Molecular detection and genotyping of microsporidia species in chickens in Turkey

Microsporidia are obligate intracellular pathogens that infect various hosts including invertebrates and vertebrates. Despite the importance, knowledge on the prevalence and molecular characteristics of microsporidia in chickens is limited, and no data are available for Turkey. A total of 300 fecal samples from chickens in the Central Anatolia Region of Turkey were analyzed by using a nested polymerase chain reaction assay targeting the rRNA internal transcribed spacer (ITS) region for the common microsporidia species. Corresponding PCR amplicons from the positive samples were sequenced for genotyping. Enterocytozoon bieneusi was identified in 22 (7.3 %) samples, whereas Encephalitozoon spp. was not detected. The prevalence of E. bieneusi was 63.6 % in Kayseri and 36.4 % in Nevsehir provinces, and 8.8 % in soft fecal samples and 9.7 % in diarrhoeic samples. No infections were found in Kirsehir Province. Significant differences were found for the distribution of E. bieneusi among provinces and fecal conditions. Infections were found only in free-range chickens. As a result of ITS region sequencing, two genotypes were characterized. The novel genotype ERUNT1 (n = 21), belonging to zoonotic group 1, was the most common genotype throughout the study area. The other known genotype, ERUSS1 (n = 1), had a restricted distribution and was previously detected in cattle and sheep in the same region. Our study provides the first data on microsporidia species from chickens in Turkey. None of these genotypes have been reported in humans; thus, the risk potential for public health is limited but needs further investigation.     

Gene organization, alternate splicing and expression pattern of porcine visfatin gene

Visfatin is a newly discovered visceral fat-specific adipocytokine. It is upregulated in obesity and exerts insulin-mimetic effects in various tissues in human and mouse. We reported here the cloning and characterization of porcine visfatin, its three alternate splicing variants. Sequence analysis indicated that variant 1 is the predominant form among species, which contains an open reading frame of 1473 bp encoding a 52-kDa protein of 491 amino acids. While the other two variants were predicted to encode two 3' truncated proteins due to early termination. The nucleotide and amino acid sequences deduced from variant 1 were conservative across species. The porcine visfatin gene was composed of 11 exons at least and had exactly the same exon/intron structure as the human orthologs. Nested PCR showed that variants 1 and 3 were ubiquitously expressed in porcine tissues and that variant 2 was expressed in most tissues examined with exception of testis and liver. The discovery of the three variants of visfatin in porcine would be useful to the further investigation of the function of the visfatin gene.     

Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens

The aim of this study was to develop and validate an ELISA for detecting chicken antibodies to Eimeria tenella. An initial comparison of merozoite and sporozoite antigen preparations revealed few differences in their ability to monitor the onset, kinetics and magnitude of the antibody response suggesting that both antigens would be equally useful for development of an ELISA. Furthermore the cross-reactivity of these antigens with sera from birds infected with chicken Eimeria species was similar. The merozoite antigen was selected for further evaluation because it was easier to prepare. Discrimination between sera from birds experimentally infected with E. tenella and birds maintained in an Eimeria-free isolation facility was excellent. In sera collected from free-range layers and commercial broilers there also appeared to be clear discrimination between infected and uninfected birds. The ELISA should prove useful for monitoring infectivity in vaccination programmes in layer and breeder flocks and for assessing the effectiveness of biosecurity measures in broiler flocks.     

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens

Background: A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs (miRNAs) targeting the affected genes. To this end we injected fertile eggs with either 0.5 μg of recombinant murine leptin or vehicle (PBS) before incubation. Results: Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides (TG) and total cholesterol (Tch) were decreased, whereas the serum levels of TG, Tch and apolipoprotein B (ApoB) were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 (SREBP-1c), SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and cholesterol 7α-hydroxylase 1 (CYP7A1) was significantly up-regulated, as was the protein content of both SREBP-1c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR. Conclusions: These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.     

