CUTANEOUS DEMODICOSIS AND UV-INDUCED SKIN NEOPLASIA IN TWO GOELDI'S MONKEYS (CALLIMICO GOELDII).

Two nonrelated Goeldi's monkeys (Callimico goeldii) from the same enclosure developed multifocal alopecia with hyperkeratotic to ulcerative skin lesions on the lower abdomen and inner thighs. Necropsy samples of the first animal showed hyperplastic dermatitis together with in situ carcinoma and intralesional Demodex organisms. The second monkey developed similar lesions 2.5 yr later. Skin scrapings and biopsies also revealed Demodex mites within hyperplastic dermatitis. Long-term treatment with ivermectin, imidacloprid-moxidectin, and sarolaner resolved the demodicosis but skin lesions progressed to actinic keratosis and carcinoma. Both cutaneous neoplasia and demodicosis are rarely described in New World monkeys and these are the first reported cases in Goeldi's monkeys. Since the animals had access to ultraviolet (UV) light, as recommended for indoor-housed callitrichids, the skin tumors were likely UV-induced and the mites have settled particularly within impaired regions. Thus, apparent demodicosis can indicate cutaneous immunosuppression and might alert caretakers to adjust the UV regime.

Intervertebral disc herniation in two coatis (Nasua nasua) and postoperative laminectomy membrane formation.

Magnetic resonance imaging revealed spinal cord compression due to intervertebral disc herniation of Hansen type I and II in the thoracolumbar vertebral column in two middle-aged coatis (Nasua nasua) with chronic progressive paraparesis. Surgical treatment included hemilaminectomy and partial corpectomy in one and dorsal laminectomy in the other coati. Both coatis recovered well after surgery. One showed unremarkable gait 6 and 15 months post surgery, while the other one suffered from recurrence of paraparesis leading to euthanasia because of deterioration of neurological signs 20 months after the first surgery. Necropsy revealed formation of a laminectomy membrane compressing the spinal cord. Histopathological signs of spinal cord injury and findings of degenerative processes in the intervertebral disc were comparable to those described in dogs. In conclusion, this case report shows for the first time that surgical intervention seems to be a useful and safe treatment in chronic intervertebral disc herniation in coatis, but relapses are possible.

Unusual Outbreak of Fatal Clostridiosis in a Group of Captive Brown Pelicans ( Pelecanus occidentalis).

Fatal clostridial infections and clostridial toxicoses are common in birds. Most fatalities are associated with toxin production and progress rapidly, often within 24 hours of infection. We describe an unusual and protracted course of disease in 6 captive brown pelicans ( Pelecanus occidentalis), which was believed to result from toxicosis by toxovar A produced by a mixed infection with Clostridium sordellii and Clostridium perfringens. Although the first death in the group occurred 3 days after signs of illness were documented, the remaining birds died over a 38-day period despite aggressive antibiotic and supportive therapy. Although the birds presented with classic signs of botulism, Clostridium botulinum was not identified in any tissues or environmental samples. Postmortem findings in all pelicans included extensive subacute myonecrosis, enteritis, and nonsuppurative hepatitis. Alpha-toxins and sordellilysin genes from C perfringens and C sordelli isolates, respectively, were detected via polymerase chain reaction. The source of the pathogenic bacteria was sediment within a water basin inside the affected birds' enclosure.

Tracheal Resection in a Secretary Bird ( Sagittarius serpentarius) with Granulomatous, Foreign-body Induced Tracheitis.

A 24-year-old female secretary bird ( Sagittarius serpentarius) was presented with acute, mild dyspnea occurring only during feeding times. Despite initial conservative therapy consisting of antibiotics and antifungal, antiparasitic, and anti-inflammatory drugs, the dyspnea worsened progressively, resulting in severe respiratory distress. Radiographs of the trachea suggested stenosis in the caudal one-third of the trachea. Tracheal endoscopy revealed an obstruction of approximately 90% of the tracheal lumen, in addition to mild suspected aspergillosis of the air sacs. Tracheal resection and anastomosis were performed, during which 1.5 cm of abnormal trachea was removed. Histopathologic examination showed severe granulomatous tracheitis, most likely induced by foreign body material. Respiratory signs resolved immediately postoperatively. Antibiotic and anti-inflammatory therapy continued for another 7 days and the bird was treated with antifungals for a total of 45 days. The bird recovered uneventfully. We encourage tracheal resection and anastomosis for severe tracheal stenosis even in aged, large birds of prey that are managed in large aviaries.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

Successful nonsurgical artificial insemination and hormonal monitoring in an Asiatic golden cat (Catopuma temmincki).

