Djelovanje različitih razrjeđivača na svojstva spermija i plodnost križanih svinja u sjevernoistočnoj Indiji.

The study was conducted to deduce the efficacy and suitability of three extenders: Beltsville thawing solution (BTS), Kiev and Modena extenders, on the preservation and fertility of boar semen. Semen ejaculates with more than 70% sperm motility from six Hampshire boars were used and preserved at 17 °C. The extended semen was evaluated for motility, viability, acrosomal and plasma membrane integrity from day 0 to 5 of preservation. There was a significant (P<0.01) reduction in sperm motility and viability between days 0 to 4, but no significant difference between day 0 and 2. BTS and Modena maintained significantly (P<0.05) higher sperm motility and viability as compared to Kiev. Plasma membrane integrity did not differ significantly up to day 2, but was significantly reduced on days 3 (P<0.05) and 4 (P<0.01). BTS and Modena extenders had significantly (P<0.05) higher plasma membrane integrity. By day 5, the percentage of mean sperm with intact acrosome was 80.4 ± 2.5, 81.2 ± 2.3 and 75.7 ± 2.7, in BTS, Modena and Kiev, respectively, with significant (P<0.05) increases in acrosomal damage after two days of storage, irrespective of all extenders. Farrowing rate and litter size did not differ significantly during the initial period of storage, but there was a significant difference after day 3 (P<0.05) in all extenders used. BTS and Modena maintained a farrowing rate of more than 60% on day 5, in contrast to 50% with the Kiev extender. The study concluded that both BTS and Modena are better extenders for short term storage of boar liquid semen as compared to the Kiev extender, and could be used efficiently in crossbreeding of pigs of north-eastern India.Istraživanje je provedeno kako bi se utvrdila učinkovitost i prikladnost beltsvilskog (BTS), kijevskog i modenskog razrjeđivača za očuvanje i plodnost nerastova sjemena. Upotrijebljeni su ejakulati sjemena od 6 hempširskih nerastova s pokretljivošću spermija većom od 70%, koji su sačuvani na 17 °C. Razrijeđeno sjeme procjenjivano je od 0. do 5. dana s obzirom na pokretljivost, živost te cjelovitost akrosomalne i plazmine membrane. Tijekom razdoblja od 0. do 4. dana došlo je do signifikantnog (P<0,01) sniženja pokretljivosti i živosti spermija, no signifikantne razlike između nultog i drugog dana nisu utvrđene. U usporedbi s kijevskim razrjeđivačem, BTS i modenski razrjeđivači su održavali signifikantno (P<0,05) višu pokretljivost i životnost spermija. Cjelovitost plazmine membrane nije se signifikantno razlikovala do 2. dana, ali je signifikantno snižena tijekom 3. dana (P<0,05) i 4. dana (P<0,01). Cjelovitost plazmine membrane bila je signifikantno veća (P<0,05) pri uporabi BTS i modenskog razrjeđivača. Do 5. dana, prosječni postotak spermija s nepromijenjenim akrosomom iznosio je kod BTS razrjeđivača 80,4 ± 2,5, kod modenskog razrjeđivača 81,2 ± 2,3, a kod kijevskog razrjeđivača 75,7 ± 2,7. Pri tome je, bez obzira na razrjeđivač, od 2. dana utvrđen signifikantni porast (P<0,05) spermija s oštećenim akrosomima. U početnom razdoblju stopa prasenja i veličina legla nisu se signifikantno razlikovale, no nakon 3. dana kod svih upotrijebljenih razrjeđivača razlike su bile signifikantne (P<0,05). Peti dan stopa prasenja kod BTS i modenskog razrjeđivača održala se preko 60% za razliku od kijevskog razrjeđivača kod kojeg je stopa prasivosti iznosila 50%. Istraživanjem se zaključuje da BTS i modenski razrjeđivači mogu, u odnosu na kijevski razrjeđivač, bolje poslužiti za kratkotrajno pohranjivanje nerastovog tekućeg sjemena, te kao takvi biti i učinkovitiji u križanjima svinja sjevernoistočne Indije

Changes in expression of monocarboxylate transporters, heat shock proteins and meat quality of Large White Yorkshire and Ghungroo pigs during hot summer period

