U. Hetzel

Application of ultrashort laser pulses for intrastromal refractive surgery

Abstract · Background: Recently, laser systems have become available which generate ultrashort laser pulses with a duration of 100–200 femtoseconds (fs). By generating micro-plasmas inside the corneal stroma with fs pulses, it is possible to achieve a cutting effect inside the tissue while leaving the anterior layers intact. The energy threshold to generate a micro-plasma with fs pulses is some orders of magnitude lower than it is for picosecond or nanosecond pulses. This results in a strong reduction of the thermal and mechanical damage of the surrounding tissue. · Methods: With a titanium:sapphire fs laser system, the cutting effect on corneal tissue from freshly enucleated porcine eye globes was investigated with different pulse energies. The irradiated samples were examined by light and electron microscopy. The laser-induced pressure transients and the laser-induced bubble formation were analysed with a broadband acoustic transducer and by flash photography.  · Results: With fs laser pulses, the extent of thermal and mechanical damage of the adjacent tissue is in the order of 1 µm or below and therefore comparable with the tissue alterations after ArF excimer laser ablation. Using pulse energies of approximately 1–2 µJ and a spot diameter of 5–10 µm, intrastromal cuts can be performed very precisely in order to prepare corneal flaps and lenticules. · Conclusion: Femtosecond photodisruption has the potential to become an attractive tool for intrastromal refractive surgery.

Cutaneous leishmaniosis in a horse in southern Germany caused by Leishmania infantum.

This report describes a case of cutaneous leishmaniosis in a horse in southern Germany. Diagnosis is based on histopathology, immunohistochemistry and electron microscopy. The protozoan was identified as Leishmania infantum via PCR and restriction fragment length polymorphism. The horse did not show specific Leishmania antibodies. The lesions healed completely within 6 months without any specific treatment. Since neither the infected horse nor its dam had ever left their rural area, autochthonous infection in Germany cannot be excluded. Factors possibly influencing the epidemiological situation are discussed.

Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease

ABSTRACT Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Kochs postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.

Emerging canine angiostrongylosis in northern England: five fatal cases

Angiostrongylus vasorum is a metastrongylid nematode parasite that resides in the pulmonary arteries and right ventricle of dogs and other canids. Since its discovery in France in 1866 by Baillet ([Rosen and others 1970][1]), A vasorum has been reported in dogs in Europe (Denmark, France, Germany,

Repair of extensive articular cartilage defects in horses by autologous chondrocyte transplantation.

Damaged adult articular cartilage has very limited capacity to heal. Autologous chondrocyte transplantation (ACT) has been used clinically and studied in experimental animals in an attempt to provide biologically based cartilage regeneration. This study evaluated cartilage repair following ACT in a large animal model over a period of 2 years. Articular cartilage defects (10 mm in diameter, full-thickness) were created in the minor load-bearing area on the lateral talus of tibiotarsal joints of eight adult horses. In each animal, the right joint was repaired using autologous chondrocytes injected beneath the periosteum, as in the original ACT procedure (Brittberg, M., A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson N. Engl. J. Med. 331:889–895, 1994); the left joint remained untreated to serve as a control. Clinical and pathological evaluation was within the range of normal for all horses at both time points. Compared to untreated defects, ACT resulted in significantly improved defect filling with a well-integrated neocartilage and comparable expression of cartilage-specific markers. The histological score (Peterson, L., T. Minas, M. Brittberg, A. Nilsson, E. Sjogren-Jansson, and A. Lindahl Clin. Orthop. 374:212–234, 2000) (10.4 ± 0.9 for ACT and 5.6 ± 3.9 for controls, all animals, p = 0.016) indicated that ACT contributed to the reparative process. For the first time, the efficacy of ACT was demonstrated in a large animal model supporting the potential of ACT for cartilage regeneration in patients.

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.

