Freezing of stallion semen with addition of glycine betaine

The effect of addition of glycine betaine to a lactose-EDTA freezing medium on the post-thaw motility of stallion semen was determined. The first three semen-rich fractions of nine stallions were collected with an open-end Krakow artificial vagina on consecutive weekdays. Semen was frozen using the Hannover method with freezing media containing glycine betaine in various concentrations from 0 to 5%. After thawing, sperm motility was analysed both by a light microscope and by a Hamilton-Thorn Motility Analyser. Total and progressive post-thaw motilities of semen containing 0.25-3% glycine betaine did not differ significantly from the total and progressive post-thaw motilities of semen frozen without glycine betaine. The total and progressive post-thaw motilities of semen containing 4 or 5% glycine betaine were significantly lower (P < 0.001) than those of semen without glycine betaine. In conclusion, glycine betaine did not show any beneficial effect on the post-thaw motility of stallion semen when semen was frozen using the Hannover method.     

Hepatic encephalomyelopathy in a calf with congenital portosystemic shunt (CPSS)

A 4-month-old female Holstein Friesian calf was referred to the Veterinary Teaching Hospital, University of Berne, Switzerland for evaluation of ataxia, weakness, apathy and stunted growth. Clinical examination revealed generalized ataxia, propioceptive deficits, decreased menace response and sensibility. Postmortem examination did not reveal macroscopic changes of major organs. Histologically, the brain and the spinal cord lesions were characterized by polymicrocavitation, preferentially affecting the white matter fibers at the junction of grey and white matter and by the presence of Alzheimer type II cells. The liver revealed lesions consistent with a congenital portosystemic shunt, characterized by increased numbers of arteriolar profiles and hypoplasia to absence of portal veins. The pathological investigations along with the animal history and clinical signs indicated a hepatic encephalomyelopathy due to a congenital portosystemic shunt.     

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.     

Eel acetylcholinesterase inhibition studies with heteroarylphosphinates

The inhibition of eel acetylcholinesterase by the 4-nitrophenyl esters of 2-furyl(methyl)-, methyl(2-thienyl)-, di-2-furyl-, and di-2-thienylphosphinic acid (I, II, III, and IV, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for alkyl/heteroaryl and diheteroaryl esters of phosphinic acids. The largest k_i value (19,330 M^?1 sec^?1) was observed for the reaction of I with the enzyme. The order for the remaining three is II > IV > III. There is no direct relationship between the hydrolysis rates of the esters and their anticholinesterase activities on eel acetylcholinesterase. Likewise, there is no direct relationship between their anticholinesterase activities and the LD₅₀ values in rats.     

A faster inoculation assay for Armillaria using herbaceous plants

Armillaria (honey fungus) is a virulent necrotrophic pathogen that causes Armillaria root disease. Conventional Armillaria inoculation assays use young saplings as hosts and consequently are cumbersome, frequently conducted outdoors, and take many years from establishment to analysis of infection. We have developed and evaluated a faster inoculation assay for Armillaria that uses herbaceous plants as hosts, is carried out in controlled conditions, and reduces experimental durations to 3 months. Plant species of known susceptibility to Armillaria and comparisons between virulent A. mellea and opportunistic A. gallica were used to validate the assay. Mortality and diagnostic symptoms of Armillaria root disease such as epiphytic rhizomorphs and mycelial fans were used to assess levels of infection. We also attempted to reduce assay preparation time by substituting woody inocula with agar inocula, but typical symptoms of Armillaria root disease were only observed on plants infected with woody inocula. Through our assay, we identified five new potential herbaceous hosts of Armillaria: Kniphofia hirsuta, Hordeum vulgare, Lobelia cardinalis, Nicotiana tabacum and Helenium hoopesii – further expanding the extensive list of plants with susceptibility to Armillaria and suggesting infection of herbaceous species may be more widespread than currently acknowledged.     

