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PubMed1995

Borrelia in pigeons: no serological evidence of Borrelia burgdorferi infection.

In order to clarify the supposed involvement of urban pigeons (Columba livia livia) in the epidemiology of Lyme disease, the presence of antibodies against Borrelia burgdorferi and Borrelia anserina in pigeons' sera, collected in 12 areas of northern and central Italy, was evaluated. This evaluation was carried out using a classic immunofluorescence assay (IFA), a surface immunofluorescence assay (SIFA) and a standard Western Blot (WB) assay. A total of 104 out of 3,186 (3.26%) serum samples were positive for both spirochetes when tested by IFA, with titres ranging from 1/40 to 1/1280. All positive specimens showed the same or a higher reactivity against B. anserina than against B. burgdorferi. Of the IFA positive samples, 30 were tested by WB and SIFA to evaluate further the specificity of the antibody response, i.e. to try to clarify against which spirochete the antibodies were raised. The presence of antibodies against the 23 kDa protein exclusive to B. anserina, and against epitopes which are not surface-exposed and which are common to B. anserina and B. burgdorferi, was assessed by WB and SIFA. No serological evidence that B. burgdorferi can infect pigeons was found.

Animals↗
PubMed2005

Surgical treatment of femoral artery infected false aneurysms in drug abusers.

BACKGROUND: Post-traumatic femoral artery infected false aneurysms (pfa-IFA) in drug abusers are very common in modern societies, but their surgical management remains controversial. METHODS: A review was undertaken of the English-language literature between 1967 and 2004 for relevant articles describing at least four cases of pfa-IFA in drug-addict populations. The available surgical treatment options are discussed. RESULTS: Recent surgical therapeutic reports favour aneurysm ligation and excision (Lig-Exc) and local debridement (Ld) with observation-selective (delayed) revascularization in cases where limb viability is threatened, or Lig-Exc and Ld alone without vascular reconstruction. The former method carries the risk of delayed decision on attempted extremity salvage (12.1% amputation rate), accepting early (13.5%) and late (7.5%) claudication rate, and although the latter method has much lower early and late amputation rates (5.7 and 6.3%, respectively), it results in a high percentage of claudication and disability (early, 54.4%; late, 44.3%). Immediate (routine) revascularization using either in situ or extra-anatomic bypass has also been associated with high complication rates. Even when it occurs through non-infected tissue planes, the risk of graft infection (early, 21.1%; late, 32.4%) is of great concern, and the possibility of sepsis (together with anastomotic dehiscence (14%) and even amputation) is high (early, 9.8%; late, 11.3%). Reversing the order of revascularization produces zero early complication rates, but long-term follow up reveals that 5.5% of patients have graft infection and 5.5% have had amputation. The follow up rates reported in the literature are poor (only 31.7% completed), and are also sometimes inaccurate. CONCLUSIONS: No surgical treatment for pfa-IFA has been proved to be safe in terms of the overall surgical complications. Longer follow-up periods are needed to provide accurate results.

Amputation, Surgical↗
PubMed2005

Incomplete Freund's adjuvant enhances locomotor performance following spinal cord injury.

Following spinal cord injury (SCI), the pathological sequelae which ensue through the secondary mechanisms of degeneration produce myelin deposits which are potent inhibitors of endogenous neuroregeneration. We have enhanced the immune-mediated response following a hemisection lesion by immunizing adult C57Bl/6 female mice against the inhibitor of neurite outgrowth Nogo-A(623-640) peptide. Moderate anti-Nogo-A(623-640) antibody titre levels were obtained by using Montanide as the adjuvant. However, this antibody response was not obtained using incomplete Freund's adjuvant (IFA). Significant benefit in locomotor performance was demonstrated only in animals which were vaccinated with IFA and not with Montanide. No further benefit could be demonstrated with the Nogo-A(623-640) peptide beyond that seen for adjuvant alone. These data imply that generating antibodies against Nogo-A(623-640) in vivo alone is not sufficient to enhance locomotor recovery and that subcutaneous injection of IFA prior to SCI can enhance locomotor performance.

