Presence of bacteria and bacteriophages in full-scale trickling filters and an aerated constructed wetland.

Aerated Constructed Wetlands are a state-of-the-art design that provides a different physical and chemical environment (compared to traditional passive wetland designs) for the wastewater treatment processes and, thus, may have different pathogen removal characteristics. In order to establish the fate of bacterial and viral indicators, a field study was carried out at a Sewage Treatment Works (STW) in the UK (serving 20,000 pe). The STW consists of primary and secondary sedimentation tanks and trickling filters (TF) as the biological stage. A large (1,160 m2) pilot aerated Vertical Flow Constructed Wetland (AVFCW) was constructed at the STW as tertiary stage receiving » of the total flow rate, i.e., 1250 m3/day. Effluent quality of the AVFCW complied with national and international standards for environmental discharge and reuse. For the first time, two sets of bacterial (Faecal coliforms, E.coli and intestinal enterococci) and viral indicators (Somatic coliphages, F-RNA specific bacteriophages and human-specific B. fragilis GB124 phages) were simultaneously investigated in an AVFCW and TF. High elimination rates were detected (up to 3.7 and 2.2 log reduction for bacteria indicators and phages, respectively) and strong correlations between the two sets were found. The superior efficiency of the aerated Constructed Wetlands in microbiological contamination removal compared to passive wetland systems was established for the first time, which may have implications for process selection for wastewater reuse. This field study therefore provides new evidence on the fate of bacteriophages and a first indication of their potential use for performance evaluation in TF and aerated Constructed Wetlands. It also demonstrates that the combination of TF with aerated constructed wetlands could be a novel and effective treatment scheme for new STW or for the upgrade of existing STW.

Coccidiostatic effects of tannin-rich diets in rabbit production.

The potential anti-eimerial effect of tannin containing resources such as sainfoin and carob in rabbits was tested on does at pre-weaning and to growing rabbits in their feed. The trial began at parturition (D0), when 24 does and their litters were assigned into three groups. They were fed either with a control (Group CO), a carob (containing 10% carob pods meal) (Group CP) or a sainfoin diet (containing 34% dehydrated sainfoin pellets) (Group SA). All diets were made isoproteic and isoenergetic and also balanced for crude fibre but differed by their tannin content. Weaning occurred at D37, and growing rabbits remained in the same cage until D51. Then, they were transferred to fattening cages until the end of the trial (D104) and slaughtering. Weight gain of young rabbits among the three groups (mean = 31.2 g/day) did not differ statistically. The mortality rates were 10% (SA), 15% (CP) and 20% (CO), respectively, but the differences were not statistically significant. Post-weaning economical feed conversion ratio (FCR) was reduced between rabbits of group SA compared to CO and CP groups. Faecal oocyst count (FOC) in group SA was 60% lower than in CO and CP groups. Areas under the curve (AUCs) calculated between sampling days and FOC, after transfer to fattening cages, was 62% lower in group SA than in CO and CP groups. The main Eimeria species identified (from D59 to D83) was Eimeria magna (53% of oocysts). AUCs for E. magna did not differ according to diet. In conclusion, the diet containing sainfoin reduced oocyst excretion of Eimeria spp. by 60%, and improved the economical FCR.

The effect of sainfoin (Onobrychis viciifolia) and carob pods (Ceratonia siliqua) feeding regimes on the control of lamb coccidiosis.

Recent research has suggested that plants containing condensed tannins may offer a promising alternative approach for the control of coccidiosis in lambs and goat kids. The present study aimed to examine the potential effect of condensed tannins in sainfoin (Onobrychis viciifolia) and carob pods (Ceratonia siliqua) incorporated in sheep rations against lamb coccidiosis. The above tannin-rich sources were studied in three independent feeding trials in which the animals (naturally infected by Eimeria spp. ewes and their lambs) were allocated (i) in the control group and received a tannin-free diet (lucerne hay), or (ii) in the treatment groups and received a tannin-rich diet based on sainfoin hay (in trials 1 and 2), or in carob pod meal and a combination of carob pod meal and sainfoin hay (in trial 3). In total, 95 newborn lambs (and their 73 ewes) were enrolled in all trials which started a month before lambing and ended 8-10 weeks after lambs were born (at weaning). The course of coccidial infection was monitored in lambs by faecal oocyst counts and consistencies which were recorded at weekly intervals. Moreover, lambs total weight gain was evaluated at the end of each trial. During all trials, 100 % of the animals got naturally infected by Eimeria species and the infection burden was higher in trials 2 and 3 compared to trial 1 but in all cases, severe signs of diarrhoea were not observed. Tannin-rich diets were well accepted by the animals not affecting their feed intake and body weight gain when compared to the controls. The results suggest that incorporation of both tannin-rich resources (especially sainfoin) in sheep rations can reduce Eimeria oocyst excretion rates by the lambs, which can decrease subsequently the contamination of the farm environment with the parasite. However, the high variability noted on the results is not allowing us to draw any definite conclusions at least until the potential of those plants is further investigated.

