P>0.05 in both cases). Taken together, the present results suggest that rats learn to avoid the inferior colliculus stimulation when light is used as the warning stimulus. However, this learning process does not occur when the neutral stimulus used is an acoustic one. Electrical stimulation of the inferior colliculus may disturb the signal transmission of the stimulus to be conditioned from the inferior colliculus to higher brain structures such as amygdala

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Effects of lead and/or zinc exposure during the second stage of rapid postnatal brain growth on delta-aminolevulinate dehydratase and negative geotaxis of suckling rats

Lead has been shown to produce cognitive and motor deficits in young rats that could be mediated, at least in part, by inhibition of the zinc-containing heme biosynthetic enzyme delta-aminolevulinate dehydratase (ALA-D). In the present study we investigated the effects of lead and/or zinc treatment during the second stage of rapid postnatal brain development on brain, kidney and blood ALA-D specific activity, as well as the negative geotaxis behavior of rats. Eight-day-old Wistar rats were injected intraperitoneally with saline, lead acetate (8 mg/kg) and/or zinc chloride (2 mg/kg) daily for five consecutive days. Twenty-four hours after treatment, ALA-D activity was determined in the absence and presence of DL-dithiothreitol (DTT). The negative geotaxis behavior was assessed in 9- to 13-day-old rats. Treatment with lead and/or zinc did not affect body, brain or kidney weights or brain- or kidney-to-body weight ratios of the animals. In spite of the absence of effect of any treatment on ALA-D specific activity in brain, kidney and blood, the reactivation index with DTT was higher in the groups treated with lead or lead + zinc than in the control group, in brain, kidney and blood (mean ± SEM; brain: 33.33 ± 4.34, 38.90 ± 8.24, 13.67 ± 3.41; kidney: 33.50 ± 2.97, 37.60 ± 2.67, 15.80 ± 2.66; blood: 63.95 ± 3.73, 56.43 ± 5.93, 31.07 ± 4.61, respectively, N = 9-11). The negative geotaxis response behavior was not affected by lead and/or zinc treatment. The results indicate that lead and/or zinc treatment during the second stage of rapid postnatal brain growth affected ALA-D, but zinc was not sufficient to protect the enzyme from the effects of lead in brain, kidney and blood.

Presence of the RHD pseudogene and the hybrid RHD-CE-Ds gene in Brazilians with the D-negative phenotype

The molecular basis for RHD pseudogene or RHDpsi is a 37-bp insertion in exon 4 of RHD. This insertion, found in two-thirds of D-negative Africans, appears to introduce a stop codon at position 210. The hybrid RHD-CE-Ds, where the 3' end of exon 3 and exons 4 to 8 are derived from RHCE, is associated with the VS+V- phenotype, and leads to a D-negative phenotype in people of African origin. We determined whether Brazilian blood donors of heterogeneous ethnic origin had RHDpsi and RHD-CE-Ds. DNA from 206 blood donors were tested for RHDpsi by a multiplex PCR that detects RHD, RHDpsi and the C and c alleles of RHCE. The RHD genotype was determined by comparison of size of amplified products associated with the RHD gene in both intron 4 and exon 10/3'-UTR. VS was determined by amplification of exon 5 of RHCE, and sequencing of PCR products was used to analyze C733G (Leu245Val). Twenty-two (11%) of the 206 D-negative Brazilians studied had the RHDpsi, 5 (2%) had the RHD-CE-Ds hybrid gene associated with the VS+V- phenotype, and 179 (87%) entirely lacked RHD. As expected, RHD was deleted in all the 50 individuals of Caucasian descent. Among the 156 individuals of African descent, 22 (14%) had inactive RHD and 3% had the RHD-CE-Ds hybrid gene. These data confirm that the inclusion of two different multiplex PCR for RHD is essential to test the D-negative Brazilian population in order to avoid false-positive typing of polytransfused patients and fetuses.

Absence of peripheral blood mononuclear cells priming in hemodialysis patients

As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC) priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP) and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6) cells). Lipopolysaccharide (5 µg/ml) did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.

Priming effects of a peripheral visual stimulus in simple and go/no-go tasks

The early facilitatory effect of a peripheral spatially visual prime stimulus described in the literature for simple reaction time tasks has been usually smaller than that described for complex (go/no-go, choice) reaction time tasks. In the present study we investigated the reason for this difference. In a first and a second experiment we tested the participants in both a simple task and a go/no-go task, half of them beginning with one of these tasks and half with the other one. We observed that the prime stimulus had an early effect, inhibitory for the simple task and facilitatory for the go/no-go task, when the task was performed first. No early effect appeared when the task was performed second. In a third and a fourth experiment the participants were, respectively, tested in the simple task and in the go/no-go task for four sessions (the prime stimulus was presented in the second, third and fourth sessions). The early effects of the prime stimulus did not change across the sessions, suggesting that a habituatory process was not the cause for the disappearance of these effects in the first two experiments. Our findings are compatible with the idea that different attentional strategies are adopted in simple and complex reaction time tasks. In the former tasks the gain of automatic attention mechanisms may be adjusted to a low level and in the latter tasks, to a high level. The attentional influence of the prime stimulus may be antagonized by another influence, possibly a masking one.

Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves

It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 ± 94 µg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 ± 0.016 and 1.19 ± 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 µM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 ± 58 to 650 ± 100 µg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

Factors associated with bacteremia due to multidrug-resistant Gram-negative bacilli in hematopoietic stem cell transplant recipients

The epidemiology of bacteremia developing during neutropenia has changed in the past decade, with the re-emergence of Gram-negative (GN) bacteria and the development of multidrug resistance (MDR) among GN bacteria. We conducted a case-control study in order to identify factors associated with bacteremia due to multidrug-resistant Gram-negative (MDRGN) isolates in hematopoietic stem cell transplant recipients. Ten patients with MDRGN bacteremia were compared with 44 patients with GN bacteremia without MDR. Bacteremia due to Burkholderia or Stenotrophomonas sp was excluded from analysis (3 cases), because the possibility of intrinsical resistance. Infection due to MDRGN bacteria occurred in 2.9% of 342 hematopoietic stem cell transplant recipients. Klebsiella pneumoniae was the most frequent MDRGN (4 isolates), followed by Pseudomonas aeruginosa (3 isolates). Among non-MDRGN, P. aeruginosa was the most frequent agent (34%), followed by Escherichia coli (30%). The development of GN bacteremia during the empirical treatment of febrile neutropenia (breakthrough bacteremia) was associated with MDR (P < 0.001, odds ratio = 32, 95% confidence interval = 5_190) by multivariate analysis. Bacteremia due to MDRGN bacteria was associated with a higher death rate by univariate analysis (40 vs 9%; P = 0.03). We were unable to identify risk factors on admission or at the time of the first fever, but the occurrence of breakthrough bacteremia was strongly associated with MDRGN bacteria. An immediate change in the antibiotic regimen in such circumstances may improve the prognosis of these patients.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.