Inactivation of Mycobacterium avium subsp. paratuberculosis in milk during commercial pasteurisation

Four studies have been published relating to the inactivation of Mycobacterium avium subsp. paratuberculosis (Map) by commercial HTST pasteurization. Three of these were large surveys of commercially pasteurized milk at processing/retail level in the UK and Ontario, Canada, and the fourth a pasteurization study involving naturally infected milk and commercial-scale pasteurizing plant. Evidence that Map is capable of surviving commercial pasteurization was obtained in two of the studies: viable Map was cultured from 50 ml aliquots of commercially pasteurized milk after decontamination with 0.75% cetylpyridinium chloride for 5 h and then culture on Herrold's egg-yolk medium without antibiotics. In both studies culture did not commence until 24-72 h post-pasteurization and samples were stored at 4 degrees C in the interim period. In the other two milk surveys smaller volumes of milk were tested (1-5 ml and 15 ml) and no firm evidence of surviving Map was obtained. The three milk surveys differed in other respects - chemical decontamination, culture media used and use of antibiotics. Recent findings suggest that sub-lethally heat-injured Map in pasteurized milk have the potential to recover viability if stored at 4 degrees C for 48 h between heating and testing.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.

Fecundity and reproductive strategies in deep-sea incirrate octopuses (Cephalopoda: Octopoda).

Coleoid cephalopods show flexibility in their reproductive strategies or mode of spawning, which can range from simultaneous terminal spawning over a short period at the end of the animal’s life to continuous spawning over a long period of the animal’s life. Although a simultaneous terminal spawning strategy is typical of shallow water temperate octopuses, it is not known whether deep-sea octopods would have the same reproductive strategy. The reproductive strategies and fecundity were investigated in nine species of deep-sea incirrate octopuses: Bathypolypus arcticus, Bathypolypus bairdii, Bathypolypus ergasticus, Bathypolypus sponsalis, Bathypolypus valdiviae, Benthoctopus levis, Benthoctopus normani, Benthoctopus sp., and Graneledone verrucosa (total n = 85). Egg-length frequency graphs and multivariate analysis (principal components analysis) suggest that B. sponsalis has a synchronous ovulation pattern and therefore a simultaneous terminal spawning strategy. Although a simultaneous terminal spawning strategy is most likely for B. levis and B. normani, the egg-length frequency graphs and multivariate analysis also suggest a greater variation in egglengths which could lead to spawning over an extended period.

Studies on the immunoglobulin E responses to Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen.

Studies on the immunoglobulin (Ig)E immune responses to the gastric nematode, Teladorsagia circumcincta, have demonstrated a major high molecular weight allergen (HMWTc). Cross reactive allergens of similar MW were demonstrated for Trichostrongylus colubriformis and Cooperia curticei, but not for Haemonchus contortus. Purification of HMWTc was achieved by gel-filtration chromatography, and nonreducing SDS-PAGE and Western blot analysis revealed two closely associated bands with a molecular weight of approximately 140-150?kDa. Reduction showed four IgE reactive bands of 120, 50, 45 and 30?kDa, and deglycosylation abrogated the immunoreactivity of the 120 and 30?kDa bands. Ultrastructural immunolocalization by electron microscopy revealed that the IgE reactivity was confined to the cuticular surface of the infective (L3) larvae. ELISA studies to determine the IgE anti-HMWTc responses in lambs during their first grazing season, demonstrated significantly higher IgE antibody in lambs with low accumulative faecal egg count (FEC) compared to animals with high accumulative FEC. These studies provide evidence for a protective function of IgE antibody in Teladorsagia infections in lambs.

Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Sexual dimorphism in amphipods: the role of male posterior gnathopods revealed in Gammarus pulex

In the mate-guarding amphipod, Gammarus pulex, the enlarged male posterior gnathopods have been variously suggested to function to grasp and subdue the female, to be used as weapons in fights between males, to signal to the female the male presence and stimulate moult accelaration, egg development or egg extrusion. These hypotheses were tested in a series of experiments, the results of which reveal an unexpected function. Ablation of the posterior gnathopods of males showed that they were neither necessary for, nor advantageous in, establishment and/ or maintenance of precopula mate guarding, with or without competition with intact males. Furthermore, these appendages do not function to advance female moult, or stimulate egg development or extrusion. However, only males with intact posterior gnathopods were able to copulate. We also show that females require a full copulation of several bouts to extrude eggs. We conclude that the function of the posterior gnathopods is to facilitate copulation and suggest future studies focus on the selective pressures acting on copulating males.

Deposition and depletion of five anticoccidials in eggs

Anticoccidials are compounds that are widely used as feed additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a certain time interval for broilers and pullets but not for laying hens. It was shown in the past that carry-over at the feeding mill is found to be the main reason for the presence of residues in eggs. An animal experiment was set up to investigate the effect of carry-over at the feeding mill on the presence of residues of anticoccidials in eggs. For the compounds diclazuril, robenidine, halofuginone and nicarbazin in combination with narasin, two concentration levels were tested: the maximum allowed concentration for broilers (100%) and a concentration corresponding to 5% carry-over during feed preparation. Also dimetridazole was included in the experiment but only at one concentration level. Eggs were sampled during treatment (14 days) and for a period of 30 days after withdrawal of the anticoccidial-containing feed. Residues were determined, and deposition and depletion curves were generated. Analyses were performed by ELISA and LC-MS/MS. For all compounds, substantial residues could be found in the 5% groups, which points out the risk of carry-over at the feeding mill. The distribution of the residues between egg yolk and white was determined by analyzing both fractions.