Objective Present study explores the effect of hot summer period on the glycolytic rate of early post-mortem meat quality of Ghungroo and Large White Yorkshire (LWY) pig and comparative adaptability to high temperature between above breeds by shifting the expression of stress related genes like mono-carboxylate transporters (MCTs) and heat shock proteins (HSPs). Methods Healthy pigs of two different breeds, viz., LYW and Ghungroo (20 from each) were maintained during hot summer period (May to June) with a mean temperature of about 38°C. The pigs were slaughtered and meat samples from the longissimus dorsi (LD) muscles were analyzed for pH, glycogen and lactate content and mRNA expression. Following 24 h of chilling, LD muscle was also taken from the carcasses to evaluate protein solubility and different meat quality measurements. Results LWY exhibited significantly (p<0.01) higher plasma cortisol and lactate dehydrogenase concentration than Ghungroo indicating their higher sensitivity to high temperature. LD muscle from LWY pigs revealed lower initial and ultimate pH values and higher drip loss compared to Ghungroo, indicating a faster rate of pH fall. LD muscle of Ghungroo had significantly lower lactate content at 45 min postmortem indicating normal postmortem glycolysis and much slower glycolytic rate at early postmortem. LD muscle of LWY showed rapid postmortem glycolysis, higher drip loss and higher degrees of protein denaturation. Ghungroo exhibited slightly better water holding capacity, lower cooking loss and higher protein solubility. All HSPs (HSP27, HSP70, and HSP90) and MCTs (MCT1, MCT2, and MCT4) in the LD muscle of pigs inclined to increase more in Ghungroo than LWY when exposed to high temperature. Conclusion Effect of high temperature on the variation of HSPs and MCTs may play a crucial role in thermal tolerance and adaptation to different climatic conditions, pH regulation, muscle acidification, drip loss, protein denaturation and also in postmortem meat quality development

Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background: In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported. Methodology/Principal Findings: Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug wasfound to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF a, IFN c and iNOS activity increased with the concomitant decrease in IL 10 and TGF b level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity. Conclusions: This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar

KMP-11 DNA Immunization Significantly Protects against L. Donovani Infection but Requires Exogenous IL-12 as an Adjuvant for Comparable Protection against L. major

As vaccine potential of cross-species protection by a candidate antigen is less explored, in this study we compared cross-specific protective efficacy of KinetoplastidMembrane Protein-11 (KMP-11) as aDNAvaccine alone and in conjunction with exogenous IL-12 administration in experimental BALB/c model against two most widely prevalent forms of clinical diseases caused by Leishmania major (LM) and Leishmania donovani (LD). Whereas, KMP-11 DNA vaccination alone showed significant potential in terms of resolution of splenic and hepatic parasite burden against virulent LD challenge, it showed considerably less efficacy (<70% reduction) against virulent LM challenge in terms of presence of parasite in lymph node. Remarkably exogenous IL-12 administration in the form of IL-12 p35/p40 expression vectors or recombinant protein along with KMP-11 DNA had exactly opposing effect on protection against LM and LD. Exogenous IL-12 administration significantly increased residual LD-burden but enhanced the protective efficacy ofKMP-11DNAvaccine against LMcompared toKMP-11 immunization alone. Elucidation of effector mechanism showed KMP-11 DNA induced protection against LDwas associated with the generation of mixed Th1/Th2 response, while KMP-11/IL-12-induced comparable protection against LMwas associated with high IgG2a titre indicative of a polarized Th1 response. Exogenous IL-12 administration resulted in robust gamma interferon (IFN-�) production and suppression of IL-4 from CD4+ T cell against both LM and LD. Nevertheless protective immune responsewas only compromised against LD infection where frequency of anti-KMP-11 CTL responsewas significantly reduced after exogenous IL-12 administration. Our study provides a comparative evaluation of effector mechanisms in the assessment of cross-specific protection by KMP-11 and KMP-11/IL-12 immunization against these two prevalent forms of leishmaniasis

Hybrid Cell Vaccination Resolves Leishmania donovani Infection by Eliciting a Strong CD8+ Cytotoxic T-Lymphocyte Response with Concomitant Suppression of Interleukin-10 (IL-10) but Not IL-4 or IL-13▿ ‡

There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens

Kinetoplastid Membrane Protein-11 DNA Vaccination Induces Complete Protection against Both Pentavalent Antimonial-Sensitive and -Resistant Strains of Leishmania donovani That Correlates with Inducible Nitric Oxide Synthase Activity and IL-4 Generation: Evidence for Mixed Th1- and Th2-Like Responses in Visceral Leishmaniasis1

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-�, TNF-�, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis

Sub-Optimal dose of Sodium Antimony Gluconate (SAG)-Diperoxovanadate Combination Clears Organ Parasites from BALB/c Mice Infected with Antimony Resistant Leishmania Donovani by Expanding Antileishmanial T-Cell Repertoire and Increasing IFN-c to IL-10 Ratio

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (SbR) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients