Detection of mycobacteria and chlamydiae in granulomatous inflammation of reptiles: a retrospective study.

A retrospective study on reptile tissues presenting with granulomatous inflammation was performed to detect the possible presence of mycobacteria and chlamydiae in these lesions. Ninety cases including 48 snakes, 27 chelonians, and 15 lizards were selected. Mycobacteria were detected by Ziehl-Neelsen (ZN) staining and a broad-range polymerase chain reaction (PCR) followed by DNA sequencing. To detect chlamydiae, immunohistochemistry with monoclonal antibodies against chlamydial lipopolysaccharide (LPS) and a Chlamydiales order-specific PCR and sequencing were applied. Acid-fast bacilli were found in 14 cases (15.6%) by ZN staining and in 23 cases (25.6%) by PCR. Sequence analysis revealed the presence of Mycobacteria other than Mycobacterium tuberculosis complex (MOTT). Chlamydial LPS antigen was observed within granulomas from five samples (5.6%), whereas the PCR screen revealed 58 positive cases (64.4%). Of these, 9 cases (10%) showed 98-99% similarity to Chlamydophila (Cp.) pneumoniae and 49 cases (54.4%) displayed a high similarity (88-97%) to the newly described "Chlamydia-like" microorganisms Parachlamydia acanthamoebae and Simkania negevensis. Results from this study confirm, on the one hand, that MOTT are probably the most important infectious etiology for granulomatous inflammation in reptiles. On the other hand, they indicate that chlamydia infects reptiles and that Cp. pneumoniae should be considered an etiological agent of granulomatous lesions of reptiles. Because both MOTT and Cp. pneumoniae are human pathogens, the potential of zoonotic transmission from reptiles to humans has to be considered. In contrast, the significance of Chlamydia-like isolates remains completely open, and further studies are needed to evaluate their role.

Molecular characterization of feline interleukin 16: chemotactic activity and effect on feline immunodeficiency virus infection and/or replication.

Interleukin 16 (IL-16) has been shown to diminish HIV and SIV replication through inhibition of HIV and SIV mRNA transcription. To evaluate its role in the FIV cat model, we cloned and expressed feline IL-16 and determined its ability to induce chemotaxis as well as to inhibit FIV replication in cultured PBMCs. Sequence comparison of rfIL-16 with human, African green monkey, rhesus macaque, and mouse IL-16 showed 84.2, 84.5, 84.4, and 79.4% identity at the nucleotide sequence level and 93, 91.5, 90.7, and 87.2% identity at the amino acid sequence level, respectively. Biocharacterization of rfIL-16 revealed potent induction of chemotaxis (p < 0.05). In addition, p24 production from feline PBMCs infected with FIV Zurich 2 in vitro was decreased up to 87% (p < 0.05). These data demonstrate biologic and antiviral functionality of rfIL-16.

Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate.

Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin. Total RNA was extracted from defined regions of single 50 microm sawed sections. These RNA preparations are suitable for quantitative PCR analysis of mRNA of different cytokines. In addition, the universally expressed housekeeping GAPDH mRNA proved to be useful as an amplification control and to correct for the degree of RNA degradation, which may vary considerably among samples. Reverse transcribed mRNA was amplified and quantitated in Real-Time PCR using a fluorescein labeled internal TaqMan probe.

Quantitative real-time PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks.

A TaqMan PCR was established for identification and quantitation of members of the Ehrlichia phagocytophila group in experimentally infected cows and in Ixodes ricinus ticks. The TaqMan PCR identified a 106-bp section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for members of the E. phagocytophila group, which include E. phagocytophila, Ehrlichia equi, and the agent of human granulocytic ehrlichiosis. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. phagocytophila. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The numbers of ehrlichiae in leukocytes of the two cows experimentally infected with E. phagocytophila were measured daily by TaqMan PCR and had a course similar to that of the percentages of infected leukocytes determined daily by light microscopy. The prevalence of infected free-living ticks, which were collected from areas where bovine ehrlichiosis is endemic and from regions with sporadic occurrences of granulocytic ehrlichiosis in dogs and horses, was identical as determined by nested PCR and TaqMan PCR.