Sunflower-seed oil,rapidly-degradable starch,and adiposity up-regulate leptin gene expression in lactating goats

We conducted experiments to evaluate the effects of lipid supplementation and the nature of starchy concentrate on the regulation of leptin synthesis in lactating goats. Multiparous goats in mid- to late lactation received diets based on different forages and containing plant oil or seeds rich in either 18:1c9, 18:2n-6 or 18:3n-3 corresponding to 3%–7% dry matter (DM) as lipid supplements, or diets based on concentrate as either rapidly or slowly degradable starch. The isoenergetic replacement of a part of the concentrate by either oleic sunflower-seed oil, formaldehyde-treated linseeds, or linseed oil did not modify leptinemia and the leptin mRNA concentration in adipose tissues, suggesting a lack of effect of 18:1c9, 18:3n-3, or their biohydrogenation products. Conversely, leptinemia and the leptin mRNA abundance were increased (by 20% and 140%, respectively, P < 0.05) in goats fed sunflower-seed oil under a grassland hay-based diet but not a maize silage-based diet, at similar energy intakes and adiposity. Thus, 18:2n-6 per se may up-regulate leptin gene expression, but the effect could be blunted by other fatty acids formed during the ruminal digestion of sunflower-seed oil when combined with maize silage. Consumption of rapidly but not slowly degradable starch increased (by 17%, P < 0.05) leptinemia. Moreover, during lactation, plasma leptin was positively correlated (P < 0.05) to adiposity parameters and negatively correlated to fiber intake. The results suggest that leptinemia responds poorly to nutritional factors in lactating goats, thus highlighting the physiological need to sustain hypoleptinemia during lactation.     

Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration

This paper reviews data on leptin gene expression in adipose tissue (AT) and mammary gland of adult ruminants, as well as on plasma leptin variations, according to genetic, physiological, nutritional and environmental factors. AT leptin mRNA level was higher in sheep and goat subcutaneous than visceral tissues, and the opposite was observed in cattle; it was higher in fat than in lean selection line in sheep; it was decreased by undernutrition and increased by refeeding in cattle and sheep, and not changed by adding soybeans to the diet of lactating goats; it was increased by injection of NPY to sheep, and by GH treatment of growing sheep and cattle. Insulin and glucocorticoids in vitro increased AT leptin mRNA in cattle, and leptin production in sheep. Long daylength increased AT lipogenic activities and leptin mRNA, as well as plasma leptin in sheep. Mammary tissue leptin mRNA level was high during early pregnancy and was lower but still expressed during late pregnancy and lactation in sheep. Leptin was present in sheep mammary adipocytes, epithelial and myoepithelial cells during early pregnancy, late pregnancy and lactation, respectively. Plasma leptin in cattle and sheep was first studied thanks to a commercial “multi-species” kit. It was positively related to body fatness and energy balance or feeding level, and decreased by β-agonist injection. The recent development of specific RIA for ruminant leptin enabled more quantitative study of changes in plasma leptin concentration, which were explained for 35–50% by body fatness and for 15–20% by feeding level. The response of plasma leptin to meal intake was related positively to glycemia, and negatively to plasma 3-hydroxybutyrate. The putative physiological roles of changes in leptin gene expression are discussed in relation with published data on leptin receptors in several body tissues, and on in vivo or in vitro effects of leptin treatment.     

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens

Background

A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs (miRNAs) targeting the affected genes. To this end we injected fertile eggs with either 0.5 μg of recombinant murine leptin or vehicle (PBS) before incubation.

Results

Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides (TG) and total cholesterol (Tch) were decreased, whereas the serum levels of TG, Tch and apolipoprotein B (ApoB) were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 (SREBP-1c), SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and cholesterol 7α-hydroxylase 1 (CYP7A1) was significantly up-regulated, as was the protein content of both SREBP-1c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR.

Conclusions

These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.     

The effects of feeding and fasting on gastrointestinal sounds in adult horses

The effect of changes in feed intake on auscultatable gastrointestinal sounds has not been systematically studied. Disagreement also is present in the literature about variation in sounds according to the quadrant of auscultation. Gastrointestinal sounds were recorded over the center of the left dorsal, left ventral, right ventral, and right dorsal quadrants and over the middle of the right abdominal flank. During 24 hours (n = 4) or 48 hours (n = 5) of fasting, there was a reduction in the intensity of gastrointestinal sounds as assessed by analysis of sound recordings. There was also a reduction in the number of mixing-like and propulsive-like sounds heard by 2 blinded observers. After refeeding, there was a marked increase in sound. Sound intensity varied among abdominal quadrants, but blinded observers did not notice significant differences in the number of mixing-like sounds. The left dorsal quadrant was quieter than others during fasting and refeeding. The right ventral quadrant appeared to be least affected by fasting, and sounds were louder over the right ventral and right middle quadrants than over the others. The blinded observers' perceptions of sound correlated poorly with one another and with objective measures of sound intensity. This experiment demonstrates the effectiveness of computerized analysis of abdominal sound in detecting a reduction in the intensity of gastrointestinal sounds during fasting and their return during refeeding. The left dorsal quadrant was quieter than other quadrants, likely because of its position over the small colon. There was considerable observer variation in the number of intestinal sounds heard.     