Adjuvants, Immunologic↗
PubMed1994

Spinal cord SP release and hyperalgesia in monoarthritic rats: involvement of the GABAB receptor system.

1. Monoarthritis was induced in Lewis rats by interdermal injection in the left hind paw of a suspension of Mycobacterium tubercolusis in mineral oil (500 micrograms 100 microliters-1). Controls were injected with 100 microliters mineral oil. 2. Withdrawal latencies to thermal stimuli of the inflamed paw, the contralateral and both paws of control rats were measured at daily intervals after injection by the plantar test. 3. After detection of the pain threshold, rat spinal cords were removed and horizontal dorsal slices were mounted in a 3-compartment bath to measure electrically-evoked release of substance P-like immunoreactivity (SP-LI). 4. The inflamed paw of monoarthritic rats exhibited a lower pain threshold to thermal stimuli than the contralateral paw of the same animals and both paws of control rats. Inflamed paw hyperalgesia was maximal two days after injection, and declined gradually between 7 to 21 days with no evidence of excitability of withdrawal reflexes after 28 days. 5. During the 28 days study, monoarthritic rats gained less weight than control rats. 6. Electrical stimulation of the dorsal roots attached to rat isolated spinal cord slices induced a significant increase (174 +/- 18% of basal outflow which was 30.3 fmol 8 ml-1, n = 5) in SP-LI release. 7. One-week after induction of inflammation no differences in the amount of SP-LI released from the spinal cord of incomplete Freund's adjuvant-treated rats (IFA) and Freund's adjuvant-treated rats (CFA) were detected. Two weeks after, CFA spinal cord tended to release more SP-LI than IFA cords and, 21 days after injection, the spinal cord of CFA rats released significantly more peptide than IFA rats (17.8 +/- 2.8 fmol ml-1, n = 12 and 6.9 +/- 3.2 fmol ml-1, n = 9, respectively).8. Twenty-one days after treatment, the evoked release from monoarthritic rat spinal cords was increased by 263 + 42% (n = 3) in the presence of the GABAB receptor antagonist, CGP 36742 (100 micro M)which also significantly potentiated monoarthritis-induced hyperalgesia up to 45 min after injection(100 mgkg-1, i.p.).9. These findings may provide a basis for a novel approach to chronic pain therapy but also an explanation for the lack of analgesia produced by the GABAB agonist, baclofen, in chronic as compared to acute pain.

Animals↗
PubMed1982

Sensitivity and specificity of the indirect fluorescent antibody test in the study of four murine coccidia.

The sensitivity and specificity of the indirect fluorescent antibody (IFA) test for the detection of serum antibodies were examined in mice that were infected with Eimeria falciformis, E. ferrisi, E. papillata, or E. vermiformis. For the study of each species, five groups of mice were given graded inoculation doses of 10, 10(2), 10(3), 10(4), or 10(5) sporulated oocysts in a primary infection. The sixth group was infected with three sequential doses of 1.5 x 10(3), 1.5 x 10(4), and 1.5 x 10(5) sporulated oocysts per mouse at two- to three-week intervals. All groups of infected mice developed serum antibodies. Sera were titrated by the IFA test with purified sporozoites. Strong fluorescence and high IFA titers were observed with homologous reactions mainly with the sera from mice infected with the higher inoculation dose levels in primary infections and from those given three sequential inoculation doses. Immunological cross reaction among the four species of Eimeria occurred at dilutions of 1:10 to 1:160. Very weak or no fluorescence of free sporozoites was observed with sera from noninfected mice, and there was no fluorescence of sporozoites contained in intact sporocysts.

Animals↗
PubMed2003

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain.

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.

Adult↗
PubMed1977

Serological responses in experimental bovine tuberculosis.

