Divergent selection for ascites incidence in chickens.

Chicken lines that were either resistant or susceptible to ascites syndrome were developed by using a hypobaric chamber to induce the disease. Birds were reared in a hypobaric chamber that simulated high altitude by operating under a partial vacuum, which thereby lowered the partial pressure of oxygen. Ascites mortality data from birds reared under hypobaric chamber conditions were used to select siblings to be used for breeding. The response to selection for the susceptible (SUS) and resistant (RES) lines of chickens was very rapid from the base population, which exhibited an incidence of ascites of 75.3%. Extremes in the incidence of ascites were observed in generation 8, with line SUS exhibited an average incidence of ascites of 95.1%, and in generation 9, with line RES exhibited an average incidence of ascites of 7.1%. The incidence of ascites in the relaxed line remained relatively stable and currently has a general incidence of ascites of 60%. The heritability estimates +/- SE for ascites were estimated to be 0.30 +/- 0.05 and 0.55 +/- 0.05 for lines SUS and RES, respectively. Changes in the incidence of ascites appeared to be associated with livability. By generation 10, selection for ascites in line RES increased livability by 11.5 d, whereas in line SUS, livability was decreased by 8 d. Although divergent selection for ascites resulted in a reduction in d 42 BW for both the SUS and RES lines, the SUS line was approximately 163 g heavier than the RES line. Negative genetic correlations between ascites and the right ventricle:total ventricle (RV:TV) ratio were observed in both the SUS and RES lines; however, no significant change in the RV:TV ratio was observed for birds reared under normal conditions in either line. The current data raise questions about the validity of using the RV:TV ratio as an indicator trait in a selection program designed to reduce the incidence of ascites. Overall, direct selection for resistance to ascites by using sire family performance appeared to be an effective means of reducing the incidence of ascites. However, simultaneous selection for BW should be applied to counterbalance the losses in correlated BW.

Hypobaric hypoxia in ascites resistant and susceptible broiler genetic lines influences gut morphology.

Genetic selection based on rapid growth rates, improved feed conversion, and increased body weights has led to a predisposition to ascites in broiler populations. Sire-family selection was applied to a commercial elite line to produce divergent lines of ascites-resistant (RES) and ascites-susceptible (SUS) broilers by the 8th generation. One objective of this research was to determine the effects of hypobaric hypoxia on gut morphology in these genetic lines. In two separate trials, pedigree broiler chickens were randomly assigned to cages in a hypobaric chamber (simulated 2,900 m above sea level) or a matching local altitude chamber (390 m above sea level). Ascites incidence was characterized by heart enlargement and fluid accumulation in the abdominal cavity. At the end of the study on d 42, all surviving birds were killed and evaluated for the presence of ascites and 2-cm sections from the duodenum and lower ileum were collected from 5 chickens per line, per altitude for each trial for morphometric analysis. At a high altitude, ascites incidence was lower in the RES line (20.9 and 3.7%) than in the SUS line (86.4 and 66.9%, Trials 1 and 2, respectively). No ascites was observed at a local altitude. Under hypoxic conditions, duodenum villus surface area was higher (P < 0.05) in the RES line (181.3 +/- 16.8 and 219 +/- 10.9 microm) compared with the SUS line (130.1 +/- 10.5 and 134.3 +/- 9.3 microm; Trials 1 and 2, respectively). No differences in ileum villus morphology were observed for any of the parameters measured. The reduced surface area in the duodenum of birds selected for ascites susceptibility suggests reduced enteric function and may provide clues as to why these birds have increased incidence of ascites.

Effect of prebiotic on gut development and ascites incidence of broilers reared in a hypoxic environment.

