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PubMed2006

Seasonal variations in detecting Borrelia burgdorferi sensu lato in rodents from north eastern Austria.

Austria is well known as an endemic area of Lyme borreliosis. To assess the annual variation of rodent populations that may host agents of Lyme borreliosis we collected rodents in northeastern Austria. Life traps were set out every six weeks during a year consecutively in one each of the three different zones (Hohenau, Ernstbrunn, Vienna Woods) that cover the main habitat characteristics of small mammals in northeastern Austria. Rodents were collected and identified. Samples of heart, urine bladder and brain were removed under aseptic conditions for cultivation of borrelia. Samples of heart muscle were additionally used for molecular detection of borrelia by Real-Time polymerase chain reaction. PCR was performed with borrelia universal primers and with species-specific primers. 938 mice were caught, most frequently Apodemus flavicollis (44%), followed by Clethrionomys glareolus (35%), Microtus arvalis (9%), A. sylvaticus (7%) and Mus musculus (6%). Significant differences were seen in the total number of catch per area (Hohenau, Ernstbrunn, Vienna Woods equal 10:9:2) and in the distribution of the various rodent species in the respective areas. Borrelia strains were grown from only 65 (7%) animals, and more frequently isolated from bladder wall than from heart muscle, and only once from brain. Heart specimens of 223 animals were positive by borrelia PCR (24%), most frequently of the rodent species A. flavicollis (43%) and C. glareolus (38%). Borrelia afzelii was most frequently identified, followed by B. burgdorferi sensu stricto, B. garinii and by mixed infection of B. afzelii with B. burgdorferi sensu stricto. B. garinii was most frequently detected in heart samples of A. sylvaticus (about 20%). In about 3% of PCR positive samples the identification of one of the three mentioned genospecies of borrelia could not be ascertained with the test panel used. The results confirm the rodent species A. flavicollis, A. sylvaticus, M. arvalis and C. glareolus as reservoir animals for B. afzelii, B. garinii and B. burgdorferi sensu stricto, agents of Lyme borreliosis. Notable is the salient presence of B. garinii in heart specimens of A. sylvaticus.

Animals↗
PubMedDate not supplied

Enterovirus infections in Germany: comparative evaluation of different laboratory diagnostic methods.

BACKGROUND: The diagnosis of an enterovirus infection may be achieved through direct virus detection from fecal or cerebrospinal fluid (CSF) samples by virus isolation or PCR. Serologically, a significant rise in antibody titer may be detected and different enteroviral types can be differentiated using the neutralization assay. PATIENTS AND METHODS: We investigated the contribution of these different laboratory parameters to the diagnosis of enterovirus infections occurring in the Frankfurt am Main area during the years 1997 to 1999, including an echovirus 30 outbreak in a group of children with aseptic meningitis in 1997. Samples were referred from 1,013 patients; virus isolation was attempted from 579 CSF specimens and from 400 stool samples. 208 CSF samples were tested by PCR. RESULTS: During the echovirus 30 outbreak we identified 22.3% of samples as positive, almost exclusively echovirus 30. In 1998 only 7.1% of samples were positive and a rather broad range of agents was isolated. In 1999 10.4% were positive, predominantly coxsackie B5 and echovirus 11. We could show that in acute enterovirus infections, virus detection by cell cuLture and PCR is superior to serological methods (neutralization assay and IgM assay). For virus isolation, there was a higher rate of positives from stool compared to CSF (1997: 27.8% versus 25%; 1998: 14.4% versus 3%; 1999: 17.9% versus 8.5%). When comparing PCR and virus isolation from the CSF, the former yielded a higher rate of positive results but was not clearly superior to virus isolation from CSF. CONCLUSION: The recommended method for the diagnosis of acute enterovirus infections is virus isolation from feces. In cases of suspected aseptic meningitis virus isolation and PCR are valuable for the direct detection of virus in CSF.

Acute Disease↗
PubMed1990

Detection of interleukin 1 beta but not tumor necrosis factor-alpha in cerebrospinal fluid of children with aseptic meningitis.

