Molecular phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont

The morphology of Onchocerca lupi, responsible for canine ocular onchocercosis, is unique within the genus. Earlier analyses of the 5S ribosomal RNA gene spacer region sequence of the parasite and the 16S ribosomal RNA gene sequence of its Wolbachia endosymbiotic bacteria (Rickettsiales) supported the morphological and biological arguments that O. lupi is a distinct species. However, the exact phylogenetic position of O. lupi and its endosymbiont could not be unambiguously determined. Herein we report analyses based on the mitochondrial cytochrome oxidase I (COI) gene of the filarial species and the Wolbachia surface protein (wsp) and the bacterial cell-cycle ftsZ genes of their wolbachiae. Our results indicate that O. lupi separated from other Onchocerca spp. early in evolution. This is in line with the previous morphological analysis demonstrating that O. lupi is an atypical Onchocerca species showing both primitive and evolved characters. The phylogenetic trees generated for the COI sequences of filariae and the wsp and ftsZ sequences of their wolbachiae were congruent with each other, which supports the hypothesis that nematodes and their Wolbachia endobacteria share a long co-evolutionary history.     

Identification of bluetongue virus serotype 2 (Corsican strain) by reverse-transcriptase PCR reaction analysis of segment 2 of the genome

In October 2000, bluetongue virus was detected on the French island of Corsica. The disease was also reported in Sardinia, Calabria, Sicily and on the Spanish islands of Majorca and Minorca. This paper describes the use of molecular techniques for a rapid identification and serotype determination of serotype 2 of the virus. The nucleotide sequences of segments 2 and 7 of the genome of the Corsican strain were determined and its phylogenetic relationships are described.     

Morphometric analysis of the corneal endothelium of Yacare caiman (Caiman yacare) using scanning electron microscopy

The objective of this study was to examine the endothelial surface morphology and to perform morphometric analysis of the corneal endothelial cells of Yacare caiman (Caiman yacare) using scanning electron microscopy. Morphometric analysis with regard to polygonality, mean cell area, cell density and coefficient of variation of mean cell area was performed. Cell areas were measured using image analysis software. The normal corneal endothelium of Yacare caiman consisted of polygonal cells of uniform size and shape with interdigitations of the cell borders. Microvilli appeared as protrusions on the cellular surface. The average cell area was 270 +/- 24 microm(2) and the endothelial cell density was 3704 +/- 324 cells/mm(2). The coefficient of variation of cell area was 0.22. This study demonstrates that the Yacare caiman corneal endothelium is similar to those described in other vertebrates.     

Morphometric and quantitative evaluation of the NADH-diaphorase positive myenteric neurons of the jejunum of streptozotocin-diabetic rats supplemented with acetyl-L-carnitine

Summary In this study we investigated the effect of the acetyl-L-carnitine (ALC) supplementation on the myenteric neurons of the jejunum of rats made diabetic at the age of 105 days by streptozotocin (35 mg/kg body weight). Four groups were used: non-diabetic (C), non-diabetic supplemented with ALC (CC), diabetic (D), diabetic supplemented with ALC (DC). After 15 weeks of diabetes induction the blood was collected by cardiac puncture to evaluate glycaemia and glycated haemoglobin. Next the animals were killed and the jejunum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The neuronal counts were made in 80 microscopic fields, in tissue samples of five animals of each group. The profiles of the cell bodies of 1000 neurons per group were analysed. Diabetes induced a significant increase in the area of the cell body and decrease in the number of NADH-diaphorase positive myoenteric neurons. ALC suplementation to the diabetic group promoted smaller hypertrophic effects and less neuronal loss than in the myoenteric neurons of the diabetic rats, and in addition diminished the body weight decrease and reduced the fasting glycaemia.     

Low‐density lipoproteins and milk serum proteins improve the quality of stallion sperm after vitrification in straws

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low‐density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25‐ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post‐warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.     

Measurement of vitamin A,vitamin E,selenium, and L-lactate in dogs with and without osteoarthritis secondary to ruptured cranial cruciate ligament

This study compared vitamin A, vitamin E, selenium (Se), and L-lactate in blood and synovial fluid in 2 groups of 6 dogs; a control group (without OA) and an osteoarthritic group with spontaneous cranial cruciate ligament rupture and OA. Concentrations of vitamin E were significantly higher in serum than in synovial fluid in both OA (P = 0.006) and control (P = 0.0008) groups. Vitamin E concentration in synovial fluid was significantly higher in the OA group than in the control group (P = 0.009). Concentrations of Se were significantly higher in serum than in synovial fluid in both OA (P = 0.003) and control (P = 0.0006) groups. There were no significant differences in levels of Se, vitamin A, and L-lactate between the 2 groups. This is the first study to show an increased concentration of vitamin E in the synovial fluid of dogs with OA compared with dogs that did not have OA.     

