Characterization by whole-cell hybridization of bacterial populations associated with shrimp hatchery biofilms

The impact of shrimp larvae development, as well as water and food inputs upon the increase of bacterial populations within the bacterial community of hatchery tank biofilms, was studied. For this study, a total of 68 biofilm samples were collected from concrete tanks at three larvae production times in a commercial shrimp hatchery. Seventeen samples were taken at each larval development stage (Zoea I, Mysis I, postlarvae 1 and postlarvae 16), as well as 37 samples from water, shrimp nauplii and food, introduced into the shrimp hatchery tanks. Culturable and direct bacterial counts were performed and 16S‐rRNA‐targeted oligonucleotide probes were used to quantify the presence of specific bacterial groups. An average of 27–70% of DAPI total cell counts were detected with the EUB338 probe, while the GAM42a probe signal ranged from 1% to 11%. Vibrio‐like bacteria (VLB) counts in TCBS agar ranged from <10 to 10¹ VLB/cm⁻², with a tendency to increase at the last postlarvae stage. The most significant external source of bacteria registered with GAM42a probe and TCBS agar were found in live Artemia nauplii, used as food; nevertheless, biofilms remain with low counts of these groups.     

Benthic microbial indicators of fish farm impact in a coastal area of the Tyrrhenian Sea

We studied the impact of organic loads due to the biodeposition of a fish farm in a non-impacted coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry and benthic microbial community were investigated from July 1997 to February 1998 on monthly basis at two stations: one was located under the fish farm, while the second was about 1 km away, and served as a reference site. The presence of the cage induced rapid changes in the benthic conditions: the sediments were rapidly (after 6 weeks) reduced. A significant accumulation of biopolymeric carbon was observed beneath the cage both 2 weeks after the initial cage deployment, on non-impacted sediments, and 5–7 months after, and appeared to be related to the fish farm production cycle. The density of microbial communities beneath the fish farm increased only during the first month of farming activity (July) and partially 7 months after cage disposal (i.e. in January), when there was a significant sediment organic enrichment. Additional evidence of rapid impact of the fish farm on the benthic bacterial communities is provided by the increase in the numbers of autofluorescent microbial cells. The photosynthetic eukaryotic cells displayed a highly reduced contribution to total autofluorescent microbial density, indicating that their decrease is related with biopolymeric carbon accumulation, and probably was due also to “shadow effect” induced by suspended material, coming from the fish farm, on the environment below. We propose here, to apply the ratio of culturable heterotrophic bacteria to microbial direct counts (CFU/MDC) to detect fish farm impact. In cage sediments, organic enrichment and the consequent modification of the characteristics of the benthic environment, determined an increase in aerobic heterotrophic bacteria and vibrio density indicating that they are efficient colonizers of organic-rich sediments. Densities of Escherichia coli and Enterococci were not significantly higher than in the reference site, and are likely of terrestrial origin. Thus they cannot be used as specific indicators of fish farm impact.     

PREVALENCE OF COMPUTED TOMOGRAPHIC SUBCHONDRAL BONE LESIONS IN THE SCAPULOHUMERAL JOINT OF 32 IMMATURE DOGS WITH THORACIC LIMB LAMENESS

Osteochondrosis is a common developmental abnormality affecting the subchondral bone of immature, large breed dogs. The purpose of this retrospective study was to describe CT lesions detected in scapulohumeral joints of 32 immature dogs undergoing CT for thoracic limb lameness. Eight dogs (14 scapulohumeral joints) had arthroscopy following imaging. Thirteen dogs (19 scapulohumeral joints) were found to have CT lesions, including 10 dogs (16 scapulohumeral joints) with subchondral bone lesions and 3 dogs with enthesopathy of the supraspinatus tendon. In one dog, subchondral bone lesions appeared as large oval defects within the mid‐aspect of the glenoid cavities, bilaterally. These lesions resembled osseous cyst‐like lesions commonly identified in the horse. This is the first report of such a presentation of a subchondral bone lesion in the glenoid cavity of a dog. In all dogs, small, focal, round or linear lucent defects were visible within the cortical bone at the junction of the greater tubercle and intertubercular groove. These structures were thought to represent vascular channels. Findings from this study support the use of CT as an adjunct modality for the identification and characterization of scapulohumeral subchondral bone lesions in immature dogs with thoracic limb lameness.     

