Potential of cinnamaldehyde essential oil as a possible antimicrobial against fowl typhoid in layers.

Fowl typhoid (FT) is an economically significant bacterial disease of layers leading to a drastic drop in egg production. Due to increased public health concerns about antibiotics in poultry feed, a search for new safe antimicrobials for treating fowl typhoid is crucial. The antimicrobial effect of cinnamaldehyde essential oil (CnEO) against fowl typhoid in layers was investigated in this experiment. The 60-week-old BV300-layer birds (n = 100) were divided into five groups: the non-challenged control group A, only cinnamaldehyde-treated group B (CnEO @ 1:8000 dilutions through drinking water for 60 days), the challenged group C, challenged plus cinnamaldehyde therapy group D (CnEO @ 1:8000 dilutions through drinking water from 16 to 30 dpi), and challenged plus antibiotic therapy group E (chloramphenicol @ 1 gm/5lit through drinking water from 16 to 30 dpi). Hens from all challenged groups were challenged with Salmonella Gallinarum (VTCCBAA588) @ 1 × 108 CFU/ml orally. Various parameters such as clinical signs, mortality, egg production and egg weight, colony-forming unit (CFU) count of cecal content, eggshell surface, and egg yolk were evaluated all through 60 days of an experimental trial. Results indicated that, in the case of the cinnamaldehyde therapeutic group, there was a significant improvement in egg production, mild clinical signs, lower feed conversion ratio (FCR), and a significantly lower bacterial count in ceca and on the eggshell surface compared to the control challenge group. Thus, CnEO @ 1:8000 dilutions through drinking water can be a potential antimicrobial for controlling fowl typhoid.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells.

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4-6 mm; <0.5 ng/mL) (ii) F2 (7-9 mm; 0.5-5 ng/mL) (iii) F3 (10-13 mm; 5-40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

Abundance of adiponectin mRNA transcript in the buffalo corpus luteum during the estrous cycle and effects on progesterone secretion in vitro.

Adiponectin is an adipocyte derived cytokine implicated in energy homeostasis, insulin resistance and is involved in the regulation of reproduction both centrally and peripherally in animals. The present study was conducted to investigate adiponectin (ADIPOQ) and its receptors ADIPOR1 and ADIPOR2 abundance of mRNA transcript and protein in different stages of corpora lutea (CL) development during the estrous cycle of water buffalo and to determine the effect of adiponectin on cultured luteal cells of water buffalo (Bubalus bubalis). The results indicate adiponectin, ADIPOR1, and ADIPOR2 were present in buffalo corpora lutea (CL) throughout the estrous cycle. The abundance of adiponectin and its receptors was greater in the early and regressing and was less in mid- and late-stages of CL functionality. Adiponectin and its receptors were localized in the cytoplasm of small and large luteal cells. Furthermore, luteal cells were cultured in the in-vitro culture system and were treated with 1 and 10 µg/mL dose of adiponectin for 48 h. Adiponectin at both doses decreased (P < 0.05) progesterone (P4) secretion from cultured luteal cells and also suppressed the abundance of factors involved in P4productionv [Steroidogenic Acute Regulatory Protein (STAR), cytochrome P45011A1 (CYP11A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at the 10 µg/mL dose as compared to adiponectin non-supplemented cells]. In conclusion, results of the present study indicate adiponectin and its receptors are present in bubaline CL and adiponectin inhibits P4 production in cultured luteal cells. The findings indicate adiponectin affects luteal dynamics and reproductive functions in water buffalo.

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo.

Adiponectin is an adipocyte-derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in-vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin-like growth factor I (IGF-I) at 10 ng/ml for 48 hr after obtaining 75%-80%s confluency. Adiponectin at 10 µg/ml increased IGF-I-induced estradiol (E2 ) and progesterone (P4 ) secretion and FSH-induced E2 secretion from GC and also increased the abundance of factors involved in E2 and P4 production (cytochrome P45019A1 [CYP19A1] and 3-beta-hydroxysteroid dehydrogenase [3ß-HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.

Pathological and molecular identification of porcine cysticercosis in Maharashtra, India.

Porcine cysticercosis, caused by metacestodes of Taenia solium is an important emerging zoonotic disease with public health and economic significance. Pigs acquire the disease through consumption of Taenia solium eggs excreted by human tapeworm carriers. The present study was conducted to investigate the prevalence of porcine cysticercosis in Nagpur and Mumbai region of Maharashtra, India by P/M examination of carcasses followed by histopathology of affected organs in infected animals and molecular identification of cysts for confirmation. Out of 1000 pigs examined during slaughter, three pigs were found to be heavily affected with T. solium cysts giving a prevalence of 0.3%. Histological section of brain in infected animals revealed marked vascular congestion of meninges, mild neuronal degeneration, perivascular cuffing and gliosis while the liver showed the infiltration of mononuclear cell, predominantly eosinophils throughout the parenchyma. Some degree of calcification was observed in the cysts lodged in liver while calcification was not evident in case of cysts lodged in brain, tongue, diaphragm and skeletal muscle. Molecular identification by PCR using two sets of oligonucleotide primers against LSU rRNA gene and Mt-Cox1 gene of T. solium confirms the cysts to be that of T. solium. The molecular diagnostics methods have been considered for validation in conjunction with P/M inspections, parasitological and histopathological examinations. The study confirms the presence of porcine cysticercosis in the two regions and demands proper sanitary measures to minimize the risk of infection from zoonoses and food safety point of view.

