Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Selection for tetracycline-resistant Chlamydia suis in treated pigs

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.     

Cytotoxic ellagitannins from Reaumuria vermiculata

Three ellagitannins and one disulfated flavonol were isolated from the aerial parts of Reaumuria vermiculata L. Besides that, 16 known compounds were characterized as well. The structures of all compounds were elucidated on the basis of spectroscopic data including 1D and 2D NMR and ESI HR-FTMS. The in vivo antioxidant activity using the oxygen radical absorbance capacity (ORAC) method, of the extract, its column fractions and two of the isolated ellagitannins was accomplished. In addition, a possible cytotoxicity of the extract and two of the new ellagitannins on HaCaT human keratinocytes and the activity of both compounds against the prostate cancer cell line (PC-3) were also assessed, whereby a potent cytotoxicity with IC(50) less than 1μg/ml was determined for both compounds. Besides, the extract exhibited a potential cytotoxic effect against four different solid tumor cell lines, namely liver (Huh-7), colorectal (HCT-116), breast (MCF-7) and prostate (PC-3). The IC(50)s were found to be substantially low (ranged from 1.3±0.15 to 2.4±0.22μg/ml) with relatively low resistance possibility reaching to 0% in the case of Huh-7 cell.     

Evaluation of a minimally invasive non-calibrated pulse contour cardiac output monitor (FloTrac/Vigileo) in anaesthetized dogs

ObjectiveTo evaluate the accuracy of a new cardiac output monitor (FloTrac/Vigileo), originally designed for humans, in dogs. This pulse contour cardiac output monitoring system cannot be calibrated and measures cardiac output (

t) from a standard arterial catheter.Study designProspective experimental trial.AnimalsEight adult Beagle dogs weighing 13.1 (9.8–17.1) kg [median (range)].MethodsAnaesthesia in the dogs was maintained using isoflurane. A pulmonary artery catheter and a metatarsal arterial catheter (22 gauge) were placed. Cardiac output was measured simultaneously 331 times by thermodilution and FloTrac technique. A broad spectrum of

t measurements was achieved through alterations of isoflurane concentration, administration of propofol boluses and dobutamine infusions. Agreement between the methods was quantified with Bland Altman analysis and disagreement was assessed with linear mixed models.ResultsMedian (10th and 90th percentile) cardiac output as measured with thermodilution was 2.54 (1.47 and 5.15) L minute^?1 and as measured with FloTrac 8.6 (3.9 and 17.3) L minute^?1. FloTrac measurements were consistently higher with a mean bias of 7 L minute^?1 and limits of agreement of ?3.15 to 17.17 L minute^?1. Difference between the methods was most pronounced in high

t measurements. Linear mixed models showed an estimated difference between the two methods of 8.05 (standard error 1.18) L minute^?1 and a significant interaction between mean arterial pressure and method. Standard deviation (4.45 higher) with the FloTrac method compared to thermodilution was increased.ConclusionCompared to thermodilution measurements, the FloTrac system was influenced to a higher degree by arterial blood pressure, resulting in consistent overestimation of cardiac output.Clinical RelevanceThe FloTrac monitor, whose algorithms were developed based on human data, cannot be used as an alternative for thermodilution in dogs.     

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla_TEM-52 gene and two of them also harboured bla_TEM-1b. The bla_CTX-M-14 gene was identified in three isolates (in association with bla_TEM-1b in one of them), and bla_CTX-M-32 was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58(C → T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim–sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.     

Risk factors for highly pathogenic H7N1 avian influenza virus infection in poultry during the 1999-2000 epidemic in Italy

In 1999-2000, Italian poultry production was disrupted by an H7N1 virus subtype epidemic of highly pathogenic avian influenza (HPAI). The objectives of the present study were to identify risk factors for infection on poultry farms located in regions that had the highest number of outbreaks (Veneto and Lombardia) and the impact of pre-emptive culling as a complementary measure for eradicating infection. A Cox regression model that included spatial factors, such as the G index, was used. The results confirmed the relationship between risk of infection and poultry species, production type and size of farms. The effectiveness of pre-emptive culling was confirmed. An increased risk of infection was observed for poultry farms located near an infected farm and those at altitudes less than 150m above sea level. The measures for the control and eradication of AI virus infection need to consider species differences in susceptibility, the types of production and the density of poultry farms in the affected areas.     

Removal of Chromium, Copper, and Nickel from an Electroplating Effluent Using a Flocculent Brewer’s Yeast Strain of Saccharomyces cerevisiae

The release of heavy metals in aquatic systems due to the discharge of industrial wastewaters is a matter of environmental concern. Heat-inactivated cells of a flocculent strain of Saccharomyces cerevisiae were used in the bioremediation, in a batch mode, of a real electroplating effluent containing Cu, Ni, and Cr. In this approach, no previous reduction of Cr(VI) to Cr(III) was required. Cr(VI) was selectively removed (98%) by yeast biomass at pH 2.3. At this pH, Cr(VI) is mainly in the form of HCrO ₄ ^? and yeast surface is surrounded by H⁺ ions, which enhance the Cr(VI) interaction with biomass binding sites by electrostatic forces. Subsequently, pH of the effluent was raised up to 6.0; this pH maximizes the efficiency of cations removal since at this pH the main binding groups of yeast cells are totally or partially deprotonated. The passage of effluent through a series of sequential batches, at pH 6.0, allowed, after the third batch, the removal of Cu(II), Ni (II), Cr total, and Cr(VI) in the effluent to values below the legal limit of discharge. The strategy proposed in the present work can be used in plants for the treatment of heavy metals rich industrial effluents containing simultaneously Cr(VI) and Cr(III).     

Differential effects of alpha1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.     

Serological survey of Toxoplasma gondii infection in domestic cats from northeastern Portugal

Cats are very important hosts in the epidemiological cycle of Toxoplasma gondii, a zoonotic protozoan parasite that can infect humans and many other animal species worldwide. We report a serological survey of antibodies to T. gondii in domestic cats from northeastern Portugal, by means of the modified agglutination test. Three cats had titres of 20 (3.9%), 18 had titres of 40 (23.7%) and 55 animals had titres of > or =800 (72.4%). Results of three seropositive kittens with less than 4 months were not considered for determining the seroprevalence of infection, which was found to be 35.8% (73/204). Differences in the seroprevalence levels were not statistically significant between males (35.6%) and females (36.0%) or pure non-European (26.7%) and European or mixed-breed cats (39.6%). Animals aged 36-71 months and 72-180 months had the highest seroprevalences of infection, i.e. 51.7% and 51.2%, respectively, which significantly differ from the values observed in cats with 2-11 months (14.6%) and 12-35 months (26.3%). Infection levels were also significantly different between cats that lived totally indoors (7.7%) and those that had access to outdoors (45.4%), as well as between cats living alone (13.8%) and those that had contact with other cats (39.4%). Seroprevalence values in cats fed only commercial canned or dried food (22.9%) and animals whose diet included raw or undercooked viscera and/or meat (53.5%) were also significantly different. Furthermore, considering only 108 cats, differences of seropositivity to T. gondii were significant between feline immunodeficiency virus infected and non-infected animals, but this was not observed for feline leukaemia virus. Age, habitat and diet were identified as risk factors for the feline T. gondii infection by logistic regression analysis. Some control measures are suggested based on these findings.