Anthelmintic activity of some Mediterranean browse plants against parasitic nematodes.

The anthelmintic properties of tannin-rich plants are being explored as an alternative to chemical drugs. Most data have been acquired on legume forages, but only few on browse plants. The present study aimed to (i) screen the in vitro effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii) verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP) and (iii) verify the in vivo effects of extracts from 4 plants. Significant inhibition was shown in vitro using a larval migration inhibition (LMI) assay for all extracts except that from Olea europaea var. koroneiki. After adding PVPP, the LMI values were restored to control levels for all plants except Pistacia lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity. In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day 0, groups G1-G5 received H. contortus and Trichostrongylus colubriformis larvae and G6 remained uninfected. The various diets were distributed from Days 14 to 45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg excretion, packed cell volumes (PCVs) and inorganic phosphate were measured weekly throughout the entire experimental period. At slaughter, the worms were enumerated and their fecundity assessed. Consumption of the 4 browser plants did not provoke differences in pathophysiological measurements but there were significant decreases in egg excretion, mainly explained by significant decreases in worm fecundity for both species, without any statistical difference in worm numbers.

Surplus activated sludge dewatering in pilot-scale sludge drying reed beds.

A pilot-scale experiment on dewatering of surplus activated sludge (SAS) is presented, where two pilot-scale vertical flow, sludge drying reed beds (SDRBs), planted with Phragmites australis are used. The bottom of the beds is filled with cobbles, connected to the atmosphere through perforated PVC ventilation tubes, in order to achieve oxygen diffusion through the overlying porous medium that is colonized by roots and an abundant nitrifying biomass. Two layers of gravel, of decreasing size from bottom to top, make the drainage layer where the reeds are planted. The two beds were fed according to the following cycle: one week feeding with SAS at rates one 30 kg/m(2)/year and the other 75 kg/m(2)/year, and resting for three weeks. The results show that planted SDRBs can effectively dewater SAS from domestic sewage, the produced residual sludge presents a high dry weight content, the degree of volume reduction depends upon the initial SAS concentration and can be of the order of 90%, and decomposition of organic matter and high levels of mineralization can be achieved. Furthermore, the percolating water is not septic. The fertilizer value of the treated SAS, which contains no added chemicals, is comparable to that of SAS treated by other methods.

Molecular genetic analysis of populations of Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica, in outbreak populations from Greece and Morocco.

Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae), is the most important cause of traumatic myiasis in the southern Palaearctic region. Larval stages are obligate parasites and the wounds caused by infestations are very similar to those caused by Old and New World screwworm flies. During the last decade, W. magnifica appears to have expanded its range to parts of northern and central Morocco, and to Crete, Greece. Specimens of W. magnifica were collected in Morocco and Crete either as larvae (preserved in 80% ethanol) or as adults (dry-pinned). Comparison specimens were collected in Spain, Hungary and mainland Greece. A DNA fragment containing the 3' 715 base pairs of the mitochondrial cytochrome b gene was amplified by polymerase chain reaction from each of 132 larvae or adults of W. magnifica and the amplicons were directly sequenced and analysed phylogeographically. Twelve cytochrome b haplotypes were detected. All haplotypes from Morocco belonged to a lineage that included specimens from the Iberian peninsula, and restricted mixing of central and northern populations in Morocco was demonstrated. Cytochrome b haplotyping combined with an analysis of larval size provided clear evidence of multiple infestations of hosts in all geographical areas, with one quarter of wounds containing larvae from two to at least four females. More than 80% of specimens from Crete contained a haplotype predominating in mainland Greece and Hungary. Our survey indicated that wohlfahrtiosis was more widespread in northern and central Morocco than previously recorded by government veterinarians. However, the prevalence of wohlfahrtiosis was low (< 1%). The high genetic diversity of Moroccan populations is consistent with longterm endemicity, rather than recent introduction. Crete showed a higher prevalence of wohlfahrtiosis (< or = 15%) and less genetic diversity of W. magnifica, which is consistent with a recent introduction. The western and eastern Mediterranean lineages may have been isolated in different Pleistocene ice-age refugia, from which there has been limited post-glacial dispersal.

