Cloning, expression and purification of feline proinsulin

Feline proinsulin was cloned and expressed using a bacterial expression system. It was then purified from inclusion bodies using size exclusion chromatography and further processed including reduction of the protein. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin can be used for therapeutic and diagnostic purposes.     

Molecular marker-based genetic linkage map of a diploid banana population (Musa acuminata Colla)

A well-saturated genetic linkage map is valuable for fundamental and applied genetic research. Genetic linkage maps of two half-sib diploid banana populations were constructed using allele-specific-polymerase chain reactions (AS-PCRs), diversity array technology (DArT), and simple sequence repeat (SSR) markers. Molecular maps were produced for each parent using the pseudo-testcross mapping strategy. The first maternal map (6142-1, 81 individuals) consisted of 231 markers divided as followed: 121 DArT, 106 SSR and 4 AS-PCR markers in 15 linkage groups (LGs) covering 670?cM. The second maternal map (6142-1-S, 58 individuals) contained a total of 152 markers including 71 DArTs, 79 SSRs, and 2 AS-PCRs mapped to 16 LGs that spanned 698?cM. The combined paternal map (139 individuals) comprised 316 markers (196 DArTs, 117 SSRs and 3 AS-PCRs) distributed over 15 LGs with a total map length of 1,004?cM. While distorted segregation of some markers was observed in all maps, this was much more frequent for the male parent. Homology between maps was assessed using common markers. While there was generally good congruity with regard to marker order across maps, incongruity in other cases may reflect chromosomal rearrangement events such as inversions, translocations, or deletions. The new banana map can provide a better understanding of the Musa genome and could be used for the identification of economically important traits and improvement of breeding strategies.     

Discrepancy between minimal inhibitory concentration to enrofloxacin and mutations present in the quinolone-resistance determining regions of Mycoplasma gallisepticum field strains

This study evaluated the in vitro activity of diphenyl diselenide against 19 Pythium insidiosum isolates and the in vivo therapeutic response of rabbits with experimentally induced pythiosis. In vitro: susceptibility tests were performed using the broth macrodilution method in accordance with the CLSI document M38-A2. The criteria for interpretation were as follows: MIC-1 and MIC-2 (inhibition of 90% and 100% of mycelium growth, respectively) and the minimum fungicide concentration (MIC-3). In vivo: twenty rabbits were divided into four groups with five animals each and treated for 40 consecutive days: groups 1 and 2 (experimentally induced pythiosis) were treated with diphenyl diselenide (10mg/kg/day) and canola oil (1 mL/kg/day), respectively; groups 3 and 4 (controls) were treated with canola oil (1 mL/kg/day) and diphenyl diselenide (10mg/kg/day), respectively. Toxicity was evaluated using biochemical and haematological parameters. In vitro susceptibility tests showed that 89.4% of isolates had a MIC-1 ≤ 0.5 μg/mL, 84.2% of isolates had a MIC-2 ≤ 1.0 μg/mL and 94.7% of isolates had a MIC-3 ≤ 2.0 μg/mL. The in vivo assay suggested that this compound has a fungistatic activity, and the biochemical and haematological parameters indicated that there was no renal, hepatic or haematological toxicity. The comparison of the unsaturated iron binding capacity levels between animals with and without pythiosis suggested the involvement of iron metabolism in the pathogenesis of pythiosis. This study demonstrated the absence of detectable toxicity caused by diphenyl diselenide and the in vitro fungicidal and in vivo fungistatic activities of this drug, which makes it an option for future therapeutic approaches in the treatment of pythiosis.     

GLUT4 but not GLUT1 expression decreases early in the development of feline obesity

The increase in obesity in people and pets has been phenomenal. As in man, obesity in pets is a risk factor for many diseases including diabetes mellitus. Recently, tissue-specific regulation of glucose metabolism in fat and muscle tissue has been identified as an important factor for insulin sensitivity and it has been hypothesized that glucose uptake into tissues is altered in obesity causing insulin resistance. The purpose of this study was to determine the expression of the glucose transporter proteins GLUT4 and GLUT1 in muscle and fat from lean and obese cats. Seventeen domestic felines were tested in the lean state and again after a 6-month period of ad libitum food intake which led to a significant increase in weight (P<0.0001). Obese cats showed a significantly higher area under the curve (AUC) for glucose, AUC for insulin and a significant decrease in glucose percentage disappearance per min (K-value) (P=0.013, 0.018 and 0.017, respectively) during an intravenous glucose tolerance test, but no change in baseline glucose or glycosylated hemoglobin concentrations. GLUT4 expression was decreased in biopsies of both muscle (P=0.002) and fat (P=0.001) in the obese animals. GLUT4 in muscle and fat significantly and negatively correlated with the insulin AUC (r²=0.36, P=0.004 and r²=0.18, P=0.040, respectively). GLUT1 expression showed no significant change in the obese cats in either tissue. It is concluded that the changes in GLUT4 are early derangements in obesity and occur before glucose intolerance is clinically evident.     

