SC and TK inbred chicken strains display differential protective immunity to coccidiosis, SC being more resistant and TK susceptible to disease. In this study, the association between interleukin (IL)-2 and disease phenotype was assessed by cytokine quantification in serum, duodenum, cecum, and spleen cell cultures of SC and TK chickens experimentally infected with Eimeria tenella. In general, after primary infection, SC and TK strains produced equivalent amounts of IL-2 in all sources examined. However, after secondary infection, SC animals displayed significantly greater IL-2 levels in serum and the duodenum compared with strain TK. IL-2 production after reinfection with Eimeria may be an important factor contributing to the genetic differences in coccidiosis between SC and TK chickens and provides a rational foundation for cytokine-based immunotherapeutic approaches to disease control strategies.

The application of a tetrazolium salt, WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt to the lymphocyte proliferation assay in the chicken system was evaluated. Proliferation of concanavalin (Con A)-induced splenic lymphocytes and peripheral blood lymphocytes (PBL) was evaluated with WST-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Coefficients of correlation (r) between these two reagents were 0.98 and 0.97 in splenic lymphocytes and PBL, respectively. In general, the sensitivity of the WST-8 assay was significantly higher than that of the MTT assay, and the standard deviations of the WST-8 assay were significantly lower than those of the MTT assay. The WST-8 assay was fast and highly reproducible and provided a good indication of mitogen-induced proliferation of spleen cells induced by Con A. With the use of the WST-8 assay, splenic mitogenic response of chickens infected with Eimeria decreased transiently at 7 days but increased significantly at 10 days after primary infection compared with that of uninfected chickens. Additionally, the measurement of interleukin (IL)-2 production with WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of Eimeria tenella-immune spleen cells with Con A. After E. tenella infection, splenic IL-2 production increased significantly at 7 days post-primary and at 2 days post-secondary infection. The WST-8 assay is fast, simple, and more reproducible and sensitive than the MTT assay. This study demonstrates the effectiveness of the WST-8 assay to assess cell-mediated immune response of chickens in normal and disease states.

Five hundred sixty-nine Salmonella were isolated out of 4745 samples from poultry products, poultry, and poultry environment in 1999 and 2000 from the Pacific northwest. These Salmonella were identified to their exact source, and some were serogrouped, serotyped, phage typed, and tested for antibiotic sensitivity. Food product samples tested included rinse water of spent hens and broilers and chicken ground meat. Poultry environment samples were hatchery fluff from the hatcheries where eggs of grandparent broiler breeders or parent broiler breeder eggs were hatched and drag swabs from poultry houses. Diagnostic samples were of liver or yolk sac contents collected at necropsy from the young chicks received in the laboratory. Of these samples tested, 569 were Salmonella positive (11.99%). Ninety-two Salmonella were serogrouped with polyvalent somatic antisera A-I and the polymerase chain reaction. Somatic serogroups B and C comprised 95.25% of all the Salmonella. Out of a total of 569 positive samples, 97 isolates of Salmonella were serotyped. A total of 16 serotypes and an unnamed Salmonella belonging to serogroup C1 were identified. The Salmonella serotypes were heidelberg (25.77%); kentucky (21.64%); montevideo (11.34%); hadar and enteritidis (5.15% each); infantis, typhimurium, ohio, and thompson (4.12% each); mbandaka and cerro (3.09% each); senftenberg (2.06%); berta, istanbul, indiana, and saintpaul (1.03% each); and an unnamed monomorphic Salmonella (2.06%). Ninety-two Salmonella were tested for drug sensitivity with nine different antimicrobials. All of the 92 Salmonella were resistant to erythromycin, lincomycin, and penicillin except one sample (S. berta), which was moderately sensitive to penicillin. All of the tested Salmonella were susceptible to sarafloxacin and ceftiofur. The percentages of Salmonella susceptible to sulfamethoxazole-trimethoprim, gentamicin, triple sulfa, and tetracycline were 97.83%, 92.39%, 86.96%, and 82.61%, respectively.

