Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains / La genómica comparada de Brucella abortus y Brucella melitensis revela el intercambio de genes, los factores de virulencia y la diversidad de SNP entre las cepas estándar, vacunales y de campo

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.(AU)

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

Improvement of bone microarchitecture in methylprednisolone induced rat model of osteoporosis by using thiolated chitosan-based risedronate mucoadhesive film.

OBJECTIVE: In this study, we investigated the potential of thiolated chitosan-based mucoadhesive film, loaded with risedronate sodium in the treatment of osteoporosis. SIGNIFICANCE: Risedronate sodium is a bisphosphonate derivative having very low bioavailability when administered through the oral route. Moreover, the adverse effects associated with the drug when administered through GIT necessitate an alternative and feasible route which can improve its bioavailability and therapeutic efficacy. METHODS: Thiolation of chitosan was interpreted by different analytical techniques. The mucoadhesive films were prepared by the solvent evaporation method and evaluated for drug content analysis, swelling degree, mucoadhesive parameters, and permeation characterization. For the screening of preclinical efficacy and pharmacodynamic parameters, a methylprednisolone induced osteoporotic rat model was used. The trabecular microarchitecture and biochemical markers were evaluated for determination of bone resorption. RESULTS: The different analytical characterization of synthesized thiolated chitosan revealed that chitosan was successfully incorporated with thiol groups. The formulation containing 2:1 ratio of thiolated chitosan and HPMC-4KM was found to have the maximum swelling degree, mucoadhesive strength with a good force of adhesion and better in vitro permeability compared to the marketed formulation. With respect to trabecular microarchitecture, the drug-loaded film formulation showed superior and promising results. Furthermore, the film formulation also improved the serum level of biomarkers better than the marketed formulation. CONCLUSIONS: The results significantly suggest that risedronate loaded novel mucoadhesive film formulation could be a logical approach in the therapeutic intervention of osteoporosis.

Microglia play a major role in direct viral-induced demyelination.

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.

Establishment of the need for oestrogen during implantation in non-human primates.

Deprivation of oestrogen during post-ovulatory mated cycles in proven fertile female bonnet monkeys by tamoxifen, aromatase inhibitor or oestrogen antiserum resulted in inhibition of pregnancy establishment in all three groups of animals. However, more than 85% of the animals became pregnant within three exposures to proven fertile males in the control group. These results suggest the requirement for oestrogen in pregnancy establishment in primates. Based on this conclusion, it is suggested that use of a suitable and potent anti-oestrogenic compound can be exploited as an alternative approach to contraception.

Studies on the luteolytic, oestrogenic and follicle-stimulating hormone like activity of plant Rhaphidophora pertusa (Roxb.).

The phytochemical, pharmacological and toxicological feature of plant Rhaphidophora pertusa (Roxb.) was done. Phytosteroids, flavonoids, tannins and glucosides were detected in the plant extracts. In cross-bred (Zebu X Holstein-Friesian or Jersey) dairy cows, subsequent to prostaglandin (PG) induced oestrus, to each group (n=4), cloprostenol (PG control) 100 microg i.m. on day 10, the rice gruel (vehicle) was fed on day 10 or the fresh stem (1 kg/animal/day) in rice gruel on day 9, or days 9 and 10, or days 9-11 of the oestrous cycle. Each group received subcutaneously either 5% gum acacia suspension or the plant ethyl acetate or methanol extract (1g in gum acacia) on days 8 (to bannur ewes) or 10 (to dairy cows) of the oestrous cycle. In PG control cows or ewes, there was induction of oestrus in 48 h and a fall in serum progesterone concentration. The feeding of fresh stem in the rice gruel or the s.c. administration of the plant extract did not induce oestrus or significantly (P>0.05) alter the serum progesterone, bilirubin, calcium, creatinine, phosphorus, magnesium and glucose concentrations or the total erythrocyte and leucocyte count, differential leucocyte count and haemoglobin concentration. The plant did not cause any toxicity in the cow or ewe. In immature rats, the aqueous or methanol (hot or cold) extract did not cause any follicle-stimulating hormone (FSH)-like activity. The methanol extract increased the uterine weight in ovariectomised rats. This suggested the presence of oestrogenic activity in the plant. In conclusion, the present study revealed the presence of oestrogenic activity in the plant and the absence of luteolytic or FSH-like or toxic activity.

Reversible immunocontraception in male monkeys immunized with eppin.

Various forms of birth control have been developed for women; however, there are currently few options for men. The development of male contraceptives that are effective, safe, and reversible is desired for family planning throughout the world. We now report contraception of male nonhuman primates (Macaca radiata) immunized with Eppin, a testis/epididymis-specific protein. Seven out of nine males (78%) developed high titers to Eppin, and all of these high-titer monkeys were infertile. Five out of seven (71%) high-anti-Eppin titer males recovered fertility when immunization was stopped. This study demonstrates that effective and reversible male immunocontraception is an attainable goal. This method of immunocontraception may be extended to humans.

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor.

Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.

Effect of chronic administration of 7alpha-methyl-19-nortestosterone on serum testosterone, number of spermatozoa and fertility in adult male bonnet monkeys (Macaca radiata).

