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ABSTRACT: Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.
Poultry Science 10/2009; 88(9):1825-31. · 1.73 Impact Factor
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ABSTRACT: Cloacal swabs were taken from migratory hunter-killed, nonmigratory, nesting waterfowl and migratory shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Samples were processed in embryonated eggs followed by hemagglutination (HA), Directigen, and real-time reverse transcription-PCR tests. Sequence analysis of the hemagglutinin (H) gene of the H10N7 Alabama isolate revealed that it was closely related (98%) to recent isolates from Delaware and Canada, but only 90% related to an H10N7 isolated 30 yr ago. Four isolates had 94 to 97% similarity to published H1N1 isolates including one from swine. No H5 or H7 isolates were found. One sample was highly pathogenic in embryos, produced a high HA titer, and was positive for both avian influenza (AIV) and Newcastle disease virus or avian paramyxovirus (APMV)-1. In recent (2008) sampling, more (14%) AIV, APMV, or both were isolated than in 2006 to 2007 (1% isolation rate). The higher isolation rate during 2008 may be attributed to optimized sample collection, storage in dry ice, new egg incubator, healthier eggs, time or habitat for isolation, species sampled, migratory status of birds, and more experience with detection procedures. An additional egg passage resulted in increased viral titer; however, no HA-negative samples became HA positive. The chance of transmission of APMV or low-nonpathogenic AIV from wild waterfowl to commercial poultry is possible. However, the chance of transmission of H5 or H7 AIV isolates from waterfowl to commercial farms in Alabama, Georgia, or Florida is unlikely. Therefore, continual testing of these birds is justified to ensure that H5 or H7 AIV are not transmitted to commercial poultry.
Poultry Science 05/2009; 88(4):851-5. · 1.73 Impact Factor
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ABSTRACT: Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.
Avian Diseases 07/2007; 51(2):597-600. · 1.46 Impact Factor
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ABSTRACT: This article reviews transmissible proventriculitis in poultry from 1971 to 2006. The disease is important in commercial broilers worldwide, resulting in reduced profits. The aetiology of this disease is unknown and different clinical presentations often result in a confused or complicated diagnosis. The lesion of enlarged proventriculus is often referred to as proventriculitis. However, the term proventriculitis can only be used correctly when there is microscopic evidence of inflammation of the proventriculus glands. Infectious and non-infectious causes of proventriculitis, with major emphasis on the infectious or transmissible causes, are reviewed.
Avian Pathology 05/2007; 36(2):87-91. · 1.71 Impact Factor
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ABSTRACT: Avian reoviruses (ARVs) can result in disease and economic losses in the poultry industry. Vaccines against ARV may not provide full protection and can cause adverse reactions. The coding sequence of the sigma C protein from strain S1133 of avian reovirus was expressed in Schizasaccharomyces pombe. Sigma C protein expression was demonstrated by Western blotting, and the protein was evaluated for its ability to protect specific-pathogen-free (SPF) chickens against challenge with the virulent S1133 strain. Serologic and challenge-infection data showed the efficacy of the recombinant vaccine administered orally each week for 3 consecutive wk. Sigma C protein induced antibody, as determined by enzyme-linked immunosorbent assay. Percentage (%) protection induced by the low dose (125 microg purified yeast-expressed sigma C protein/chicken) or the high dose (250 microg purified yeast-expressed sigma C protein/chicken) was 64 and 91, respectively. The commercial vaccine administered once or twice provided 82% protection. Results supported the feasibility of a plant-derived vaccine for use in poultry immunization schemes.
Avian Diseases 07/2005; 49(2):281-4. · 1.46 Impact Factor
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ABSTRACT: VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.
Biotechnology Letters 06/2004; 26(10):787-92. · 1.68 Impact Factor
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ABSTRACT: VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis
thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciensby electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.