Modern broilers have been genetically selected for an increased growth rate and improved feed conversion, but they are also more susceptible to ascites. Ascites occurs when there is an imbalance between available oxygen and the oxygen demand of the broiler. We hypothesized that promoting neonatal gut development with a prebiotic, such as Aspergillus meal (Prebiotic-AM), would enhance gut efficiency, decrease the oxygen demand of the gut, and reduce ascites incidence. In this study, we compared the effect of Prebiotic-AM on ascites incidence and gut development in commercial broilers reared at a local altitude (390 m above sea level) and a simulated high altitude (2,900 m above sea level). Half of the birds received a National Research Council recommended corn-soybean ration, and the other half received the same ration supplemented with 0.2% Prebiotic-AM. These 2 groups were further divided into a local altitude group and a simulated high altitude group for a total of 4 treatment combinations. Tissues were collected on d 1, 3, 7, 14, and 21 from the duodenum and lower ileum and placed in 10% buffered formalin for morphometric analysis. At a simulated high altitude, ascites incidence was 68% for birds fed the Prebiotic-AM supplement compared with 92% ascites incidence in birds given the control feed. The simulated high altitude decreased (P < 0.05) gut development, but prebiotic-treated birds reared in hypoxic conditions had similar gut development to control birds reared at local altitude. These data suggest that a feed ration supplemented with Prebiotic-AM may reduce the effect of hypoxia on broiler gut development and ascites incidence.

Differential expression of cardiac muscle mitochondrial matrix proteins in broilers from ascites-resistant and susceptible lines.

Ascites is a metabolic disorder of modern broilers that is distinguished by cardiopulmonary insufficiency in the face of intense oxygen demands of rapidly growing tissues. Broilers with ascites exhibit sustained elevation of pulmonary arterial pressure and right ventricular hypertrophy, the end result of which is heart failure. It has been shown that mitochondrial function is impaired in broilers with ascites. In the current study, mitochondrial matrix protein levels were compared between ascites-resistant line broilers and ascites-susceptible line broilers with and without ascites using two-dimensional (2-D) gel electrophoresis. One hundred seventy-two protein spots were detected on the gels, and 9 of the spots were present at different levels in the 4 groups of broilers. These 9 protein spots were selected for identification by mass spectrometry. Two of the spots were found to contain single mitochondrial matrix proteins. Both mitochondrial matrix proteins, the dihydrolipoamide succinyltransferase component of the 2-oxoglutarate dehydrogenase complex and the alpha-subunit of mitochondrial trifunctional enzyme, were present at higher levels in ascites-resistant line broilers with ascites in the present study. The elevated levels of 2 key proteins in aerobic metabolism in ascites-resistant line broilers with ascites observed in the present study suggests that the mitochondria of broilers with this disease may respond inappropriately to hypoxia.

Stress response differences and disease susceptibility reflected by heterophil to lymphocyte ratio in turkeys selected for increased body weight.

Three genetic lines of turkeys were compared for their responses to Escherichia coli challenge following dexamethasone injection (Dex) or E. coli challenge preceding transport stress (TS). The turkey lines were a slow growing line selected for increased egg production (Egg line), a fast growing line selected for increased 16-wk BW (F line), and a commercial line (Comm line). At 14 wk of age, the Dex group was treated with 3 injections of 2 mg of Dex/kg of BW followed by airsac challenge with 100 cfu of E. coli. The TS group was given the same E. coli challenge at 1 x 10(4) cfu/bird without Dex treatment, and was subjected to transport stress, including 12 h of holding time in a transport vehicle, 8 d after the challenge. All treated birds and untreated control birds were bled at the same time, which was 1 d after transport and 9 d after challenge with E. coli. The main effect mean (MEM) total leukocyte counts (WBC) and the percentages of eosinophils (Eos) and basophils (Baso) were the same for all 3 lines; however, the MEM percentages of heterophils (Het) and monocytes (Mono) and the heterophil/lymphocyte ratio (H/L) were lower and the percentage of lymphocytes (Lym) was higher in the Egg line compared with the 2 fast-growing lines. Both stress treatments increased WBC, Het, and H/L and decreased Lym in all 3 lines; however, these effects were significantly greater in both fast growing lines compared with the Egg line. Sixteen-week BW was unaffected by either treatment in the Egg line and was decreased by both treatments in the Comm line and by the Dex treatment in the F line. Main effect mean airsacculitis score (AS) was not affected by line and was significantly increased by TS and Dex treatments. Neither treatment affected AS of the Egg line birds, whereas Dex treatment increased AS of the F line, and both Dex and TS increased AS of the Comm line. Mortality was significantly higher in the Comm line compared with the Egg line and was intermediate in the F line. The differences between these lines in their disease resistance and physiological response to stress in 2 stress models suggests that increasing selection for BW of turkeys is accompanied by changes in the stress response resulting in increased susceptibility to opportunistic bacterial infection.

