Rough vaccines in animal brucellosis: structural and genetic basis and present status

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.     

Purification of xylitol obtained by fermentation of corncob hydrolysates

Hydrolysates obtained by autohydrolysis-posthydrolysis of corncobs were detoxified with charcoal, concentrated, supplemented with nutrients, and fermented with Debaryomyces hansenii. After biomass removal, the fermented media contained 0.1137 kg of nonvolatile components (NVC)/kg of liquor, which corresponded mainly to xylitol (0.6249 kg/kg of NVC) but also to minor amounts of inorganic components (measured as ashes), proteins, nonfermented sugars (xylose and arabinose), uronic acids, arabitol, and other nonvolatile components (ONVC). The media were subjected to further processing (sequential stages of adsorption, concentration, ethanol precipitation, concentration, and crystallization) to obtain food-grade xylitol. Adsorption experiments were carried out at various solid-to-liquor ratios. Under selected conditions (1 kg of charcoal/15 kg of liquors), the xylitol content increased to 0.6873 kg/kg of NVC, and almost total decoloration was achieved. The resulting liquor was concentrated by evaporation to increase its NVC content to 0.4032 kg/kg of liquor (corresponding to a xylitol concentration of 0.280 kg/kg of liquor), and ethanol was added to precipitate a part of the NVC (mainly proteins, but also uronic acids, ashes, and other nonvolatile compounds). Refined liquors (containing 0.7303 kg of xylitol/kg of NVC) were concentrated again, and ethanol was added (to reach 40-60% volume of the stream) to allow crystallization at -10 or -5 degrees C. Under selected conditions, 43.7% of xylitol contained in the initial fermentation broth was recovered in well-formed, homogeneous crystals, in which xylitol accounted for 98.9% of the total oven-dry weight. Material balances are presented for the whole processing scheme considered in this work.     

Analysis of organophosphorus pesticides in honeybee by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry

Pesticides applied in extended agricultural fields may be controlled by means of bioindicators, such as honeybees, in which are the pesticides bioaccumulate. Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) experiments with positive (PI) and negative (NI) ion modes were optimized for the analysis of 22 organophosphorus pesticides in honeybee samples. The extraction required 3 g of sample, which was extracted with acetone. The extract was purified with coagulating solution and reextracted with Cl(2)CH(2). Pesticides studied could be detected by both ionization modes except for parathion, parathion-methyl, and bromophos, which did not give signals in PI mode, and triazophos, which was not detected in NI mode. Fragmentation voltage and vaporizer temperature were optimized to achieve the highest sensitivity. The spectra profile of each pesticide in PI mode showed the [M + H](+) ion as the main signal, whereas in NI mode only fragment ions were shown. The detection limit obtained in selected ion monitoring mode ranged from 1 to 15 microg kg(-1). The average recoveries from spiked honeybees at various concentration levels (0.5-5 mg kg(-1)) exceeded 65% with relative standard deviations of 4-15%. The method was applied to real samples, in which residues of coumaphos and dimethoate were detected.     

Structural and ultrastructural study of the intracranial portion of the oculomotor, trochlear and abducent nerves in dog

The right intracranial portion of the oculomotor, trochlear and abducent nerves were removed from six adult German shepherd dogs and analysed by light and electron microscopy. In all cases the nerve sectional area was calculated. Unmyelinated and myelinated fibres were analysed and number, diameter and cross-sectional area were calculated. In myelinated fibres, also calculated were the corresponding axon area and diameter, and myelin sheath thickness. The mean number of myelinated fibres was 8543.50 +/- 1231.85 being the unmyelinated 1402 +/- 241.58 in the oculomotor nerve; 1509 +/- 223.17 and 287.67 +/- 72.28 in the trochlear nerve and 2473.00 +/- 211.41 and 231.25 +/- 92.67 respectively in the abducent. The mean diameter was 10.23 +/- 0.68 microm in myelinated and 0.43 +/- 0.21 for unmyelinated in oculomotor nerve, 10.53 +/- 0.55 microm and 0.33 +/- 0.04 for the trochlear, and 10.45 +/- 1.27 microm and 0.47 +/- 0.09 in the abducent nerve respectively. This study reveals that oculomotor, trochlear and abducent nerves of the dog show structural and ultra-structural features similar to the same nerves in other species.     

Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro

Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10mg/L), Al (2,5, 25 and 50mg/L), Cd (1, 10, 20 and 40mg/L), Pb (1, 10, 20 and 50mg/L) and Cr (1 and 10mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1mg/L of mercury, 10mg/L of cadmium and 50mg/L of lead. Decreased phagocytosis was also observed at 5mg/L of mercury, 50mg/L of aluminium and 10mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals.     

