Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis - ultrastructure and morphogenesis.

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family.

Susceptibility of koi x crucian carp and koi x goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD).

Hybrids of koi, Cyprinus carpio x crucian carp, Carassius carassius and koi x goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV-3) and developed KHV disease (KHVD). While hybrids of koi x goldfish were partly resistant to mortality following infection by immersion, most koi x crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi x goldfish and koi x crucian carp hybrids as well as in SPF carp.

Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M.

Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

[Comparison of methods for detection of an infection with different isolates of infectious haematopoietic necrosis virus (IHNV)]. / Vergleich von Methoden zum Nachweis einer Infektion mit verschiedenen Isolaten des Virus der Infektiösen Hämatopoetischen Nekrose (IHNV).

The infectious haematopoietic necrosis (IHN) is beside the viral haemorrhagic septicemia (VHS) one of the viral fish diseases that have a considerable economic impact on German aquaculture. The measures actually in force are focused on control and spread prevention of the disease within the borders of the European Union (EU). The detection and confirmation of an outbreak is performed according to the pertinent EU legislation which allow the application of methods like the virus neutralisation test (VNT), the immunofluorescence test (IFT) and the enzyme-linked immunosorbent assay (ELISA). Besides the classic virological serology methods, further tests for the identification and confirmation of the fish pathogen like i.e. PCR and DNA probe techniques are recommended by the OIE. To compare diagnostic methods as ELISA, cell cultivation and RT-PCR, rainbow trout of the strain "Isle of Man" were infected with six IHNV strains. Samples were taken on day 7 (viraemia period) and at the day 28 of the trial. The ground organs were inoculated into EPC cells (Epithelioma papulosum Cyprini cells) and examined by ELISA as well as after RNA extraction by RT-PCR. Besides the determination of the isolate as well as the virulence for 20 g trout, significant differences in the demonstration of the viruses were observed. While the RT-PCR demonstrated to be the most sensitive method, antigen ELISA and virus cultivation results showed in dependence of the IHNV isolate that not all viruses were identifiable under the chosen experimental condition in the same manner.

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV): kinetics of infection in lymphatic organs and lung.

Pigs were infected by the oronasal route with European isolates of the porcine reproductive and respiratory syndrome virus (PRRSV; I10 and Cobbelsdorf). The kinetics of infection in lymphatic organs and the lung were analysed by immunofluorescence detection of virus antigen, re-isolation of the virus and reverse transcription--polymerase chain reaction (RT-PCR) for PRRSV-specific RNA. The kinetics of PRRSV infection proceeded in three phases, irrespective of the varying infestation of lymphatic organs within the first days post-infection (p.i.). First, an early acute infection of lymphatic organs developed within the first week and was characterized by a high number of antigen-positive macrophages. Second, a delayed acute infection of the lung was observed, which was most pronounced during the second and third week p.i. when a high number of infected alveolar macrophages was observed. The acute infection of lymphatic organs had resolved at this time. Infected cells in the lung were predominantly located in pneumonic lesions. Third, a persistent infection was demonstrated by RT-PCR and immunohistology when the experiments were terminated at day 49 p.i. The virus persisted in lymphatic organs, especially in the tonsils, and in the lung. At this stage, indications for a re-occurrence of acute infection were observed in restricted areas of the lung.

Does porcine reproductive and respiratory syndrome virus potentiate classical swine fever virus infection in weaner pigs?

Fifteen 6-week-old crossbred weaners weighing about 12 kg each were randomly divided into three groups of five animals each. One group of pigs was inoculated first with porcine reproductive and respiratory syndrome (PRRS) virus and then 3 days later with CSF virus. The second group received classical swine fever (CSF) virus, while the third group was inoculated with PRRS virus only. The aim of the experiment was to determine whether a primary PRRS virus infection influences the clinical outcome of experimentally induced CSF in young pigs. The PRRS virus infected weaners developed mild respiratory symptoms and recovered completely. All five weaners which were inoculated with CSF virus only showed severe clinical signs typical of the acute form of CSF. One pig had to be killed 15 days post-inoculation (p.i.); the remaining four died between the 18th and 22nd day p.i. The clinical course of the animals inoculated with both viruses was slightly different from that of the pigs that received only CSF virus. Four out of five pigs from the PRRS/CSF group became febrile and viraemic earlier than the animals which received CSF virus only. These pigs had to be killed 15-17 days post CSF virus inoculation. One animal in this group survived the acute phase of CSF and recovered completely. It was concluded that the observed divergences of the clinical courses would not have been noticed under field conditions. Therefore these findings cast doubt on the relevance of PRRS virus infection potentiating significantly the clinical outcome of CSF in young pigs.

Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease.

Pigs were infected with the porcine respiratory and reproductive syndrome virus (PRRSV) by the oronasal route. We studied the development of histological lesions, sites of virus infection and of inflammatory infiltrates by quantitative evaluation of reactive cells. The animals developed a multifocal interstitial pneumonia. Clinical signs of pneumonia were observed from day 7 to 21. In the first stage, an acute alveolitis was found, which was characterised by a hyperplasia of type II pneumocytes within the septa and an accumulation of macrophages in the alveolar spaces. Within 2-4 days p.i., virus infected cells were prominent in lymphatic organs, but their number declined rapidly during the following days. In the following period, the number of virus antigen positive cells increased in the lung. An interesting discrepancy existed between the relatively small number of virus specific cells and the degree of intensive pneumonia. As a first step to analyse mechanisms leading to the induction of pneumonia, we studied transcriptional expression of cytokines and other immunomodulatory molecules by semiquantitative RT-PCR.