Changes in the tibial growth plates of chickens with thiram-induced dyschondroplasia.

Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival. The results showed that thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were "up-regulated," suggesting that thiram has pro-angiogenic activity. However, TUNEL assay showed that thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation. It was concluded that thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes.

Alternatives to antibiotics: utilization of bacteriophage to treat colibacillosis and prevent foodborne pathogens.

Bacteriophages are viruses that infect and kill bacteria. Bacteriophage do not infect animal and plant cells, which makes them a potentially safe alternative to antibiotics. We have been conducting research on the efficacy of bacteriophage to prevent and treat colibacillosis in poultry. Bacteriophages that were lytic to a non-motile, serotype 02 isolate of Escherichia coli were isolated from municipal wastewater treatment plants and poultry processing plants. This E. coli isolate is pathogenic to poultry, causing severe respiratory and systemic infections. Two bacteriophage isolates were selected for use in studies designed to determine the efficacy of these bacteriophage to prevent and treat severe colibacillosis in poultry. Colibacillosis was induced by injecting 6 x 10(4) cfu of E. coli into the thoracic air sac when birds were 1 wk of age. Initial studies demonstrated that mortality was significantly reduced from 85 to 35% when the challenge culture was mixed with equal titers of bacteriophage, and the birds were completely protected when the challenge culture was mixed with 10 pfu of bacteriophage. In subsequent studies, we have shown that an aerosol spray of bacteriophage given to birds prior to this E. coli challenge could significantly reduce mortality even when given 3 d prior to the E. coli challenge. Our research on treating colibacillosis in poultry has demonstrated that an intramuscular injection of bacteriophage given 24 or 48 h after the birds were challenged rescued the birds from this severe E. coli infection. We have demonstrated that bacteriophage can be used to prevent and treat colibacillosis in poultry and may provide an effective alternative to antibiotic use in animal production.

Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers.

A study was conducted to evaluate the therapeutic efficacy of bacteriophage and the antibiotic enrofloxacin individually and in combination to treat colibacillosis. The experimental design was a 2 x 2 x 2 factorial with 8 treatments and 4 replicate pens of 10 birds. The treatments were 1) control, 2) unchallenged birds treated with bacteriophage, 3) enrofloxacin, or 4) the combination; 5) birds challenged with Escherichia coli, and birds challenged with E. coli and treated with 6) bacteriophage, 7) enrofloxacin, or 8) the combination of bacteriophage and enrofloxacin. Birds in the E. coli challenged treatments were challenged at 7 d of age by injecting 10(4) cfu of E. coli into the thoracic air sac. The antibiotic treatment was initiated immediately after the birds were challenged and consisted of 50 ppm enrofloxacin in the drinking water for 7 consecutive days. The bacteriophage treatment consisted of a single intramuscular injection of 2 different bacteriophage (10(9) pfu) administered immediately after the E. coli challenge. Mortality in the birds challenged with E. coli and untreated was 68%, and the bacteriophage and enrofloxacin treatments significantly decreased mortality to 15 and 3%, respectively. There was total protection in birds that received both the bacteriophage and enrofloxacin representing a significant synergy. The decrease in mortality with enrofloxacin (3%) was significantly better than the decrease in mortality with bacteriophage (15%). Airsacculitis lesion scores and lesion incidence in surviving birds were significantly less in the enrofloxacin treatment compared with the bacteriophage treatment. Both bacteriophage and enrofloxacin provided effective treatments of colibacillosis, and the synergy between these 2 treatments suggests that bacteriophage combined with antibiotic treatment has significant value.