Epidemiologic investigation of an outbreak of goose parvovirus infection in Sweden

An outbreak of goose parvovirus (GPV) infection on a Swedish goose farm in the spring of 2004 increased the mortality rates from 2% in the early unaffected hatches to 90% and 99% respectively in the two hatches following virus introduction and 40% in goslings hatched later in the same breeding season. In this paper we describe the clinical observations, diagnostic procedures, and epidemiologic investigation carried out to elucidate the source of the infection. The diagnosis was confirmed by serology, virus isolation, and sequence analysis of a 493-bp-long fragment of the VP1 gene. Phylogenetically the causative virus was closely related to pathogenic GPV strains isolated in 2003 and 2004 from Poland and the United Kingdom, respectively. The Swedish isolate exhibited less homology with pathogenic strains from Hungary and Asia and with attenuated vaccine strains. The epidemiologic investigation showed that the virus was first introduced to a contract farm (farm A) and then was transferred with newly hatched goslings to the farm that had submitted the birds for necropsy (index farm). The exact time and source of the virus introduction to farm A could not be determined with absolute certainty. Possible sources of the infection included backyard goose eggs that had been delivered to farm A for subcontract incubation and hatching, wild geese that frequented the flock of breeding geese on pasture on farm A, and a clutch of Canada goose eggs (Branta canadensis) that had been produced by wild geese and was hatched in the same machine as the eggs produced by farm A.     

Effects of transcutaneous electrical nerve stimulation on the fatty acid profile of rabbit longissimus dorsi muscle (preliminary report)

This study was designed to investigate whether transcutaneous electrical nerve stimulation (TENS) of the longissimus dorsi muscle (MLD) of rabbits induces specific proportional changes in the muscle fatty acid composition. Ten 4-week-old Pannon White rabbits were exposed to TENS treatment two times a day, with the following settings: 30 Hz, 20 micros impulse length, 10 mA, 2 x 20 min. After a treatment period of 50 days rabbits were slaughtered and the fatty acid composition of the MLD was determined by gas chromatography. The TENS treatment increased the proportions of linoleic (C18:2 n-6), linolenic (C18:3 n-3) and gondoic acids (C20:1 n-9), compared with the control group. The level of palmitic (C16:0), stearic (C18:0), oleic (C18:1 n-9) and eicosapentaenoic (C20:5 n-3) acids significantly decreased. The proportion of total unsaturated fatty acids significantly increased. On the basis of the results obtained, TENS may have similar effects on the muscle fatty acid profile like physical training. Based on the supposal that the composition of membrane structure was also affected, the electrical stimulation of muscles may have further consequences, e.g. on membrane properties.     

Evaluation of metabolic detoxifying enzyme activities and insecticide resistance in Frankliniella occidentalis

The western flower thrips Frankliniella occidentalis (Pergande) is a very significant pest of a number of different agricultural crops in the south-east of Spain. The importance of thrips as a pest is not due mainly to the direct damage inflicted on the plant, but to the loss in commercial value which occurs as a consequence of the development of dark spots caused by the tomato spotted wilt virus (TSWV) which they transmit. The economic threshold is therefore almost zero, which enhances the problems of resistance management. The present work is part of a global project that attempts to evaluate the status of insecticide resistance in field populations of thrips obtained from several agricultural crops. We have studied, in either individual or pooled insects, some enzyme systems classically related to detoxification of insecticides: esterase and glutathione-S-transferase (GST). The activity of these enzymes from laboratory populations selected with various classes of insecticides has also been measured using several appropriate substrates. An increase in GST mean activity was found in two field-collected strains. Differences in frequency distributions of esterase and GST activities were found for both field-collected strains and for a laboratory strain selected with acrinathrin. These activities were compared with those of a wild-type reference strain.     

Occurrence, Distribution and Epidemiology of Grapevine Yellows in Spain

The main viticultural production areas in Spain were surveyed in 1994, 1995 and 1996 for the occurrence and incidence of Grapevine Yellows diseases associated to phytoplasmas. Samples from 300 plants showing symptoms of phytoplasma infection were collected from grapevine fields in the Spanish regions of Aragón, Catalonia and Navarra and analysed by PCR with specific primers for a non-ribosomal DNA of stolbur/Bois Noir (BN) and of Flavescence dorée (FD) phytoplasma. Nested PCR with universal primers P1/P7 and fU5/rU3 was also used. In the survey conducted in 1994 and 1995 only BN/stolbur phytoplasma was detected. The incidence of symptomatic plants was low in five plots of Catalonia from 3% to 18% in 1994 and 1995, respectively, and high in two plots of Navarra, from 60% to 80%. In the survey conducted in 1996 the incidence of symptomatic plants in Catalonia increased (6–80%) due to the presence of FD in five plots in the Northeastern Catalonia. An epidemiological study was carried out in two BN-affected plots of two regions from 1994 to 1997, with the evaluation of potential vectors and of host plants. The stolbur phytoplasma was found in individuals from different insect species belonging to the families Cicadellidae and Delphacidae. Some wild plants naturally infected with stolbur phytoplasma around the infected grapevines were: Convolvulus arvensis, Lavandula officinalis, Polygonum convolvulus, Solanum nigrum, and Thymus officinalis. The incidence of the disease in one BN-infected grapevine plot increased from 3.4% in 1994 to 18.40% in 1997.