The use of T‐DNA tagging to isolate mutants of Colletotrichum gloeosporioides and Colletotrichum acutatum with reduced virulence against Hevea brasiliensis

Agrobacterium tumefaciens‐mediated transformation (ATMT) is being increasingly recognized as an effective insertional mutagenesis tool in studies of filamentous fungi for gene discovery and functional analysis. We developed and optimized ATMT for 2 Colletotrichum species, Colletotrichum gloeosporioides and Colletotrichum acutatum, the causative agents of Colletotrichum leaf disease in rubber trees in Southern China. A. tumefaciens strain AGL‐1 carrying an ILV1 gene and a green fluorescent protein gene were used to transform the conidia of these 2 Colletotrichum species. The transformation efficiency was correlated with the co‐cultivation duration and bacterial cell concentrations, which reached 300–400 transformants per 1 × 10⁶ conidia after optimization. Southern blot analysis indicated that about 60.0% of the C. gloeosporioides transformants and 46.2% of the C. acutatum transformants had a single copy of T‐DNA in their genomes. Fungal genomic DNA segments flanking the T‐DNA were identified in the transformants through thermal asymmetrical interlaced polymerase chain reaction followed by sequencing. The flanking sequences from 4 C. acutatum and 7 C. gloeosporioides transformants showed moderate or weak homology to the NCBI database entries. Some sequences matching those reported virulence‐related genes. The results suggest that the T‐DNA inserted mutants banks constructed are useful for the discovery of new or important genes and to elucidate their function in the 2 Colletotrichum species.     

Comparison of continuous infusion with intermittent bolus administration of cefotaxime on blood and cavity fluid drug concentrations in neonatal foals

Hewson, J., Johnson, R., Arroyo, L. G., Diaz‐Mendez, A., Ruiz‐López, J. A., Gu, Y., del Castillo, J. R. E. Comparison of continuous infusion with intermittent bolus administration of cefotaxime on blood and cavity fluid drug concentrations in neonatal foals. J. vet. Pharmacol. Therap.  36 , 68–77. Healthy neonatal foals were treated with cefotaxime by bolus (40 mg/kg IV q6h for 12 doses; n = 10) or by infusion (loading dose of 40 mg/kg IV followed by continuous infusion of a total daily dose of 160 mg/kg per 24 h for 3 days; n = 5). Population pharmacokinetics was determined, and concentrations in cavity fluids were measured at steady state (72 h). Highest measured serum drug concentration in the bolus group was 88.09 μg/mL and minimum drug concentration (C_min) was 0.78 μg/mL at 6‐h postadministration (immediately before each next dose), whereas infusion resulted in a steady‐state concentration of 16.10 μg/mL in the infusion group. Mean cefotaxime concentration in joint fluid at 72 h was higher (P = 0.051) in the infusion group (5.02 μg/mL) compared to the bolus group (0.78 μg/mL). Drug concentration in CSF at 72 h was not different between groups (P = 0.243) and was substantially lower than serum concentrations in either group. Insufficient data on pulmonary epithelial lining fluid were available to compare the methods of administration for cefotaxime in this cavity fluid. Results support continuous drug infusion over bolus dosing in the treatment for neonatal foal septicemia to optimize time that cefotaxime concentration exceeds the minimum inhibitory concentration of common equine pathogens.     

Pulmonary pharmacokinetics of tulathromycin in swine. Part I: Lung homogenate in healthy pigs and pigs challenged intratracheally with lipopolysaccharide of Escherichia coli

The objective of the study was to assess the pharmacokinetics of tulathromycin in lung tissue homogenate (LT) and plasma from healthy and lipopolysaccharide (LPS)‐challenged pigs. Clinically healthy pigs were allocated to two dosing groups of 36 animals each (group 1 and 2). All animals were treated with tulathromycin (2.5 mg/kg). Animals in group 2 were also challenged intratracheally with LPS from Escherichia coli (LPS‐Ec) 3 h prior to tulathromycin administration. Blood and LT samples were collected from all animals during 17‐day post‐tulathromycin administration. For LT, one sample from the middle (ML) and caudal lobes (CL) was taken. The concentration of tulathromycin was significantly lower in the ML after the intratracheal administration of LPS‐E. coli (P < 0.02). In healthy pigs and LPS‐challenged animals, the distribution of the drug into the lungs was rapid and persisted at high levels for 17‐day postadministration. The distribution of the drug within the lung seems to be homogenous, at least between the middle and caudal lobes within dosing groups. The concentration versus time profile of the drug and pharmacokinetic parameters in two different lung areas (middle and caudal lobe) were consistent within the groups. The clinical significance of these findings is unknown.     

Auditory brainstem responses in the normal beagle

Summary

The latencies of the peaks in brainstem responses and the threshold response were determined in 18 healthy beagles.

In the first series of measurements the dogs were sedated and the stimulus was delivered via an in‐the‐ear transducer. The latencies, the threshold levels, and the influence of the stimulus repetition rate on the latencies were measured. Using a miniature electret microphone in the outer ear canal near the tympanic membrane, it was found that at a level setting corresponding to 0 dB human level (HL) the major peak in damped oscillation during condensation reached a sound pressure level (SPL) of about 27 dB and the secondary rarefaction peak reached 24 dB SPL.

In the second series of measurements the dogs were not sedated and the stimulus was delivered via a headphone.

The wave forms, the mean latencies for peaks I to V as a function of the stimulus level, and the threshold of each wave are presented from both series. In the first series the latency values at 80 dB HL (107 dB SPL) were 1.21, 1.97, 2.67, 3.12 and 3.61 ms for peaks I, II, III, IV and V, respectively. The thresholds for peaks I to V were 47.5 ± 9.5, 47.5 ± 11.5, 41.3 ± 13.0, 63.3 ± 17.4 and 28.0 ± 9.7 dB HL, respectively. The difference in peak latency between the first and the second series was 0.065 ms. This difference corresponded to the difference in length of the acoustic pathways.