Evidence of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in Switzerland.

A total of 1,667 Ixodes ricinus ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the Ehrlichia phagocytophila group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of Ehrlichia. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in I. ricinus in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses.

A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.

Molecular cloning and expression of feline interleukin-16.

Human IL-16 (hIL-16) is a homotetrameric cytokine with chemotactic properties towards cells expressing the CD4 receptor. This chemotactic cytokine plays an important role in attracting cells of the immune system to the site where CD8+ T-cells were activated for example by a foreign antigen. In addition to the chemotactic activity, hIL-16 also induces expression of IL-2 receptor, increasing the responsiveness to IL-2 and therefore implying a role for specific expansion of the CD4+ T-cell population in an area of induced inflammation. In this report we describe the cloning, sequencing and the expression of feline IL-16 (fIL-16). At the nucleotide level, fIL-16 shows 84.6 and 84.5%, on the amino acid level 93 and 91.5% identity to the human and African green monkey (agm) IL-16, respectively.

Experimental oral transmission of Ehrlichia phagocytophila to calves.

Three groups of four calves were used to determine whether Ehrlichia phagocytophila could be transmitted orally to calves via infected milk. Groups 1 and 2 consisted of four-week-old calves and group 3 of newborn calves. The calves in group 1 were fed for several days with milk from cows infected experimentally with E phagocytophila. The calves in groups 2 and 3 were fed 200 ml of whole blood containing E phagocytophila organisms; for group 2 the blood was added to milk before being fed, and for group 3 the blood was added to colostrum before being fed within three hours after birth. Blood samples for haematological, serological and cytological examination, and for polymerase chain reaction (PCR) were collected from all the calves, starting on the first or only day of administration and then every four days for four weeks. The calves of groups 1 and 2 showed no clinical, haematological or serological changes, and there was no direct or indirect evidence of the agent. In contrast, all the calves in group 3 had mild pyrexia and seroconverted on day 8, and in one of them E phagocytophila organisms were visible in leucocytes, and the PCR on the buffy coat was positive on day 8.

Detection of Ehrlichia phagocytophila DNA in Ixodes ricinus ticks from areas in Switzerland where tick-borne fever is endemic.

A total of 1,523 adult Ixodes ricinus ticks were collected from regions where bovine ehrlichiosis is endemic and were examined for Ehrlichia phagocytophila via PCR. Of the ticks from cattle with ehrlichiosis, the ticks from healthy cattle, and the free-living ticks, 26.5% (18 of 68), 4.4% (35 of 802), and 0.8% (5 of 653), respectively, were positive.

Identification of a granulocytic Ehrlichia strain isolated from a horse in Switzerland and comparison with other rickettsiae of the Ehrlichia phagocytophila genogroup.

This case report describes a 12-year-old Arabian mare with granulocytic ehrlichiosis. Clinical signs included fever, apathy, anorexia, icterus, limb edema, and reluctance to move. Examination of buffy coat smears revealed Ehrlichia organisms in neutrophils and eosinophils. A band of 1,428 bp was amplified from DNA of leukocytes via nested PCR and was identified as part of the Ehrlichia 16S rRNA gene. It differed from the gene sequences of Ehrlichia phagocytophila and E. equi at two and three positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% identical to that of the agent of human granulocytic ehrlichiosis.

Granulocytic ehrlichiosis in two dogs in Switzerland.

This case report describes two dogs with granulocytic ehrlichiosis. Dog 1 was a male Labrador retriever with clinical signs of lymphosarcoma. Dog 2 was a female Airedale terrier, whose clinical signs included apathy, pyrexia, diarrhea, and abdominal pain. Examination of blood smears revealed Ehrlichia organisms in the neutrophils of both dogs. There was thrombocytopenia in both dogs, and dog 2 also had leukopenia. In both dogs, bands of identical length were amplified from DNA of leukocytes via nested PCR. The bands had identical nucleotide sequences, which differed from the gene sequences of Ehrlichia equi and E. phagocytophila in three and two positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% homologous to that of a human granulocytic ehrlichia.