Supporting the extensive dairy sheep smallholders of the semi-arid region of Crete through technical intervention.

The objective of this field study was to depict the extensive system of dairy sheep farming in the semi-arid environment of the island of Crete and to assess the potential margins of improvement through technical intervention. Forty-three family-run farms keeping a total of 13,870 sheep were surveyed in seven representative areas of the island. Several parameters were dealt with, concerning socio-economy, flock management and productivity. Study areas differed widely regarding feeds supplied per sheep, land cultivated for feeds, grazing land utilized and housing space. A range of parameters were recorded on flock size and their production characteristics such as births, fertility and number of lambs weaned. Milk yield and parameters associated with milk quality, such as somatic cell counts and total microbial flora, were also recorded. Technical intervention was directed towards removal of non-productive animals, programming of matings, balancing of diets, management of grazing lands and health care. Ewe fertility and numbers of lambs weaned per ewe, as well as harvested milk and milk quality (based on somatic cell counts and microbial load of milk) were also significantly improved. Information derived from this study stresses the important role of extension services to small farm sustainability and contributes to our knowledge of the dairy sheep farming systems in countries around the Mediterranean and elsewhere.

Wohlfahrtiosis in sheep and the role of dicyclanil in its prevention.

Wohlfahrtia magnifica is the main agent of traumatic myiasis in the southern Palaearctic zone. It was recorded in outbreak situations in Crete, Greece, for the first time in 1999, causing widespread production losses and considerable concern in the livestock sectors. Most commonly applied curative insecticides, organophosphates and pyrethroids, can kill larvae of W. magnifica, but they do not provide long-term protection from infestation or re-infestation, which would facilitate wound healing. The objective of the present work was to study the seasonal dynamics of sheep wohlfahrtiosis in Crete and to determine the prophylactic efficacy of dicyclanil, an insect growth regulator, against natural infestations by larvae of W. magnifica. Six sheep flocks were studied, three from a semi-intensive husbandry system and three from an extensive husbandry system. Two flocks were kept as untreated controls and also were used to study the disease dynamics. Dicyclanil was strategically applied just to males and young non-milking females in four treatment flocks; milking females in these flocks were not treated. The untreated flocks demonstrated seasonal patterns in case numbers that were associated with changes in climate and husbandry activities, especially reproduction and shearing. Cases were most common on the genitalia (60%) and a greater proportion of males than females were infested. The prophylactic efficacy of dicyclanil in males was up to 91.3% over the entire trial period. Hence the incidence in treated males at 22 weeks, when final infestations were recorded, was 5-10% compared to 45-55% in untreated controls. Moreover, the application of dicyclanil to a limited number of animals per flock (males and non-milking females=c. 15-20% of the flock) significantly reduced the incidence of wohlfahrtiosis even in the untreated animals. Hence, final overall incidences in the treated flocks ranged from 4.5 to 5.5% compared to 13.3-13.4% in the control flocks.

Field trial of the efficacy of dicyclanil for the prevention of wohlfahrtiosis of sheep.

Two groups of 40 ewes from each of two flocks were used in a field trial of the efficacy of dicyclanil to protect them against a natural infestation by Wohlfahrtia magnifica larvae, when administered topically as a spray-on formulation. The animals of one group in each flock were treated with dicyclanil and those of the other group were left untreated as controls. The trial lasted 31 weeks and the animals were inspected daily for wohlfahrtiosis. The incidences of wohlfahrtiosis ranged from 17.5 per cent to 20 per cent in the control groups and from 2.5 per cent to 5 per cent in the treated groups, and 77.8 per cent of the infestations were in the genitalia. When compared with the control groups, dicyclanil achieved a 100 per cent reduction of infestation in the treated groups of both flocks for at least 24 weeks, and an 80 per cent reduction for 31 weeks.