Five two-year-old heifers were each inoculated intravenously with 0.02 mg M. bovis strain AN5. Clinical, haematological and microbiological observations were made during the course of the experiment and antibody levels were measured before and after infection by means of the indirect immunofluorescent antibody (IFA) and bentonite flocculation tests. All cattle developed M. bovis infection varying in severity from peracute tuberculous pneumonia resulting in death within 33 days to chronic progressive generalised tuberculosis. Only cattle developing peracute or acute forms of tuberculosis showed marked haematological changes characterised by leucopenia with lymphopenia. Bacteraemia was detected in the two cattle with peracute tuberculosis 22 days after infection. Anti-mycobacterial antibody was detected after infection in all cattle but fluctuated markedly during the course of the disease. Of a total of 61 serum samples examined from all cattle after inoculation with M. bovis, only 38 were positive to the IFA test and 30 to the bentonite flocculation test. Only 18 were positive to both tests at any one time. IgM was the predominant type of anti-mycobacterial antibody detected by the IFA test and this was found to cross-react with M. avium in almost every sample.

Animals↗
PubMed1990

[A rapid method for serodiagnosis of Japanese encephalitis using persistently infected C6/J-121 cells].

The usefulness of persistently infected C6/36 (C6/J-121) cells with Japanese encephalitis virus (JEV) for a rapid serodiagnostic test was examined with the sera of men, swine and laboratory animals by indirect immunofluorescent antibody (IFA) technique. Detection of specific antibodies was completed within 3 to 5 hours. Nonspecific fluorescence frequently observed in other IFA tests was few. The sensitivity of antibody detection and the serological specificity of IFA (IgG) were similar to those of hemagglutination-inhibition (HI) test. For the detection of IgM antibodies in sera of JE-patients and naturally JEV infected swine, this method was found to be simpler and more sensitive and rapid than the conventional HI test which required 2-ME treatment of the sera.

Animals↗
PubMed1991

[Monoclonal antibodies to herpes simplex viruses antigens--specificity and utility in rapid serotyping of clinical isolates].

Sixteen hybridomas secreting antibodies to HSV-1 and 22 hybridomas secreting antibodies to HSV-2 were derived from fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with each respective virus. Four of the former 16 hybridomas and seven of the latter 22 hybridomas were subcloned and injected into pristane-primed mice to obtain high titers of monoclonal antibodies. Antigen specificity of these monoclonal antibodies were determined by the Western blotting (WB) assay. Two out of four monoclonal antibodies that showed selective reactivity for HSV-1 in IFA, reacted with HSV-1 specific proteins; #1 reacted with 100 KD and 70 KD proteins and #4 with a 150 KD protein, respectively, while the remaining two antibodies reacted only with a 50 KD protein that is type-common antigen. On the other hand, two out of seven antibodies which showed selective reactivity for HSV-2 in IFA, reacted with HSV-2 specific proteins: #5 with a 100 KD protein and #10 with three proteins of 30, 25, and 20 KD, and the other two antibodies reacted with a 50 KD protein that is a type-common antigen. The remaining three antibodies, two of which were found to be immunoglobulin type IgM, reacted with neither HSV-1 nor HSV-2 antigens in WB assay. In order to determine their utility in serotyping, 11 monoclonal antibodies were examined by IFA test for reactivity to cells that were infected with 20 HSV-1 or 16 HSV-2 isolates which had been typed by neutralization test.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

[Studies on tsutsugamushi diseases in Gifu Prefecture. 5. Characterization of monoclonal antibodies to prototype strains of Rickettsia tsutsugamushi and immunological grouping of newly isolated strains using the antibodies].