[Characterization of viral hemorrhagic septicemia (VHS) virus isolates in trout]. / Charakterisierung von Isolaten des Virus der Viralen Hämorrhagischen Septikämie (VHS) der Forellen.

Virus diseases of fish can seriously impair the economy of aquacultur. Control and prevention of fish diseases in the European Union (EU) are focussed on the viral haemorrhagic septicaemia (VHS) and the infectious haematopoeitic necrosis (IHN). The diagnosis of VHS and IHN is performed in the Federal Republic of Germany (FRG) on the basis of the legislation of the EU. Since 1994 we received an increasing number of VHS virus (VHSV) isolates which did not react with a commercially available anti-VHSV monoclonal antibody (MAb) in the indirect immuno fluorescence test. With our own MAb ID8, however, as well as with additional diagnostic methods these virus isolates could be identified. These isolates of rainbow trouts were designated as VHSV type "Wi". Electron microscopically all stages of rhabdovirus maturation could be detected. Morphologically the isolates were undistinguishable from other rhabdoviruses. By immuno electron microscopy using the MAb ID8 rhabdovirus nucleocapsid structures were demonstrated. The virulence of the new VHSV type Wi was not different from that of a VHSV isolate with conventional reaction patterns as well as of a VHSV laboratory strain.

[Experimental reproduction of the respiratory form of infection with the agent of epidemic late abortion of swine]. / Experimentelle Reproduktion der respiratorischen Verlaufsform der Infektion mit dem Erreger des Seuchenhaften Spätabortes der Schweine.

Porcine reproductive and respiratory syndrome has been observed in a large swine-farm. Diagnostic investigations were undertaken. Piglets infected with homogenate from the lung of a sick gilt developed biphasic fever up to 42 degrees C, inappetence and apathy. A few piglets showed red-blue discolored parts of the ears and of the skin in the abdominal region. A cytopathogenic agent has been isolated on cultivated alveolar-macrophages. Infection of another group of piglets with macrophages-propagated agent resulted in similar clinical signs too. Organs of euthanatized piglets have been examined anatomical, histological and with the fluorescent antibody technique. Specific fluorescence could be seen in tonsils, bronchial lymph nodes, spleen and in pathological-changed parts of the lungs. Antibodies against PRRS-virus were detected in blood samples of experimentally infected pigs.

[Hemodynamic changes during the induction phase of halothane-nitrous oxide anesthesia in childhood]. / Hämodynamische Veränderungen während der Einleitungsphase von Halothan-Lachgas-Narkosen im Kindesalter.

The haemodynamic effects of halothane nitrous oxide anaesthesia were studied in 15 children aged 4-13 using measurements of heart rate, blood pressure and impedance cardiography. The preinduction (control) measurements were compared with measurements obtained one minute after inhalation of nitrous oxide and oxygen, at the beginning of the stage of excitation, at the beginning of the stage of tolerance and after the injection of 0.02 mg/kg atropine. During the induction period blood pressure decreased significantly, while heart rate remained unchanged. Cardiac output was significantly decreased at the stages of excitation and tolerance. Virtually no change in total peripheral resistance occurred. After atropine significant increases in heart rate, mean arterial blood pressure and total peripheral resistance were measured. Cardiac output was increased compared with the pre-atropine values, but not with the control value. Impedance cardiography is a useful non-invasive method of measuring changes of stroke volume during paediatric anaesthesia.

[Blood pressure regulation in neurosurgical interventions in the lumbar area in the seated patient]. / Zur Blutdruckregulation bei neurochirurgischen Eingriffen im Lumbalbereich am sitzenden Patienten.

It has been proved studies of the physiology of the circulatory system that a sufficient regulatory mechanism is present during intervertebral disk operations on the sitting patient. So this positioning variant can be well recommended from an anaesthesiological point of view. Patients without a sufficient blood pressure regulation should be excepted from this positioning variant.

[The effect of various ventilation conditions on evoked potentials of the visual cortex of the cat in distention of the stomach]. / Der Einfluss verschiedener Ventilationsstufen auf evozierte Potentiale des visuellen Kortex der Katze bei Magendehnung.

In 10 cats anaesthetized using chloralose and with muscular relaxation by gallamine and continuous artificial ventilation in acute experiments photically evoked potentials (PEP) were recorded from the visual area of the cortex under different intragastric pressures and varying tidal volumes. It was the aim of this study to examine whether an exteroceptive stimulus (light flash) in the visual analyzer, which is influenced by an interoceptive stimulation (gastric pressure), may also be modified by different tidal volumes. Times, amplitudes and slope parameters, blood pressure and heart rate of the animals were studied. The PEP of the visual cortex was modified under the influence of the interoceptive stimuli. It could, however, be shown that already hypo- and hyperventilation alone may cause alterations in the latency period of the PEP. They arise in a different manner in individual parts of the measured parameters of the PEP and do not change these values in a typical manner. Additional dilatation of the stomach may suppress the phenomena caused by alterations in the tidal volume. Thus, attention should be paid to both blood gases and acid base balance in animals in studies on viscero-cerebral interrelationships as well as to interactions between different analyses using the method of evoked potentials.