Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115 bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish.

A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA.

The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Following a tick bite: double infections by tick-borne encephalitis virus and the spirochete Borrelia and other potential multiple infections

In Central Europe and large parts of Asia, tick-borne-encephalitis (TBE) and Lyme borreliosis caused by the spirochetal bacterium of the genus Borrelia are among the most common diseases transmitted by the bite of a tick. When in regions with overlapping TBE virus and Borrelia endemicity, a tick bite causes the victim to become ill, it is important that appropriate serological and other laboratory investigations form part of the differential diagnosis. Account must always be taken of the fact that a tick bite may be followed by a double infection with the TBE virus and Borrelia. For this reason, a comprehensive diagnostic work-up aimed at detecting co-infection by both pathogens, even when the tick bite occurs in an endemic region for both pathogens but the initial clinical symptoms suggest an infection with only one of the two pathogens. The present article discusses a number of published cases of a co-infection with TBE virus and Borrelia and other potential multiple infections.     

Intra-tumoral gene delivery of feIL-2, feIFN-gamma and feGM-CSF using magnetofection as a neoadjuvant treatment option for feline fibrosarcomas: a phase-I study

Despite aggressive pre- or postoperative treatment, feline fibrosarcomas have a high relapse rate. In this study, a new treatment option based on immune stimulation by intra-tumoral delivery of three feline cytokine genes was performed. The objective of this phase-I dose-escalation study was to determine a safe dose for further evaluation in a subsequent phase-II trial. Twenty-five client-owned cats with clinical diagnosis of fibrosarcoma - primary tumours as well as recurrences - entered the study. Four increasing doses of plasmids coding for feIL-2, feIFN-gamma or feGM-CSF, respectively, were previously defined. In groups I, II, III and IV these doses were 15, 50, 150 and 450 microg per plasmid and a corresponding amount of magnetic nanoparticles. Two preoperative intra-tumoral injections of the magnetic DNA solution were followed by magnetofection. A group of four control cats received only surgical treatment. Side effects were registered and graded according to the VCOG-CTCAE scale and correlated to treatment. Statistical analyses included one-way anova, post hoc and Kruskal-Wallis tests. ELISA tests detecting plasma feIFN-gamma and plasma feGM-CSF were performed. One cat out of group IV (450 microg per plasmid) showed adverse events probably related to gene delivery. As these side effects were self-limiting and occurred only in one of eight cats in group IV, this dose was determined to be well tolerable. Altogether six cats developed local recurrences during a 1-year observation period. Four of these cats had been treated with dose IV. Regarding these observations, a subsequent phase-II trial including a representative amount of cats should be tested for the efficacy of dose IV as well as dose III.     

CliniTox: the computer-based information system for poisoning in farm animals

Poisonings can cause reduced performance and severe economic loss in farm animals. A swift and targeted action is required from veterinarians. We have established a computer-based decision support system for poisonings in ruminants and pigs. The system offers access to the most recent information available in clinical toxicology of farm animals. Towards this goal, relevant reports from the peer-reviewed literature were evaluated and organised according to the requirements of a structured database. The information provided for each toxic substance includes a summary of its chemical and physical properties, sources, commercial uses or natural occurrences, toxicokinetic data, mechanisms of action, threshold doses, clinical symptoms with brief case reports, sampling and analytical results, post-mortem abnormalities, differential diagnoses, therapeutic guidelines and references to the literature. This decision support system has been programmed with two user-friendly search functions: a search tool that allows the choice of clinical and pathological findings, and another function that serves to find a substance using its chemical name, the class of compounds to wich it belongs, a possible source or one of its main applications. CliniTox can be accessed directly via our webserver (http://www.clinitox.ch).     

Effects of various applications of lipopolysaccharides on blood parameters of pigs

In five experiments, lipopolysaccharides (LPS) of Escherichia coli O26:B6 and O111:B4 were applied intravenously, intramuscularly, subcutaneously or intrabronchially in doses of 5000-15,000 U/kg body mass to a total of 47 weaner pigs and compared with the application of sodium chloride. Different parameters of blood cells were investigated, including cell numbers, in vivo interleukin secretion, radical formation, phagocytosis capacity and IL-6 as well as TNFalpha formation ex vivo. Non-specific effects and dependencies on the type of application and LPS dose are discussed.     

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.     

The change of acute phase protein haptoglobin in cattle during spontaneous labor and Caesarean section with or without torsio uteri intrapartum

The change of haptoglobin concentration in cattle with dystocia after torsio uteri which lead to Caesarean section is regarded in contrast to haptoglobin concentrations in cattle at parturitions with undisturbed puerperium and Caesarean section without torsio uteri intra partum. Animals with natural parturition had low haptoglobin concentrations with slight changes, for a slightly increased haptoglobin concentration was seen as physiologic. Also surgical treatment such as Caesarean section did not result in changes of haptoglobin concentration. Thus, the curve of animal patients with anomalies concerning position or with a too large fetus (relatively or absolutly) was nearly identical to the curve of animals with regular parturitions over the first four days after surgery. Not before the fifth day after surgery, a moderate increase of haptoglobin could be observed. Animals with dystocia after torsio uteri intra partum showed significantly higher haptoglobin concentrations in contrast to controls over the time of investigation. The correlation of haptoglobin concentrations in animal patients with torsio uteri bin concentration shortly after surgery an additionally significant increase of haptoglobin concentration could be observed in later fertile cattles but not in later infertile cattle with torsio uteri. In two cows with torsio uteri and ovariohysterectomy haptoglobin concentrations decreased rapidly. Haptoglobin concentration may serve as an indicator for the degree of impairment of the uterus during a parturition as well as a predictor for regenerative potential.