Ovarian morphology and internal vis-à-vis non internal laying in relation to triacylglycerol, hormones and their receptors concentration around the age of sexual maturity in broiler breeder hens.

1. Ovarian morphology, serum hormone concentrations of 17-ß-estradiol, progesterone, testosterone, tri-iodothyronine (T3), thyroxine (T4) and triacylglycerol (TAG) were investigated at 23 and 26 weeks of age in broiler breeder hens provided with ad libitum access to feed. Progesterone, oestrogen-ß, thyroid-α and -ß receptor mRNAs were also quantified in the infundibulum at the same ages. 2. A large variation in the ovarian morphology was observed at 23 weeks of age including hens with undeveloped ovaries, non-laying hens with post ovulatory follicles (POF) and a predominance of non-laying hens without a POF. 3. Serum concentrations of triglyceride, 17-ß-estradiol and progesterone at 23 weeks of age were lower in hens with an undeveloped ovary compared with other groups of hens, whereas testosterone, triiodothyronine and thyroxin were higher. 4. At 26 weeks of age, the average number of hierarchical yellow follicles in normal layers was 7.64 ± 0·41 whereas in internal layers, the follicular numbers were significantly greater at 8.66 ± 0·53. The higher follicular numbers in internal layers were associated with higher serum triglyceride and progesterone concentrations. 5. Oestrogen receptor-ß and thyroid receptor-ß mRNA was up regulated in the infundibulum of internal layers compared with normal laying hens at 26 weeks of age.

Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken.

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates various biological functions through three different receptor subtypes (Mel-1a, Mel-1b, and Mel-1c). However, the distribution of different melatonin receptor subtypes in chicken reproductive tissues was not known. In the present investigation, the partial sequences of ovarian melatonin receptor subtypes (Mel-1a, Mel-1b, and Mel-1c) were characterized. Further, the expression profile of melatonin receptor subtypes in the granulosa and theca layers of different preovulatory and postovulatory follicles (POF) were studied by semi-quantitative RT-PCR. The expression of all three subtypes of melatonin receptors were observed in the ovary of domestic chicken. Analysis of partial sequences of ovarian melatonin receptors revealed that the melatonin subtypes were identical to the brain receptors. In small white ovary follicles, we observed only the expression of mel-1b receptors, but not mel-1a or mel-1c receptors. In yellow follicles, all the three subtypes of receptors expression were noticed. Interestingly, we observed the expression of mel-1a receptor only in thecal layer, but not in granulosa layer. In contrast, mel-1b and -1c receptors were expressed in both granulosa and thecal layer. During the regression of POF, we observed significant upregulation of melatonin receptors (mel-1a and 1c) expression, that downregulated in the later stages of regression. We assume that the expression of melatonin receptors might have been influenced by the atresia or apoptosis of different follicular layers in POF. Our findings suggest that the differential distribution of melatonin receptor subtypes might have distinct downstream cellular functions in the ovarian tissues.

Effect of in ovo threonine supplementation on early growth, immunological responses and digestive enzyme activities in broiler chickens.

1. The effects of injecting threonine in ovo on early growth, some immunological responses and the activity of digestive enzymes of broiler chicks were investigated. Fertile eggs were distributed into 6 groups, each of 60. These were: untreated control, sham control, 10, 20, 30 or 40 mg threonine. Threonine was dissolved in 0.5 ml sterile saline and inoculated into the yolk sac of the 14-d-old embryo through the narrow end of the egg. 2. The ratio of chick to egg weight was 1.6% higher in the group given 30 mg threonine and at 28 d of age chicks receiving threonine were 29 to 79 g heavier than untreated controls. 3. Food conversion ratio until 7 d after hatching was improved in those chicks receiving 10, 20 or 40 mg threonine but there was no significant effect on the activities of amylase, pepsin or trypsin. 4. The humoral response to sheep red blood cells was significantly greater in those groups receiving 10, 20 or 30 mg threonine supplementation than in untreated controls. 5. The response to phytohaemagglutinin-P, a measure of the cell-mediated immune response, was not affected, however. 6. It is concluded that injections of 20 to 30 mg threonine into yolk sac can improve post-hatching growth and humoral responses of broiler chicks.