Role of wild ruminants in the epidemiology of bluetongue virus serotypes 1, 4 and 8 in Spain

ABSTRACT: Although the importance of wild ruminants as potential reservoirs of bluetongue virus (BTV) has been suggested, the role played by these species in the epidemiology of BT in Europe is still unclear. We carried out a serologic and virologic survey to assess the role of wild ruminants in the transmission and maintenance of BTV in Andalusia (southern Spain) between 2006 and 2010.A total of 473 out of 1339 (35.3%) wild ruminants analyzed showed antibodies against BTV by both ELISA and serum neutralization test (SNT). The presence of neutralizing antibodies to BTV-1 and BTV-4 were detected in the four species analyzed (red deer, roe deer, fallow deer and mouflon), while seropositivity against BTV-8 was found in red deer, fallow deer and mouflon but not in roe deer. Statistically significant differences were found among species, ages and sampling regions. BTV RNA was detected in twenty-one out of 1013 wild ruminants (2.1%) tested. BTV-1 and BTV-4 RNA were confirmed in red deer and mouflon by specific rRT-PCR.BTV-1 and BTV-4 seropositive and RNA positive wild ruminants, including juveniles and sub-adults, were detected years after the last outbreak was reported in livestock. In addition, between the 2008/2009 and the 2010/2011 hunting seasons, the seroprevalence against BTV-1, BTV-4 and BTV-8 increased in the majority of provinces, and these serotypes were detected in many areas where BTV outbreaks were not reported in domestic ruminants. The results indicate that wild ruminants seem to be implicated in the dissemination and persistence of BTV in Spain.     

The use of Verticillium dahliae and Diplodia scrobiculata to induce resistance in Pinus halepensis against Diplodia pinea infection

The pathogenic fungi Verticillium alboatrum and Diplodia scrobiculata were assayed for biological control of Diplodia pinea on Aleppo pine (Pinus halepensis) in Catalonia (north-eastern Spain). Young shoots were pre-treated with inoculations of either V. dahliae or D. scrobiculata, by placing colonized agar plugs on wounds made by removing a single needle fascicle. An inoculation with D. pinea was performed 15 days later. Two months after the shoot inoculations, the canker length on the stems was measured and the percentage of shoot dieback calculated. Verticillium dahliae and D. scrobiculata were found to significantly reduce the canker length of D. pinea (P < 0.05) when compared with positive controls. Diplodia pinea was slightly more sensitive to V. dahliae than to D. scrobiculata, but no significant differences (P > 0.05) were observed in the mean canker length between the two treatments. Trees pre-inoculated with V. dahliae resulted in 31.12% shoot dieback, while those pre-inoculated with D. scrobiculata resulted in 32.18% shoot dieback, compared with positive controls (42.85%).     

Cycle‐Characteristic Odour of Cow Urine Can Be Detected by the Female Face Fly (Musca autumnalis)

Due to declining dairy cow fertility rates, there is great interest in developing tools for oestrus detection. Compounds in the volatile profile of oestrous cows are suggested as oestrus‐specific, but consistent results have not been presented. Certain haematophagous arthropods can discriminate stages of the mammalian reproductive cycle based on host volatiles. This study investigated whether the face fly, Musca autumnalis de Geer (Diptera: Muscidae), can discriminate between urine from cows in oestrus and urine collected during the luteal phase. Individual flies were tested in a two‐choice behavioural assay with choice between odour of oestrous or luteal urine and water (control). Flies chose the control arm significantly more when exposed to oestrous urine than when exposed to luteal urine. Analysis of volatiles showed that 1‐hexadecanol (cetyl alcohol) was released in greater amounts from oestrous urine than from urine collected during the luteal phase. In a dose response assay, flies were significantly attracted by 0.01 ng of 1‐hexadecanol but significantly repelled by 0.1 ng, a pattern consistent with fly responses to urine. In conclusion, M. autumnalis can discriminate between oestrous and luteal urine, and this may be mediated by differences in 1‐hexadecanol concentration.