We characterized 8 monoclonal antibodies (MAbs) to Karp, Kato, and Gilliam strains of Rickettsia tsutsugamushi, and analysed 17 isolates from patients with Tsutsugamushi disease using these MAbs. These were divided into 3 strain-specific (Kp/D11, Kt/2D9, and Gi/E4) and 5 cross-reactive MAbs (Kp/1F11, Kp/1C10, Kp/C6, Kt/3B2, and Kt/3C2). All MAbs recognized characteristic protein antigens using the indirect fluorescent-antibody test (IFA) and proteinase K treatment. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that Kato-specific MAb Kt/2D9 recognized a polypeptide with a molecular mass of 54 kilodalton (kDa) of the homologous strain, and cross-reactive MAbs Kp/1F11, Kp/C6, and Kt/3B2 recognized those of 46-47 kDa, 46-47 KDa, and 60 kDa, respectively to the homologous and heterologous strains. MAbs Kp/1C10 which exhibited a high IFA titer against the Karp strain and only low titers against heterologous strains recognized only the 110 kDa polypeptide of the homologous strain. MAb Kt/3C2 which reacted with both Karp and Kato strains recognized a 54 to 56 kDa polypeptide band of the two prototype strains as well as several other polypeptides, however, each molecular mass was present in only one of two strains. Testing by the plaque reduction technique showed another characteristic of MAb Kt/3C2 to neutralize both Karp and Kato Strains. Fourteen isolated strains from patients in the south and west regions of Gifu Prefecture, the Shimokoshi stain isolated in Niigata Prefecture, and Kawasaki and Kuroki stains isolated in Miyazaki Prefecture were examined for reactivities to 8 MAbs by IFA to classify their antigenicities. No isolated strains reacted with Karp-specific Kp/D11, Kato-specific Kt/2D9, or Gilliam-specific Gi/E4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

[Detection of antibody to varicella zoster virus by immune adherence hemagglutination].

Anti varicella-zoster virus (VZV) antibodies were detected by an immune adherence hemagglutination (IAHA) test, and were compared with the CF test, IFA test and ELISA test, respectively. Type O, Rh-positive RBC for IAHA was obtained from five healthy volunteers. All five RBCs had sufficient sensitivity as the indicator cell. Optimum incubation temperature was 37 degrees C in the serum and in the complement. The complement was obtained from guinea pig sera, and the most suitable concentration was 1;100. The convalescent VZV antibody titers were similar to the values obtained from any of the methods previously mentioned. However, mean titers measured by CF were about two-to fourfold lower than in the values of IAHA. Seroconversion rates of the live VZV vaccine, as detected by CF and IFA were relatively low (CF; 76%, IFA; 56%). In contrast, those obtained from ELISA and IAHA showed perfectly (100%). These results indicate that IAHA has sufficient sensitivity and specificity in the detection of VZV antibodies. In addition, the IAHA test is thought to be a rapid and easy test to perform in ordinary laboratories.

Antibodies, Viral↗
PubMed1998

[Determination of anti-Bartonella henselae antibody by indirect fluorescence antibody test--comparison of two types of antigen: non-cocultivated B. henselae and cocultivated B. henselae with Vero cells].

Serum anti-Bartonella henselae IgG and IgM antibody titers for the diagnosis of cat scratch disease (CSD) were determined by indirect fluorescence antibody (IFA) tests. B. henselae as antigen were harvested either by cocultivating with Vero cells (cocultivated B. henselae) or by cultivating without them (non-cocultivated B. henselae). Based on the results on 110 healthy adults, cut off values were set at 1:32 for IgG, and < 1:20 for IgM antibodies. According to these criteria, IgG antibody was positive in 2.7% of the 110 adults, while nobody was positive for IgM antibody. The titers did not change depending on the types of antigen used. On the other hand, IgG antibody titers against cocultivated B. henselae tended to be higher than those against non-cocultivated B. henselae in 33 CSD suspected patients; 75.8% of the patients were anti-B. henselae IgG positive when tested with cocultivated B. henselae as antigen, while only 48.5% of the same patients gave positive results with non-cocultivated B. henselae. Anti-B. henselae IgM antibody was positive in 24.2% of the 33 CSD suspected patients against both types antigen. Vero cells themselves seemed to nonspecifically bind some IgM (but not IgG). We recommended cocultivated B. henselae as antigen for IgG IFA, and non-cocultivated B. henselae for IgM IFA in the serological tests of CSD.

