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Biomedical subjects

C M Leutenegger

Publications and source records attributed to C M Leutenegger.

At least 19 recordsLinked to original sources
PubMed2006

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

Animals↗
PubMed2004

Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus).

Bartonella species are emerging pathogens that have been isolated worldwide from humans and other mammals. Our objective was to estimate the prevalence of Bartonella infection in free-ranging African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Blood and/or serum samples were collected from a convenience sample of 113 lions and 74 cheetahs captured in Africa between 1982 and 2002. Whole blood samples available from 58 of the lions and 17 of the cheetahs were cultured for evidence of Bartonella spp., and whole blood from 54 of the 58 lions and 73 of the 74 cheetahs tested for the presence of Bartonella DNA by TaqMan PCR. Serum samples from the 113 lions and 74 cheetahs were tested for the presence of antibodies against Bartonella henselae using an immunofluorescence assay. Three (5.2%) of the 58 lions and one (5.9%) of the 17 cheetahs were bacteremic. Two lions were infected with B. henselae, based on PCR/RFLP of the citrate synthase gene. The third lion and the cheetah were infected with previously unidentified Bartonella strains. Twenty-three percent of the 73 cheetahs and 3.7% of the 54 lions tested by TaqMan PCR were positive for Bartonella spp. B. henselae antibody prevalence was 17% (19/113) for the lions and 31% (23/74) for the cheetahs. The prevalence of seropositivity, bacteremia, and positive TaqMan PCR was not significantly different between sexes and age categories (juvenile versus adult) for both lions and cheetahs. Domestic cats are thus no longer the only known carriers of Bartonella spp. in Africa. Translocation of B. henselae seronegative and TaqMan PCR negative wild felids might be effective in limiting the spread of Bartonella infection.

Acinonyx↗
PubMed2003

Evidence for modulated immune response to Anaplasma phagocytophila sensu lato in cats with FIV-induced immunosuppression.

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease in which some patients experience unusual opportunistic infections. In this study, cats infected with the HGE agent, Anaplasma phagocytophila s.l., had clinical granulocytic ehrlichiosis (GE), anti-nuclear antibodies and increased IFN-gamma mRNA. In FIV-immunosuppressed cats with GE, there was upregulated IL-10 transcription but not IFN-gamma. Cats with FIV had poor response to vaccines, regardless of GE status. This preliminary report demonstrates that cats with FIV-infection make a good model of ehrlichiosis in an immunocompromised host, and that viral immunosuppression may not increase the severity of ehrlichiosis but may attenuate immune responses to the pathogen.

Anaplasma phagocytophilum↗
PubMed2003

The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.

TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.

Animals↗
PubMed2002

HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6.

The most common viral disease of cats worldwide is the infection with feline herpesvirus 1 (FeHV-1). This infection may be followed by Herpetic stromal keratitis (HSK), which is supposed to have an immunopathological basis. Experiments using herpes simplex viruses (HSV) in mouse models indicated that HSK may be treated by topical application of the interleukin 10 (IL-10) gene. The objective of this study was the construction of human herpes simplex virus type 1 (HSV-1)-based amplicon vectors expressing feline interleukin genes and delivery of these genes into cells of feline origin. HSV-1-based amplicon vectors encoding either the enhanced green fluorescent protein, the feline IL-6 or the feline IL-10 under control of the HSV-1 immediate-early 4/5 promotor were constructed, packaged into amplicon particles, transduced into feline cells, and tested for RNA synthesis and biological activity. Feline cells were successfully transduced by HSV-1-based amplicon particles and RNA specific for the transgene was detected already at 2h post transduction, with a maximum at 24h. The recombinant feline IL-10 was functionally active as demonstrated by the reduction of both IL-12 p40 and interferon-gamma-mRNA production in Pansorbin stimulated feline peripheral mononuclear cells. Similarly, the recombinant feline IL-6, which was secreted into the supernatant of transduced cells, was able to support the growth of the IL-6-dependent murine B cell hybridoma 7TD1. HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6. It will be of high interest to study the effects of these tools in vivo.