Effect of dietary Vitamin E on the cloacal gland, foam and semen characteristics of male Japanese quail.

This experiment was to investigate the effects of increasing the level of dietary Vitamin E (Vit. E) on cloacal gland size, foam production and semen characteristics of male Japanese quail (Coturnix coturnix Japonica). One hundred and eighty male Japanese quail chicks (day old) were randomly distributed to three dietary treatments for a period of 25 weeks. Each treatment comprised of three replicates each containing 20 chicks. The basal diet contained 15 IU Vit. E/kg and the two experimental diets were supplemented with 150 and 300 IU Vit. E/kg (diets T2 and T3, respectively). DL alpha-tocopherol acetate was used as the source of Vit. E. All chicks were provided feed and water ad libitum. Foam characteristics, in terms of frequency of foam discharge (24h), cloacal gland index and foam weight were significantly higher (P<0.05) in T2 group. Body weight, testes weight (left and right) and plasma testosterone concentrations did not differ significantly. Semen characteristics (semen volume, sperm motility, % live sperm, % hatchability and sperm concentrations) did not differ significantly (P>0.05). Percentages of abnormal and dead spermatozoa were significantly (P<0.05) lower and fertility was higher (P<0.05) in the T2 group. From this study, it can be concluded that moderate supplementation of dietary Vit. E may be beneficial for foam production, cloacal gland and improve the semen characteristics in male Japanese quail.

Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis.

The mechanism(s) used by Mycobacterium tuberculosis to establish disease in the human host are not well understood. The virulent M. tuberculosis H37Rv strain and its avirulent derivative M. tuberculosis H37Ra provide an attractive system for the identification of virulence-specific genes of the tubercle bacillus. Differentially expressed genes in the virulent strain of M. tuberculosis (dev genes) were identified by screening a plasmid gene bank of H37Rv with a cDNA probe that was enriched in dev transcripts by subtraction of RNAs common to H37Ra. Individual dev clones coded for RNA transcripts that were differentially expressed in H37Rv in comparison to H37Ra. In contrast, mRNAs and stable RNAs that were commonly expressed in both the strains were present in equivalent amounts. The identification and cloning of dev genes marks the first step in defining bacterial gene(s) involved in the pathogenesis of M. tuberculosis.

Functional analysis of transcription of the Mycobacterium tuberculosis 16S rDNA-encoding gene.

A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study. RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth. Transcription start points (tsp) were mapped by primer extension analysis of RNA from M. tuberculosis H37Rv and M. tuberculosis H37Ra. An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M. tuberculosis H37Rv and H37Ra. One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter. The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E. coli. The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene (cat) in a shuttle plasmid vector, pSD7. The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M. smegmatis. The M. smegmatis transformant exhibited a CAT activity of 16,669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength. Two tsp utilized in M. tuberculosis were also employed in M. smegmatis. The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.

An M. tuberculosis DNA fragment contains genes encoding cell division proteins ftsX and ftsE, a basic protein and homologues of PemK and small protein B.

A 4-kb fragment of the M. tuberculosis chromosome was identified which contains several genes including those involved in cell division and possibly macrophage survival. DNA sequence analysis revealed open reading frames (ORFs) encoding putative proteins bearing significant homology with proteins FtsX and FtsE associated with cell division in E. coli, with PemK protein which inhibits cell division in E. coli harboring plasmid R100 and with SmpB protein of Salmonella typhimurium implicated in its survival within macrophages. The ftsX gene is conserved among mycobacteria belonging to the M. tuberculosis Complex. Furthermore, ftsX-specific transcripts were prevalent in equivalent amounts in M. tuberculosis H37Rv and H37Ra as analyzed by RT-PCR and primer extension. Transcription start points (tsp) a and b map in the region upstream of the FtsX ORF whose promoter activity was established by (i) a promoter-fusion experiment and (ii) by mapping the 5' ends of transcripts derived from the promoter-fusion construct. FtsX transcription is modulated as a function of mycobacterial growth and division status, maximum expression being observed in log phase cells. Growth-related expression of ftsX may provide a basis for developing a marker to distinguish actively replicating M. tuberculosis cells from quiescent mycobacteria.

Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy.

AIMS: To evaluate the usefulness of the devR based polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in lymph node aspirates and tissues of lymphadenitis and to compare PCR with conventional diagnostic techniques. SUBJECTS AND METHODS: Coded specimens of fine needle aspirates and biopsies from 22 patients with tuberculous lymphadenitis, 14 patients with non-tubercular lymphadenitis, and nine patients with granulomatous lymphadenitis were processed and subjected to analysis by PCR, smear microscopy, M tuberculosis culture, histology, and cytology. RESULTS: Tuberculous lymphadenitis was correctly diagnosed by PCR in 18 patients, by culture in five patients, by histology in 13 patients, and by cytology in seven patients. PCR gave two false positive results in 14 patients with non-tubercular lymphadenitis. The sensitivity of the conventional techniques was significantly higher with biopsies (17 of 22 specimens; 77%) than with fine needle aspirates (nine of 22 specimens; 41%). However, the sensitivity of PCR was not significantly higher with biopsies (68%) in comparison with fine needle aspirates (55%). The sensitivity of either biopsy PCR or fine needle aspirate PCR was not significantly different from that of either histology combined with culture or cytology combined with culture. The overall combined specificity of PCR was 86%. Mycobacterium tuberculosis DNA was detected in six of nine patients with granulomatous lymphadenitis. CONCLUSION: PCR is the most sensitive single technique available to date for the demonstration of M tuberculosis in specimens derived from patients with a clinical suspicion of tuberculous lymphadenitis. The value of PCR lies in its use as an adjunct test in the diagnosis of tuberculous lymphadenitis, particularly in those patients where conventional methods fail. Because fine needle aspiration is not an invasive procedure, it is the procedure of choice, and PCR should be performed initially on these samples. Excisional biopsy histology and PCR should be recommended only for patients in whom fine needle aspirate PCR is negative or when there is discrepancy with the clinical impression.

Novel use of guanidinium isothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material.

Nucleic acid amplification technologies offer great promise for the rapid, sensitive and specific diagnosis of tuberculosis. However, the isolation of inhibitor-free DNA from biological specimens is a bottleneck of the PCR assay. Here we describe a simple method for the isolation of PCR-amplifiable DNA of Mycobacterium tuberculosis from all types of samples of pulmonary and extrapulmonary origin tested. Briefly, it involves concentration of the bacilli by high-speed centrifugation, removal of PCR inhibitors by a wash solution containing guanidinium isothiocyanate and the release of bacterial DNA by heating in the presence of detergents and Chelex-100 resin. The entire process is accomplished within approximately 3 h. The method has been validated on 780 samples of human, bovine and guinea pig origin including sputum, cerebrospinal fluid, pulmonary fluids, pus, fine needle aspirate, tissue, blood and milk.

Purification of calf thymus RNA polymerase II for in vitro transcription studies.

A procedure for the purification of a large amount of RNA polymerase II (RNAP II) from calf thymus is described. This procedure results in an approximately 1400-fold purification with 40% yield of enzyme in 60 h. The partially purified enzyme is highly suitable for in vitro transcription studies in a cell-free system utilizing HeLa S-100. This method for RNAP II purification would find significant applications in in vitro studies for the analysis of factors modulating eukaryotic transcription.

Fatal atypical mycobacterial infection in a cardiac transplant recipient.

