Cold-induced enhancement of avian uncoupling protein expression, heat production, and triiodothyronine concentrations in broiler chicks

The relationships among avian uncoupling protein (avUCP) mRNA expression, heat production, and thyroid hormone metabolism were investigated in 7-14-day-old broiler chicks (Gallus gallus) exposed to a low temperature (cold-exposed chicks, CE) or a thermoneutral temperature (TN). After 7 days of exposure, CE chicks exhibited higher heat production (+83%, P < 0.01), avUCP mRNA expression (+20%, P < 0.01), and circulating triiodothyronine (T-3) levels (+104%, P = 0.07) for non-statistically different body weights and feed intake between 3 and 7 days of exposure as compared to TN chicks. Plasma thyroxine (T-4) concentration was clearly decreased in CE chicks (-33%, P = 0.06). The lower hepatic inner-ring deiodination activity (-47%) and the higher renal outer-ring deiodination activity (+75%) measured in CE compared to TN chicks could partly account for their higher plasma T3 concentrations. This study describes for the first time the induction of avUCP mRNA expression by low temperature in chickens, as it has been previously shown in ducklings, and supports the possible involvement of avUCP in avian thermogenesis. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

Performance, incidence of metabolic disturbances and endocrine variables of food-restricted male broiler chickens

1. This experiment was carried out to evaluate the productive and physiological consequences of a slight but long term food restriction of male broiler chickens from 2 commercial strains.2. Cobb-500 and Ross chickens were submitted to a 20% food restriction from 8 to 21 d of age. Strain, food programme and their interactive effects were analysed in terms of consequences upon performance, mortality, incidence of sudden death syndrome (SDS) and ascites syndrome (AS), index of right cardiac hypertrophy and plasma concentrations of hormones related to metabolism and growth (T-3, T-4, T-3:T-4 ratio, IGF-I and GH).3. Although some catch-up growth was observed by refeeding previously restricted birds after 22 d of rearing, food restriction decreased (P less than or equal to 0.05) body weight at market age (42 d) irrespective of the strain, but improved (P less than or equal to 0.05) food conversion.4. The incidence of mortality was not high in non-restricted birds but SDS and AS caused more than 50% of deaths. Hypertrophic cardiac index was observed in chickens of both strains after 4 weeks of age and was higher in ad libitum fed birds.5. During the period of food restriction, plasma T-3 and IGF-I concentrations decreased whereas plasma T-4 and GH concentrations increased compared to those of the age-matched ad libitum fed counterparts. During the subsequent ad libitum feeding period, few differences in circulating hormone concentrations were observed, except for the higher mean CH litres in previously food-restricted chickens at 35 d of age.6. These results indicate that even a non-severe food restriction negatively affects body weight of 42-d-old male broilers but these are benefits with improved food efficiency and diminished mortality from metabolic disturbances. The hormone results suggest that the degree of food restriction applied was not severe because there was a very fast adaptive response with small and transient alterations in T-3, T-4 and GH plasma concentrations during the period of compensatory growth.

SEROLOGICAL RESPONSE OF CHICKENS TO INFECTION WITH SALMONELLA-GALLINARUM S-PULLORUM DETECTED BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.

Isolation of Campylobacter jejuni from viscera and bile secretion of broiler chickens with diarrhea

Since chickens are important reservoirs of Campylobacter jejuni and their meat is the most frequent route of transmission for human campylobacteriosis, the purpouse of the present study was to investigate the presence of Campylobacter in viscera of chickens with diarrhea, evaluating the frequency of isolation of this microorganism from organs considered to be preferential for its isolation. A total of 107 visceral samples from chickens with diarrhea from different farms in the Ribeirao Preto region, SP, were examined for the presence of Campylobacter jejuni. The material consisted of 73 livers and 34 spleens, plus 29 bile secretion samples. The frequency of Campylobacter jejuni isolation was 54.79% for the liver samples, 35,29% for the spleens and 6,89% for the bile secretion samples. The data suggest that, under the conditions of the present study, the liver may be the organ of choice for the isolation of Campylobacter in the presence of diarrhea and liver involvement in chickens.

Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers

The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

Ileal and caecal microbial populations in broilers given specific essential oil blends and probiotics in two consecutive grow-outs

Specific essential oil (EO) blends and probiotics used as feed additives have been shown to promote healthy digestive microbials resulting in improved poultry production. Two consecutive experiments were conducted with broilers fed corn-soybean meal diets to determine comparative effects of feed additives on ileal and caecal microbial populations (MP). Ross 708 broilers were placed in 84 pens with previously used litter and treatments maintained in the same pens for both experiments. Eight treatment groups were fed diets containing: Bacitracin methylene disalicylate (BMD) as positive control (PC); no additives as negative control (NC); three probiotics: BC-30; BioPlus 2B (B2B); and Calsporin; and the essential oil blends Crina Poultry Plus (CPP) at 300 or 150 ppm in the first experiment; and CPP at 300 ppm and Crina Poultry AF at 100 ppm in experiment 2. Starter and grower diets contained the ionophore (Coban). Ileal and caecal samples were collected at 43 days of age from male broilers. The DNA of microbial populations was isolated from digesta samples and analysed by denaturing gradient gel electrophoresis to generate percentage similarity coefficients (%SC) from band pattern dendrograms. Differences were observed in ileal and caecal populations depending on treatment, respectively, and especially between experiments. Broilers fed diets with probiotics had very similar MP. The EO CPP at 300 ppm resulted in ilea! MP similar to those observed in chickens fed probiotics. We concluded that antibiotic treatment affected ileal, but no caecal MP. More pronounced changes in ileal and caecal MP were seen in broilers at 43 days of age following probiotic and essential oil treatments.

