Differential expression of Toll-like receptor mRNA in White Leghorn and indigenous chicken of India

In the present experiment, the expression profile of Toll-like receptor mRNA in indigenous and pure line chickens was studied. The expression of TLR3, TLR4, TLR5 and TLR7 were quantified in heterophils of Aseel, Kadaknath, Naked neck, Dwarf and White Leghorn lines by Quantitative Real-time PCR. White Leghorns expressed significantly (P < 0.01) higher levels of TLR3 mRNA compared to other lines. TLR4 and TLR5 mRNA were significantly highly expressed in Kadaknath line. Among the TLRs investigated TLR5 was more expressed in all lines studied. TLR7 was highly expressed in indigenous chicken Aseel and Kadaknath than other lines. Dwarf chicken expressed significantly (P < 0.01) lower levels of all TLRs investigated. On the basis of the present study we conclude that the differential expression of TLR mRNA in the heterophils of indigenous and other chicken breeds might contribute to their variable disease resistance/susceptibility.     

Evaluation of two Indian native chicken breeds for reproduction traits and heritability of juvenile growth traits

The present study was conducted to evaluate two Indian native chicken breeds, namely, Aseel and Kadaknath for fertility, hatchability, genetic parameters of juvenile growth traits, and semen quality traits at the onset of sexual maturity. The fertility was similar in Aseel (86.96%) and Kadaknath (85.15%); however, a relatively higher hatchability was observed in Kadaknath (77.94%) than Aseel (70.74%). Heritability estimates of body weights at 4 weeks of age were almost similar in Aseel (0.37) and Kadaknath (0.39), while the estimate of body weight at 6 weeks of age was higher in Aseel (0.42) than Kadaknath (0.31). The heritability estimate of shank length at 6 weeks of age was lower in Aseel (0.16) compared to Kadaknath (0.35). The age at first egg in the flock was comparable in Aseel (148 days) and Kadaknath (150 days). Aseel breed with significantly (P ≤ 0.001) higher body weight, absolute and relative testes weights had significantly higher semen volume (P ≤ 0.05) and sperm motility (P ≤ 0.01) but had lower seminal plasma cholesterol level (P ≤ 0.05) as compared to Kadaknath. It can be concluded that there is a scope for genetic improvement of these two native breeds for juvenile growth traits since heritability estimates of these traits were relatively high.     

Significance of nitric oxide concentration in plasma and uterine secretes with puerperal endometritis in dairy cows

Endometritis is an inflammation of the endometrial lining of the uterus without systemic signs, which is associated with chronic postpartum infection of the uterus with pathogenic bacteria. Nitric oxide (NO) is an inflammatory mediator that among other effects causes smooth muscle relaxation and mediated cytoimmunity and inflammation toxicity. To see if the nitric oxide concentration in plasma and uterine secrets is related with postpartum endometritis, NO concentrations in plasma and uterine secrets were measured in dairy cows with puerperal endometritis (clinical endometritis (n = 60) and subclinical endometritis (n = 58)). Cows with clinical or subclinical endometritis showed higher concentrations of NO in both plasma and uterine secrets when compared with normal cows and the highest concentrations of NO in plasma and uterine secrets were found in dairy cows with clinical endometritis. Expression level of NOS2 mRNA in endometrial biopsies from cows with puerperal endometritis was also higher and the highest expression of NOS2 mRNA was found in cows with clinical endometritis. The results showed that concentrations of NO in plasma and uterine fluid are related with the degree of endometritis which may be useful to diagnose the endometritis in dairy cows.     

Expression of toll‐like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO‐affected horses

Reasons for performing study: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables. It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll‐like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)‐8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. Hypothesis: TLR2 and IL‐8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. Objectives: To determine TLR 2 , IL‐8 and A20 mRNA expression in lungs of stabled and pastured RAO‐affected and control horses. Methods: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR 2 , IL‐8 and A20 mRNA expression quantified by qRT‐PCR in 6 RAO‐affected and 6 control horses, during and after exposure to hay and straw. Results: Airway obstruction and neutrophils were increased in RAO‐affected horses during stabling. While stabling increased IL‐ 8 , TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. Potential relevance: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal‐induced airway inflammation. Equine epithelial‐derived A20 may be involved in modulation of airway inflammation.     

Follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular characteristics and intrafollicular concentrations of nitric oxide and ascorbic acid during ovarian acyclicity in buffaloes. Ovaries were collected from 56 acyclic and 95 cyclic buffaloes at slaughter, surface follicle number was counted and follicles were classified into small (5.0–6.9 mm), medium (7.0–9.9 mm), and large (≥10.0 mm) size categories based on their diameter. Follicular fluid was aspirated and assayed for nitric oxide, ascorbic acid, estradiol, and progesterone. Acyclic buffaloes had a higher (P < 0.05) number of medium-sized follicles and a lower (P < 0.001) number of large follicles than the cyclic ones. In acyclic animals, the number of large follicles was lower (P < 0.01) than in medium size category which in turn was lower (P < 0.001) than the number of small follicles. In contrast, the number of medium and large follicles was not different (P > 0.05) in the cyclic control. However, the number of small-sized follicles was higher (P < 0.001) compared to the other two categories. The incidence of large-sized follicles was lower (P < 0.05) in acyclic buffalo population compared to the cyclic control. Evaluation of estrogenic status demonstrated that all the follicles of acyclic buffaloes are estrogen-inactive (E ₂/P ₄ ratio < 1). Small- and medium-sized follicles of acyclic buffaloes had higher concentrations of nitric oxide (P < 0.05 and P < 0.001, respectively) and lower concentrations of ascorbic acid (P < 0.05 and P < 0.01, respectively) than the corresponding size estrogen-active follicles of their cyclic counterparts. In conclusion, this study indicates that follicular development continues during acyclicity in buffaloes. Although follicles in some acyclic buffaloes attain a size corresponding to morphological dominance, they are unable to achieve functional dominance, perhaps due to an altered balance of intrafollicular nitric oxide and ascorbic acid and, as a result, these follicles instead of progressing to ovulation undergo atresia.     

Toll‐like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll‐like receptors (TLR) mediate innate immune mechanisms via the production of pro‐inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30–50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll‐like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN‐G), and interleukin (IL)‐12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL‐6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro‐inflammatory cytokine mRNA expression.     

Differential gene expression of antimicrobial peptides β defensins in the gastrointestinal tract of Salmonella serovar Pullorum infected broiler chickens

Salmonella enterica serovar Pullorum causes substantial mortality in chicks as well as results in persistent infection and vertical transmission in layer birds. An effective innate immune response in the early stages of infection could reduce bacterial colonization and mortality in chicks and persistency of infection in later stages. β Defensins (AvBDs) are now considered as one of the key components of innate immunity in avian species. In the present study, we quantified the mRNA expression levels of AvBDs (1–14) by real-time PCR in the gastrointestinal (GI) tissues (duodenum, jejunum, ileum and caecum) of 3-day-old broiler chicks after 24 h of oral infection with Salmonella Pullorum. Quantitative real-time PCR analysis revealed significant (P < 0.05) upregulation of AvBD3, 4, 5, 6 and 12 and a significant (P < 0.05) down regulation in the expressions of AvBD10, 11, 13 and 14 in one or few GI tissues, while no significant changes were observed for AvBD1, 2, 7, 8 and 9 gene expressions in any of the GI tissues investigated upon infection with S. Pullorum. Most substantial change in gene expression was found for AvBD5, being significantly (P < 0.01) upregulated in most of the GI tissues investigated. The differential expression levels of β defensins shed light on tailored innate immune response induced by S. Pullorum during the early stages of infection in chicks.     

Polymorphism of <Emphasis Type="Italic">GDF</Emphasis>9 gene and its association with litter size in goats

Growth differentiation factor 9 (GDF9) was studied as a candidate gene for high prolificacy in goats. The polymorphism of exon 1 and flanking of GDF9 gene was detected by PCR-SSCP in five goat breeds with different prolificacy. Three genotypes (AA, AB and BB) were detected in goat breeds joined and two silent mutations (c.183A>C and c.336C>T) were identified in comparison genotype AA with genotype BB. Heterozygous genotype AB and wild type BB were detected in all five goat breeds and homozygous genotype AA was only detected in Jining Grey goats. The frequencies of genotypes AA, AB and BB were 0.18, 0.42 and 0.40 in Jining Grey goats, respectively. The genotype distribution was different (P < 0.01) between high prolificacy breed (Jining Grey goat) and low prolificacy breeds (Boer, Wendeng Dairy, Liaoning Cashmere and Beijing native goats). The Jining Grey goat does with genotype AA and AB had 0.72 (P < 0.01) and 0.56 (P < 0.01) kids more than those with genotype BB, respectively. The does with genotype AA had 0.16 (P > 0.05) kids more than those with genotype AB. These indicated that the allele A may have certain correlation with prolificacy in Jining Grey goats.     

The fungal T-2 toxin alters the activation of primary macrophages induced by TLR-agonists resulting in a decrease of the inflammatory response in the pig

T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. The objective of this work was to evaluate the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM). Cytotoxic effects of T-2 toxin on PAM were first evaluated. An IC₅₀ of 19.47 ± 0.9753 nM was determined for the cytotoxicity of T-2 toxin. A working concentration of 3 nM of T-2 toxin was chosen to test the effect of T-2 toxin on TLR activation; this dose was not cytotoxic and did not induce apoptosis as demonstrated by Annexin/PI staining. A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1 beta, TNF-alpha, nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. By contrast, the activation of TLR7 by ssRNA was not modulated by T-2 toxin pre-treatment. In conclusion, our results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases.     