Hormonal approaches to male contraception that are based on the suppression of LH secretion require androgen replacement treatment to maintain sexual behaviour and secondary sexual characteristics. Androgen supplementation not only involves large and frequent doses of testosterone esters but also results in undesirable effects on the prostate gland. In an attempt to avoid such problems, a synthetic androgen, 7alpha-methyl-19-nortestosterone (MENT), which is much more potent than testosterone, has been developed. In the present study, MENT was administered at different doses (25, 50, 100, 300 and 1000 microg day(-1)) either alone or in combination with oestradiol via Silastic implants for a specified period to adult male bonnet monkeys (Macaca radiata). Blood and semen samples were collected at specific intervals and analysed for serum testosterone and seminal parameters, respectively. The results of the present study clearly indicate that administration of MENT at all doses tested results in suppression of the nocturnal surge of testosterone (by day 3), as well as a decrease in the number of spermatozoa (by day 45). Co-administration of oestradiol resulted in a reduction in the dose of MENT required to suppress the nocturnal surge. None of the male bonnet monkeys treated with MENT were able to impregnate females, clearly demonstrating the efficacy of MENT in blocking fertility in male bonnet monkeys.

Electroejaculation and seminal parameters in bonnet monkeys (Macaca radiata).

In the present study, our experience in the collection of semen from adult bonnet monkeys and some of the characteristics of semen are reported. Semen samples were collected by penile stimulation every 15 days over a period of 3 years. The penile method of ejaculation was more consistent with an increased volume of semen. A distinct sesonality in the sperm count of the bonnet monkey was observed. Sperm counts were high during the months of August to October and started declining from November onwards. The sperm counts were significantly (P < 0.05) higher during the mid and post-monsoon (July to November) periods than the pre- and early monsoon (March to June) periods.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata).

Administration of Tamoxifen via the Alzet pump at a rate of 50 micrograms hr-1 for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Use of norethisterone and estradiol in mini doses as a contraceptive in the male. Efficacy studies in the adult male bonnet monkey (Macaca radiata).

Administration of norethisterone (NET) or NET + estradiol benzoate using an Alzet minipump or as once-a-month intramuscular injection of their depot forms, NET-enanthate (NET-EN) and estradiol valerate (E-val), resulted in azoospermia in all monkeys (n = 13) within 60 to 150 days of treatment. Although addition of depot form of testosterone (T, 20 mg/month) to the regimen restored the behavioral response typical of a normal male, it did not reverse the azoospermic state. Serum T (heightened nocturnal) levels were significantly reduced (> 85%, p < 0.001) in all the treated groups. Evidence for blockade in spermatogenesis following treatment was obtained by DNA flow cytometry. Following withdrawal of treatment, the T level was restored to normalcy within 15 days but 120 days more were required for the animals to exhibit normal sperm counts. In conclusion, the efficacy of once-a-month injection of relatively low doses of NET-EN + E-Val to bring about azoospermia in monkeys, in a relatively short time, has been demonstrated. As the results are uniform and reproducible, it appears desirable that this steroid regimen be tested in man for its contraceptive efficacy.

PIP: Monthly intramuscular injection with an Alzet minipump of depot norethisterone enanthate and estradiol valerate (E-val) produced azoospermia in 13 adult male bonnet monkeys within 60-150 days. Although azoospermia was achieved earlier when E-val was added to the injection, this agent can be eliminated once azoospermia occurs. Addition of the depot form of testosterone (20 mg/month) restored the sexual behavioral response but did not reverse azoospermia. Serum testosterone levels were significantly reduced in all treatment groups. DNA flow cytometry revealed evidence for blockade in spermatogenesis after treatment. There were no changes in the serum lipid profile. The testosterone level returned to normal within 15 days after the end of treatment, but normal sperm counts were not observed for another 120 days. Since the steroid formulation investigated in this study has been used effectively in women for over a decade, tests of its contraceptive efficacy in men now seem warranted.

Breeding of bonnet monkeys (Macaca radiata) in captivity.

The results of studies on the breeding of South Indian bonnet monkeys (macaca radiata) over 20 years at our institution are presented. The menstrual cycle and hormonal changes were similar to those reported for Macaca mulatta. It was noted that summer amenorrhoea could be eliminated by housing the monkeys in rooms supplied with humidified air. Although a fertility index (proportion of animals becoming pregnant within three exposures to a proven fertile male) of 60 to 65% was achieved by random breeding, an index of 80 to 85% was achieved by controlled breeding (prior monitoring of serum estradiol-17 beta concentration on days 7, 8, 9, and 10).

Successful recovery of preimplantation embryos by nonsurgical uterine flushing in the bonnet monkey.

A major limitation to progress in primate embryology is the lack of an adequate supply of preimplantation embryos. We describe a method for recovering preimplantation-embryos in bonnet monkeys (Macaca radiata ) using a nonsurgical uterine flushing technique similar to the one previously employed in rhesus monkeys. Forty cyclic females were screened for cervical cannulation, and 10% of these had an impassable cervix. Eleven females suitable for cannulation were selected, and 27 menstrual cycles were monitored over a 5-mo period. Seventy-one percent of the cycles showed estrogen peaks, which were observed between Days 9 and 14 of the cycle. Following natural mating, uterine flushings were performed on Days 5 to 8 of pregnancy (Day 0 = the day following the estrogen peak). Of the 27 recovery attempts, 9 (33.3%) resulted in the recovery of ovulation products, including those of an unfertilized oocyte and empty zona (2 cases), retarded cleavage-stage (4 to 8-cell) embryos (4 cases), morula (1 case) and blastocysts (2 cases). These results show, for the first time, that the nonsurgical uterine flushing technique can be successfully performed to recover uterine-stage preimplantation embryos from bonnet monkeys.