Differential expression of mitochondrial electron transport chain proteins in cardiac tissues of broilers from pulmonary hypertension syndrome-resistant and -susceptible lines.

Pulmonary hypertension syndrome (PHS) is a metabolic disease associated with the rapid growth rate of modern broilers. Broilers susceptible to PHS experience sustained elevation of pulmonary arterial pressure leading to right ventricular hypertrophy and ultimately heart failure. Previous studies have shown that mitochondrial function is defective in broilers with PHS; they use oxygen less efficiently than broilers without PHS. In this study mitochondrial electron transport chain (ETC) protein levels were compared in cardiac tissues from PHS resistant and susceptible line broilers using quantitative immunoblots. Seven of 9 anti-mammalian mitochondrial ETC protein antibodies tested exhibited cross-species reactivity. Six ETC proteins were differentially expressed in the right ventricles of broilers raised under simulated high altitude conditions (2,900 m above sea level). Four ETC proteins were present at higher levels in resistant line birds without PHS than in resistant line birds with PHS or in susceptible line birds with or without PHS. One ETC protein was present at higher levels in broilers without PHS than in broilers with PHS in both lines, and one ETC protein was present at lower levels in susceptible line birds without PHS than in susceptible line birds with PHS or in resistant line birds with or without PHS. Interestingly, differential expression of mitochondrial ETC proteins was not observed in the right ventricles of broilers raised at local altitude (390 m above sea level) nor was it observed in the left ventricles of broilers exposed to simulated high altitude. These results suggest that higher levels of mitochondrial ETC proteins in right ventricle cardiac muscle may be correlated with resistance to PHS in broilers.

The effects of water supplementation with vitamin E and sodium salicylate (Uni-Sol) on the resistance of turkeys to Escherichia coli respiratory infection.

The objective of this study was to determine the prophylactic efficacy of two commercial products, soluble vitamin E and soluble sodium salicylate (Uni-Sol), in an Escherichia coli respiratory challenge. The drinking water of male turkey poults was nonsupplemented or supplemented with either vitamin E or Uni-Sol or a combination of both at dosages recommended by the manufacturer. There were 110 birds in each of the four treatments, housed in four floor pens per treatment. At 5 wk of age, birds in half of the pens were challenged with an air sac inoculation of approximately 50 colony-forming units of E. coli. Water treatment commenced 5 days before challenge and continued for 2 wk after challenge, when birds were necropsied. All water treatments prevented the decrease in body weight due to E. coli challenge; however, either vitamin E or Uni-Sol alone, but not the combination of the two, decreased body weight in nonchallenged controls. Either vitamin E or Uni-Sol treatment alone, but not the combination of the two, significantly decreased mortality and air sacculitis scores of challenged birds, and all treatments decreased the isolation rates of E. coli from the liver. All treatments protected liver, spleen, and bursa weights (relative to body weight) from the effects of E. coli challenge, and Uni-Sol alone or vitamin E with Uni-Sol protected relative heart weights from the effect of challenge. Uni-Sol treatment alone increased the main effect mean total leukocyte counts and the number and percent of lymphocytes. Uni-Sol in combination with vitamin E increased the number of lymphocytes of challenged birds. Uni-Sol alone decreased the main effect mean heterophil/lymphocyte ratio (H/L) ratio, whereas vitamin E alone increased the H/L ratio of challenged birds. These results indicate that treatment of turkey poults with vitamin E or Uni-Sol prior to and during the stressful events that can lead to colisepticema may decrease disease incidence and mortality.

Comparative efficacy of different dithiocarbamates to induce tibial dyschondroplasia in poultry.

