Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-polymerase chain reaction

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS-PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.     

Studies about the mechanism of internalization by mammary epithelial cells of Escherichia coli isolated from persistent bovine mastitis

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).     

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.     

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.     

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.     

Impact of invA-PCR and culture detection methods on occurrence and survival of salmonella in the flesh, internal organs and lymphoid tissues of experimentally infected pigs

This study evaluated the suitability of invA gene amplification by PCR as an effective means of detecting Salmonella species in pigs experimentally infected with S. Typhimurium DT104. A controlled infection study using 24 pigs was performed in order to compare efficacy, precision and detection rates of the invA-based PCR method originally described by Rahn, K. De Grandis, S.A., Clarke, R.C., McEwan, S.A., Galan, J.E., Ginocchio, C., Curtiss, R. 3rd, C.L. Gyles, (Mol. Cell. Probes 1992; 6: 271-279) as a new in-house invA-based PCR method for the specific detection of Salmonella spp. in pork and different tissue samples of slaughter pigs. Finally, PCR results were compared with culture detection rates obtained by isolation procedures following the ISO 6579:2000, the 'gold standard'. After slaughtering, 14 different tissue samples of each pig were investigated to verify the usefulness of the two invA-based PCR methods in different matrices of slaughter pigs. The results demonstrate that the application of the widely used invA-based primer pair (139 + 141) may result in questionable products if samples gained from selective enrichment in the Rappaport-Vassiliadis medium were investigated. These questionable products can lead to false-positive results, if no additional hybridization procedure is attached or if unspecialized persons use this method in routine laboratory practice. The newly developed in-house PCR method used is based on the 3'-prime region of invA, especially designed and harmonized for the detection of Salmonella in different matrices of slaughtered pigs after bacterial enriched broth culture. In this study, this PCR revealed no questionable products and, furthermore, the specificity of the amplificate could be tested by means of the restriction enzyme NdeI. In comparison with the culture detection procedure, the new PCR method has a sensitivity of 100% and a specificity of 96%. Thus, this method might be used as a meaningful tool in eliminating Salmonella-positive carcasses at slaughterhouse level and thus, keeping them out of the food chain.     

Shedding consistency of strongyle-type eggs in Dutch boarding horses

Faeces of 484 horses were sampled twice with an interval of 6 weeks while anthelmintic therapy was halted. Faecal eggs counts revealed that 267 (55.2%) horses had consistently low numbers of eggs per gram faeces (EPG) (EPG < 100 or = 100), 155 (32.0%) horses had consistently high EPGs (EPG > 100). Horses with consistently high EPGs were more often mares with access to pasture, aged less than 6 or more than 23 years, that were dewormed at intervals longer than 6 months, and were treated for the last time more than 3 months before the start of the study. Horses with consistently low EPGs were more often male horses with no or limited access to pasture, that were dewormed at maximally 6-month intervals, and were aged between 6 and 23 years. The results are an indication that some horses have consistently low EPGs and perhaps could be used as non-treated animals in a selective anthelmintic treatment scheme aimed at the prevention of the development of anthelmintic resistance.