Biotechnology Letters 01/2004; 26(10):787-792. · 1.68 Impact Factor
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ABSTRACT: An experimental vaccine was prepared by mixing commercial reovirus vaccine with antibody. Vaccines were inoculated into 18 day-old commercial broiler embryos at 0.1 of the recommended dose. At 3, 6, 9, 12 and 15 days post in ovo vaccination (PIOV), serum was collected and antibody against reovirus analyzed by an enzyme-linked immunosorbent assay (ELISA). At the same time, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEF). At day 15 PIOV, chickens were challenged with a virulent reovirus S1133 strain. At day 25 PIOV, birds were euthanized and weighed. Efficacy was based on safety, antibody reaction, and percent (%) protection. Reovirus vaccine alone (vac) or complexed with antibody (AB) did not affect hatchability, morbidity, and mortality. Best protection was with the 2 experimental vaccine groups (73% for vac+1/16 dilution of AB) and (64% for vac+1/4 dilution of AB) provided by the vaccine mixed with 1:16 dilution of antibody. No vaccine resulted in an increase in antibody; however, titers of all challenged groups rose after challenge. Vaccine virus was detectable at day 3 PIOV in group 3 (vac+1/16AB) and 4 (vac) chickens. In contrast, vaccine virus detection was delayed until day 9 PIOV in group 2 (vac+1/4AB) chickens. The experimental antibody complex vaccine servers as a good starting point for development as a commercial vaccine.
Journal of Animal and Veterinary Advances. 01/2003;
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ABSTRACT: Two monoclonal antibodies (MAb), E9 and H3, prepared against avian reovirus (ARV) S1133, were used in an immuno-dot assay to detect ARV antigens from cell culture and from tendon tissue samples of chickens. The limit of viral antigens detected was 8 ng using both MAb probes. The probes detected 10 ARV isolates representing at least two serotypes or pathotypes. The results indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from six unrelated avian viruses. The ARV antigens in tendon tissue samples were detected by both probes, and it is possible, therefore, to use either of the two MAb probes for detection of ARV infections.
Journal of Virological Methods 06/2000; 86(2):115-9. · 2.01 Impact Factor
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ABSTRACT: A nested polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis identified and differentiated avian reoviruses (ARVs). PCR products amplified from the S1 gene segment of ARV of USA isolates were 738 and 342 bp, respectively. PCR products were conformed by Southern and dot blot hybridizations. The amplified cDNA fragments were cloned into the pUC18 vector and subjected to DNA sequencing. The nucleotide and deduced amino acid sequences of four USA (S1133, 1733, 2408, and CO8) and two Australian isolates (RAM-1 and SOM-4) were compared. Results of paired difference analysis and a predicted dendrogram revealed that USA isolates were closely related, but different from, Australian isolates. The deduced amino acid sequences of the N-terminal region of ARV sigma C showed a heptapeptide repeat of hydrophobic residues in all ARV isolates.
Journal of Virological Methods 06/1997; 65(2):159-67. · 2.01 Impact Factor
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ABSTRACT: The sigmaC-encoding cDNA of avian reovirus (ARV) 1733 strain was amplified, cloned and sequenced using double nested polymerase chain reaction (PCR). The ARV sigmaC protein is a minor component of the outer capsid that induces type-specific neutralization antibodies. Four overlapping sigmaC-encoding cDNA fragments were obtained. Together, the four fragments represented the whole coding sequence. The nucleotide and deduced amino acid sequences of sigmaC-encoded gene of U.S. (S1133 and 1733) and Australian isolates (RAM-1 and SOM-4) were compared. The U.S. isolates were closely related, but different from Australian isolates. The degree of differences between the U.S. and Australian isolates was over 44.89% at both the nucleotide and deduced amino acid levels and suggested that the virus is evolving separately in different continents. The deduced amino acid sequences of ARV sigmaC indicated a heptapeptide repeat in the N-terminal region of ARV sigmaC existed in all ARVs. The results suggested that ARV sigmaC is structurally related to mammalian reovirus (MRV) sigma1.