Nucleotide and predicted peptide sequence of feline interleukin-12 (IL-12).

Feline Interleukin-12 (IL-12) is a heterodimeric glycoprotein consisting of two disulfide linked subunits of about 40 kD (p40) and 35 kD (p35). It is a pleiotropic cytokine mediating biological activities on T- and NK-cells. One important function is the induction of a Th1 immune response. Here we report the cloning and sequencing of feline IL-12, the expression of the p40-protein in E. coli and production of monoclonal antibodies. At the nucleotide level, feline IL-12 shows between 87-90%, on the amino acid level between 82-87% identity to the bovine and human IL-12, respectively.

Laboratory findings in cows after experimental infection with Ehrlichia phagocytophila.

The goal of this study was to assess various hematological variables in 10 cows after experimental infection with Ehrlichia phagocytophila. Blood samples were collected at regular intervals for examination of leukocytes for Ehrlichia organisms and for determination of hematological and biochemical variables. In addition, PCR amplification was performed throughout the disease period on blood and milk samples for the detection of E. phagocytophila organisms. The time of seroconversion and the duration of serum titers indicating positivity were determined by indirect immunofluorescence. For all cows, E. phagocytophila organisms were first detected microscopically in leukocytes 5 to 8 days postinfection and could be demonstrated for a period of 6 to 14 days. For all cows, the appearance of E. phagocytophila organisms in leukocytes coincided with transient erythropenia, leukopenia, and thrombocytopenia and a decrease in hematocrit and hemoglobin concentration. For five lactating cows, E. phagocytophila organisms were identified in leukocytes of milk samples during the acute phase of the disease, which, we believe, has not previously been reported. E. phagocytophila DNA was detected in blood samples by nested PCR from 1 to 2 days before to 2 to 12 days after the organisms were identified microscopically. In milk samples, E. phagocytophila DNA was detected for an average of 11 days.

Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli.

The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit. After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity. In addition, a contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to methylmalonyl-CoA decarboxylation. The small epsilon subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex.

Sequence of the sodium ion pump methylmalonyl-CoA decarboxylase from Veillonella parvula.

The genes encoding methylmalonyl-CoA decarboxylase from Veillonella parvula were cloned on plasmids using oligonucleotides derived from N-terminal amino acid sequences as specific probes. The entire DNA sequence of the methylmalonyl-CoA decarboxylase genes together with upstream and downstream regions was determined. The genes encoding subunits alpha (mmdA), delta (mmdD), epsilon (mmdE), gamma (mmdC), and beta (mmdB) of the decarboxylase were clustered on the chromosome in the given order. The previously unnoted epsilon-chain (M(r) 5,888) was clearly shown to be a subunit of the decarboxylase by correspondence of the N-terminal amino acid sequence with that deduced from the DNA sequence of mmdE. The alpha-subunit was 60% identical with the carboxyltransferase domain of rat liver propionyl-CoA carboxylase, the beta-subunit showed 61% sequence identity with the beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae, and the biotin-containing gamma-subunit was 29-39% identical with biotin-domains of other biotin enzymes. The delta-subunit of methylmalonyl-CoA decarboxylase and the gamma-subunit of oxaloacetate decarboxylase did not show significant sequence homology. The gross structure of both proteins, however, was similar, consisting of a hydrophobic membrane anchor near the N terminus, a proline/alanine linker, and a remarkable accumulation of charged amino acids in the C-terminal part. The sequence of the small epsilon-subunit could be aligned to the C-terminal region of the delta-subunit downstream of the proline/alanine linker, where the two subunits were 47% identical. Of considerable interest for the mechanism of Na+ transport are the long stretches of complete sequence identity between the hydrophobic beta-subunits of methylmalonyl-CoA decarboxylase and oxaloacetate decarboxylase and the presence of two conserved aspartic acid residues within putative membrane-spanning helices.