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.

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.

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.

Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57-63%) as opposed to albumen, while 71-80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low µg kg -1 concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.

Diel variation in egg-laying by the freshwater fish louse Argulus foliaceus (Crustacea : Branchiura)

Removal of deposited eggs could be a useful control strategy for the damaging fish ectoparasite Argulus foliaceus, but focused control requires knowledge of egg-laying patterns. Here, we investigated diel changes in the egg-laying behaviour of a natural population of A. foliaceus. Data were collected from 17-28 May 2004. Days were divided into 3 time periods: 06:00-14:00, 14:00-22:00 and 22:00-06:00 h. Significantly more egg clutches were laid from 06:00-14:00 h than during the other 2 time periods, which were not significantly different from each other. Significantly more egg clutches per hour were laid during hours of daylight as compared to hours of darkness. Significantly more egg clutches were laid in the top 1 m of the water column than at the bottom, and this was consistent throughout all 3 time periods. It is suggested that the increase in egg-laying activity during daylight hours may be due to a higher motivation to search for hosts during the night and an increased ability to locate silhouetted egg-laying sites during the day. These data can provide information useful for egg removal and control strategies.

Seasonal and vertical patterns of egg-laying by the freshwater fish louse Argulus foliaceus (Crustacea : Branchiura)

Argulus foliaceus is a damaging fish ectoparasite for which new control measures are being developed based on egg-removal, In an attempt to develop further understanding of seasonal and vertical egg-laying patterns in this parasite, egg-laying activity was monitored over the period 14 April to 17 November 2003 in 2 rainbow trout Oncorhynchus mykiss fisheries in Northern Ireland, UK. At Site 1, egg-laying was continuous from 21 April to 17 November, when water temperature was above 8 to 10 degrees C. At Site 2, egg-laying was continuous from 4 June to 29 October. In the early months of the season, egg-laying was recorded mainly within the top 1 m of the water column; however, a significant shift to deep water egg-laying was recorded between 7 July and 17 November at Site 1 and between 20 August and 29 October at Site 2. Egg clutches were preferentially laid at depths of up to 8.5 m during this time (Site 2), a feature of egg-laying hitherto unappreciated. Temperature and dissolved oxygen did not differ significantly among depths, but there was an increase in water clarity over time. However, the precise environmental triggers for deep water egg-laying are still unclear. These new insights into the reproductive behaviour of this species will be useful in developing control methods based on egg-removal.

Development of an ELISA screening test for nitroimidazoles in egg and chicken muscle

An antibody was generated that can bind metronidazole (MNZ), a nitroimidazole drug used in veterinary medicine to treat poultry for coccidiosis and histomoniasis. A direct competitive enzyme-linked immunosorbent assay (cELISA) is described. It was used to characterise binding of this antibody to a number of nitroimidazole drugs. It displayed cross-reactivity with dimetridazole (DMZ), ronidazole (RNZ), hydroxydimetridazole (DMZOH), and ipronidazole (IPZ).

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was

The `Chicken and Egg' of Subjective and Social Factors in Desistance from Crime

It is now widely acknowledged that progression from persistent offending to desistance from crime is the outcome of a complex interaction between subjective/ agency factors and social/environmental factors. A methodological challenge for desistance researchers is to unravel the differential impacts of these internal and external factors and the sequence in which they come into play. Towards this, the present investigation draws on a prospective study of 130 male property offenders, interviewed in the 1990s (the Oxford Recidivism Study), and followed up 10 years later. The analysis supports a `subjective—social model' in which subjective states measured before release have a direct effect on recidivism as well as indirect effects through their impact on social circumstances experienced after release from prison.

Fasciola hepatica: Histology of the testis in egg-producing adults of several laboratory-maintained isolates of flukes grown to maturity in cattle and sheep and in flukes from naturally infected hosts

A total of 8 calves approximately 6 months old and 22 lambs of similar age were infected with metacercariae of Fasciola hepatica of various laboratory-maintained isolates including: Cullompton (sensitive to triclabendazole) and Sligo, Oberon and Leon (reported as resistant to triclabendazole). Ten to 16 weeks after infection, flukes were harvested from these experimental animals and the histology of the testis tissue was examined in a representative sample of flukes from each population. Adult wild-type flukes were also collected from 5 chronically infected cattle and 7 chronically infected sheep identified at post-mortem inspection. The testis tissue of these flukes was compared with that of the various laboratory-maintained isolates. Whilst the testes of the wild-type, Oberon and Leon flukes displayed all the usual cell types associated with spermatogenesis in Fasciola hepatica (spermatogonia, spermatocytes, spermatids and mature sperm), the Cullompton flukes from both cattle and sheep showed arrested spermatogenesis, with no stages later than primary spermatocytes represented in the testis profiles. The presence of numerous eosinophilic apoptotic bodies and nuclear fragments suggested that meiotic division was anomalous and incomplete. In contrast to the wild-type flukes, no mature spermatozoa were present in the testes or amongst the shelled eggs in the uterus. A high proportion of the eggs collected from these flukes hatched to release normal-appearing miracidia after an appropriate incubation period, as indeed was the case with all isolates examined and the wild-type flukes. It is concluded that the eggs of Cullompton flukes are capable of development without fertilization, i.e. are parthenogenetic. The implications of this for rapid evolution of resistant clones following an anthelmintic selection event are discussed. Amongst the Sligo flukes examined, two subtypes were recognised, namely, those flukes with all stages of spermatogenesis and mature spermatozoa present in the testes (type 1), and those flukes with all stages of spermatogenesis up to spermatids present, but no maturing spermatozoa in the testes (type 2). Each sheep infected with the Sligo isolate had both type 1 (approximately 60%) and type 2 (approximately 40%) flukes present in the population. Spermatozoa were found amongst the eggs in the uterus in 64% of flukes and this did not necessarily reflect the occurrence of spermatozoa in the testis profiles of particular flukes, suggesting that cross-fertilization had occurred. The apparent disruption of meiosis in the spermatocytes of the Cullompton flukes is consistent with reports that Cullompton flukes are triploid (3n = 30), whereas the Sligo and wild-type flukes are diploid (2n = 20). In the Sligo flukes the populations are apparently genetically heterogenous, with a proportion of the flukes unable to produce fully formed spermatozoa perhaps because of a failure in spermiogenesis involving elongation of the nucleus during morphogenesis. (C) 2008 Elsevier B.V. All rights reserved.