Journal of Virological Methods 02/1997; 63(1-2):203-8. · 2.01 Impact Factor
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ABSTRACT: Type X collagen is produced exclusively in hypertrophic chondrocytes of the growth plate of the proximal tibiotarsus and is believed to play an important role during normal development from chondrogenesis to osteogenesis. Chondrocytes of chickens with tibial dyschondroplasia (TD) fail to attain full hypertrophy and the amount of type X collagen, being a marker of hypertrophy, is likely to be reduced. It is not clear whether transcriptional regulation is functional for expression of the type X collagen gene in TD birds. Nucleotide sequence of the type X collagen gene promoter was determined by sequencing PCR-based DNA clones. Nucleotide identity of this fragment between the normal and TD carriers was 97.6%. Both normal and TD birds were similar in a putative transcription start site, the site of TATAA box, and neither had a CCAAT box. However, there were two gaps in TD carriers, four gaps in normals, and five nucleotide substitution sites. By rapid amplification of cDNA ends by PCR (RACE-PCR), transcription of the gene was assessed using total RNA and mRNA from both normal chondrocytes and TD lesions at 3 and 4 wk of age. The RACE-PCR product for type X collagen mRNA was detectable in both normal and TD birds at two stages. No difference was found between them. This result does not support the hypothesis that transcriptional regulation of type X collagen gene is important in TD development of chickens. Variations in the promoter region did not affect transcription of type X collagen gene in TD carrier chickens.
Poultry Science 07/1996; 75(6):691-4. · 1.73 Impact Factor
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ABSTRACT: Lines selected for high (H) and low (L) incidence of tibial dyschondroplasia (TD) for eight generations and a randombred control (C) line of broiler chickens were fingerprinted by random amplification of genomic DNA mixed from 20 individuals of each line with 20 oligonucleotide primers. Among these 20 primers, 15 could distinguish the H from the L line, 14 the H from the C line, and 13 the L from the C line. Band sharing (BS), on the average over 20 primers, was .7 for the H vs L comparison and .8 for both H vs C and L vs C comparisons. The levels of BS calculated from individuals was .6 between the H and L line, .7 between the H and C line, and .7 between the L and C line. The ranking of BS values obtained from individual DNA samples was consistent with that obtained from the mixed DNA samples. Genomic distance between divergently selected lines (H vs L) was larger than that between the divergently selected lines and randombred line (H vs C and L vs C). Individual variation within lines was detected in spite of eight generations of selection. Results showed that eight generations of divergent selection for TD incidence in broiler chickens had resulted in genetic variation among lines. The procedure of random amplified polymorphic DNA assay using mixed DNA samples could be used to evaluate genetic distance among lines of chickens.
Poultry Science 09/1995; 74(8):1253-8. · 1.73 Impact Factor
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ABSTRACT: Reverse transcription with polymerase chain reaction (PCR) followed by restriction endonuclease analysis detected genetic variations among serotype I isolates of infectious bursal disease virus (IBDV). Using a set of synthetic primers derived from the large genome segment of APHIS-IBDV, the hypervariable region (AccI-SpeI fragment) located in the VP2 gene was amplified. With all strains, a cDNA fragment of approximately 643 bp was amplified, indicating that there were no apparent deletions or insertions in this region among isolates. Fragments amplified from 9 isolates were digested with 14 restriction enzymes. Restriction fragment profiles generated by restriction enzymes NaeI, StuI, TaqI, and SacI, showed genetic variations among isolates. This study provided a simple and sensitive method for detection of genetic variations among isolates that are closely related serologically and could not be differentiated using current serologic methods.