A worm's best friend: recruitment of neutrophils by Wolbachia confounds eosinophil degranulation against the filarial nematode Onchocerca ochengi

Onchocerca ochengi, a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus. Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia, but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.

First autochthonous case of canine ocular onchocercosis in Germany

were identified as nematodes belonging to the genus Onchocerca (Chitwood and Lichtenfels 1972). It was not possible to identify them to species level. Until now, the Onchocerca species known to be native to Germany are Onchocerca gutturosa and Onchocerca lienalis in cattle (Zahner and Schulz-Key 1990), and Onchocerca flexuosa and Onchocerca tarsicola in cervids (Bain and Schulz-Key 1974, Schulz-Key and Bain 1976). Additionally, Onchocerca cervicalis and Onchocerca reticulata in horses are thought to be present in Germany, but there are no available data on the species. Onchocerca lupi (Rodonaja 1967), the Onchocerca species of canids, has not been reported in this country. The maximum diameter of the female worms found in the present case was similar to that of O gutturosa or O lienalis, but other morphological criteria differed significantly from these species. Moreover, microfilariae of O lienalis (Eberhard 1979) and other related nematodes of the genus Onchocerca (for a review see Szell and others 2001b) occurring in Europe are considerably larger than those found within the uteri of the nematodes and in the surrounding tissues of the dog in the present case. One reason for the small size of the microfilariae in this case might be inappropriate conditions for a noncanid Onchocerca species in the dog acting as an accidental host. In fact, aberrant Onchocerca species infections have been reported in dogs (Orihel and others 1991, Gardiner and others 1993) and human beings (Beaver and others 1974, AliKhan 1977, Sreter and others 2002b). On the other hand, aberrant infections usually involve only a few worms, which do not become sexually mature (Komnenou and others 2002). In the present case, the presence of gravid females and microfilariae in the connective tissue suggests that the dog was a suitable definitive host for the nematode species involved. FIG 1: (a) Overview of transverse sections of a gravid female Onchocerca species in the periocular tissue of a dog. Haematoxylin and eosin. Bar=100 μm. (b) Detail of (a), showing the paired uterus (ut) filled with microfilariae (mf), ovary (ov) and intestine (in). Haematoxylin and eosin. Bar=200 μm (a)

Parapoxvirus Infection in Harbor Seals (Phoca vitulina) from the German North Sea

In the summer of 2000, proliferative lesions of the skin and oral mucosa were observed in 26 young harbor seals (Phoca vitulina) from a rehabilitation center in Schleswig-Holstein, Germany. Verrucose, roundish nodules, approximately 1–2 cm in diameter, were presented in the oral cavity, especially on the tongue. Some animals developed similarly sized spherical dermal elevations with ulceration on flippers, chest, neck, and perineum. Necropsy of one animal showed multifocal, verrucose nodules in the oral cavity and a mild tonsillitis. Histologically, the nodules were characterized by ballooning degeneration of the outer parts of the spiny layer and stratum granulosum, with large eosinophilic cytoplasmic inclusions and a perivascular to interstitial lymphohistiocytic infiltration accompanied by fibroblastic proliferation and neovascularization. Negative staining of mucosal tissue homogenates demonstrated parapoxvirus-like particles. The presence of parapoxvirus was confirmed by polymerase chain reaction, using primers specific for parapoxvirus of ungulates. By in situ hybridization, using a parapox-specific, digoxigenin-labeled DNA probe, abundant parapoxvirus DNA-positive epithelial cells were detected in the stratum granulosum and the outer parts of the spiny layer. There was no parapoxvirus-positive signal in the adjacent submucosa. Although DNA analysis revealed that the causative agent can clearly be distinct from terrestrial parapoxviruses, lesions resembled parapoxvirus infections in other terrestrial species, and the pattern of virus DNA distribution indicated a direct effect of the virus on keratinocytes. In contrast, changes in the corium may be considered an indirect response mediated by the virus or the immune system.

The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica

Background Liver fluke infection of livestock causes economic losses of over US