Estimating exposed pulp lengths of tusks in the African elephant (Loxodonta africana africana)

Captive and wild African elephants frequently suffer tusk fractures. Several institutions shorten the tusks of captive elephants to reduce fractures and injury as a result of behaviour within enclosures. Fracturing or coronal amputations that expose pulp lead to pain for the elephant. Estimating coronal pulp lengths may thus help to minimise the risk of pulp exposure during amputations. We aimed to determine the length of the pulp beyond the lip margin from an external tusk characteristic. Tusks collected from elephants in Namibia and the Kruger National Park had similar morphological relationships. This statistical property allowed us to correct for missing data in our data sets. Pulp volume and pulp length correlated with tusk circumference at the lip. Even so, the circumference at the lip could not predict the length of the pulp in the crown external to the lip. Our findings suggest that tusks, irrespective of sex or age, amputated further than 300 mm from the lip should not expose pulp.     

Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria , obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2 , shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis -aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.     

Spermatocrit and spermatozoa density in Atlantic cod (Gadus morhua): correlation and variation during the spawning season

The utility of spermatocrit (the proportion of solid packed material in semen after centrifugation) as an indicator of spermatozoa density and male spawning stage was tested in Atlantic cod (Gadus morhua). Semen was collected from captive male cod over three spawning seasons. Spermatocrit was positively and significantly correlated with spermatozoa density measured with a Coulter counter (Multisizer), but not with spermatozoa counts in a haemacytometer. Spermatocrit increased significantly as the spawning season progressed. However, day of season explained only 35% of the variation because spermatocrit varied among individual males. Spermatozoa size remained unchanged throughout the sampling period and was not correlated with spermatocrit, indicating that variation in spermatocrit was due to variation in spermatozoa number and not their size. Spermatocrit is a good estimator of sperm density but is not reliable as indicator of spawning stage because of the variation among individual males.     

Atypical bacterial gill disease: a new form of bacterial gill disease affecting intensively reared salmonids

An unusual form of bacterial gill disease (BGD) was identified which affected five species of cultured salmonids from Canada (i.e. rainbow trout, chinook salmon and Atlantic salmon), Norway (i.e. brown trout) and Chile (i.e. coho salmon). All outbreaks occurred at low water temperatures (< 10 °C) and with clinical presentations distinct from classical BGD, which is caused by Flavobacterium branchiophilum. In contrast to classical BGD, fish did not show marked respiratory distress with flaring of the opercula, the animals did not orientate at the surface of the water column near inflow water or at the margins of the tanks, and the feed response of the fish was varied. While mortality was increased, it was not precipitous as in classical BGD. Eight outbreaks were examined in greater detail using histopathology, scanning electron microscopy, bacteriology and immunohistochemistry. Large numbers of small bacterial rods were seen adhering to the lamellar epithelium of affected gills from all outbreaks. Histologically, the lamellar epithelium appeared swollen, often with evidence of single cell degeneration and exfoliation. In more severe instances, the formation of lamellar synechiae was seen, usually associated with sequestration of bacteria between fused lamellae. By contrast with typical BGD, overt epithelial hyperplasia, lamellar fusion and filamental clubbing were not common sequelae to infection; instead, the end result was shortened and somewhat stubby lamellae covered with swollen epithelial cells. The predominant bacterium recovered from affected gills was a small, Gram-negative, motile, fluorescent pigment-producing rod that shared phenotypic characteristics with Pseudomonas fluorescens. Polyclonal antisera prepared against three representative isolates indicated a weak antigenic similarity among them. Immunohistochemistry corroborated this finding, in that the antisera reacted strongly with gill sections containing the homologous bacteria, but not against morphologically similar bacteria in heterologous sections. A Gram-negative, yellow pigmented bacterium (YPB), identified as Flavobacterium psychrophilum, was also recovered, but only from the gills in the Ontario outbreaks. Antiserum prepared against this YPB indicated an antigenic similarity among isolates recovered from the Ontario outbreaks, but immunohistochemistry failed to recognize antigenically related bacteria on the gills of fish from the other outbreaks. Based on the unusual clinical presentation and the histopathological appearance of the gills, in conjunction with the absence of filamentous bacteria associated with and recovered from affected gills, the present authors have called this condition ‘atypical bacterial gill disease’ or ABGD.