Journal of Virological Methods 08/1994; 48(2-3):281-91. · 2.01 Impact Factor
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ABSTRACT: The kinetics of serum antibody responses in broiler chickens against Cryptosporidium baileyi were studied. Broilers were inoculated intratracheally with 250,000 C. baileyi oocysts at 1, 7, or 14 days of age. Antibody was quantified by an enzyme linked immunosorbent assay. Anti-cryptosporidial serum immunoglobulins (IgM and IgG) were detected 9 days post-inoculation (DPI) in birds inoculated at 1 or 7 days of age with oocysts and 4 DPI when 14-day-old birds were inoculated. Results also reaffirmed age related susceptibility, with day-old birds being more susceptible than 7-day, and 14-day-old birds were not susceptible to clinical disease. The susceptibility to infection correlated with the amount and duration of the IgM response. Day-old inoculated birds developed a higher, longer-lasting response than 7 or 14-day-old infected birds.
Avian Pathology 10/1993; 22(3):525-32. · 1.71 Impact Factor
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ABSTRACT: Experiments were conducted to determine the efficacy of simultaneous administration at 1 day of age of the herpes virus of turkeys vaccine (HVT) and viral tenosynovitis (VT) vaccine. Day-old broilers from commercial breeders, which were immunized against both MD and VT, were used. The vaccines were injected at full dosage or diluted 1 to 4. Challenge with either Marek's disease virus (MDV; intraperitoneal) or VT virus (footpad) was at 7 days of age. At 7 wk of age, birds were weighed, killed, and examined for gross lesions. Either vaccine given at full dosage alone, or in combination, rendered birds resistant to homologous viral challenge. However, when either vaccine was diluted and administered alone, the efficacy of the VT but not the HVT vaccine was reduced, resulting in increased lesions and mortality and decreased weight gain after VT challenge. When the HVT vaccine was diluted and combined with undiluted VT vaccine, interference occurred, resulting in reduced efficacy of the HVT vaccine (increased tumors and mortality and reduced body weight). In contrast, combining the HVT at full dosage and the diluted VT vaccine given alone. The data indicate that caution should be maintained when mixing HVT and VT vaccines. For maximum efficacy, both vaccines should be mixed undiluted when administered simultaneously.
Poultry Science 10/1989; 68(9):1213-7. · 1.73 Impact Factor
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ABSTRACT: Clinical signs of respiratory tract disease were observed in chickens that were inoculated intratracheally with 1 x 10(6) oocysts of Cryptosporidium baileyi at 2 or 14 days of age (10 chickens/group), but not in chickens inoculated at 28 or 42 days of age (10 chickens/group). Orally inoculated chickens in all age groups (10 chickens/group) did not develop clinical signs of disease. Orally and intratracheally inoculated chickens in all age groups were infected, as determined by the finding of cryptosporidia in tissue sections of the trachea, bursa of Fabricius, and cloaca, and by the recovery of oocysts from their feces. Chickens inoculated at 2 and 14 days of age excreted oocysts for a longer period and had greater numbers of cryptosporidia in their tissues, compared with chickens inoculated at 28 and 42 days of age.
