Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

Fecundity of Nephrops norvegicus from the Irish Sea

Potential fecundity, number of oocytes in the mature ovary, and realized fecundity, number of eggs extruded and attached to the pleopods of female Nephrops, caught at the start of the incubation period were estimated for females from the eastern and western Irish Sea grounds. Potential fecundity was found to differ significantly between eastern and western Irish Sea stocks, while realized fecundity did not differ between areas. Inter-year comparison of realized fecundity, and effective fecundity (the number of mature eggs on the pleopods of females at the end of the incubation period) in the western Irish Sea stocks revealed no significant variation over time. Egg loss during the transition from oocytes in the ovary to mature eggs increased with female size, ranging from 40% at 25 mm carapace length (CL) to 65% at 40 mm CL. No relationship was found between egg diameter or volume and female size.

Estimation of survival rate and extinction probability for stage-structured populations with overlapping life stages

The development of methods providing reliable estimates of demographic parameters (e. g., survival rates, fecundity) for wild populations is essential to better understand the ecology and conservation requirements of individual species. A number of methods exist for estimating the demographics of stage-structured populations, but inherent mathematical complexity often limits their uptake by conservation practitioners. Estimating survival rates for pond-breeding amphibians is further complicated by their complex migratory and reproductive behaviours, often resulting in nonobservable states and successive cohorts of eggs and tadpoles. Here we used comprehensive data on 11 distinct breeding toad populations (Bufo calamita) to clarify and assess the suitability of a relatively simple method [the Kiritani-Nakasuji-Manly (KNM) method] to estimate the survival rates of stage-structured populations with overlapping life stages. The study shows that the KNM method is robust and provides realistic estimates of amphibian egg and larval survival rates for species in which breeding can occur as a single pulse or over a period of several weeks. The study also provides estimates of fecundity for seven distinct toad populations and indicates that it is essential to use reliable estimates of fecundity to limit the risk of under- or overestimating the survival rates when using the KNM method. Survival and fecundity rates for B. calamita populations were then used to define population matrices and make a limited exploration of their growth and viability. The findings of the study recently led to the implementation of practical conservation measures at the sites where populations were most vulnerable to extinction. © 2010 The Society of Population Ecology and Springer.

Fasciola hepatica: Comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study. F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response. (C) 2011 Elsevier B.V. All rights reserved.

Verification of McCabe's Delta L Law for Growth of Lysozyme Crystals by Hanging Drop Method

Lysozyme is a naturally occurring enzyme in egg white and has high commercial importance due to its antimicrobial properties. The main objective of this work was to study the growth rate of lysozyme crystals isolated from egg for the first 72 hours and verify the results with McCabe’s constant crystal growth theory. Hanging drop crystallization method was used to form high purity lysozyme crystals from the embryonic stage. To this end, this work differs from an earlier work of Forsythe et al., who used seed crystals in the size range of 10 µm - 40 µm for face growth measurements at different pH values. The maximum crystal size recorded in the present work was 392.86 µm, which is within the typical size range of 50 µm - 500 µm for which constant crystal growth is expected to hold according to McCabe’s ?L law. Electron micrographs (SEM) revealed the structure and dimensions of the crystals while SDS-Page was used to measure the purity of the crystals. The SEM results showed that that lysozyme growth rate was linear and agreed with McCabe’s constant growth theory, producing a growth rate of 1.77 x 10-3 µm .s-1

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) in poultry and eggs

A survey was carried out on the occurrence of dinitrocarbanilide (DNC), the marker residue for nicarbazin, in poultry produced in Ireland during 2002-2004. Liver (n = 736) and breast muscle samples (n = 342) were tested. DNC residues were found in 40 and 26% of liver and breast muscle samples at levels greater than 12.5 and 5 mu g kg(-1), respectively. DNC residues were found at >200 mu g kg(-1) in 12 and 0% of liver and muscle samples, respectively. Samples of breast muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested and DNC residues were found in nine samples, with levels ranging between 14 and 122 mu g kg(-1). Analysis of poultry, carried out as part of official food inspection in the period 2004-2006, indicated a reduction in the number of broiler liver samples containing DNC at >200 mu g kg(-1), to approximately 7%. Low levels of DNC residues continue to be found in

Enzyme immunoassay for semicarbazide - The nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed-all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC beta) of 0.25 mu g kg(-1) when a threshold of 0.21 mu g kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 mu g kg(-1) fortified sample, n = 20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 mu g kg(-1). N-FZ-incurred muscles (12) containing SEM at 0.5-5.0 mu g kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver. (C) 2007 Elsevier BN. All rights reserved.