Preliminary comparison of different immune and production components in local and imported Saanen goats reared under a sub-tropical environment

Three objectives were included in this research work. The first objective compared different immune components in healthy mature males, mature females, and female kids of local and imported Saanen goats, reared under a sub-tropical environment. The significantly differing immune components were the blood monocyte percent, blood CD8 count, and the total white blood cell count. The second objective compared the performance of Saanen versus local does. The means of the milk yield and prolificacy of the imported Saanen does were significantly higher than those of the local does (p < 0.05). The third objective compared the immune responses (hemagglutination-HA titers) and complement fixation (CF) titers in mature does of the two breeds to chicken red blood cells (c-RBC). The HA titers showed a significant seroconversion only in imported Saanen (p < 0.05) but not in local does; however, the CF titers increased significantly at 4 weeks following priming with c-RBC in local (p < 0.05) but not in the imported Saanen does. The impact of the differences in blood immune components and responses to antigens in the compared goats on protection potential against prevalent diseases in the sub-tropical zone of the eastern Mediterranean countries is discussed.     

Differential induction of nitric oxide, degranulation, and oxidative burst activities in response to microbial agonist stimulations in monocytes and heterophils from young commercial turkeys

The toll-like receptors (TLRs) recognize microbial pathogens and pathogen-associated molecular patterns and trigger inflammatory immune responses to control the infection. Here, we examined functional innate immune responses to Salmonella enteritidis (SE, live or formalin-killed) and various TLR agonists including lipoteichoic acid (LTA) and peptidoglycan (PGN) from Staphylococcus aureus and synthetic lipoprotein Pam3CSK4 (PAM), poly I:C (synthetic double-stranded RNA analog), lipopolysaccharide (LPS) from S. enteritidis, flagellin (FGN) from S. typhimurium, loxoribine (LOX) and R837 (synthetic anti-viral compounds), and CpG oligodeoxydinucleotide (CpG ODN)by measuring antimicrobial activities including oxidative burst and degranulation in heterophils and nitric oxide production in peripheral blood monocytes. Our results demonstrate differential nitric oxide responses to TLR agonists in turkey monocytes. LTA and CpG ODN were the most potent stimuli for nitric oxide induction followed by PAM, poly I:C, and LPS, whereas FGN, PGN, LOX, R837, and control ODN stimulated little or no nitric oxide production. Live SE stimulated significantly less NO production than formalin-killed SE (FKSE). Although FKSE induced significant degranulation and oxidative burst, most TLR agonists stimulate little oxidative burst and degranulation responses in turkey heterophils.     

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.     

Mutations in bone morphogenetic protein 15 and growth differentiation factor 9 genes are associated with increased litter size in fat-tailed sheep breeds

The objective of this study was to investigate association between GDF9 and BMP15 gene polymorphism and litter size in fat-tailed sheep, a total of 97 mature ewes from four breeds (Afshari=19; Baluchi=18; Makui=30 and Mehraban=30) were genotyped for the BMP15 HinfI and GDF9 HhaI polymorphisms by PCR-RFLP technique. The highest and lowest mutant allele frequencies were found in Makui (0.27) and Afshari (0.10) sheep for the BMP15 gene and in Afshari (0.24) and Mehraban (0.18) sheep for the GDF9 gene, respectively. Litter size was significantly influenced by genotype of the ewe for two genes (P < 0.01). Heterozygous genotypes for both loci showed higher litter size than homozygous genotypes (P < 0.01). None of the individuals carried homozygous genotype for both of the GDF9 and BMP15 variants in these breeds. The individuals carrying the mutant allele for one of the investigated candidate gene still showed fertile phenotype. Thus, existence of homozygosity at one of the BMP15 and GDF9 variant is not probably able to block normal hormonal pathway of reproduction in fat-tailed sheep.     

Comparative evaluation of carcass traits and meat quality in native Aseel chickens and commercial broilers

1. A comprehensive study was conducted to analyse the meat quality attributes, composition and carcass traits in Aseel chickens and commercial broilers at market age on the basis of physiological age. A total of 20 Aseel (26 and 56 weeks) and 20 broiler (6 weeks) chickens were divided into two groups on a live weight basis, i.e. large (≥2.5 kg) and small (<2.5 kg) with 10 birds in each subgroup.