Maternal care in the rockpool amphipod Apherusa jurinei: developmental and environmental cues

There is evidence that active, pre-emergence maternal brood care in amphipod crustaceans may be associated with 'harsh' environmental conditions. We examined, in the rockpool amphipod Apherusa jurinei, behavioural activities that may function as a form of active brood care. Only ovigerous females showed 'curl' and 'stretch' activities, with consequent flushing of the brood pouch and cycling of the eggs therein. There was a significant decline in these activities as embryonic development advanced and brood care almost ceased when well-developed embryos showed a heart pulse and self-ventilation. We propose that this pattern of brood care reflects changes in the physiological requirements of embryos as they develop within the egg membrane. In addition, ovigerous females showed significantly higher levels of brood care under lowered oxygen conditions. They achieved this by increasing the average duration of the 'stretch' component, with other brood care components remaining constant. Thus, developmental and environmental cues alter the components of active brood care in distinct ways. Experimental removal showed that the physical presence of eggs in the brood pouch is important in controlling the expression of brood care activities. However, females with all of their eggs removed continued to brood at low levels, suggesting that a maternal state also controls brood care. The sophisticated expression of active maternal brood care in amphipods under 'harsh' environmental conditions such as rockpools has implications both for individual reproductive success and the distribution and abundance of brooding versus nonbrooding species. (C) 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.

Reproduction and fecundity of the Irish pollan (Coregonus autumnalis Pallas, 1776), a threatened lake coregonid

The sex ratio, age at maturation, fecundity, and seasonal development of gonads of pollan (Coregonus autumnalis) in Lough Neagh are described from samples collected between November 1997 and December 1999. Pollan reproductive ecology in the 1990s was similar to that found in the 1970s. Pollan egg size and fecundity differed between years but there were no long-term trends in fecundity despite considerable subsequent eutrophication of the lough and changes in the fish community.

Neuroactive substances and associated major muscle systems in Bucephaloides gracilescens (Trematoda : Digenea) metacercaria and adult

Cholinergic, serotoninergic and neuropeptidergic components of the nervous system were examined and compared in the progenetic metacercaria and adult gasterostome trematode, Bucephaloides gracilescens in order to provide baseline information on neuronal control of the musculature involved in egg-assembly. Enzyme cytochemistry and indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy demonstrated all three classes of neuroactive substance throughout the central and peripheral nervous systems. A comparable orthogonal arrangement of the central nervous system (CNS) and peripheral array of nerve plexuses was observed in both metacercaria and adult. Staining patterns for cholinergic and peptidergic substances showed significant overlap, while the serotoninergic system was confined to a separate set of neurons. Immunostaining for FMRFamide-related peptides (FaRPs) was strong in the CNS and peripheral innervation to the attachment apparatus of metacercaria and adult but was only found in the innervation of the ootype in ovigerous adults, implicating FaRPs in neuronal control of the muscle of the female reproductive tract during egg-assembly.

Flatworm nerve-muscle: structural and functional analysis

Platyhelminthes occupy a unique position in nerve-muscle evolution, being the most primitive of metazoan phyla. Essentially, their nervous system consists of an archaic brain and associated pairs of longitudinal nerve cords cross-linked as an orthogon by transverse commissures. Confocal imaging reveals that these central nervous system elements are in continuity with an array of peripheral nerve plexuses which innervate a well-differentiated grid work of somatic muscle as well as a complexity of myofibres associated with organs of attachment, feeding, and reproduction. Electrophysiological studies of flatworm muscles have exposed a diversity of voltage-activated ion channels that influence muscle contractile events. Neuronal cell types are mainly multi- and bi-polar and highly secretory in nature, producing a heterogeneity of vesicular inclusions whose contents have been identified cytochemically to include all three major types of cholinergic, aminergic, and peptidergic messenger molecules. A landmark discovery in flatworm neurobiology was the biochemical isolation and amino acid sequencing of two groups of native neuropeptides: neuropeptide F and FMRFamide-related peptides (FaRPs). Both families of neuropeptide are abundant and broadly distributed in platyhelminths, occurring in neuronal vesicles in representatives of all major flatworm taxa. Dual localization studies have revealed that peptidergic and cholinergic substances occupy neuronal sets separate from those of serotoninergic components. The physiological actions of neuronal messengers in flatworms are beginning to be established, and where examined, FaRPs and 5-HT are myoexcitatory, while cholinomimetic substances are generally inhibitory. There is immunocytochemical evidence that FaRPs and 5-HT have a regulatory role in the mechanism of egg assembly. Use of muscle strips and (or) muscle fibres from free-living and parasitic flatworms has provided baseline information to indicate that muscle responses to FaRPs are mediated by a G-protein-coupled receptor, and that the signal transduction pathway for contraction involves the second messengers cAMP and protein kinase C.

Ovariole number - a predictor of differential reproductive success among worker subfamilies in queenless honeybee (Apis mellifera L.) colonies

A honeybee queen normally mates with 10-20 drones, and reproductive conflicts may arise among a colony's different worker patrilines, especially after a colony has lost its single queen and the workers commence egg laying. In this study, we employed microsatellite markers to study aspects of worker reproductive competition in two queenless Africanized honeybee colonies. First, we determined whether there was a bias among worker patrilines in their maternity of drones and, second, we asked whether this bias could be attributed to differences in the degree of ovary activation of workers. Third, we relate these behavioral and physiological factors to ontogenetic differences between workers with respect to ovariole number. Workers from each of three (colony A) and one (colony B) patrilineal genotypes represented less than 6% of the worker population, yet each produced at least 13% of the drones in a colony, and collectively they produced 73% of the drones. Workers representing these genotypes also had more developed follicles and a greater number of ovarioles per ovary. Across all workers, ovariole development and number were closely correlated. This suggests a strong effect of worker genotype on the development of the ovary already in the postembryonic stages and sets a precedent to adult fertility, so that