Listeria goaensis sp. nov.

Two Listeria-like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5 % sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7-99.7 % nucleotide identity to other Listeria species and had less than 92 % nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria. Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15 : 0, anteiso C15 : 0, iso C16 : 0, C16 : 0, iso C17 : 0 and anteiso C17 : 0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria. WGS-based average nucleotide identity and in silico DNA-DNA hybridization values were lower than the recommended cut-off values of 95 and 70 %, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeriagoaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).

Multiple cortical brain abscesses due to Listeria monocytogenes in an immunocompetent patient.

Listeria monocytogenes is an intracellular organism which is well recognised for its ability to cause meningeal infections in neonates, immunosuppressed, debilitated and elderly individuals. 1 Other less common central nervous system (CNS) infections caused by Listeria spp. include rhomboencephalitis, cerebritis and abscesses in the brain, brain stem and spinal cord. The neuroradiological appearance of Listeria brain abscesses is similar to other types and may also mimic primary or metastatic brain tumours. 2 , 3 We report a case of Listeria brain abscesses in a patient who was being treated for atypical parkinsonism. A good clinical outcome was achieved after appropriate antimicrobial therapy.

Comparative diagnostic efficacy of recombinant LLO and PI-PLC-based ELISAs for detection of listeriosis in animals.

The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92) having either a history of abortion, emaciation and/or apparently healthy animals. Isolation of Listeria was attempted from the blood samples using USDA-FSIS method. On screening of test sera by rLLO-based ELISA, antibodies against anti-listeriolysin O (ALLO) were observed in goats (22.46%), bovines (15.10%) and pigs (16.31%). As advocated, after adsorption of positive serum samples with streptolysin O (SLO), the seropositivity for ALLO was marginally reduced (p>0.05) in goats (21.73%) and bovines (10.38%), whereas, in pigs the reduction (5.43%) was significant (p<0.05). On the contrary, rPI-PLC-based ELISA revealed higher non-specific seropositivity for antilisterial antibodies in goats (45.65%), bovines (31.13%) and pigs (8.69%). Further, on comparing the seropositivity with isolation rate, of the 16 animals that were culturally-positive for L. monocytogenes, 15 showed ALLO positivity in unadsorbed as well as SLO-adsorbed sera by rLLO-based ELISA, however, rPI-PLC-based ELISA could detect seropositivity in only 5 animals. Moreover, rPI-PLC-based ELISA also showed seropositivity in those animals (7/30) that were culturally positive for other Listeria spp. In conclusion, rLLO can serve as a better antigen than rPI-PLC in ELISA for the serodiagnosis of listeriosis in animals; however, prior adsorption of test sera with SLO is required to avoid false positive results.

Molecular characterization of genotype XIIIb Newcastle disease virus from central India during 2006-2012: Evidence of its panzootic potential.

Newcastle disease virus (NDV) is the causative agent of Newcastle disease (ND) in many avian species. ND is a serious problem in developing countries, causing huge loss in the poultry industry. Although there are reports of continuous outbreaks of ND leading to serious losses to the poultry farming, very less is known about the genetic characteristics of its strains circulating in different parts of India. In the present study, we have five isolates of NDV reported from different outbreaks in Central India between the years 2006-2012. Deduced amino acid sequence of the F protein cleavage site and phylogenetic analysis of all the five isolates showed circulation of NDV genotype XIIIb. All the isolates showed a unique virulent cleavage site 112RRQKR↓F117. The close genetic similarity of all the isolates suggested circulation of the virulent NDV strains of the same ancestor in and around central India. Continuous isolation of genotype XIIIb NDV strains within the country suggests its panzootic potential. The study will be useful to understand the circulating strains of NDV and plan a vaccination strategy for poultry in India.

Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241₋255 PESRKKLEKALLAWA and NS31902₋1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996₋1003 YEPRDSYF, NS21223₋1230 STVTGIFL and NS5A3070₋3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS1223₋1230 STVTGIFL and NS31902₋1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.

Assessment of the phenotype and functionality of porcine CD8 T cell responses following vaccination with live attenuated classical swine fever virus (CSFV) and virulent CSFV challenge.

Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3(+) CD4(-) CD8(hi) T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3(+) CD4(+) CD8α(+)) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44(hi) CD62L(-) and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ(+) CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.

Neuroendocrine tumor in the lung of a captive black spider monkey (Ateles paniscus).

This paper describes a neuroendocrine (NE) tumor of the lung that was observed during the necropsy of a 14-year-old female black spider monkey (Ateles paniscus) with sudden death. Grossly, multifocal firm and coalescing nodular masses were observed in the lung. The histological examination showed the tumor to be an typical NE tumor with polygonal cells grouped in small solid aggregates, with regularly sized, spherical, centrally placed nuclei with modest, lightly granular cytoplasm suspended in a fibrovascular stroma. The immunohistochemical examination revealed the tumor to be positive for cytokeratin, chromogranin A and synaptophysin, and negative for CD56. To the best of our knowledge, this is the first report of NE tumor in the lung of the black spider monkey.