Tumor necrosis factor-alpha and interleukin 1 beta have been shown to be mediators of meningeal inflammation in animal models of bacterial meningitis. The presence of both cytokines in cerebrospinal fluid (CSF) of patients with bacterial meningitis has been documented recently. In this study, we measured concentrations of interleukin 1 beta and tumor necrosis factor-alpha in CSF samples from 36 patients with nonbacterial (aseptic) meningitis, 13 of whom had culture-proved enteroviral meningitis, and from 14 control patients. None of the samples from patients with aseptic meningitis and from the controls had detectable tumor necrosis factor activity in CSF. Thirty-two (89%) of 36 patients with aseptic meningitis had detectable interleukin 1 beta in CSF (mean +/- SEM, 48 +/- 11 pg/mL). These concentrations were significantly smaller than those previously reported in patients with bacterial meningitis (944 +/- 128 pg/mL). Only 2 of the 14 control patients had detectable CSF interleukin 1 beta concentrations of 21 and 42 pg/mL. A significant correlation was evident between interleukin-1 beta concentrations and white blood cell counts in the CSF of patients with aseptic meningitis. Our data suggest that the initial events of CSF inflammation in children with aseptic meningitis are different than those in patients with bacterial meningitis, and the participation of these two cytokines, especially tumor necrosis factor-alpha, is less critical to the process.

Adolescent↗
PubMed1995

Bacteremia in conjunction with endodontic therapy.

This study characterizes oral microorganisms believed to have spread from the root canal into the blood stream during and after endodontic therapy of teeth with Asymptomatic apical periodontitis. Microbiological samples were taken under aseptic conditions from the root canal of 26 single-rooted teeth in 26 patients. In the endodontic treatment of 13 of the patients (Group 1), the first 3 reamers, sizes 15, 20 and 25, were used to a level 2 mm beyond the apical foramen. In the other 13 patients (Group 2), the instrumentation ended inside the root canal 1 mm short of the apical foramen. Blood samples were taken from the patients during the instrumentation and 10 min after the treatment was completed. Anaerobic microorganisms were isolated from all root canals. In 7 patients of Group 1, Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia and Saccharomyces cerevisiae were recovered from the blood. In 4 patients of Group 2, P. intermedia, Actinomyces israelii, Streptococcus intermedius and Streptococcus sanguis were isolated from the blood. Biochemical tests and antibiograms revealed that the isolates from the root canal and blood had identical profiles within the patients, strongly suggesting that the microorganisms isolated from the blood had the root canal as their source.

Actinomyces↗
PubMed2006

Natural presence of Campylobacter spp. in various internal organs of commercial broiler breeder hens.

Campylobacter are known to cause acute bacterial gastroenteritis in humans. Poultry products have been implicated as a significant source of these infections. Six experiments were performed to determine whether Campylobacter could be isolated naturally from the primary and secondary lymphoid organs, liver/gallbladder, and ceca of commercial broiler breeder hens. Broiler breeder hens were acquired from different commercial sources during the early, middle, and late lay cycles. The birds were euthanatized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, the thymus, spleen, and liver/gallbladder were aseptically removed prior to removal of the ceca. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. In this study Campylobacter were found in 11 of 43 thymii, eight of 43 spleens, four of 43 liver/gallbladders, and 30 of 43 ceca. Overall, 28 of 53 isolates from the above samples were Campylobacter coli and 25 of 53 isolates were found to be Campylobacter jejuni.

Animals↗
PubMed2002

The role of inducible nitric oxide synthase in aseptic loosening after total hip arthroplasty.

