Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.     

Larval sea lamprey release two unique bile acids** to the water at a rate sufficient to produce detectable riverine pheromone plumes

The discovery that the olfactory system of the anadromous sea lamprey is extremely sensitive to two unique bile acids (petromyzonol sulfate [PS] and allocholic acid [ACA]) produced by stream-resident larval conspecifics has lead us to hypothesize that these compounds function as a migratory pheromone. Here, we test whether lamprey release these bile acids to the water in quantities sufficient for them to function as a long distance attractant. Five experiments were conducted. First, high performance liquid chromatography (HPLC) of liver extracts from all three life history stages of this species established that only larvae produce PS and ACA; parasites and maturing adults produced no identifiable bile acids. Large quantities of PS and ACA were found in larval gall bladders. Second, HPLC analyses of larval lamprey holding waters established that recently-fed larvae held in the laboratory release these bile acids to the water, with PS being released at a rate of approximately 16 ng h^–1 animal^–1 and ACA at 5 ng h^–1 animal^–1. Fasted animals released little bile acid. Third, an investigation of bile acid release routes demonstrated that larvae release bile acids primarily via their feces. Fourth, a study of the stability of ACA and PS in river water found both to have a half-life of a day. Finally, theoretical extrapolations using these data suggest that PS and ACA are present in picomolar concentrations in lamprey streams, a concentration within the detection range of adults. In conclusion, these data demonstrate for the first time in a fish that bile acid release rates and modes are adequate for these compounds to have pheromonal function.     

Anaesthesia, sedation and transportation of juvenile Menidia estor (Jordan) using benzocaine and hypothermia

Anaesthesia, sedation and transportation of the Atherinopsid, Menidia estor, were investigated using benzocaine, hypothermia and combinations of the two. The optimum dose rates of benzocaine for sedation of juvenile Chirostoma estor were 15 and 18 mg L⁻¹. When hypothermia was used alone, stable sedation of C. estor was induced at 15 and 12°C, with no mortalities and full recovery after about 8 min. There was synergism when benzocaine and hypothermia were combined, with the optimal combination being at 15°C and 12 mg L⁻¹ benzocaine. Successful transportation was conducted over 3.5 and 8.5 h using combined benzocaine and hypothermia. Aloe vera extract used in the transportation medium reduced scale loss. This work has, for the first time, defined a systematically derived system of safe, long distance, live transportation of this species and others of the genus Menidia.     

Growth and sensory characteristics of organically reared broilers differing in strain,sex and age at slaughter

Abstract

The effects of strain, sex and age on live weight, slaughter weight and sensory characteristics of organically reared broilers in orchards are presented. A total of 450 broilers of three different strains (I 657, Light Sussex, New Hampshire) were reared in an organic research orchard. Half of each strain was slaughtered at 91 days and the other half at 120 days. All broilers were weighed and a clinical welfare assessment was made at slaughter. A trained sensory panel evaluated the breast meat in relation to flavour, smell and texture characteristics defined by the panel. At both 91 and 120 days of age the commercial breed I 657 was significantly heavier than the slower growing strains. Males were significantly heavier than the females across strains. Weight ratios between males and females were nearly the same at both slaughter ages in I 657, whereas weight ratios increased significantly at 120 days in Light Sussex and New Hampshire. No overall effect of strain was found on the flavour and smell of the breast meat. However, the age-related changes in tenderness and toughness differed significantly between strains, since the commercial strain tended towards a tougher and less tender consistency with age, whereas the opposite was the case for the slower growing pure breeds. The positive flavour of salt was significantly improved at 120 days across strains with females having a saltier flavour than the males with age. The positive flavour of sweet corn was improved in the meat from the males, whereas the positive smell of sweet corn was significantly improved in both males and females. No negative effects of age were found.

In conclusion, broilers were tastier when slaughtered at 120 days compared with 91 days. The development in meat tenderness was related to strain, and weights at slaughter were related to both strain and sex.     

Evaluation of changes in antimicrobial susceptibility patterns of Pasteurella multocida subsp multocida isolates from pigs in Spain in 1987-1988 and 2003-2004