Since it is reported to be difficult to establish Asiatic golden cat (Catopuma temmincki) breeding pairs in captivity as a result of overaggressive behavior of the male, artificial insemination (AI) may be a desired option by which to achieve pregnancy. This approach was chosen in the present case involving a nulliparous, 6-yr-old female cat that was inseminated transcervically during a naturally occurring estrus, which therefore required only a single general anesthetic procedure. On day 4 of estrus behavior, the male was anesthetized and semen was collected via urethral catheterization (UC) to recover spermatozoa in high concentration followed by electroejaculation (EE) to obtain additional semen and seminal fluid. The fresh UC semen, totaling 180 microl in volume and containing spermatozoa showing 55-70% sperm motility, was inseminated 2.5 hr later via a commercial cat urinary catheter passed through the cervix into the uterus. Immediately afterwards, the EE fraction (100 microl) was inseminated deeply into the dorsal medial fold of the vagina. The GnRH analogue Receptal (0.75 ml, i.m.) was given during anesthesia in an attempt to induce ovulation. Increasing fecal concentrations of progesterone after AI and a significant rise in fecal prostaglandin F2alpha metabolite (PGFM) concentrations (P < 0.0001) from day 45 post-AI indicated that the cat had conceived, and it produced healthy twin cubs after an 84-day gestation.

Fatal Balamuthia mandrillaris infection in a gorilla - first case of balamuthiasis in Germany.

BACKGROUND: A 12-year-old female western lowland gorilla died in a zoological garden in Germany after exhibiting general neurological signs. METHODS: Balamuthia mandrillaris was identified as causative agent by indirect immunofluorescent staining of brain sections and confirmed by PCR and respective sequencing. RESULTS: The animal suffered from a chronic progressive necrotizing amebic meningoencephalitis. CONCLUSION: This is the first case of Balamuthia amebic encephalitis in Germany.

A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla).

Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2-10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

Tyrosine phosphorylation of annexin A2 regulates Rho-mediated actin rearrangement and cell adhesion.

Cell adhesion and motility require a dynamic remodelling of the membrane-associated actin cytoskeleton in response to extracellular stimuli that are primarily transmitted through receptor tyrosine kinases. In a cellular model system for tyrosine phosphorylation-based growth factor signaling, we observed that annexin A2 is tyrosine-phosphorylated upon insulin receptor activation. The phosphorylation precedes peripheral actin accumulations and subsequent cell detachment. These morphological changes are inhibited by annexin A2 depletion and require Rho/ROCK signaling downstream of tyrosine-phosphorylated annexin A2. A phospho-mimicking annexin A2 mutant is sufficient to drive peripheral actin accumulation and the resulting cell detachment in the absence of insulin stimulation. Thus, a tyrosine phosphorylation switch in annexin A2 is an important event in triggering Rho/ROCK-dependent and actin-mediated changes in cell morphology associated with the control of cell adhesion.

Reliable generation of stable high titer producer cell lines for gene therapy.

OBJECTIVE: Retroviral vectors represent one of the most robust technologies for in vivo expression of heterologous gene sequences and are still the most commonly used vectors in clinical gene therapy trials. The production of high titer retroviral preparations, however, can be a problematic procedure for certain constructs. METHODS: GALV- or RD114-pseudotyped retroviral particles carrying selectable fluorescence markers or drug resistance genes, such as the green fluorescent protein (GFP) or the O(6)-methylguanine-DNA-methyltransferase (MGMT) mutants, were used to stably transduce Phoenix-(FNX-)eco cells. Thereafter, a polyclonal population of producer cells was generated by enriching cells with high marker gene expression. In addition, single producer clones were selected by limiting dilution. RESULTS: Retroviral titers were increased 1-2 logs by enriching for a polyclonal population of producer cells, and selection of single producer clones allowed another 1- to 2-log increase in titers. Using this method, reproducibly high titer viral preparations allowing efficient transduction of hematopoietic stem cells were generated. CONCLUSION: A reliable and time-effective method to generate stable high titer producer cells based on the FNX-cell line for problematic retroviral vector constructs is described.

The transcriptional activator ClgR controls transcription of genes involved in proteolysis and DNA repair in Corynebacterium glutamicum.