American Journal of Veterinary Research 09/1988; 49(8):1412-4. · 1.27 Impact Factor
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ABSTRACT: Seven-day-old conventional broilers were inoculated either orally or intratracheally (IT) with 2.5 X 10(5), 5.0 X 10(5), or 2.0 X 10(6) oocysts of Cryptosporidium baileyi (32 birds for each dosage level per group; 192 birds total). Thirty-two birds served as unninoculated controls. Mean weekly weight gain and feed conversion were determined during a 5-week period. Carcass pigment was graded using a Roche Color Fan. Fecal oocysts were calculated from random cage samples 6, 8, 11, 13, 15, 18, 20, 22, and 25 days after inoculation (DAI). Effects of C. baileyi on immune responses were examined for Newcastle disease virus-hemagglutination inhibition (NDV-HI) antibody, infectious bursal disease virus-enzyme-linked immunosorbent assay (IBDV-ELISA) antibody titers and delayed hypersensitivity (DH) in half of the birds in each group. Disease or death from cryptosporidiosis did not result from oral inoculation of C. baileyi. Signs of respiratory disease, consisting of rales, sneezing, and dyspnea were observed in all IT-inoculated birds 7 to 21 DAI. Seven deaths occurred in the IT-inoculated groups 14 to 21 DAI. At necropsy, lung parenchyma was gray, firm, and wet in the ventral region. Air sacs contained a foamy, white to gray, mucoid fluid. Histologic lesions in the air sacs and bronchi were epithelial hyperplasia, discharge of mucocellular exudate to the mucosal surface, thickening of the mucosa by cellular infiltrates, loss of cilia, and dilation of mucous glands. Weight gains for IT-inoculated birds were lower (P less than .05) than controls from 14 to 21 DAI, although weight gains for the 5-week period were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)
Poultry Science 04/1987; 66(3):442-9. · 1.73 Impact Factor
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ABSTRACT: Aflatoxin (AF)-contaminated ground corn was mixed with a commercial swine ration to yield 2 concentrations (500 mg of AFB1/kg of feed [A] and 300 mg of AFB1/kg [B]) and was fed to 2 groups of pigs. Groups A and B were fed the AF-containing ration, whereas control group C was fed the same commercial ration mixed with ground corn devoid of AF. A comparative analysis of the average weight gain per pig in each of the treatment groups, compared with that in the control group, indicated a significantly (P less than 0.01) greater weight gain in the control group. The average feed conversion rate was also significantly (P less than 0.01) lower in group A pigs, compared with that in the control group. The humoral immune response to Erysipelothrix rhusiopathiae, measured by enzyme-linked immunosorbent assay, did not reveal a significant difference among groups; there were no consistent differences observed in the proliferative responses of lymphocytes to mitogens. In contrast, a significant (P less than 0.05) reduction in complement titers was observed, whereas an increase in serum immunoglobulin G and M values occurred in the AF-treated group A, compared with that in group C. Gross enlargement of the liver, substantiated by histologic evidence of toxic damage to the hepatic parenchyma, revealed that AF at concentrations of 500 mg/kg of feed was toxigenic and produced an adverse effect on the growth rate, feed efficiency, and general well-being of young pigs.
American Journal of Veterinary Research 10/1986; 47(9):2062-7. · 1.27 Impact Factor
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ABSTRACT: The effect of crude aflatoxin (AF) on the growth, performance, and immune response of turkeys and broilers was studied. Crude AF, produced from a natural outbreak of Aspergillus flavus on corn, was ground and mixed in rations to contain either 0, 100, 200, 400, or 800 ppb of aflatoxin B1 (AFB1). Turkeys (Experiment 1) and broilers (Experiment 2) were used in identical experimental designs. In each, 200, 14-day-old birds were divided equally by sex into five groups of 40 and were fed one of five AF diets for 35 days. In Experiment 1, crude AF greater than or equal to 400 ppb was highly toxic to turkeys. These levels produced signs and lesions of aflatoxicosis as well as a significant decrease in weight gain and feed conversion during 5 weeks. In addition, microscopic lesions, indicative of aflatoxicosis, were evident as low as 100 ppb, and significant decreases in cell-mediated immunity were noted in the 200 ppb group birds. Experiment 2 indicated that chickens were less susceptible to crude AF than turkeys. Neither morbidity nor mortality occurred in broilers. Gross lesions consistent with AF toxicity were evident in birds given 800 ppb and microscopic lesions were observed in birds given 100 ppb. Feed conversion was significantly increased in the 800 ppb broilers only. Cell-mediated immunity, measured by a delayed hypersensitive skin test, was significantly decreased in broilers receiving AF at 200 ppb or greater. Neither humoral immunity nor the development of the acquired immunity to Newcastle disease or fowl cholera vaccination were decreased in turkeys or broilers given AF.
Poultry Science 10/1985; 64(9):1678-84. · 1.73 Impact Factor
