Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues

The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin‐fixed, paraffin‐embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)‐based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat‐shock protein (hsp65) gene. PCR‐restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false‐negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (～30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR‐based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax‐embedded and formalin‐fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies.     

Effects of recombinant equine somatotropin on wound healing, carbohydrate and lipid metabolism, and endogenous somatotropin responses to secretagogues in geldings.

The primary purpose of this experiment was to assess the possible beneficial effects of recombinant equine somatotropin (reST) administration on wound healing in adult geldings. The effects of the 21-d reST treatment on carbohydrate and lipid metabolism and on endogenous ST characteristics were monitored as well. Single, full-thickness skin incisions (7.62 x 7.62 cm) were made in the pectoral region of all geldings on d 0. Treated geldings received reST at 20 microg/kg BW i.m., and control geldings received vehicle (10 mM sodium borate) at equivalent volumes daily from d 0 (immediately after surgery) through d 20. Tracings of the wounds were made with acetate transparencies, and wound areas were calculated via a digital analyzer. In addition to once-daily blood samples collected at specified days throughout the treatment period, an i.v. glucose tolerance test was performed on d 16, and three assessments of endogenous ST secretion were performed in the 2 d immediately following the end of treatment: epinephrine administration during the morning of d 21, an exercise test during the afternoon of d 21, and i.v. aspartic acid infusion on d 22. There was no effect (P > . 1) of reST treatment on wound healing as assessed by changes in wound areas. Daily plasma ST, IGF-I, glucose, and insulin concentrations were higher (P < .05) and urea-nitrogen concentrations were lower (P < .001) in geldings receiving reST relative to controls. Glucose, NEFA, and insulin concentrations were all higher (P < .01) in reST-treated geldings before glucose infusion on d 16, and the responses to glucose were greater (P < .05) as well. Epinephrine administration increased (P < .02) ST concentrations in control geldings on d 21 but not in reST-treated geldings; a similar suppressive effect of reST treatment was observed for the ST response to exercise (P < .001). After aspartic acid infusion on d 22, reST-treated geldings had a much smaller (P < .001) ST response than did control geldings. In conclusion, reST administered to geldings at 20 microg/kg BW i.m. caused hyperglycemia, hyperinsulinemia, insulin insensitivity, mobilization of fatty acids, and an apparent negative feedback on the pituitary's ST response to various stimuli known to induce ST secretion. However, there was no beneficial effect of reST treatment with the wound model used in this experiment.     

Bovine abortions associated with Bacteroides fragilis fetal infection.

Two Santa Gertrudis cattle from a herd of 105 aborted within a 24-hour period. Bacteroides fragilis was isolated from tissues of each aborted fetus. Histopathologic lesions included placentitis and bronchopneumonia in which gram-negative, rod-shaped organisms were visible. The diagnostic workup failed to reveal other causes of abortion. Anaerobes are rarely implicated in bovine abortions, and no other report was found that described abortion in cattle due to B. fragilis.     

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm‐water fish for which no vaccines are commercially available. In this study, a whole cell formalin‐inactivated vaccine was developed for the first time using the highly virulent isolate STIR‐GUS‐F2f7 and the oil‐based adjuvant Montanide? ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post‐vaccination, all fish were i.p. injected with 4.0 × 10³ CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.     

Effects of age, season and active immunization against estrogen on serum prolactin concentrations in stallions

Prolactin concentrations in stallion serum were measured by radioimmunoassay based on antiserum generated against equine prolactin and radioiodinated canine prolactin. Prolactin concentrations in serum collected from 152 stallions at a slaughterhouse were higher (P less than .01) in summer than in winter (5.7 +/- .15 vs 2.0 +/- .17 ng/ml). Moreover, there was an effect of age (P less than .02) in the analysis of variance; there was no interaction between age and season. In general, prolactin concentrations increased with age up to 3 to 5 years. Samples of serum collected from five control and five estrogen-immunized stallions from 6 to 26 months of age also were assessed for prolactin concentration. Active immunization against estrogen had no effect (P greater than .10) on prolactin concentrations. In contrast, whether analyzed by age or by month of the year, prolactin concentrations varied with time (P less than .01) in these stallions. Averaged over both groups, concentrations were high during summer and low during winter and were also gradually increasing with time as indicated by a linear trend (P less than .01) in the data. It is concluded that serum prolactin concentrations in stallions 1) are seasonal, as in many other seasonally breeding species, 2) in general are highest in older stallions and lowest in young stallions and 3) are not affected by active immunization against estrogens.     

Patch dynamics and the design of nature reserves

Island biogeographic theory has been applied to the design of nature reserves. However, immigration, which is important in maintaining species equilibrium on true islands, will not contribute significantly to the maintenance of equilibrium on reserves in the future because of the disappearance of recolonisation sources. Consequently, extinction becomes the dominant population process, and the internal disturbance dynamics become the critical design feature of reserves. The design of reserves should be based on ‘minimum dynamic area’, the smallest area with a natural disturbance regime which maintains internal recolonisation sources and hence minimises extinctions. Determination of minimum dynamic area must be based on knowledge of disturbance-generated patch size, frequency, and longevity, and the mobilities of the preserved species. These features have not all been explicitly considered in the previous island biogeographic design recommendations.     

Linkage between S alleles and pigmentation genes in marrow-stem kale,brassica oleracea

Pigmentation genes can be used as markers to facilitate the recognition of plants, homozygous for S alleles. Such plants are necessary in kale breeding for the production of 100 per cent hybrid seed. Close linkage has been found in marrow-stem kale between S alleles and the genes controlling hypocotyl and stem colour, spotting of anthers and marking of sepals and fruit. Of these, anther spotting is one of the most useful markers in kale.