Parentage assignment of stocked black sea bream <Emphasis Type="Italic">Acanthopagrus schlegelii</Emphasis> in Hiroshima Bay using microsatellite DNA markers

The genetic contribution of 51 broodstock, comprising 29 females and 22 males, reared at Hiroshima City Marine Products Promotion Center for the production of stocked black sea bream was monitored during two consecutive years using seven microsatellite DNA loci. The high discrimination ability of these markers was reflected in the polymorphic identification content (PIC=0.831), the exclusion probability (Q≈1), and the low probability of identity index (I=3.635⁻¹⁰). The total number of breeders contributing to the mating process was estimated at 32 (62.7%) in 2000 and 30 (58.8%) in 2001. On pedigree reconstruction, 69.3% of the offspring were successfully assigned to a single broodstock pair. Loss of alleles accounted for 16.9% during seed production; nevertheless, 90.9% of males and 69.0% of females participated in the mating process. Based on microsatellite genetic tagging, 58.9% of the fish sampled during the two months after release were identified as hatchery stock, presenting no significant differences from wild conspecifics in either fork length or body weight.     

β-cyclodextrin and caffeine complexes with natural polyphenols from olive and olive oils: NMR, thermodynamic, and molecular modeling studies

Complexes of β-cyclodextrin (β-CD) and caffeine (Caf) with biophenols present in olive and olive oil (tyrosol, hydroxytyrosol, homovanillic acid, 3,4-dihydroxyphenylacetic acid, and protocatechuic acid) were investigated by NMR spectroscopy and thermodynamical-molecular dynamic studies to verify the formation of supermolecular aggregates. The obtained results indicated that the investigated biophenols form inclusion complexes with β-CD in a molar ratio of 1:1 in aqueous solution having binding constant values from 10- to 40-fold bigger than those of the corresponding complexes with Caf. Then, β-CD preferentially encloses the biophenol molecule, decreasing its bitter taste and, at the same time, preserving it against chemical and physical decomposition reactions that occur during storage.     

First Isolation of Rotavirus Associated with Neonatal Diarrhoea in Guanacos (Lama guanicoe) in the Argentinean Patagonia Region

Group A rotavirus (RV) and coronavirus (CV) are common viral pathogens associated with neonatal diarrhoea in numerous animal species. The purpose of this work was to investigate the presence of these viral agents in two farm populations of captured guanacos (Lama guanicoe) in the Argentinean Patagonia region, that developed severe diarrhoea outbreaks. Stool and serum samples were analysed for RV and bovine CV antigen and antibody by enzyme‐linked immunosorbent assay. Rotavirus was detected in faeces from two new‐born guanacos with acute diarrhoea, one in each farm. After electrophoretic analysis, each isolated strain, showed a distinctive long dsRNA electropherotype characteristic of group A rotaviruses (4:2:3:2). In addition, 95% (38 of 40) of the sampled animals were positive for RV antibodies, suggesting a high prevalence of RV infection in the populations tested. No evidence of CV circulation by antigen or antibody analysis was observed. To our knowledge, this is the first report of the detection and isolation of RV associated with neonatal diarrhoea in Lama guanicoe.     

Producing progeny from endangered birds of prey: treatment of urine-contaminated semen and a novel intramagnal insemination approach.

Wild raptors brought into an ex situ environment often have poor semen quality that is further compromised by urine contamination. Generally, it is believed that in birds, artificial insemination into the cloaca or caudal vagina of females requires large doses of high-quality spermatozoa to maximize fertility. In an effort to define and overcome some of the challenges associated with reproduction in wild raptors, the objectives of this study were to 1) evaluate the frequency, impact, and remediation of urine contamination in fresh ejaculates for the purpose of maintaining sperm motility and viability in vitro, and 2) develop a deep insemination method that allows low numbers of washed sperm to be placed directly into the magnum to increase the probability of producing fertilized eggs. The species evaluated include golden eagle (Aquila chrysoetos), imperial eagle (A. adalberti), Bonelli's eagle (Hiernaetus fasciatus), and peregrine falcon (Falco peregrinus). Semen samples were collected and pooled by species, and a minimum of 25 pooled ejaculates per species were evaluated for urine contamination, pH, sperm viability, and sperm motility; the samples were either unwashed or washed in neutral (pH 7.0) or alkaline (pH 8.0) modified Lake's diluent. Female golden eagles and peregrine falcons were inseminated via transjunctional, intramagnal insemination with washed spermatozoa from urine-contaminated samples. Urine contamination occurred in 36.8 +/- 12.8% (mean +/- SEM) golden eagle, 43.1 +/- 9.1% imperial eagle. 28.7 +/- 16.1% Bonelli's eagle, and 48.2 +/- 17.3% peregrine falcon ejaculates. The pH in urine-contaminated semen samples ranged from 6.48 +/- 0.3 to 6.86 +/- 0.2, and in noncontaminated samples it ranged from from 7.17 +/- 0.1 to 7.56 +/- 0.1. Sperm viability and motility were reduced (P < 0.05) in all species for unwashed vs. washed sperm after 30 min incubation at room temperature. Two peregrine falcon chicks and one golden eagle chick hatched after intramagnal insemination. This study demonstrates that urine contamination, a common and lethal acidifier in manually collected raptor ejaculates, can be circumvented by immediate, gentle seminal washing. Furthermore, these processed sperm, when deposited by transjunctional intramagnal insemination, can produce live young.     