We collected 16 samples of the membrane which surrounds loose hip prostheses from patients undergoing revision operations for aseptic loosening. To serve as the control group, samples of the synovial tissue and the fibrous capsular tissue were collected from 11 patients undergoing primary hip arthroplasties. Analyses of the expression levels of inducible nitric oxide synthase (iNOS), tumour necrosis factor-alpha (TNF-alpha), and cytosolic phospholipase A2 (cPLA2) mRNAs were performed by a reverse transcription polymerase chain reaction, and the content of nitrite was measured by the Griess reaction using sodium nitrite as the standard. The expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs in the membranes were significantly higher than those in the control samples (p < 0.05). The expression levels of iNOS mRNA and the nitrite content in the membranes significantly correlated with those of TNF-alpha and cPLA2 mRNAs, respectively. In addition, the expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs were significantly higher in membranes from cementless than in those from cemented implants (p < 0.05). Our results suggest that the expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs in the membranes are regulated by closely-related mechanisms and that these have a significant role in aseptic loosening.

Adult↗
PubMed2002

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples.

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was < or = ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle 'one step' NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a 'one step' RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.

Base Sequence↗
PubMed1993

Association of Mycobacterium paratuberculosis infection with reduced mastitis, but with decreased milk production and increased cull rate in clinically normal dairy cows.

Approximately 45 Holstein cows that were Mycobacterium paratuberculosis-positive on the basis of fecal culture results were maintained at any one time in a 210-cow dairy herd. Farm management participated in the New York State Paratuberculosis Eradication Program. Paratuberculosis-positive cows were grouped separately from paratuberculosis-negative cows, but they were otherwise managed identically. During a 1-year study, 180 paratuberculosis-negative cows and 113 clinically normal paratuberculosis-positive cows were identified. Quarter milk samples (n = 6,100) were aseptically collected for microbiologic culture of mastitis pathogens from paratuberculosis-negative cows, and 3,129 quarter samples were obtained from paratuberculosis-positive cows. Dairy Herd Improvement Association (DHIA) records were used to monitor milk somatic cell count linear scores, mature equivalent milk production, new mastitis infections, and chronic mastitis infections. For second-lactation cows greater than 100 days in milk production, and increasing with age beyond that point, paratuberculosis-positive cows had lower mature equivalent milk production than did negative herdmates. Rates of new and chronic mastitis infections, as measured by DHIA linear scores were significantly (P < 0.05, P = 0.05, respectively) lower in cows with nonclinical paratuberculosis. Infected cows were culled from the herd at a faster rate than were paratuberculosis-negative herdmates. Therefore, paratuberculosis was associated with financial loss attributable to reduced milk production and increased culling of infected cows.

Animals↗
PubMed2005

Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-life broiler breeder hens.

Campylobacter and Salmonella are known to cause acute bacterial gastroenteritis in humans. Raw poultry products have been implicated as a significant source of these infections. Five trials were conducted to determine whether Campylobacter and Salmonella spp. exist naturally in the mature and immature ovarian follicles of late-life broiler breeder hens. Broiler breeder hens ranging from 60 to 66 wk of age were obtained from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. Overall, Campylobacter was found in 7 of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca. Campylobacter was found in at least one of each sample of mature follicles and in ceca in each of the five trials. Salmonella was found in 0 of 55 immature follicles, 1 of 47 mature follicles, and 8 of 55 ceca. In this study, the recovery rate of Salmonella from late-life broiler breeder hen ovarian follicles was relatively low. However, the recovery rate of Campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs. Determining how Campylobacter contaminated these ovarian follicles and how many chicks could be colonized from this source are the next steps in helping to elucidate a better understanding of this ecology and the control of Campylobacter in poultry production.

Animals↗
PubMed1988

Interleukin-2 levels in serum and cerebrospinal fluid of multiple sclerosis patients.

A sensitive enzyme-linked immunosorbent assay method was employed to measure interleukin-2 (IL-2) levels in cerebrospinal fluid (CSF) and sera from 30 patients with multiple sclerosis (MS) and 8 patients with other neurological diseases. Detectable levels of IL-2 were found in 6 sera and 9 CSF samples of 21 patients with acute relapse of MS. However, only 3 patients showed measurable IL-2 both in CSF and in serum. IL-2 was not detected in specimens from 9 patients with chronic-progressive MS, whereas high levels were found in 2 CSF samples from patients with aseptic meningitis. Our data suggest that systemic activation of a T-cell population is present in some MS patients; moreover, an active immune mechanism involving IL-2 production takes place within the central nervous system.