OBJECTIVE: To determine the susceptibility of strains of Pasteurella multocida subsp multocida isolated from lung specimens of pigs with pneumonia to 20 antimicrobials and to evaluate the emergence of resistance to those antimicrobials in Spain during the past 2 decades. SAMPLE POPULATION: 63 isolates recovered from 1987 to 1988 and 132 isolates recovered from 2003 to 2004. PROCEDURE: A broth microdilution method was used to determine minimal inhibitory concentration (MIC) range and values for MIC50 and MIC90. Resistance of a strain to an antimicrobial agent was determined by use of the breakpoint value when available. RESULTS: Isolates were generally susceptible to penicillin, ampicillin, ceftiofur, gentamicin, apramycin, neomycin, spectinomycin, chlortetracycline, erythromycin, tilmicosin, enrofloxacin, and florfenicol, and most isolates were resistant to clindamycin, tylosin tartrate, and tiamulin regardless of the time period. A substantial increase in resistance to sulfa-chlorpiridazine, sulfadimethoxine, sulfathiazole, and trimethoprim-sulfamethoxazole was observed, and a minor increase in resistance to oxytetracycline was also detected. Several multiresistance patterns were observed, most frequently among isolates recovered in the 2003 to 2004 interval. CONCLUSIONS AND CLINICAL RELEVANCE: Ceftiofur, florfenicol, and enrofloxacin are recommended for treatment of infections caused by P multocida subsp multocida in Spain. Increased frequency of resistance to oxytetracycline and sulfonamide drugs may be a contraindication for their use.     

Evidence of increased Hepatitis E virus exposure in Lao villagers with contact to ruminants

Although pigs are the main reservoir, ruminants have also been shown to be susceptible to hepatitis E virus (HEV). We investigated zoonotic transmission of HEV in rural settings of Lao People's Democratic Republic (Lao PDR) where humans are in close contacts with ruminants and where pigs are rare. Villagers with (n = 171, risk group) and without (n = 155, control group) cattle were recruited in seven villages in Vientiane Capital. Owners of pigs were excluded. Blood, as well as information on socio‐demographics, animal contact, dietary habits and awareness of zoonoses were collected to assess risk factors. Blood and rectal swabs were collected from cattle (n = 173) and other ruminants (27 goat, 5 buffaloes) to measure anti‐HEV antibody and virus prevalence. A similar anti‐HEV antibody seroprevalence was found in cattle (6.8%) and other ruminants (8%). HEV RNA was detected in none of the animal rectal swabs and human sera. Anti‐HEV IgG seroprevalence was higher in cattle farmers than in the control group (59.1% vs. 43.9%, p = 0.008) and increased significantly with age. Other risk factors included male gender, close contact with cattle and consumption of undercooked meat. We find that HEV is highly endemic in rural Laos and provide first evidence that HEV circulates in free‐roaming ruminants with open access to village water sources. Despite some awareness about hygiene, villagers are likely constantly exposed to zoonotic diseases by dietary and lifestyle habits. Cattle farmers had a higher risk of HEV infection than other villagers. Our study highlights the need to raise the awareness of the rural population about water‐ and food‐borne pathogens, and about the role of cattle as a possible source of infection. The knowledge gained on local risk factors and husbandry conditions should guide future awareness raising campaigns and promote appropriate hygienic measures including handwashing and the consumption of safe food and water.     

Infectivity of pestivirus following persistence of acute infection

Bovine viral diarrhoea virus (BVDV) is an endemic pathogen worldwide and eradication strategies focus on the identification and removal of persistently infected (PI) animals arising after in utero infection. Despite this, acute infections with BVDV can persist for months or years after the removal of the PI source despite repeated screening for PIs and tight biosecurity measures. Recent evidence for a prolonged duration of viraemia in the testicles of bulls following acute BVDV infection suggests the possibility of a form of chronic persistence that may more closely resemble the persistence strategies of hepatitis C virus (HCV). To investigate the potential for virus transmission from infected and recovered cattle to virus naïve hosts we established an acute infection of 5 BVDV-naïve calves and monitored animals over 129 days. Infectious BVDV was detected in white blood cells between days 3 and 7 post-challenge. The animals seroconverted by day 21 post-infection and subsequently were apparently immune and free from infectious virus and viral antigen.Animals were further monitored and purified white blood cells were stimulated in vitro with phytohaemagglutinin A (PHA) during which time BVDV RNA was detected intermittently.Ninety-eight days following challenge, blood was transferred from these apparently virus-free and actively immune animals to a further group of 5 BVDV-naïve calves and transmission of infection was achieved. This indicates that BVDV-infected, recovered and immune animals have the potential to remain infectious for BVDV-naïve cohorts for longer than previously demonstrated.     

CD27 expression discriminates porcine T helper cells with functionally distinct properties

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α^- T helper cells but divides CD8α⁺ T helper cells into a CD27⁺ and a CD27^- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α^-CD27⁺ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were found in blood, spleen and liver. Both CD8α⁺CD27⁺ and CD8α⁺CD27^- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α^-CD27⁺, CD8α⁺CD27⁺ and CD8α⁺CD27^- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α⁺CD27^- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α⁺CD27^- T helper cells were mostly CCR7^- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α⁺CD27⁺ T helper cells, which also had a proliferation rate similar to naïve CD8α^-CD27⁺ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α⁺CD27⁺ T helper cells displayed a phenotype and functional properties of central memory cells.