Animals↗
PubMed2002

[New molecular biology detection methods for tick-borne infectious agents].

Tick-borne zoonotic pathogens are well known in many areas all over the world. Among the tick-borne transmitted diseases in Switzerland, Lyme disease caused by Borrelia burgdorferi, ehrlichiosis caused by various species of Ehrlichia and tick-borne encephalitis caused by the tick-borne encephalitis virus (TBEV) are the most important zoonotic diseases. Early diagnosis and treatment is necessary to prevent fatal infections and chronic damage to various tissues. Due to the variety of uncharacteristic clinical signs, tick-borne diseases are not easily recognized. Diagnosis is based on clinical findings, a record of tick exposure, and direct or indirect detection of the pathogen. Here we discuss briefly the most important tick-borne infections and their diagnosis with emphasis on a new molecular diagnostic tool--the real-time TaqMan PCR--and its importance for the diagnosis of tick-borne pathogens.

Animals↗
PubMed2002

Does training affect mRNA transciption for cytokine production in circulating leucocytes?

Physical exertion is a stimulus for the upregulation of cytokine production including IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-10 and TNF-alpha in horses. To investigate that hypothesis, we initiated training of 5 stall-rested Thoroughbreds. Blood samples were drawn before and weekly during training. The relative transcription of mRNA within the leucocytes was measured using real time TaqMan quantitative PCR. The training protocol was walking (3 min), trotting (3 min) and cantering/galloping (6 min) increasing in intensity weekly (6 to 12 m/s) and culminating in an intense exercise period. Comparisons of mRNA concentrations were made using a repeated measures ANOVA on ranks and a Student-Newman-Keuls pair-wise multiple comparison (P<0.05). The training programme or intense exercise bout did not affect IL-2, IL-4, IL-6 and IL-10. IL1-beta and TNF-alpha transcription increased on Day 23. TNF-alpha peaked on Day 23 and IL1-beta on Day 30. Neither demonstrated a response to intense exercise. IL-1ra decreased significantly on Day 9; rose significantly from Day 9 to Days 16 and 23; remained significantly elevated through Days 30 and 37 over Day 9, and rose very slightly after intense exercise on Day 56. Alterations in leukocyte cytokine responses may influence susceptibility to infectious disease, metabolic responses to exercise or exercised induced syndromes.

Analysis of Variance↗
PubMed2002

Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR.

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R(2) = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

Animals↗
PubMed2001

Infection of mice with the agent of human granulocytic ehrlichiosis after different routes of inoculation.

Population kinetics of the agent of human granulocytic ehrlichiosis (aoHGE) were examined after needle and tickborne inoculation of C3H mice. Blood, skin, lung, spleen, liver, kidney, brain, lymph node, and bone marrow samples were analyzed by using real-time polymerase chain reaction (PCR) at various intervals after inoculation, using a p44 gene target. The highest number of copies of the p44 gene target occurred in blood and bone marrow samples, emphasizing aoHGE leukocytotropism. Numbers of copies of the p44 gene target in other tissues reflected vascular perfusion rather than replication. Needle-inoculated infected mice had earlier dissemination, but kinetics of infection in both groups were parallel, with declining rates of infection by day 20 and recovery in some mice on days 20-60 after inoculation. On the basis of an aoHGE lysate ELISA, mice seroconverted by day 10 after inoculation. Therefore, real-time PCR is useful for quantitative studies with the aoHGE in experimental infections, and results showed that needle inoculation can be used to study the aoHGE infection because of its similarity to tickborne inoculation.

Animals↗
PubMed2001

Virulence differences between two field isolates of feline immunodeficiency virus (FIV-APetaluma and FIV-CPGammar) in young adult specific pathogen free cats.