A 37-year-old female underwent heart transplantation for giant cell myocarditis. The patient died within three-and-a-half months of cardiac transplantation. Postmortem specimens from the heart and lung showed multiple necrotizing granulomas with numerous acid-fast bacilli. Polymerase chain reaction done on both the postmortem samples confirmed the presence of atypical mycobacterial infection. This fatal case of atypical mycobacteriosis in a cardiac transplant patient is reported for its rarity.

Development of a 23S rRNA-based PCR assay for the detection of mycobacteria.

The partial nucleotide sequence of a recombinant plasmid containing the 23S rRNA gene of Mycobacterium tuberculosis was determined and an assay was developed for amplifying 23S rRNA gene sequences of mycobacteria. The PCR-based non-radioactive test enabled us to distinguish Mycobacterium from other closely related genera and was sensitive enough to detect 2 bacterial genome equivalents. The assay was extended to the detection of mycobacterial DNA in uncultured clinical specimens; 23S rRNA sequences were detected in thirty four of forty eight (70.8%) sputum and cerebrospinal fluid (CSF) specimens by the PCR assay, whereas direct smear examination and culture methods demonstrated a positivity rate of 29.2% and 16.7% respectively for the same specimens. A RNA-based PCR assay with a detection limit of 1 genome equivalent was also developed. These PCR assays should prove useful for the early and rapid detection of mycobacterial infection in uncultured clinical specimens.

Utility of a Mycobacterium tuberculosis GC-rich repetitive sequence in the diagnosis of tuberculous pleural effusion by PCR.

A GC-rich repetitive sequence (GCRS) of Mycobacterium tuberculosis was identified in our laboratory which displayed a high homology with GC-rich sequences of M. tuberculosis and M. bovis. A PCR assay based on the amplification of the proximal 150 bp of GCRS and its detection by non-radioactive hybridization was developed. The accuracy of the GCRS-based PCR assay was evaluated in a clinical setting for the detection of mycobacterial DNA in pleural fluids for the diagnosis of tuberculosis (TB) using clinical criteria and pleural biopsy histology as gold standard. In a blind study, a total of 67 pleural fluid samples (38 tuberculous and 29 nontuberculous) were analysed by PCR and the results were compared with pleural biopsy, Ziehl-Neelsen staining and culture. Mycobacteria could not be detected by either smear or culture techniques in any of the pleural fluids samples. Out of 38 tuberculous pleural effusions, 24 were positive by PCR (63.2% sensitivity). When PCR results were compared with pleural biopsy histology, an increased sensitivity of 73.3% was obtained. Out of the 29 nontuberculous pleural effusions, 2 false positive results were obtained accounting for an overall specificity of 93.1%. The GCRS-based PCR assay thus has a definite role in the diagnosis of tuberculous pleural effusion in contrast to smear and/or culture techniques.

Development of a new method for sperm RNA purification in the chicken.

Currently RNA transcripts are being used as male fertility biomarker for many mammalian species, but research work on chicken is at halt because classical RNA isolation methods are not effective for chicken spermatozoa. Hence, attempts have been made to optimize RNA isolation protocol from chicken sperm by using different methods, and to confirm the presence of sperm-specific transcripts of PRM and PLCZ1. Semen samples were centrifuged at low speed for removing debris like uric acid. Further, 1mL diluted semen was gently placed over 40% PureSperm or 45%/90% Percoll, and centrifuged to remove somatic cells and immature diploid spermatocytes. RNA was isolated from sperm by using RNAzol or TRIzol reagent or RNeasy Micro kit with certain modification, and RNA quantity and quality were evaluated. RNA isolated by using RNAzol or RNeasy Micro Kit yielded good quantity and quality of RNA for downstream applications compared to TRIzol. 40% PureSperm was found effective in removing somatic cells. RT-PCR results showed that sperm RNA samples were negative for CD4 and PTPRC. All the sperm RNA samples were positive for PRM and PLCZ1, markers of sperm RNA.

Decreased C3 Activation by the devR Gene-Disrupted Mycobacterium tuberculosis Strain in Comparison to the Wild-Type Strain.