Assessment of cypermethrin and doramectin for controlling wohlfahrtiosis in Crete.

The fleshfly, Wohlfahrtia magnifica, became an animal health problem in Crete, Greece, during 1999, causing outbreaks of wohlfahrtiosis throughout the main livestock producing regions of the island. The aim of this study was to determine the efficacy of commercially available drugs in order to design control strategies against wohlfahrtiosis. The test substances were cypermethrin and doramectin sc. The test animals were randomly divided between three groups (30 in each): one treated with cypermethrin pour-on, one treated with doramectin and one untreated controls. The trial lasted 45 days and the animals were inspected daily for natural infestations by W. magnifica. The first infestations were recorded on the 5th, 13th and the 23rd day in the control, cypermethrin and doramectin groups, respectively. There was a statistically significant difference between the survival curves for doramectin and the control group up to day 40, but not at the end of the study.

Assessment of progesterone concentration using enzymeimmunoassay, for early pregnancy diagnosis in sheep and goats.

The objective of this study was to determine a value of serum progesterone (P4) concentration, assessed using an enzymeimmunoassay (EIA), for the early distinction between pregnant and non-pregnant ewes and goats. Adult, non-lactating ewes of Chios (n=53), Berrichon (n=30) and Sfakia (n=45) breeds were synchronized during the breeding season with progestagens and gonadotrophins and mated to fertile rams (Experiment I). Adult, lactating goats of Swiss breeds (Alpine and Saanen, n=104) and indigenous Greek breed (n=45) were synchronized during the transitional season with progestagens, PGF2alpha and gonadotrophins. Cervical artificial insemination (AI) with fresh semen was applied once, 42-44 h after sponge removal (Experiment II). Jugular blood samples were collected on day 19 after sponge removal (ewes) or on day 21 after AI (goats) and serum P4 concentration was determined by EIA. Progesterone concentrations >/=1.0, >/=1.5, >/=2.5 and >/=4.0 ng/ml were tested as indicative of pregnancy. Pregnancy diagnosis was verified on birth. In the case of sheep, using a discriminatory level of 2.5 ng/ml, overall accuracy of pregnancy diagnosis was 91.4% and predictive value of negative and positive diagnoses were 98.3 and 85.3%, respectively. In the case of goats, predictive value of negative diagnosis was 95.8 and 94.0% and predictive value of positive diagnosis 71.3 and 71.7%, for 1.5 and 2.5 ng/ml, respectively; overall accuracy was 79.2% using either level. The other discriminatory levels tested did not improve these results. A significant positive correlation was observed between P4 concentration and the number of lambs or kids born, and further analysis indicated that this relationship is not a simple linear function. Based on the results of this study, P4 concentrations of 2.5 ng/ml in the case of ewes and 1.5-2.5 ng/ml in the case of goats, determined with EIA, are proposed as discriminatory levels between pregnant and non-pregnant animals, at an interval of one oestrous cycle after service.

Use of progestagen-gonadotrophin treatments in estrus synchronization of sheep.

The main objective of this study was to investigate the effectiveness of certain progestagen-gonadotrophin treatments on synchronization of estrus in sheep. In Experiment I, 30 Chios ewes were treated at the beginning of the breeding season with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days and a single i.m. treatment of either FSH (Group 1,10 IU, n = 8; Group 2, 5 IU, n = 8; Group 3, 2.5 IU, n = 8) or eCG (Group 4, 400 IU, n = 6) at the time of sponge removal. Ten days after sponge removal laparotomy was performed to record ovarian response. Clinical estrus was observed in more (though not at a significant level) FSH treated than eCG treated sheep (62.5% versus 33.3%). Administration of 400 IU eCG resulted in the highest mean number of CL perewe ovulating (2.8 +/- 0.2), with administration of 10 IU FSH producing the next best results (2.1 +/- 0.3). Statistically significant differences in the mean number of CL per ewe ovulating were found only between ewes in Group 3 (1.7 +/- 0.4) and Group 4 (2.8 +/- 0.2) (P < 0.05). In Experiment II, 53 Chios and 30 Berrichon ewes were treated during the mid-breeding season with MAP intravaginal sponges for 12 days and a single i.m. treatment of either 10 IU FSH (27 Chios and 16 Berrichon ewes) or 400 IU eCG (26 Chios and 14 Berrichon ewes), at the time of sponge removal. Ewes that were in estrus on Days 2-4 and 19-23 after sponge removal were mated to fertile rams. No significant differences were recorded between treatment or breed groups in the proportions of ewes observed in estrus after treatment. In the Berrichon breed, FSH administration resulted in higher lambing rates (93.8% versus 57.1%, P < 0.05) and higher mean number of lambs born per ewe exposed to rams (1.4 +/- 0.2 versus 0.8 +/- 0.2, P < 0.05) than that of eCG. After treatment with eCG, the mean number of lambs born per ewe exposed to rams was higher in the Chios than the Berrichon breed (1.4 +/- 0.2 versus 0.8 +/- 0.2, P < 0.05). After treatment with FSH, the lambing rate was higher in the Berrichon than the Chios breed (93.8% versus 63.0%, P < 0.05). In conclusion, a single FSH treatment (5 or 10 IU) at the end of progestagen treatment appears to be more effective than eCG for the induction of synchronized estrus in sheep at the beginning of the breeding season, with no cases of abnormal ovarian response observed. During the mid-breeding season FSH (10 IU) appears to be equally as effective as eCG (400 IU) in respect of lambing rate and mean number of lambs born per ewe.