Expression of the structural genes encoding the ATP-dependent proteases ClpCP and Lon in Corynebacterium glutamicum and Streptomyces lividans is activated by the transcriptional regulator ClgR in response to yet unknown environmental stimuli. As it was not known whether ClgR controls expression of additional genes we used DNA microarrays in order to comprehensively define the ClgR regulon in C. glutamicum. The mRNA levels of 16 genes decreased >/= 2-fold in a DeltaclgRDeltaclpC mutant (ClgR absent) compared with a DeltaclpC mutant (ClgR present). For five genes in four operons (NCgl0748, ptrB, hflX and NCgl0240-recR) regulation by ClgR could be independently verified by primer extension analyses and confirmation of binding of purified ClgR to the regulatory regions of these operons. ptrB encodes an endopeptidase, which is consistent with the proteolytic functions of the genes already known to be under ClgR control. However, RecR is unrelated to proteolysis but required for recombinational repair of UV-induced DNA damage. Possibly ClgR-dependent activation of gene expression is triggered by environmental stresses damaging both proteins and nucleic acids, although DNA damage induced by UV radiation and mitomycin C treatment did not result in ClgR-dependent transcriptional activation of any of the newly identified ClgR regulon members. In order to functionally analyse the NCgl0748 and hflX genes we have constructed C. glutamicum strains with deletions in these genes. The DeltaNCgl0748 mutant displayed reduced growth rates in minimal and rich media. The NCgl0748 protein was shown to be localized in the cytoplasm only, while the HflX pool is equally distributed between cytoplasm and plasma membrane. In order to study the proposed degradation of ClgR by ClpCP we have constructed a conditional clpP1P2 mutant. Depletion of ClpP1 and ClpP2 in that strain resulted in the accumulation of ClgR, indicating that ClgR is in fact a substrate of the ClpCP1 and/or ClpCP2 protease in C. glutamicum.

Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes.

Annexin 2 is a Ca(2+)-regulated membrane protein and an F-actin-binding protein enriched at actin assembly sites both, on the plasma membrane and on endosomal vesicles. Here, we identify annexin 2 as a phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2))-interacting protein, thereby explaining this specific membrane association. Using the pleckstrin-homology (PH) domain of phospholipase Cdelta1 fused to yellow fluorescent protein as a marker for PtdIns(4,5)P(2), we show that annexin 2 and its ligand p11 (S100A10) are targeted to sites of PtdIns(4,5)P(2) enrichment where F-actin accumulates. At the plasma membrane, adhesion of pedestal-forming enteropathogenic Escherichia coli induces a recruitment of 1-phosphatidylinositol-4-phosphate 5-kinase (PtdIns4P 5-kinase) and an enrichment of PtdIns(4,5)P(2) and annexin 2-p11 at sites of bacterial adhesion. Induction of PtdIns(4,5)P(2)-enriched ruffles and PtdIns(4,5)P(2)-positive, actin-coated vacuoles by Arf6-mediated activation of PtdIns4P 5-kinase also leads to a concomitant accumulation of the annexin 2-p11 complex and the PH domain. Binding studies with immobilized phosphoinositides and phosphoinositide-containing liposomes reveal that the purified annexin 2-p11 complex directly and specifically binds to PtdIns(4,5)P(2) with an affinity comparable to that of the PH domain of phospholipase Cdelta1. Experiments using individual subunits identify annexin 2 as the PtdIns(4,5)P(2)-binding entity. Thus, the direct interaction of annexin 2 with PtdIns(4,5)P(2) is a means of specifically recruiting the annexin 2-p11 complex to sites of membrane-associated actin assembly.

clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor sigmaH.

The ATP-dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes. In Corynebacterium glutamicum, the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit. We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC. Deletion of clgR in the DeltaclpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes. ClgR activity itself is probably controlled via ClpC-dependent regulation of its stability, as ClgR is only present in DeltaclpC and not in wild-type cells, whereas the levels of clgR mRNA are comparable in both strains. clpC, clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR. Identification of the heat-responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for sigmaECF-dependent promoters. The ECF sigma factor sigmaH could be identified as being required for transcriptional activation of clpC, clpP1P2 and clgR in response to severe heat stress. A second heat-responsive but sigmaH-independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR. Taken together, these results show that clpC and clpP1P2 expression in C. glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli. Both the ClgR- and sigmaH-dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes.

The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes.

The Ca2+- and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor- and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2- or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.