Adult↗
PubMed1991

Some aspects of hybridoma cell cultivation.

Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyser and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.

Animals↗
PubMed1982

Microbial presence in the uterine cavity as affected by varieties of intrauterine contraceptive devices.

A group of 33 baboons was used to study the effect of various intrauterine device (IUD) assemblies on the entrance of vaginal/cervical microflora into the uterine cavity for a total experimental period of 16 months. These animals were specially prepared surgically so that intrauterine samples could be taken aseptically and percutaneously rather than transvaginally. While the insertion and presence of any of the IUD assemblies used in this study could promote intrauterine bacteria, the principal determining factor was not the device itself, but rather retrieval tail. Multifilament tails were much more effective than monofilaments. Of particular interest is the fact that many potential pathogens can be present in the uterus for long periods in a benign, almost "normal flora" fashion without producing disease.

Animals↗
PubMed2004

Effect of timing of first clinical mastitis occurrence on lactational and reproductive performance of Holstein dairy cows.

Objectives of this study were to determine the influence of timing of first clinical mastitis case occurrence on lactational and reproductive performance in high producing lactating dairy cows during the first 320 days in milk (DIM). Holstein cows, 1001, from two commercial dairy farms in California were retrospectively divided into four treatment groups according to timing of first clinical mastitis case caused by environmental pathogens: control with no recorded clinical cases of mastitis (C; n=501); first clinical mastitis prior to first postpartum AI (MG1; n=250); first clinical mastitis between first postpartum AI and pregnancy diagnosis (MG2; n=147); and first clinical mastitis after diagnosed pregnant (MG3; n=103). Clinical cases of mastitis were identified at every milking by the herd personnel based on abnormal milk or swelling of the mammary gland. A fore sample of milk was aseptically collected from every clinical case for microbiological culture. Mastitis decreased yields of milk, 3.5% fat-corrected milk, and milk components, but the effect was only observed for MG1 and MG2. Cows in the control group had lower linear somatic cell count (SCC) score throughout the lactation. Culling was increased by mastitis, and cows in the mastitis groups left the study earlier than controls. Conception rate at first postpartum AI and pregnancy rate at the end of the study were both decreased by mastitis prior to or after first AI, and MG1 and MG2 cows had extended days open. Furthermore, cows experiencing mastitis during lactation had a higher incidence of abortions. The negative effects of mastitis on reproduction were observed regardless of clinical case being caused by either Gram positive or negative bacteria. Mastitis either prior to or after first postpartum AI impairs lactation performance, increases culling, and decreases reproductive efficiency in high producing Holstein dairy cows.

Abortion, Veterinary↗
PubMed1995

Staphylococci in heifer mastitis before and after parturition.

Aseptic udder quarter secretion samples (n = 1112) from 200 mastitic and 65 non-mastitic control heifers from 160 different farms were collected for bacteriological examination and in vitro antimicrobial susceptibility testing. Coagulase-negative staphylococci (CNS) were the most frequently isolated bacteria (57.8%). The percentage of Staphylococcus aureus was 20.1, of streptococci 11.3 and of other pathogens 10.8. Staph. simulans, Staph. hyicus, Staph. xylosus and Staph. chromogenes were the most common CNS. Staph. simulans and Staph. hyicus were most frequently found in clinical samples after parturition, whereas other CNS were equally or more often found in control samples. Most CNS infections detected in the prepartum period were eliminated spontaneously or with antibiotic treatment during early lactation, but the infected quarters were more susceptible to new infection by other pathogens. Milk N-acetyl-beta-glucosaminidase activities were measured to monitor changes in inflammation. Among the CNS infections, N-acetyl-beta-glucosaminidase activities were highest for Staph. hyicus and lowest for Staph. xylosus in the lactating mammary gland of heifers. CNS were resistant to trimethoprim-sulphonamide, ampicillin and erythromycin. Staph. aureus was more resistant than CNS to beta-lactamase-sensitive penicillins.