An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver

An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-1) (n = 12) and the functional limit of detection as 3.2 ng g(-1) for egg (n = 6) and 11.3 ng g(-1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.

Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole

Lambs infected with the Cullompton triclabendazole (ICBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10 mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3 h, 24 h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3 h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24 h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24 h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h Pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24 h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24 h, demonstrating that this process is affected very rapidly pt. (C) 2011 Elsevier B.V. All rights reserved.

Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRFamide-related peptides

Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 mu M) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (less than or equal to 0.1 mu M) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations greater than or equal to 0.1 mu M. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

5-HYDROXYTRYPTAMINE-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE, ENTOBDELLA-SOLEAE

5-HT-immunoreactivity in Entobdella soleae was found to be extensive throughout both the central and peripheral nervous systems, with the strongest staining occurring in the innervation of the forebody, most notably in the paired cerebral ganglia, pharynx and adhesive pads. In the reproductive system, staining was evident throughout the numerous cell bodies and fibres innervating the musculature of the egg-assembly apparatus. The haptor contained an extensive array of serotoninergic fibres derived from the main longitudinal cords; this array was associated with the haptoral muscles and sclerites, and possibly with the ventral sensory papillae.

A novel calmodulin-like protein from the liver fluke, Fasciola hepatica

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.

Gastric emptying in patients with flatulent dyspepsia, with and without gallbladder disease

It has been suggested that the symptoms of flatulent dyspepsia are caused by a functional disturbance of the upper gastrointestinal tract. The aim of this study was to investigate delayed gastric emptying as the basis of symptoms in patients with and without gallbladder disease and after cholecystectomy. There were 13 dyspeptic patients with gallbladder disease, 12 with normal gallbladders, and 13 post-cholecystectomy patients. Gastric emptying was measured by means of a 99mTc-labelled scrambled egg meal and external scintillation counter. The rate of emptying in the symptomatic groups was compared with that in 24 asymptomatic normal control subjects and 12 non-dyspeptic patients with gallbladder disease. Delayed gastric emptying tended to occur in patients with gallbladder disease with and without dyspepsia and was not specifically associated with symptoms.

Role of Predalors, Habitat Attributes, and Spatial Autocorrelation on the Distribution of Eggs in the Northern Spectacled Salamander (Salamandrina perspicillata)

We predicted that the probability of egg occurrence of salamander Salamandrina perspicillata depended on stream features and predation by native crayfish Austropotamobius fulcisianus and the introduced trout Salmo trutta. We assessed the presence of S. perspicillata at 54 sites within a natural reserve of southern Tuscany, Italy. Generalized linear models with binomial errors were constructed using egg presence/absence and altitude, stream mean size and slope, electrical conductivity, water pH and temperature, and a predation factor, defined according to the presence/absence of crayfish and trout. Some competing models also included an autocovariate term, which estimated how much the response variable at any one sampling point reflected response values at surrounding points. The resulting models were compared using Akaike's information criterion. Model selection led to a subset of 14 models with Delta AIC(c) <7 (i.e., models ranging from substantial support to considerably less support), and all but one of these included an effect of predation. Models with the autocovariate term had considerably more support than those without the term. According to multimodel inference, the presence of trout and crayfish reduced the probability of egg occurrence from a mean level of 0.90 (SE limits: 0.98-0.55) to 0.12 (SE limits: 0.34-0.04). The presence of crayfish alone had no detectable effects (SE limits: 0.86-0.39). The results suggest that introduced trout have a detrimental effect on the reproductive output of S. perspicillata and confirm the fundamental importance of distinguishing the roles of endogenous and exogenous forces that act on population distribution.