Effects of remifentanil infusion regimens on cardiovascular function and responses to noxious stimulation in propofol-anesthetized cats

OBJECTIVE: To evaluate the effects of 2 remifentanil infusion regimens on cardiovascular function and responses to nociceptive stimulation in propofol-anesthetized cats. ANIMALS: 8 adult cats. PROCEDURES: On 2 occasions, cats received acepromazine followed by propofol (6 mg/kg then 0.3 mg/kg/min, i.v.) and a constant rate infusion (CRI) of remifentanil (0.2 or 0.3 microg/kg/ min, i.v.) for 90 minutes and underwent mechanical ventilation (phase I). After recording physiologic variables, an electrical stimulus (50 V; 50 Hz; 10 milliseconds) was applied to a forelimb to assess motor responses to nociceptive stimulation. After an interval (> or = 10 days), the same cats were anesthetized via administration of acepromazine and a similar infusion regimen of propofol; the remifentanil infusion rate adjustments that were required to inhibit cardiovascular responses to ovariohysterectomy were recorded (phase II). RESULTS: In phase I, heart rate and arterial pressure did not differ between remifentanil-treated groups. From 30 to 90 minutes, cats receiving 0.3 microg of remifentanil/kg/min had no response to noxious stimulation. Purposeful movement was detected more frequently in cats receiving 0.2 microg of remifentanil/kg/min. In phase II, the highest dosage (mean +/- SEM) of remifentanil that prevented cardiovascular responses was 0.23 +/- 0.01 microg/kg/min. For all experiments, mean time from infusion cessation until standing ranged from 115 to 140 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: Although the lower infusion rate of remifentanil allowed ovariohysterectomy to be performed, a CRI of 0.3 microg/kg/min was necessary to prevent motor response to electrical stimulation in propofol-anesthetized cats. Recovery from anesthesia was prolonged with this technique.     

Leishmania-specific isotype levels and their relationship with specific cell-mediated immunity parameters in canine leishmaniasis

In the current retrospective study, Leishmania infantum-specific IgG, IgA and IgM levels were determined by ELISA in 106 untreated dogs with clinically-patent leishmaniasis (Sx) and in 171 clinically healthy dogs (Asx) from Spain to investigate the relationship between these Ig isotypes and clinical status. In addition, we studied if different Leishmania-specific humoral pattern exists between Asx dogs with and without cellular mediated immunity (CMI). Fifty-six dogs were assessed by means of lymphoproliferation assay (LPA), interferon production (IFN) and leishmanin skin test (LST), 71 dogs by means of both LPA and IFN and 44 only by LST. Both Sx and Asx dogs produced Leishmania-specific IgG, IgA and IgM antibodies, however the levels and proportion of positive dogs for each Ig isotype were significantly higher in Sx than in Asx ones (P<0.001). Analysis of immunological profiles determined for each cellular technique (positive and negative cellular response for each technique combined with positive or negative specific humoral response) showed that Asx dogs constituted a high heterogeneous group. No correlations were observed between CMI tests and specific IgG or IgM levels. However, a significant inverse correlation was demonstrated between specific IgA levels and LPA response (Spearman's r=-0.220; P=0.035). In general, the low correlation detected between CMI tests and isotype levels might indicate that the immune response is not strongly polarized in the majority of Asx dogs. Additionally, this study suggests that dogs developing T-cell response are probably able to avoid the dissemination of the parasite at least to mucosal surfaces and, as a consequence, to produce low or background specific IgA levels. Further studies are needed to investigate the relationship between specific IgA and parasite load, especially at mucosal site.