Activation of the complement component C3 is an important step in the complement cascade, contributing to inflammatory mechanisms. Considerable research on gene-disrupted mycobacterial strains using animal models of tuberculosis infection has reported the roles of some of the mycobacterial genes during tuberculosis infection. The aim of the present study was to assess the pattern of complement activation by the devR gene-disrupted Mycobacterium tuberculosis H37Rv strain and compare with that by its wild-type strain. In vitro complement activation at the level of C3 by the gene-disrupted strain, its complemented strain, and wild-type strain was performed using solid-phase ELISA. It was observed that the ability of devR gene-disrupted M. tuberculosis H37Rv to activate C3 was significantly reduced in comparison to its wild-type strain (P < 0.05). In addition, C3 activation by the complemented devR mutant strain was almost similar to that of the wild strain, which indicated that the reduced ability to activate C3 could potentially be due to the deletion of devR gene. These findings indicate that the gene devR probably aids in complement activation and contributes to the inflammatory processes during tuberculosis infection.

Residues of fluoroquinolone drugs in the cloacal gland and other tissues of Japanese quail.

1. In this study we investigated the residues of fluoroquinolone drugs (ciprofloxacin and pefloxacin) in the cloacal gland (a site of foam synthesis) and other tissues such as breast muscle, testes, brain, kidney and plasma. 2. Fifty-four healthy male Japanese quail were selected at random from a flock, maintained under uniform husbandry conditions and divided into three groups, each of 18 birds. Group I (control) received 1 ml vehicle (normal saline 0.9% (w/v) NaCl) daily for 12 d through the intraperitoneal route. Birds of groups II and III received ciprofloxacin and pefloxacin by the same route at the rate of 10 and 12 mg/kg body weight, respectively, every day for a similar period. 3. Birds from each group were killed, at 1, 5 and 10 d after the cessation of treatment, to collect the cloacal gland together with other tissues that were analysed for residual drugs. 4. Cloacal gland retained the maximum drug residues of ciprofloxacin (60%) and pefloxacin (80%) on d 10 compared with that on d 1 after drug withdrawal. The drug residues were found 60 and 80% in ciprofloxacin and pefloxacin groups, respectively, in the cloacal gland tissue even on d 10 after withdrawal of the treatment. 5. In the ciprofloxacin-treated group, all tissues except cloacal gland contained very small amounts of the drug residues on d 10 after treatment ended. In the pefloxacin group the cloacal gland, breast muscle and kidney retained a fairly high amount of drug even on d 10 after treatment ceased. No residues of pefloxacin were detectable in testes and brain throughout. 6. In conclusion, the cloacal gland in Japanese quail acted as the largest sink for the fluoroquinolone drugs. Ciprofloxacin was more widely distributed in different tissues and persisted for a shorter period than pefloxacin.

The role of glycolysis in aflatoxin biosynthesis.

In resting mycelia of Aspergillus parasiticus NRRL 3240, all the glycolytic intermediates with the exception of glucose and phosphoenolpyruvate stimulated the de novo synthesis of aflatoxin from exogenous acetate. Intracellular levels of glycolytic intermediates were measured in the toxigenic strain, A. parasiticus NRRL 3240, and the nontoxigenic strain, A. flavus NRRL 3537. In 48-h cultures, the concentrations of the intermediates beyond the phosphofructokinase reaction were higher in toxigenic mycelial extracts than in nontoxigenic cultures. There was a marked reduction in the specific activity of phosphofructokinase with increasing age in the toxigenic strain. The specific activity of this enzyme was two- to four-fold lower in zinc-deficient mycelia of A. parasiticus. In resting cultures, the uptake rate of labeled glucose was considerably lower in zinc-deficient mycelia than in zinc-replete or nontoxigenic cultures. On the basis of the results obtained, it can be said that intense formation of aflatoxin is associated with a sharp fall in the level of phosphoenolpyruvic and pyruvic acids. This was accompanied by a significantly higher pyruvate kinase activity.