Tibial dyschondroplasia (TD) is a metabolic cartilage disease in poultry the natural etiology of which is not known. In the absence of biomarkers to monitor the initiation and progression of the naturally occurring disease, experimentally induced TD can provide a suitable venue to study the mechanism of its pathogenesis. Therefore, the objective of this study was to establish a streamlined experimental protocol to induce TD using dithiocarbamates and to determine a time course of its progression. Three different dithiocarbamates, dimethyldithiocarbamate, pyrrolidine dithiocarbamate, and tetramethyl thiuram disulfide (thiram), were tested with respect to their abilities to induce TD and affect different physiological factors. Our results show that chickens fed thiram during the first 2 wk of age showed a maximum TD index. Thiram appeared to be the most potent of the 3 dithiocarbamates with dimethyldithiocarbamate having the least ability to induce TD and pyrrolidine dithiocarbamate showing an intermediate potency. A transient exposure to thiram for a day or 2 was sufficient to markedly increase the incidence of TD and produce lasting damage as determined by the presence of severe lesions in a high percentage of birds at 2 to 3 wk after the treatment. Thiram affected the chondrocyte morphology of maturing zone cartilage evident by nuclear shrinkage and emptied chondrocyte lacunae during later times and also involutions of capillary vessels. Such changes were not seen in prehypertrophic zone chondrocytes of the same growth plates. Thiram reduced the BW, increased blood heterophil-to-lymphocyte ratios, and elevated serum corticosterone concentrations indicating physiological stress. However, there was no change in relative liver weights or blood clinical chemistry including the serum concentrations of Ca, P, and Cu in thiocarbamate-fed chickens. Induction of TD in young chickens by means of a short feeding protocol with thiram may be useful to study the mechanisms of pathogenesis of TD and to identify micronutrients that can provide protection against this disease.

Sequence analysis of bone morphogenetic protein receptor type II mRNA from ascitic and nonascitic commercial broilers.

Ascites syndrome, also known as pulmonary hypertension syndrome (PHS), is a common metabolic disorder in rapidly growing meat-type chickens. Environmental factors, such as cold, altitude, and diet, play significant roles in development of the disease, but there is also an important genetic component to PHS susceptibility. The human disease familial primary pulmonary hypertension (FPPH) is similar to PHS in broilers both genetically and physiologically. Several recent studies have shown that mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are a cause of FPPH in humans. To determine whether mutations in the chicken BMPR2 gene play a similar role in PHS susceptibility, BMPR-II mRNA from ascitic and nonascitic commercial broilers were sequenced and compared with the published Leghorn chicken BMPR-II mRNA sequence. Fourteen single nucleotide polymorphisms (SNP) were identified in the commercial broiler BMPR-II mRNA. No mutations unique to ascites-susceptible broilers were present in the coding, 5' untranslated or 3' untranslated regions of BMPR-II mRNA. The twelve SNP present within the coding region of BMPR-II mRNA were synonymous substitutions and did not alter the BMPR-II protein sequence. In addition, analysis of BMPR2 gene expression by reverse transcriptase-PCR indicated that there were no differences in BMPR-II mRNA levels in ascitic and nonascitic birds. Therefore, it appears unlikely that mutations in the BMPR2 gene were responsible for susceptibility to PHS in these commercial broilers.

Effect of cold stress on broilers selected for resistance or susceptibility to ascites syndrome.

Genetic selection for an ascites-resistant line of broilers is seen as a permanent solution to the ascites problem. Ascites-resistant and ascites-susceptible lines have been developed using sire family selection based on mortality data taken from siblings reared in a hypobaric chamber (simulated 2,900 m above sea level). The relaxed line is representative of the original commercial pureline stock randomly mated with no artificial selection pressure. The objectives of this study were to evaluate the differences between the lines when reared in floor pens and subjected to an ascites-inducing cold stress. Seven hundred eighty three straight run broilers were reared in floor pens at local elevation (390 m above sea level). Feed and water were available ad libitum. Birds were brooded at 32 C during the first week. The second week birds were maintained at 30 degrees C. Cold stress was applied for the remaining 4 wk at 14 degrees C. Mortalities were necropsied daily to determine cause of death. Birds and feed were weighed weekly. At 6 wk, five birds per pen were bled, and half the survivors in each pen (8 to 15 birds) were killed, necropsied, and scored for ascites. Blood gases, clinical chemistries, and blood cell counts were taken. Liver, spleen, split heart, and lung weights were recorded. Body weights were not different among the resistant, susceptible, and relaxed lines (P < 0.05). Feed conversion was better in the resistant line when compared to the susceptible and relaxed lines (P < 0.05). Ascites incidence, as measured by mortality and lesion score at necropsy, was higher in the susceptible and relaxed lines when compared to the resistant line; 18.8, 12.7, and 1.6% respectively (P < 0.001). Susceptible and relaxed lines showed more right ventricular hypertrophy when compared with the resistant line (P < 0.05). The results show that under severe cold stress at local altitude (390 m above sea level), the ascites-resistant line was growing as rapidly as the other lines and was as resistant to ascites as it had been under hypobaric conditions.