Synergistic effects of Miconazole and Polymyxin B on microbial pathogens

The therapeutic value of antibiotics depends on the susceptibility of the infecting microorganism and the pharmacological profile of the drugs. To assess the value of an antibiotic combination of polymyxin B and miconazole this study examined the in vitro synergistic potential of the two drugs on Gram-negative and Gram-positive bacteria and yeast. Antifungal and antibacterial activity was tested by minimum inhibitory concentration (MIC) of broth macrodilution and urea broth microdilution, by fluorescence microscopy and flow cytometry. Synergism was calculated using the fractional inhibitory concentration index (FICi). With Staphylococcus intermedius as target we found up to an eightfold reduction of the individual MICs when both drugs were combined. However, the FICi was 0.63 suggesting no real interaction between the two drugs. With Escherichia coli, Pseudomonas aeruginosa, and Malassezia pachydermatis as targets the antimicrobial drug combination reduced the MICs of polymyxin B and miconazole from fourfold to hundredfold resulting in FICi between 0.06 and 0.5 which defines a synergistic action. Thus, if polymyxin B and miconazole are combined their effect is greater than the sum of the effects observed with polymyxin B and miconazole independently, revealing bactericidal and fungicidal synergism. Our results indicate a strong therapeutic value for the combination of these antimicrobial agents against Gram-negative bacteria and yeast and a weaker value against Gram positive bacteria for clinical situations where these pathogens are involved.     

Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples

Several real-time PCR assays for quantification of PCV2 DNA (qPCR) have been described in the literature, and different in-house assays are being used by laboratories around the world. A general threshold of 10(7) copies of PCV2 per millilitre serum for postweaning multisystemic wasting syndrome (PMWS) diagnosis has been suggested. However, neither inter-laboratory nor inter-assay comparisons have been published so far. In the present study, two different qPCR probe assays used routinely in two laboratories were compared on DNA extracted from serum, nasal and rectal swabs. Results showed a significant linear association between the assays (p<0.0001), and a systematic difference of 1.4 log10 copies of PCV2 per millilitre of sample (p<0.0001). This difference indicated that the assay from laboratory 1 yielded a higher output than the one from laboratory 2. Results also showed that there was no linear association between the amount of PCV2 DNA and the amount of total DNA, neither in nasal (p=0.86) nor in rectal (p=0.78) swabs, suggesting that normalizing of PCV2 DNA load in swab samples to total DNA concentration is not suitable. The present exploratory study highlights the need for the performance of ring trials on qPCV2 protocols between laboratories. Meanwhile, the proposed thresholds for PMWS diagnosis should only be considered reliable for each particular laboratory and each particular assay.     

The pharmacokinetics and metabolism of ivermectin in domestic animal species

The pharmacokinetic properties of drugs are closely related to their pharmacological efficacy. The kinetics of ivermectin are characterised, in general terms, by a slow absorption process, a broad distribution in the organism, low metabolism, and slow excretion. The kinetics vary according to the route of administration, formulation, animal species, body condition, age, and physiological status, all of which contribute to differences in drug efficacy. Characterisation of ivermectin kinetics can be used to predict and optimise the value of the parasiticide effects and to design programmes for parasite control. This article reviews the pharmacokinetics of ivermectin in several domestic animal species.     

Limited awareness of animal influenza prevention and control among Dai Lue smallholder farmers in Southwest China

Awareness of animal influenza and its prevention and control is important for ensuring livestock health, production and welfare. In China, a country stereotyped as a major source of emerging zoonotic infectious diseases, research on the public understanding of animal influenza is limited to the Han, the main ethnic group. The present qualitative study in Southwest China investigated awareness of animal influenza among the Dai, an ethnic minority. The participants (15 men and 10 women, ages 18–83) were smallholder farmers of pigs and poultry in rural areas of Jinghong, Xishuangbanna, Yunnan Province. A mixture of interviews and group discussions took place in homes and villages. The participants were asked about their knowledge of avian influenza (H7N9), swine influenza (H1N1), precautions taken to protect against influenza, procedures when animals were sick and perceived risk of animal influenza. The data were analysed following coding and thematic analysis. The findings demonstrated a limited understanding of animal health and welfare among participants. Specifically, they were largely unaware of animal influenza (H7N9, H1N1) including its causes, symptoms, prevention and treatment. The farmers were also uninformed of the risks they faced and unknowingly engaged in behaviours which increased direct or indirect exposure to infected animals, a risk factor for human infection. They also reported poor usage of veterinary services. In order to guarantee the health, welfare and production of their livestock, immediate action is needed to enable Dai smallholder farmers to prevent and respond to animal influenza effectively and timely.