Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.     

Reference values and clinical application of magnetic peripheral nerve stimulation in cats

Magnetic stimulation of radial (RN) and sciatic (SN) nerves was performed bilaterally in 40 healthy cats. Reference values for onset latency and peak-to-peak amplitude of magnetic motor evoked potentials (MMEPs) were obtained and compared with values of electric motor evoked potentials (EMEPs) in 10/40 cats. Onset latencies and peak-to-peak amplitudes of the MMEPs of three cats with polyneuropathy (PNP) were compared to the reference values. Magnetic motor evoked responses were easily recorded in all normal cats. Significant differences were found in onset latencies between MMEPs and EMEPs, but peak-to-peak amplitudes were equal. The MMEPs of three cats with PNP can be seen as outliers in comparison to the reference values. MMEPs from the RN and SN were easily obtained and reproducible in normal cats. The technique could represent a useful adjunct in the assessment of peripheral nerve disorders.     

Prevalence of methicillin-resistant Staphylococcus aureus in mammals of the Royal Zoological Society of Antwerp, Belgium

Contrary to the numerous reports on methicillin-resistant Staphylococcus aureus (MRSA) in domestic animals, only three articles concerning zoo animals are documented in the literature. A skin infection of an African elephant (Loxodonta africana) calf was most likely acquired from an infected caretaker. Another zoo detected MRSA in the rumen content of a mouflon (Ovis aries), and, in a third facility, it was reported in a fistulous wound at the coronary band of a digit of an Indian rhinoceros (Rhinoceros unicornis). In the present study, which lasted 13 months and involved 93 different individual mammals that belonged to 40 species and 19 families housed in the Royal Zoological Society of Antwerp, Belgium, this study reports the absence of MRSA in swabs of nostrils, skins, conjunctiva, vulva, abscess, and arm rests in public spaces. Samples were enriched overnight and inoculated on a selective chromogenic medium.     

Diagnosis of Endometritis in the Bitch: A New Approach

There are a few investigations into endometritis in the bitch and its relationship with failure to conceive remains unclear. This may be because of the difficulty in collecting uterine samples for further investigations. Recently, transcervical catheterization by vaginal endoscopy has been introduced allowing the evaluation of the endometrium. In this study, uterine cytology and bacteriology were evaluated in 26 infertile bitches. Endometritis was bacterial in origin in most cases (70% of affected bitches), but these results may be underestimated, as some other pathogens (anaerobic bacteria, mycoplasms and fungi) were not investigated. Endometritis, in our opinion, should be investigated in each case of unexplained infertility in bitches. The method used here seems reliable although defining more accurate classification criteria will improve the efficiency of this non-invasive technique.     

Critical Influence of Culture Medium and Cr(III) Quantification Protocols on the Interpretation of Cr(VI) Bioremediation by Environmental Fungal Isolates

The influence of culture medium composition on chromium(VI) quantification according to diphenylcarbazide (DPC) colorimetric determination was evaluated. Considering the eventual biospeciation of Cr(VI) as a mechanism of microbial bioremediation, the possibility to quantify Cr(III) in culture medium was also explored. Yeast nitrogen base (YNB) was identified as the least interferent culture medium for Cr(VI) quantification by DPC and it was applied to compare different strategies for Cr(III) oxidation. The most appropriate oxidation protocol consisted in the reaction with 80 mM KIO₄ at room temperature for 30 min prior to DPC. Parameters like basal culture medium (vitamins + salts + oligoelements), C and N source were systematically evaluated, either independently or in combination. Results demonstrated that C source was the most interferent culture medium component, being the use of sucrose preferable to glucose. A medium arbitrarily named as YNB′ (YNB without amino acids and ammonium sulfate plus 50 g L^?1 sucrose and 0.6 g L^?1 (NH₄)₂SO₄) was defined for Cr(VI)-amended fungal cultures. Kinetics of growth, Cr(VI) removal, and nutrient consumption for isolates A. pullulans VR-8, filamentous fungus PMF-1, and Lecythophora sp. NGV-1 were obtained. The order of Cr(VI) removal efficiency was as follows: A. pullulans VR-8 > Lecythophora sp. NGV-1 > filamentous fungus PMF-1, and a similar trend was observed for biomass yield and nutrients consumption. Studies on biospeciation by means of the selected Cr(III) oxidation protocol were unsuccessful, leading to Cr(VI) values much lower than expected. It revealed that this kind of protocols should be cautiously evaluated when studying microbial Cr(VI) bioremediation.     