The effects of behavior and environmental enrichment on disease resistance of turkeys.

Turkey poults were provided with environmental enrichment for the first 14 days after hatch. At 2 days of age each bird was tested for the speed in which it would cross a T-maze. At 5 weeks of age birds were treated with dexamethasone and challenged with Escherichia coli. Enriched birds had higher body weights and incidence of disease and mortality and lower numbers of leukocytes when challenged. Challenged and enriched birds that tested FAST had higher mortality and air sacculitis scores and lower body weights than SLOW birds. Non-enriched, SLOW birds were the only ones to respond to challenge with increases in leukocyte counts and heterophil/lymphocyte ratios. These data suggest that environmental enrichment during the first 2 weeks after hatch may be detrimental to those birds with a FAST T-maze response, and that poults with a FAST response in the T-maze may be more susceptible to experimental bacterial infection.

Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection.

Two studies were conducted to determine the efficacy of either aerosol or i.m. injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens. An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m. with bacteriophage. Five birds were bled at each period. In study 1, there were 10 treatments with three replicate pens of 10 birds. The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E. coli challenge at 7 d of age, and E. coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge. In study 2 there were 11 treatments with three replicate pens of 10 birds per pen. The treatments were untreated controls, birds injected i.m. in the thigh with heat-killed or active bacteriophage, E. coli challenge at 7 d of age, PBS challenge, E. coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge. In both studies the E. coli challenge consisted of injecting 10(4) cfu into the thoracic air sac. Treatment of this severe E. coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge. The i.m. injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively. Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration. All birds injected i.m. with bacteriophage had detectable levels of bacteriophage in their blood at levels of 10(4) pfu/mL of blood up to 6 h after bacteriophage administration, and four of the five birds had detectable bacteriophage in their blood at an average level of 70 pfu/mL of blood 24 h after bacteriophage administration. The relative inefficiency of the spray treatment to the i.m. injection treatment may be due to the inability to get bacteriophage into the blood at high concentrations when the birds are sprayed versus the consistent high titers achieved with the i.m. injection of bacteriophage. These data provide support to the concept that bacteriophage may be an effective alternative to antibiotics in animal production when they are administered in a way that delivers high titers of the bacteriophage to the critical site of the bacterial infection.

Bacteriophage treatment of a severe Escherichia coli respiratory infection in broiler chickens.