2. The pH of meat did not show any significant variation between Aseel and broiler chickens. The meat from heavier birds had significantly higher pH. Shear force value and hydroxyproline contents were significantly higher in Aseel chickens. Aseel birds had significantly higher red (a*) colouration and lower lightness (L*) than broiler chickens.

3. The texture and acceptability of Aseel meat were significantly higher.

4. Scanning electron microscopy revealed that muscle fibres in Aseels were arranged in a more coiled pattern making the muscle tough. A larger amount of connective tissue was also observed between the muscle fibres compared with the broiler chickens.

5. The dressing percentage was significantly higher in larger chickens. Commercial broilers recorded significantly higher meat proportion and lower proportion of bone. The meat:bone ratio was 1.07:1.0 in Aseel and 1.31–1.0 in broiler chicken. Breast muscle content was significantly lower in smaller Aseel chickens. Aseel chicken had stronger and heavier backs and shanks. Abdominal fat percentage was significantly lower in Aseel (0.73–0.78%).

6. The study concluded that the firm texture of Aseel meat was due to the high collagen content and interlocking connective tissue between the muscle fibres. The texture and acceptability of Aseel meat was higher. Aseel cocks had strong legs, lean meat and less abdominal fat, making them a high-value meat bird in addition to their aggressive fighting ability.

     

Serological Screening for MHC (B)-Polymorphism in Indigenous Chickens

As part of a series of studies to characterize innate and specific immune responses of indigenous chicken lines, birds from Bolivia and India were screened serologically for MHC class IV (BG) polymorphism by direct haemagglutination using haplotype-specific antisera (B2, B4, B12, B13, B14, B15, B19, B21). The sample consisted of 95 Bolivian indigenous chickens and 119 hens from the four most common North Indian 'back-yard' chicken lines: Yellow Aseel (AP), Kadaknath (KN), frizzled typed (Ff-) and naked neck (NN). Of all chickens tested, the majority were haplotyped as B2, B15, B19 and B21. Of the Bolivian chickens, 89.5% could be haplotyped: 54.9% were homozygous (including 43.3% B15), and 34.6% were heterozygous (including 15.7% B15). B2-like haplotypes were not found among the Bolivian hens, and only 3.2% of these birds showed homozygous B21-like proteins. Of the Indian hens, MHC (BG)-like proteins could be detected in 60.0% of the AP birds, 6.7% of the KN birds; 40.0% of the Ff- birds; and 10.3% of the NN birds. In these lines, a total of 40.1% (AP), 6.7% (KN), 30.1% (Ff-) and 10.3% (NN) were homozygous for the B-haplotype. Only in the AP line (19.9%), and the Ff- line (9.9%) were heterozygous B-haplotypes types found. The B2 haplotype was found in all Indian chicken lines. Most Indian birds have completely unknown haplotypes, indicating a potentially interesting genetic pool. Subgrouping the Bolivian and Indian indigenous hens into monomorphic BG populations revealed individual differences based on the B-types.     

Immunomodulatory effect of Artemisia argyi polysaccharide on peripheral blood leucocyte of broiler chickens

This study was conducted to investigate the immunomodulatory effect of a water‐soluble polysaccharide extracted from Artemisia argyi (AAP) in vitro. The effect was assessed in peripheral blood leucocytes (PBLs) of broilers, which were incubated with different AAP concentrations (0, 25, 50, 100, and 200 μg/ml) for 24 hr at 37°C in a 5% CO₂ incubator. The results showed that, compared with the control group, immunoglobulin M (IgM) concentration was increased in the supernatant of the 100 μg/ml AAP‐treated group (p < .05), and immunoglobulin G (IgG) concentration was increased in the supernatant of the 200 μg/ml of AAP group (p < .05). In terms of cytokine production, production of interleukin‐1beta (IL‐1β), interleukin‐6 (IL‐6) and tumour necrosis factor‐alpha (TNF‐α) in the supernatant was enhanced in the AAP group in a dose‐dependent function, as well as enhanced mRNA expressions were showed in the cells (p < .05). The highest concentration of these three cytokines was observed in different AAP groups (IL‐1β for 25 μg/ml of AAP, IL‐6 for 100, and 200 μg/ml of AAP, and TNF‐α for 100 μg/ml of AAP respectively). The concentration of nitric oxide (NO) was increased when using AAP at the concentration of 100 μg/ml (p < .05) as compared to the control group. No significant effects on inducible nitric oxide synthase, Toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 and nuclear factor Kappa B (NF‐κB) mRNA level were observed at each concentration of AAP. In conclusion, we found that AAP can specifically promote the production of immunoglobulins (IgM and IgG), cytokines (IL‐1β, IL‐6 and TNF‐α), as well as the NO concentration in vitro, but not through the activation of the TLR4/NF‐κB signalling pathway.