Cross-reactivity of antibodies in some commercial deoxynivalenol test kits against some fusariotoxins

Cross-reactivity of antibodies in AGRAQUANT, DON EIA, VERATOX, ROSA LF-DONQ, and MYCONTROLDON designed for deoxynivalenol (DON) determination in food and feedstuffs was evaluated against nivalenol, 3-acetylDON, 15-acetylDON, de-epoxy metabolite 1 of DON, DON-3β-glucoside, T2-toxin, HT2-toxin, fusarenone X, diacetoxyscirpenol, verrucarol, and zearalenone. Cross-reactivity measurements were run in water using the 50% reduction of absorbance of the blank for ELISA kits or through direct DON determination upon using the standards of mycotoxins via ROSA LF-DONQ or MYCONTROLDON. For the tested toxin concentrations, all DON kits have low cross-reactivity toward diacetoxyscirpenol, T2-toxin, HT2-toxin, verrucarol, and zearalenone and moderate cross-reactivity toward 15-AcetylDON and fusarenone X. AGRAQUANT, DON EIA, and VERATOX kits showed high cross-reactivity in various ranking orders against DON-3-Glc, DOM-1, and 3AcDON. DON EIA showed also high cross-reactivity against nivalenol and fusarenone X. These mycotoxins could coexist in food or feedstuffs, and analytical results can be wrongly interpreted. Cross-reactivity does not allow checking the compliance with the legal norms, but it does allow an overall risk assessment for the consumers. Updating regularly the cross-reactivity evaluation of the produced batches is recommended for 3-acetylDON, nivalenol, DON-3-Glc, de-epoxy metabolite 1, and fusarenone X.     

Saponins show high entomotoxicity by cell membrane permeation in Lepidoptera

BACKGROUND: In this study, the effects of three saponins and one sapogenin with a triterpenoid or steroid structure in two lepidopteran insect cell lines, ovarian Bm5 and midgut CF‐203 cells, were analysed with regard to cell viability, cell membrane permeation, EcR responsiveness and DNA fragmentation. In addition, the entomotoxic action of Q. saponaria saponin with primary midgut cell cultures and larval stages of the cotton leafworm Spodoptera littoralis was tested. RESULTS: Both lepidopteran cell lines show a high sensitivity to all four sapo(ge)nins, with a concentration‐dependent viability loss and EC₅₀ values of 25–100 µM in MTT bioassays. A trypan blue assay with Q. saponaria saponin confirmed rapid cell membrane permeation to be a cause of cytotoxicity. Saponins caused no EcR activation in Bm5 cells, but a loss of ecdysteroid signalling was observed with IC₅₀ values of 5–10 µM . Lower saponin concentrations induced DNA fragmentation, confirming their potential to induce apoptosis. Finally, Q. saponaria saponin caused cytotoxicity in primary midgut cell cultures of S. littoralis (EC₅₀ = 4.7 µM ) and killed 70–84% of S. littoralis larvae at pupation at 30‐70 mg g^?1, while lower concentrations retarded larval weight gain and development. CONCLUSIONS: The data obtained provide evidence that saponins exert a strong activity on lepidopteran cells, presumably based on a cytotoxic action due to permeation of the cell membrane. Primary midgut cell cultures and larvae of S. littoralis showed high sensitivity to Q. saponaria saponin, indicating the insect midgut as a primary target for entomotoxicity and the potential use of saponins in the control of pest Lepidoptera. Copyright © 2012 Society of Chemical Industry     

Causes, diagnostic signs, and the utility of investigations of fever in dogs: 50 cases

This study aimed to determine the distribution of diseases causing fever in dogs in France. Dogs with fever were reviewed and 50 dogs were retrospectively assigned to disease groups. Fever profile and intensity, the time taken to reach a diagnosis, and inflammatory status were compared among groups. Almost half the dogs (48%) were diagnosed with non-infectious inflammatory diseases. No final diagnosis was reached in 14 dogs, 13 of which belonged to owners who did not wish to pursue the investigations. No association was found between disease group and the intensity of fever, fever profile, or serum C-reactive protein concentration. Cytological examinations were most frequently found to be the most important determinant for diagnosis (55.7%). This study confirms the predominance of non-infectious inflammatory diseases as causes of fever. Neither clinical nor biological factors were found to be predictive of disease group.