Adult↗
PubMed2000

[Clinical evaluation of commercial serological test for Bartonella infection].

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.

Adolescent↗
PubMed1996

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.

Animals↗
PubMed1999

Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.

Animals↗
PubMed2003

Detection by immunofluorescence assay of Bartonella henselae in lymph nodes from patients with cat scratch disease.

Laboratory diagnosis of Bartonella henselae infections can be accomplished by serology or PCR assay on biopsy samples. The purpose of our work was to assess immunofluorescence detection (IFD) in lymph node smears using a specific monoclonal antibody directed against B. henselae and a commercial serology assay (IFA) compared with PCR detection. Among 200 lymph nodes examined from immunocompetent patients, 54 were positive for B. henselae by PCR, of which 43 were also positive by IFD. Among the 146 PCR-negative lymph nodes, 11 were positive by IFD. Based on PCR results, the specificity of this new technique was 92.5%, the sensitivity was 79.6%, and the positive predictive value was 79.6%. At a cutoff titer of 64, the sensitivity of the IFA was 86.8% and the specificity was 74.1%. Diagnosis of cat scratch disease (CSD) may be improved, with a specificity of 100%, when the two tests (IFD and IFA) were negative; the sensitivity was 97.4% if one of the two tests was positive. Since PCR-based detection with biopsy samples is available only in reference laboratories, we suggest using IFD coupled with the commercial serology test for the diagnosis of CSD.

Adolescent↗
PubMed2003

Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays.

Two hundred seventy one serum samples from South Korean patients were tested to detect antibodies against Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) and Ehrlichia chaffeensis (the human monocytic ehrlichiosis agent) by indirect fluorescent-antibody assay (IFA) and the Western blot assay. These sera were collected from patients with symptoms of high fever. The rate of seropositivity for Orientia tsutsugamushi was 50.9% by IFA at the Public Health & Environmental Research Institute and National Institute of Health in South Korea. By IFA, 30 (11.1%) and 39 (14.4%) of the serum samples reacted with A. phagocytophilum and E. chaffeensis antigens, respectively. By the Western blot assays, 24 (8.9%) and 29 (10.7%) of the serum samples reacted with purified A. phagocytophilum and E. chaffeensis protein antigens, respectively. This report strengthens other evidence regarding the presence of A. phagocytophilum and E. chaffeensis infections in humans in South Korea.

Adult↗
PubMed1996

Evaluation of serological assays for identification of parvovirus B19 immunoglobulin M.

Three different enzyme immunoassays (EIAs) (Parvoscan-B19, IBL parvovirus B19, and IDEIA parvovirus B19) and one immunofluorescence assay (Biotrin Parvo B19 IFA) were evaluated for detection of parvovirus B19 immunoglobulin M (IgM) antibodies in 203 clinical serum samples. An IgM antibody capture radioimmunoassay was used as a reference test. Serum specimens obtained from patients with clinical symptoms suggestive of parvovirus B19 infections were used to evaluate the sensitivities of the assays, which were shown to be comparable for the Biotrin IFA and IDEIA (97%) and lower for the other two EIAs (90%). In order to test the specificity of the assays, clinical serum samples with IgM antibodies against other viruses were examined, as well as sera with rheumatoid factor activity and sera from healthy pregnant women. The specificities of B19 IgM antibody detection were 96% for the Biotrin IFA, 96% for IDEIA, 90% for Parvoscan, and 88% for the IBL assay. These results show that all four assays can be recommended for diagnostic purposes, although false-positive results may be seen with other acute viral infections, healthy pregnant women, and rheumatoid factor-positive samples.

Adolescent↗