Acetylglucosaminidase↗
PubMed1986

Nosocomial Mycobacterium gordonae pseudoinfection from contaminated ice machines.

Thirty-two clinical specimens submitted to the laboratory during a 12-month period from July 1980 to June 1981 were reported to be culture-positive for Mycobacterium gordonae, an organism generally considered to be a slow-growing saprophyte with natural habitats which include soil and water. Only seven similar isolates had been recovered in the preceding 4 1/2 year period. The discordance between clinical findings and the mycobacterial cultures suggested extrinsic contamination of the specimens. Contamination in the laboratory was believed unlikely because: 1) clinical samples obtained in an aseptic manner were never contaminated; 2) various surveillance cultures of reagents and deionized water used in the laboratory were negative; and 3) substitution of deionized water with sterile water did not control the outbreak. Extensive hospital-wide cultures of water sources implicated the use of ice and ice water from contaminated ice machines as the source of this pseudoepidemic. Cleaning of the ice machines resulted in a sharp decrease in the number of M. gordonae isolates. Pseudoinfection by M. gordonae from improperly maintained ice machines has not been reported before.

Adult↗
PubMedDate not supplied

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production.

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Adenoviridae↗
PubMed2003

Removal of hair surrounding the teat and associated bacterial counts on teat skin surface, in milk, and intramammary infections.

The effectiveness of monthly removal of hair surrounding teats on the reduction of teat skin surface bacteria, and the incidence of intramammary infection (IMI), was studied for 10 months in a dairy farm. A split udder design was used where hair was removed on one side, left or right, with the other side serving as a control. Controls and treatment sides were randomly applied in a systematic fashion to 218 cows. Standard milking time pre- and post-milking hygiene practices were applied to all udders during the trial. Collection of teat skin swab solutions preceded aseptic collection of milk samples, performed at monthly intervals, immediately prior to milking. Teat skin bacterial counts did not differ between control and treated teats. Incidences of IMI were similar for treatment when compared with control mammary quarters, as measured by total or by pathogen type. In a second study, the effect of hair removal on the bacterial content of milk was determined using 40 cows. Treatments and allocations were as described. Udder half milk, milk from both mammary quarters of each udder half, was combined and diverted into separate buckets. Buckets were thoroughly cleaned and sanitized between milkings. A portion of bucket milk was collected 24 h after removal of udder hair. The total milk bacterial counts, and counts of psychrotrophs and thermoduric organisms were not reduced by udder hair removal. Results do not suggest that removal of udder hair leads to an improvement in milk quality as determined by milk bacterial content in the herd studied.

Animal Husbandry↗
PubMed2001

Efficacy of a new collection procedure for preventing bacterial contamination of whole-blood donations.

BACKGROUND: Transfusion-related bacterial contamination is a serious problem. The introduction of bacteria into donations at the collection stage seems frequent, despite well-conducted phlebotomy site preparation. Additional preventive measures are required. STUDY DESIGN AND METHODS: The aim of this study was to assess the potential efficacy of excluding the first 15 mL of blood to reduce the bacterial contamination of donations. A special device allowed the aseptic collection of two samples at the beginning of donation: S1 (first 15 mL) and S2 (next 15 mL). Bacteriologic cultures of S1 and S2 were performed by using an automated system. The procedure's efficacy was measured by the proportion of positive donations in S1 that were then negative in S2. RESULTS: S1 and/or S2 were positive in 76 (2.2%) of 3385 donations. In about three-fourths of the culture-positive donations, contamination was detected in the first 15-mL sample only. Gram-positive cocci accounted for 81 percent of species, gram-positive bacilli for 14 percent, and gram-negative bacilli for 5 percent. The new procedure would have prevented the introduction of bacteria in 55 donations, reducing to 0.6 percent the risk of contamination from the first 15 mL collected. CONCLUSION: Although the final effect on blood component bacterial contamination rates cannot be derived from the study, excluding the first 15 mL of blood may reduce the rate of bacterial contamination in donations.

Adult↗