The goal of this study was to identify a strain of feline immunodeficiency virus (FIV) that would be more virulent for adult cats than the prototype FIV-APetaluma and, thereby, enhance the FIV infection model for HIV-1 related research. Diehl et al. reported that one clade C strain of FIV, FIV-CPGammar, was more virulent than other known FIV isolates. Mortalities from 58 to 100% were reported for kittens 12 weeks of age and less following intravenous inoculation. A more variable and somewhat less virulent disease course was observed in neonatal to 8-10-week-old kittens infected orally, intravaginally or intrarectally with this same isolate (Obert and Hoover, 2000). However, no studies have been done with FIV-CPGammar in adult cats. Therefore, the virulence of FIV-CPGammar for young adult cats was compared to that of FIV-APetalulma, the original FIV isolate. One group of five cats were inoculated intraperitoneally with 470 TCID(50) of FIV-CPGammar in the form of pooled plasma from acutely infected cats, while a second group was infected with plasma containing the 750 TCID50 of FIV-APetaluma. The cats were observed for 20 weeks for gross signs of disease, hematologic abnormalities, time of antibody appearance, and plasma and peripheral blood mononuclear cell (PBMC) associated virus levels. Viral RNA and proviral DNA were measured by a real-time PCR, sensitive to 50 copies per milliliter. The only outward sign of disease was lymphadenopathy, which occurred at a similar time and intensity in both groups of cats. Cats infected with FIV-CPGammar were more likely to be neutropenic and lymphopenic during the first 10-12 weeks of infection than cats infected with FIV-APetaluma. Both groups of cats showed similar overall declines in absolute mean CD4 cell counts and identical concomitant increases in CD8 cells. CD4/CD8 cell ratios were also similar. Antibody, as measured by an ELISA against recombinant FIV-TM antigen, appeared in all cats by 4 weeks post-infection. The most significant differences were in plasma viral RNA and PBMC proviral DNA levels. Cats infected with FIV-CPGammar had up to 100 times higher mean levels of viral RNA during the first few weeks of infection than cats infected with FIV-APetaluma. This difference was also mirrored in levels of proviral DNA in PBMC, which were significantly higher in the FIV-CPGammar infected cats. Plasma viral RNA and PBMC proviral DNA levels were virtually identical in both groups of cats at 20 weeks post-infection. However, proviral DNA in tissues such as thymus and popliteal lymph nodes was 10-fold or so higher in FIV-CPGammar infected cats at 20 weeks and histopathologic lesions were more severe. Based on these various parameters, we concluded that FIV-CPGammar was more virulent than FIV-APetaluma in young adult cats during the 20-week study period. However, we were not able to recreate the severe and rapidly progressive disease previously reported for kittens, suggesting an age-related resistance similar to that observed previously for FIV-APetaluma (George et al., 1993).

Animals↗
PubMed2001

Cytokine mRNA levels in isolated feline monocytes.

Real-time PCR systems were developed to quantitate cytokine expression in short-time cultivated feline monocytes. Feline-specific interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) primers as well as TaqMan probes were designed and were adapted to a quantitative PCR system which had been previously established for feline IL-10 and IL-12 p40. Quantitative analysis of cytokine messenger RNA (mRNA) transcription based on the comparison of the cytokine with the housekeeping gene feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing universally expressed mRNA. GAPDH mRNA was readily detectable in cDNA prepared from short-time cultivated peripheral blood monocytes. Cytokine mRNA was demonstrated in all samples at variable amounts. IL-1beta and TNF-alpha mRNA was constitutively expressed whereas IL-6, IL-10 and IL-12 p40 mRNA was generally expressed at a lower level and was occasionally not detected. There was a great variability of cytokine production between individual cats and at different time points in the same cat.

Animals↗
PubMed2001

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA.

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Animals↗
PubMed2001

Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus.