Use of enzyme-immunoassay for oestradiol-17beta and progesterone quantification in canine serum.

The objective of this investigation was to develop and evaluate competitive inhibition-enzyme-immunoassays for canine serum oestradiol-17beta (E(2)) and progesterone (P(4)) quantification. Sera from 56 healthy bitches at various stages of oestrus cycle and pregnancy were tested. For E(2) measurement, each sample (0.4 ml) was extracted with diethyl ether and after solvent evaporation the resultant hormone was reconstituted to one-fifth of the original sample volume in aqueous buffer. Each reconstitute (30 microl) was assayed for E(2) to estimate respective serum concentration. For P(4), each sample (10 microl) was directly assayed without extraction. The classic cyclic hormonal pattern during the oestrus cycle of the bitch was observed. The brief, sharp dominance of E(2) during the follicular phase was followed by the long-lasting dominance of P(4) during the luteal phase (late oestrus, dioestrus or pregnancy). During the anoestrus phase both hormones were found at basal levels, with the exception of E(2) during late anoestrus which appeared to be rising. Both assays had acceptable specificity (cross-reactions < or =10%), precision (coefficient of variation (C.V.) < 7%) and accuracy (E(2) recovery: 97%; P(4) recovery: 104.7%). The sensitivity of E(2) and P(4) assay was 4 pgml(-1) and 0.28 ngml(-1), respectively.

Development and evaluation of a direct enzyme immunoassay for oestrone sulphate in urine as a tool for diagnosis of early pregnancy in swine.

A competitive inhibition-type enzymeimmunoassay (EIA) has been developed using 3-hemisuccinate-oestrone-peroxidase as conjugate for direct measurement of the hormone in swine urine. The method has a minimum detection level at 0.3 ng ml(-1) and satisfactory specificity, recovery and reproducibility. In a field trial with a group of 387 sows (7 in oestrus, 16 non-pregnant and 364 pregnant sows at several stages post service), it was shown that the assay is potentially an accurate pregnancy test in assessing the viability of the fetoplacental unit from day 23 up to day 30 post service. The assay is well suited for routine testing, particularly as a swine early pregnancy diagnosis test since urine sampling is easier and does not disturb the animal, while in the present assay there is no restriction in the time of sampling and the sample storage conditions.

Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay.

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).

Comparison of different antibody-conjugate derivatives for the development of a sensitive and specific progesterone assay.

Antibodies raised against progesterone with hormone-carrier protein bridges placed at four different carbon positions were used to compare the sensitivity and specificity of homologous and heterologous enzyme-hormone conjugates. All heterologous assays were at least twice as sensitive as the corresponding homologous assays. The best results were obtained by using antibodies against 7 alpha-carboxyethyl-thioether-progesterone with 6 beta-hemisuccinate-progesterone conjugate (or hemimaleate). The sensitivity with human sera was 0.25 ng ml-1 and, the highest crossreaction 10% with 5 beta-pregnane-3,20-dione, and reproducibility, recovery and accuracy were satisfactory. The correlation coefficient with radioimmunoassay in 103 human sera tested was r = 0.915. The assay was successfully applied for the diagnosis of pregnancy in dairy cattle.