A bacteriophage to a serotype 02, nonmotile Escherichia coli was isolated from municipal waste treatment facilities and poultry processing plants. A study was conducted to determine the efficacy of multiple vs. single intramuscular (i.m.) injections of bacteriophage to treat a severe E. coli respiratory infection. The birds were challenged at 7 days of age by injection of 6 x 10(4) colony-forming units (cfu) of E. coli into the thoracic air sac followed by an i.m. injection into the thigh with either heat-killed or active bacteriophage. There were 16 treatments with three replicate pens of 10 birds. There were four control treatments, which included untreated birds, birds injected with either heat-killed or active bacteriophage, and birds challenged only with E. coli. In the remaining treatments, birds were injected with heat-killed or active bacteriophage either once immediately after E. coli challenge or immediately after challenge and at 8 and 9 days of age, once at 8 days of age or at 8, 9, and 10 days of age, and once at 9 days of age or at 9, 10, and 11 days of age. Mortality was significantly decreased from 57% to 13% in the birds given a single i.m. injection of bacteriophage immediately after E. coli challenge, and there was complete recovery in birds treated immediately after challenge and at 8 and 9 days of age, which was a significant improvement from the single injection treatment. There was a significant reduction in mortality from 57% to 10% in the birds treated with bacteriophage once at 8 days of age and those birds treated at 8, 9, and 10 days of age, with no difference between single or multiple treatments. The mortality in the single or multiple phage treated birds that started at 9 days of age was reduced from 57% to 28% and 27%, respectively, but was not statistically different from the control. These data suggest that bacteriophage can be an effective treatment when administered early in this experimental E. coli respiratory disease and that early multiple treatments are better than a single treatment. The efficacy of bacteriophage treatment diminishes as it is delayed, with no difference between single or multiple treatments. Bacteriophage may provide an effective alternative to antibiotics, but like and biotic therapy, the effectiveness of phage to rescue animals decreases the longer treatment is delayed in the disease process.

Prevention of Escherichia coli infection in broiler chickens with a bacteriophage aerosol spray.

Bacteriophage to an Escherichia coli isolate that is pathogenic in poultry were isolated from municipal sewer treatment facilities or poultry processing plants. Three studies were conducted to determine the efficacy of aerosol administration of bacteriophage to prevent an E. coli respiratory infection in broiler chickens. In all three studies the experimental design consisted of nine treatments with three replicate pens of 10 birds. Three treatments were not challenged with E. coli and consisted of unsprayed birds, birds sprayed with a diluent control, and birds sprayed with a combination of two bacteriophages. Six treatments were challenged with E. coli by injecting 10(4) cfu into the thoracic air sac when birds were 7, 8, or 10 d of age after being sprayed at 7 d of age with either a diluent control or a combination of two bacteriophages. In Studies 1 and 2, BW at 2 wk of age of all the birds challenged with E. coli, regardless of spray treatment, were decreased significantly from the unchallenged controls, except in Study 2 for the birds sprayed with bacteriophage and challenged at 10 d of age. There was a significant decrease in mortality in Studies 1 and 2 when the birds were challenged with E. coli immediately after bacteriophage administration and in Study 2 in birds challenged at 10 d of age. In Study 3 a suspected pre-existing E. coli infection resulted in mortality in the unchallenged, unsprayed controls, and in the diluent sprayed controls of 20 and 27%, respectively. The mortality in the unchallenged bacteriophage sprayed birds was 3%, representing a significant decrease. Mortality in Study 3 was significantly decreased in the bacteriophage-sprayed birds challenged with E. coli immediately or 1 d later but not 3 d after bacteriophage administration. The decrease in BW at 2 wk of age in challenged birds indicates that bacteriophage treatment did not provide complete protection; however, in all three studies mortality was significantly decreased, indicating that aerosol spray of bacteriophage may be practical for administration of bacteriophage and may provide an alternative to the use of antibiotics in poultry production.

Effect of dietary supplementation with vitamin D metabolites in an experimental model of turkey osteomyelitis complex.