Effect of immersion and inoculation in ovo of Lactobacillus spp. in embryonated chicken eggs in the prevention of Salmonella enteritidis after hatch

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Digestive enzymatic responses of chickens feed-restricted and refed as affected by age

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

Endogenous amino acid flow in the avian ileum: quantification using three techniques

The aim of the present study was to compare the protein-free diet, guanidinated casein (GuC) and enzyme hydrolysed casein (EHC) methods for the quantification of endogenous amino acid (AA) flow in the avian ileum. Growing broiler chickens (5 weeks old) were used. All three assay diets were based on dextrose, and in the GuC and EHC diets GuC or EHC were the sole source of N. Endogenous AA flows determined with the use of protein-free diet were considerably lower (P < 0.05) than those determined by the GuC and EHC methods. The, total endogenous AA flows determined by the GuC and EHC methods were almost 3-fold greater (P < 0.05) than those determined by the protein-free diet. The endogenous AA values obtained from GuC and EHC methods were similar (P >0.05), except for the flow of arginine, which was lower (P < 0.05) in the EHC method. Glutamic acid, aspartic acid, threonine and glycine were the predominant endogenous AA present in digesta from the distal ileum. The contents of methionine, histidine and cystine were lower compared with other AA. The method of determination had no effect on the AA composition of endogenous protein, except for threonine, glutamic acid, lysine, arginine and cystine. The concentrations of threonine and arginine were lower (P < 0.05) and that of lysine was higher (P < 0.05) with the EHC method compared with the other two methods. The concentration of glutamic acid was greater (P < 0.05) and that of cystine was lower (P < 0.05) in the EHC and GuC methods compared with the protein-free diet method. The results showed that the ileal endogenous flows of N and AA are markedly enhanced by the presence of protein and peptides, above those determined following feeding of a protein-free diet. It is concluded that the use of EHC and GuC methods enables the measurement of ileal endogenous losses in chickens under normal physiological conditions.

Optimum inclusion of field peas, faba beans, chick peas and sweet lupins in poultry diets. II. Broiler experiments

1. Three experiments were undertaken to determine the optimum inclusion rates of held peas, faba beans, chick peas and sweet lupins in broiler starter and finisher diets in amounts up to 360 g/kg. 2. In experiment A chickens in cages grown to 21 d on diets with field peas and faba beans gave better growth rate and feed efficiency than those with sweet lupins and chick peas. Growth rate and Food conversion ratio (FCR) improved with increasing amounts of faba beans in the diet while for chick peas growth rate and FCR declined. Digesta viscosity and excreta stickiness scores were much higher on diets with sweet lupins. Steam pelleting improved growth rate and FCR on all diets. 3. In experiment B birds were in cages and grown from 21 to 42 d. There were no differences between grain legumes (when combined for all inclusions) for growth rate, food intake or FCR. Viscosity was again much higher on the sweet lupin-based diets while the pancreas was significantly enlarged on the diets with chick peas, as observed previously in chickens grown to 21 d. Steam pelleting of diets gave a consistent and positive response for weight gain and FCR. 4. Experiment C was carried out in pens each holding 60 birds under semi-commercial conditions and grown to 4 2 d on starter and finisher diets with the same grain legumes as used previously but each at 2 rates of inclusion similar to those in commercial practice. Field peas at 200 to 300 g/kg and chick peas at 150 to 220 g/kg gave inferior growth to faba beans (150 to 180 g/kg) and sweet lupins (120 g/kg). 5. The results of these experiments allowed tentative recommendations to be made to industry for inclusion rates of these cultivars of the 4 grain legumes. These were: field peas 300 g/kg; faba beans 200 g/kg, chick peas 100 g/kg and sweet lupins

Canonical discriminant analysis applied to broiler chicken performance

The mechanisms involved in the control of growth in chickens are too complex to be explained only under univariate analysis because all related traits are biologically correlated. Therefore, we evaluated broiler chicken performance under a multivariate approach, using the canonical discriminant analysis. A total of 1920 chicks from eight treatments, defined as the combination of four broiler chicken strains (Arbor Acres, AgRoss 308, Cobb 500 and RX) from both sexes, were housed in 48 pens. Average feed intake, average live weight, feed conversion and carcass, breast and leg weights were obtained for days 1 to 42. Canonical discriminant analysis was implemented by SAS((R)) CANDISC procedure and differences between treatments were obtained by the F-test (P < 0.05) over the squared Mahalanobis` distances. Multivariate performance from all treatments could be easily visualised because one graph was obtained from two first canonical variables, which explained 96.49% of total variation, using a SAS((R)) CONELIP macro. A clear distinction between sexes was found, where males were better than females. Also between strains, Arbor Acres, AgRoss 308 and Cobb 500 (commercial) were better than RX (experimental), Evaluation of broiler chicken performance was facilitated by the fact that the six original traits were reduced to only two canonical variables. Average live weight and carcass weight (first canonical variable) were the most important traits to discriminate treatments. The contrast between average feed intake and average live weight plus feed conversion (second canonical variable) were used to classify them. We suggest analysing performance data sets using canonical discriminant analysis.