Determination of retroviral load is an important tool in the investigation of the success of therapeutic or vaccination trials in patients infected with lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) counterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detection System (TaqMan) to quantify the viral load of FIV-infected cats. Two different primer/probe systems were designed to detect a broad range of clade A FIV isolates. Both systems are characterized by excellent reproducibility, high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations have greater impact on the accuracy of the viral load estimation. To compensate for these variations, we improved the assay and developed a multiplex real-time RT-PCR, which allows simultaneous calculation of the viral copy number and the individual recovery rate in an one-tube reaction. This enables the rapid and accurate calculation of copy number independent of preassay variations. In further studies, two additional real-time RT-PCR assays were designed and used to investigate the influence of sequence variations in the binding regions for either the primers or probe. Sequence mismatches in this region had a significant effect (up to 4 logarithmic decades) on reaction efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies before determining viral load.

Animals↗
PubMed2001

Quantitative fluorogenic PCR assay for measuring ovine herpesvirus 2 replication in sheep.

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.

Animals↗
PubMed2000

Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction.

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.

Animals↗
PubMed2000

Modulation of early growth response (EGR) transcription factor-dependent gene expression by using recombinant adenovirus.

Early growth response (EGR) transcription factors link initial cytoplasmic events to long-term alterations of cellular gene expression and are induced by various stimuli. To test their roles in cell physiology, we constructed adenoviral recombinants encoding NGFI-A binding protein 2 (NAB2, a repressor of EGR1, EGR2, and EGR3), EGR1, NAB-insensitive EGR1(I293F) (EGR1*), EGR2, and the NAB-binding, repressive domain 1 (R1) of EGR1. These viruses regulated EGR-dependent expression of GFP and luciferase reporter genes in heterologous expression assays. Infection of a myoblast cell line with EGR1 and EGR1* adenovirus induced the endogenous gene for platelet-derived growth factor A chain (PDGF-A). In addition, in neuroblastoma cells, the two novel EGR1 target genes EGR3 and NAB2 were identified by using adenoviral transfer of EGR1 and EGR1*. Our results demonstrate that recombinant adenovirus is useful to regulate heterologous and endogenous EGR target gene expression, and suggest that EGR transcription factors can autoregulate themselves.

Adenoviridae↗
PubMed2000

[Serologic and clinical evidence for endemic occurrences of human granulocytic ehrlichiosis in North-Eastern Switzerland].

BACKGROUND: Tick-borne human granulocytic ehrlichiosis (HGE) has not been diagnosed in Switzerland, although the HGE agent has been identified in ticks and animal reservoirs and human infections have been suspected on the basis of serological surveys. METHODS: We retrospectively tested sera of 48 persons with antibodies to B. burgdorferi and/or tick-borne encephalitis virus for the presence of antibodies to E. phagocytophila (a surrogate marker of the agent of HGE), and reviewed their charts with regard to clinical manifestations possibly associated with a tick-borne infection. We then prospectively examined EDTA blood of 80 patients who presented with fever 7 to 21 days after a tick bite for the presence of the HGE agent (using nested PCR and microscopic examination of blood smears) and anti-E. phagocytophila antibodies. We also collected clinical data. RESULTS: The retrospective study revealed 12 persons (25%) with anti-E. phagocytophila antibody titers > or = 1:80, suggesting coinfection with HGE and either Lyme Borrelia or tick-borne encephalitis virus. Among these, 7 patients presented with clinical manifestations compatible with HGE disease. The prospective investigation identified 8 patients (10%) with anti-E. phagocytophila antibody titers > or = 1:80, and 7 of these presented with signs and symptoms suggesting HGE. The HGE agent, however, was detected neither by PCR nor by microscopic examination. CONCLUSIONS: Serological and clinical data suggest the occurrence of an HGE-like agent as well as of coinfections with HGE and B. burgdorferi or tick-borne encephalitis virus in Switzerland. However, the HGE agent was not identified in persons living in Switzerland.

Adolescent↗