Analysis of variance was used to determine whether the successive peaks in the response followed at a constant time interval, i.e., whether a shift in the first peak with a change in the stimulus level was followed by the same shift in subsequent peaks. The analysis showed a significant (P < 0.001) interaction between the inter‐peak latency differences and the effect of stimulus level. This inter‐peak latency depended on stimulus level, although the effect was small.

The use of the in‐the‐ear transducer and sedation resulted in a far more efficient procedure than the use of the headphone without sedation.     

Myocardial damage in cats that died after anaesthesia

Summary

The necropsy findings of 85 cats that died up to six weeks after administration of injectable anaesthetics are described The most obvious findings in these cats were degeneration and necrosis of heart muscle fibres followed and related to time after the administration of the anaesthetics, by infiltration of predominantly mononuclear cells and by an increase in collagenous connective tissue. The lesions were most obvious on the inner side of the myocardium, indicating that hypoxic injury occurred during anaesthesia. The possible mechanism of the damage to the heart and its role in the pathogenesis of cardiomyopathy in the cat is discussed.     

Relationship between latency of brainstem auditory‐evoked potentials and head size in dogs

Summary

Cranium and brainstem dimensions were measured in 32 postmortem dog heads. Positive correlations were found between cranium length (CL) and brainstem length (BL) (r=0.87), between cranium width (CW) and brainstem width (BW) (r=0.83), and between cranium distance (CD = CL CW/2) and brainstem distance (BD = BL+BW/2) (r=0.91). Positive correlation coefficients were also found between CL and CW (r=0.90), and between BL and BW (r=0.85). It was concluded that head size accurately reflected brainstem size. A least squares estimation of the brainstem distance (BD) from CL and CW values was BD = 10.9 + 0.16 (CL CW/2) (BD, CL and CW in mm).

Brainstem auditory evoked potentials (BAEPs) and cranium dimensions were measured in 43 dogs (86 ears) with different head size, body size, sex and age. Wave form, absolute and interpeak latencies and correlation coefficients, relating latencies to cranium dimensions and body weight, were analysed CL, CW, and CD were positively correlated with body weight (r=0.93, 0.70 and 0.93, respectively), and CL, CW, and CD were correlated with age (r=0.33, 0.52 and 0.40, respectively). BAEPs consisted of five distinct positive peaks (I to V). Secondary positive peaks following peaks I and II were seen in 60% (I') and 90% (II') of the recordings. Late waves were recorded in 90% (VI), 50% (VII), and 25% (VIII) of the recordings. Latencies increased with decreasing stimulus intensity level (from 90 dB to 10 dB hearing level, HL),especially for peaks I, II, V, and the I‐V interpeak interval Absolute and interpeak latencies were positively correlated with cranium distance and body weight. Correlation coefficients increased as wave latencies increased At 90 dB HL, the highest correlation coefficients, relating cranium distance to peak V and the I‐V interpeak latency, were 0.55 and 0.53 (P < 0.00001), respectively. Regression analysis showed that each 1 cm increase in cranium distance was accompanied by an increase of 0.006 ms in the latency of wave I, 0.03 ms for wave III, 0.05 ms for wave V, and 0.05 ms for the I‐V interpeak interval Regression analysis showed that an increase of 1 kg in body weight was accompanied by an increase of 0.001 ms in the latency of wave I, 0.005 ms for wave III, 0.011 ms for wave V, and 0.01 ms for the I‐V interpeak interval. It is concluded that head size, which accurately reflects brain size, is a relevant source (25%) of intersubject variance of BAEP latencies in the dog.     

Experimental studies on the impact of an increased dose of anionic salts on the metabolism of dairy cows

Summary

This study was initiated to investigate the influence of a daily dose of anionic salts (AS) above the valid upper limit at present on metabolism of dairy cows. Eleven non‐pregnant and non‐lactating Holstein‐Friesian crossbred cows with a permanent rumen cannula were used in a study with a controlled feeding design. The initial daily dose was 2500 meq/day, which resulted in a Dietary Cation Anion Difference (DCAD) of ‐211 meq/kg dry matter. Every seven days, the daily dose was raised by 500 meq. If a cow stopped eating, the application of AS was stopped and these cows were monitored over the next seven days. On day 30 another batch of hay, having the same DCAD but higher concentrations of minerals and energy, was fed. Blood and urine samples were taken to monitor acid‐base balance and calcium concentrations. Acid‐base balance was strongly influenced by AS. Blood pH dropped steadily and reached values around 7.23. Urine pH dropped quickly below 6 and remained at that level regardless of the increased dosage of AS. Net acid base excretion (NABE) fell continuously with the increase of the dosage of AS and reached values below ‐200 mmol/l. Calcium concentrations in the serum were nearly stable, but those in urine increased sharply and remained on an elevated level with increasing doses of AS. A few days before the individual cow's refusal of feed intake, calcium excretion in urine decreased. The majority of cows stopped eating while consuming a diet containing 3500 to 4000 meq AS except two animals who consumed up to 6000 meq/day AS but they received the better hay in the second half of the treatment period. In this time pH in blood increased slowly. NABE remained stable on a low level at ‐100 mmol/l. The results showed that with an increasing amount of AS fed the risk of clinical acidosis increased. The decreasing urine concentrations of calcium indicate a breakdown of the compensation capability of the single cow. Besides the dose of AS fed, the quality of the feed stuff might be another factor concerning the tolerance of cows against AS.