Supplementation with vitamin D3 was previously shown to protect Escherichia coli challenged birds that underwent two dexamethasone (DEX) treatments at 5 and 12 wk of age in an experimental model of turkey osteomyelitis complex (TOC). The purpose of the present study was to determine the effects of dietary supplementation with 10 microg of 1,25 dihydroxyvitamin D3 (1,25D)/ kg feed or 99 microg of 25-hydroxyvitamin D3 (25D)/kg feed on disease resistance in the same model. Birds were fed the supplemented diets continuously and ad libitum. Seven hundred twenty turkey poults were placed into 24 floor pens in a 3 x 2 x 2 design (three vitamin D treatments, two DEX treatments, two E. coli treatments, with two replicate pens per treatment). At 5 wk of age, half of the birds were treated with DEX, and half of the DEX-treated birds and half of the nontreated birds were challenged with E. coli. All mortalities and lame birds were necropsied. At 9 wk, all of the DEX- or E. coli-treated birds were given another series of DEX injections; 2 wk later 10 birds per pen were necropsied. At 12 wk, survivors of the previous challenges were given a third DEX treatment, and all birds were necropsied 2 wk later. After the first series of DEX injections, mortality was increased in the 25D-supplemented birds that were given the DEX treatment and the E. coli challenge. After the second series of DEX injections, the main effect mean BW was significantly lower in birds given 1,25D as compared to controls and 25D-supplemented birds. Mortality was higher in 1,25D-supplemented birds that were challenged with E. coli at 5 wk and treated with DEX at 9 wk as compared to 25D-supplemented birds. The 1,25D-treated birds that were treated with DEX at 5 and 9 wk and challenged with E. coli at 5 wk had higher mortality and air sacculitis scores as compared to controls and 25D-treated birds. The main effect mean mortality was significantly higher in birds given 1,25D as compared to controls and 25D-treated birds. The percentage of birds with TOC lesions was decreased from 27% to 0 by 25D and 1,25D in the groups given two DEX treatments and E. coli challenge. After the third DEX treatment, BW of 1,25D-suppplemented birds was decreased, and mortality and air sacculitis scores were increased. Bone strength was generally increased by supplementation with 1,25D, whereas 25D supplementation increased bone strength only in birds challenged at 5 wk and treated with DEX at Weeks 9 and 12. In this study, supplementation with vitamin D metabolites decreased TOC incidence in E. coli-challenged birds given two DEX treatments. However, toxic effects were observed in most supplemented DEX-treated birds and may be attributed to an additive effect of DEX treatment, E. coli septicemia, and vitamin D supplementation.

Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage (SPR02).

Bacteriophages are viruses that can infect and kill bacteria. Three studies were conducted to determine the efficacy of bacteriophage to prevent an Escherichia coli respiratory infection in broiler chickens. In the first study 3-d-old-birds were challenged with an air sac inoculation of 10(3) cfu of E. coli per mL mixed with either 10(3) or 10(6) pfu of bacteriophage, or 10(4) cfu E. coli mixed with 10(4) or 10(8) pfu of bacteriophage. In the second study, drinking water of birds to 1 wk of age was treated with 10(3) or 10(4) pfu of bacteriophage per mL and birds were air sac challenged with 10(3) cfu of E. coli, or water was treated with 10(4) or 10(6) pfu of bacteriophage per milliliter and birds were challenged with 10(4) cfu of E. coli. In the third study, birds were air sac challenged at 1 wk of age with 10(4) cfu of E. coli and given 10(5) or 10(6) pfu of bacteriophage per mL of water from 1 d of age to 2 wk of age. In Studies 1 and 2, there were two replicate pens per treatment with 10 birds per pen, and in Study 3, there were four replicate pens per treatment with 10 birds per pen. The studies were all concluded when the birds were 3 wk of age. In Study 1, BW was decreased at 1 and 2 wk of age in the birds that were challenged with 10(3) or 10(4) cfu of E. coli and was decreased at 2 wk of age in the birds challenged with 10(4) cfu of E. coli mixed with 10(4) pfu of the bacteriophage. Mortality was decreased from 80% in the birds challenged with 10(3) cfu of E. coli to 25 and 5% when mixed with 10(3) or 10(6) pfu of the bacteriophage, respectively. Mortality was decreased from 85% in birds challenged with 10(4) cfu of E. coli to 35% when mixed with 10(4) pfu of the bacteriophage, and no mortality occurred when mixed with 10(8) pfu of bacteriophage. There was essentially no protection observed in Studies 2 and 3 when the birds were challenged with 10(3) or 10(4) cfu of E. coli with bacteriophage present in their drinking water at any level. These data suggest that bacteriophage can protect birds from a respiratory challenge with E. coli, but that adding the bacteriophage to the drinking water offered no protection to the birds. The complete protection of the birds observed in Study 1 suggests that bacteriophage may possibly be developed as an alternative to antibiotic use in poultry.