Effect of supplementation on the feed intake and performance of confined and scavenging crossbred growing chickens in Burkina Faso

An experiment was conducted to evaluate the performance of crossbred growing chickens (Rhode Island Red × indigenous Burkina Faso hens) from 6 to 17 weeks of age, under five feeding/management regimes: (1) CMx(+), confined and given a mixed feed containing cracked maize and cowpea and a vitamin–mineral premix ad libitum; (2) CS(+), confined and offered ad libitum a choice of cracked maize and cowpea with the premix; (3) ScS(+), scavenging from 09:00 to 16:00 with the diet in treatment (2) available from 16:00 to 09:00; (4) ScS(−), treatment (3) but without the premix; and (5) ScO, scavenging only, with no supplements provided. Daily dry matter (DM) intake was highest for CS(+) (43.5 g), and lowest for CMx(+) (33.6 g) (p < 0.05), with intermediate intakes for ScS(+) and Sc(−) of 36.7 g and 36.2 g, respectively. The ratios of intakes of cowpea to maize were 50:50, 21:79, 27:73 and 22:78 for CMx(+), CS(+), ScS(+) and ScS(−), respectively (p < 0.05). Dietary concentrations of crude protein (CP) were 15.7%, 11.5%, 12.3% and 11.6% of DM for CMx(+), CS(+), ScS(+) and ScS(−), respectively. Average daily gains (ADG) were 8.15 g, 5.24 g, 6.03 g, 5.36 g and 4.45 g for CMx(+), CS(+), ScS(+), ScS(−) and ScO, respectively, and were significantly higher for CMx(+) (p < 0.05). Feed conversion ratio was highest for CS(+) and lowest for CMx(+). ADG of the males (6.44 g) was significantly (p < 0.05) higher than that of the females (5.86 g). Breast and thigh muscle weights were highest for ScS(+) (p < 0.05).     

Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe

Until recently, Toxoplasma gondii was considered to be clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known of the genetics of T. gondii strains from Africa. In this study, we genotyped 19 T. gondii isolates from chickens from six African countries (Egypt, Kenya, Nigeria, Congo, Mali, and Burkina Fasco) using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed four genotypes. Thirteen isolates belong to the Type III lineage, five isolates have Type II alleles at all loci except apico and they belong to the Type II lineage. One isolate from Nigeria had atypical genotype. In general, these isolates were mostly clonal Type III and II strains that predominate in North American and European. DNA sequencing at several loci for representative isolates confirmed the results of PCR-RFLP genotyping. Taken together with recent studies of T. gondii isolates from Africa, it is clear that the three clonal lineages (Types I, II and III) predominate not only in North America and Europe, but also in Africa.     

Birth weight and gender determine expression of adipogenic, lipogenic and adipokine genes in perirenal adipose tissue in the young adult sheep

Epidemiological studies have demonstrated that low birth weight is associated with an increased incidence of visceral obesity and metabolic disorders in later life. In the present study, we have determined the impact of birth weight and gender on gene expression in visceral adipose tissue (VAT) in the young adult sheep. Lambs (n=19, birth weight range 2.6-7.55 kg) were born at term and growth monitored for 22.4+/-0.2 weeks, when body composition was determined by Dual X-ray Absorptiometry (DXA) and samples of VAT and subcutaneous (SCAT) adipose tissue collected. Plasma samples were collected at post-mortem for the determination of free fatty acids (FFA), glucose and insulin concentrations. Peroxisome-Proliferator Activated Receptor-gamma (PPARgamma), glycerol-3-phosphate dehydrogenase (G3PDH), lipoprotein lipase (LPL), adiponectin and leptin mRNA expression was determined by qRT-PCR. Fractional growth rate in postnatal weeks 1-3 was inversely related to birth weight in both males and females (R2=0.22, P<0.05, n=19). PPARgamma mRNA expression in VAT, but not SCAT, was inversely related to birth weight (R2=0.60, P<0.01, n=18). In males, but not females, PPARgamma mRNA in VAT was directly related to G3PDH mRNA expression (R2=0.69, P<0.01, n=9). Plasma FFA concentrations were inversely related to birth weight in both males and females (R2=0.22, P<0.05, n=19). These findings demonstrate that low birth weight is associated with an increased expression of a key adipogenic factor in visceral adipose tissue in young adulthood. In males, this is associated with an increased expression of lipogenic genes, and this may contribute to the increased propensity for visceral obesity in low birth weight males compared to females.     

Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens

The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue.     

Polymorphism identification and cardiac gene expression analysis of the calsequestrin 2 gene in broiler chickens with sudden death syndrome

1. Sudden death syndrome (SDS) in broilers is a cardiac disease associated with ventricular tachycardia (VT) and ventricular fibrillation (VF); however, its pathogenesis at the molecular level is not precisely determined.

2. Downregulation and mutations of calsequestrin 2 (CASQ2), a major intracellular Ca²⁺ buffer, have been associated with VT and sudden cardiac death (SCD) in humans but in chickens there is no report describing CASQ2 abnormalities in cardiac diseases.

3. In order to better understand the molecular mechanisms predisposing the myocardium to fatal arrhythmia in broilers, the mRNA expression level of chicken CASQ2 gene (chCASQ2) in the left ventricle of dead broilers with SDS was determined and compared to healthy broilers using quantitative real-time PCR (qPCR). To determine the probable mutations in chCASQ2, PCR and direct sequencing were also done.

4. Results showed a reduction in chCASQ2 expression in broilers dead by SDS. Three novel mutations (K289R, P308S, D310H) which are absent in healthy broilers were observed in chCASQ2.

5. It is concluded that susceptibility to fatal cardiac arrhythmia in SDS may be associated with changes in intracellular Ca²⁺ balance due to mutation and downregulation of chCASQ2.

     

Sexual maturation in hens is not associated with increases in serum leptin and the expression of leptin receptor mRNA in hypothalamus

Background

In mammals, leptin is an attractive candidate for mediating the metabolic signal and the reproductive function via the specific receptor in hypothalamus. However, till now, the role of leptin on reproduction in birds is less well established. This experiment was conducted to elucidate the role of leptin on the onset of reproduction in bird, as a first step, to detect the changes of peripheral leptin and leptin receptor mRNA expression in hypothalamus between mature and immature hens at the same age. 120 ISA brown pullets at D60 were allocated randomly into two groups, long light (LL) group being raised under artificial light regimes with incrementally increased light phase (from 8 L:16D to 14 L:12D) and short light (SL) group raised on consistent light (8 L:16D) for 12 wk.

Results

The results showed that pullets in LL group reached sexual maturation 15 d earlier than those in SL group. Serum E₂ showed a significant increase with age, but no difference was observed between two groups. Serum leptin concentration decreased significantly from D112 to D136 in LL, and was markedly higher in LL group than that in SL at D112, while there was no significant difference between two groups at D136. Leptin receptor and GnRH-I mRNA expression in hypothalamus were significantly increased with age, yet there was no significant difference between SL and LL chickens at the same age. The expression of FSH-β and LH-β mRNA in pituitary was increased with age but did not show significant difference between LL and SL group. GnRH-I mRNA expression was very rich in pineal gland, and decreased from D112 to D136 in LL but not in SL group, and there was no difference between two groups at the same age.

Conclusions

These results indicate that the earlier sexual maturation in hens induced by long-light regime is not accompanied with an increase in serum leptin or leptin receptor gene expression in hypothalamus, or genes expression in HPG axis.     

Effect of age and sex in determining cognitive ability in Vanaraja chickens

(1) To evaluate the cognitive ability of male and female Vanaraja birds, three hundred and sixty 1-d-old sexed chickens were reared under similar conditions in three treatment groups with 4 replicates in each group: 120 females in Treatment 1, 120 males in Treatment 2 and both males and females (60 + 60) as a mixed group in Treatment 3.

(2) To assess learning ability, the birds were trained in T- and Y-mazes and tested at 3-week intervals in 4 test schedules (21, 42, 63 and 84 d). The birds were put into tonic immobility (TI) in each test schedule.

(3) In each maze test, the latency to find the feed was regarded as a successful completion of the task. In the TI-test, the time taken to stabilise on a plane surface after swinging in the hanging cradle for 20–25 s was recorded.

(4) The results indicated that male birds appeared to be cognitively superior to females in terms of learning and cognitive evolution in all the mazes, but by d 84, the females performed as well as the males. With increasing age, spatial memory gathering and processing improved. In the TI-test, the effect of sex or grouping system had no significant effect on the performance of birds at the various ages.