The relationship between antibody response to Salmonella enteritidis vaccine and internal organ burden of S. enteritidis is not fully understood. The genetic relationship, therefore, between postchallenge S. enteritidis burden and antibody response to S. enteritidis vaccine was determined in broiler breeder chicks. Sibling chicks from a broiler breeder male line were either inoculated with a pathogenic S. enteritidis or vaccinated with a commercial S. enteritidis vaccine. Spleen, liver, cecal wall, and cecal content samples from S. enteritidis–challenged chicks (n = 120) were cultured for enumeration of bacteria. Unchallenged chicks (n = 314) were vaccinated at 11 days of age, and serum samples were taken at 10 days postvaccination. Antibody response to vaccination and number of S. enteritidis in cecal content cultures were negatively correlated (−0.772), demonstrating that genetic potential for greater antibody response to S. enteritidis vaccine is associated with lesser S. enteritidis bacterial burden in cecal content of broiler breeder chicks. The findings suggest that genetic selection for vaccine antibody responsiveness can lower bacterial burden in the gut lumenal content and, thus, potentially reduce contamination of poultry products at processing.

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P ≤ 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P ≤ 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P ≤ 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.

This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.

The H5N1 type A influenza viruses that emerged in Hong Kong in 1997 are a unique lineage of type A influenza viruses with the capacity to transmit directly from chickens to humans and produce significant disease and mortality in both of these hosts. The objective of this study was to ascertain the susceptibility of emus (Dramaius novaehollandiae), domestic geese (Anser anser domesticus), domestic ducks (Anas platyrhynchos), and pigeons (Columba livia) to intranasal (i.n.) inoculation with the A/chicken/Hong Kong/220/97 (H5N1) highly pathogenic avian influenza virus. No mortality occurred within 10 days postinoculation (DPI) in the four species investigated, and clinical disease, evident as neurologic dysfunction, was observed exclusively in emus and geese. Grossly, pancreatic mottling and splenomegaly were identified in these two species. In addition, the geese had cerebral malacia and thymic and bursal atrophy. Histologically, both the emus and geese developed pancreatitis, meningoencephalitis, and mild myocarditis. Influenza viral antigen was demonstrated in areas with histologic lesions up to 10 DPI in the geese. Virus was reisolated from oropharyngeal and cloacal swabs and from the lung, brain, and kidney of the emus and geese. Moderate splenomegaly was observed grossly in the ducks. Viral infection of the ducks was pneumotropic, as evidenced by mild inflammatory lesions in the respiratory tract and virus reisolation from oropharyngeal swabs and from a lung. Pigeons were resistant to HK/220 infection, lacking gross and histologic lesions, viral antigen, and reisolation of virus. These results imply that emus and geese are susceptible to i.n. inoculation with the HK/220 virus, whereas ducks and pigeons are more resistant. These latter two species probably played a minimal epidemiologic role in the perpetuation of the H5N1 Hong Kong–origin influenza viruses.

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.

Antibiotics are used in the livestock industry not only to treat disease but also to promote growth and increase feed efficiency in less than ideal sanitary conditions. However, certain antibiotic families utilized in the poultry industry have recently been found to adversely affect bone formation and cartilage metabolism in dogs, rats, and humans. Therefore, the first objective of this study was to determine if certain antibiotics used in the poultry industry would inhibit in vitro cartilage degradation. The second objective was to determine if the antibiotics found to inhibit in vitro cartilage degradation also induced tibial dyschondroplasia in growing broilers. Ten antibiotics were studied by an avian explant culture system that is designed to completely degrade tibiae over 16 days. Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not inhibit degradation at any concentration tested. Doxycycline (200 µg/ml), oxytetracycline (200 µg/ml), enrofloxacin (200 and 400 µg/ml), ceftiofur (400 µg/ml), and salinomycin (10 µg/ml) prevented complete cartilage degradation for up to 30 days in culture. Thus, some of the antibiotics did inhibit cartilage degradation in developing bone. Day-old chicks were then administered the five antibiotics at 25%, 100%, or 400% above their recommended dose levels and raised until 21 days of age. Thiram, a fungicide known to induce experimental tibial dyschondroplasia (TD), was given at 20 ppm. Birds were then killed by cervical dislocation, and each proximal tibiotarsus was visually examined for TD lesions. The results showed that none of these antibiotics significantly induced TD in growing boilers at any concentration tested, whereas birds given 20 ppm thiram had a 92% incidence rate.

Attempts to reproduce malabsorption syndrome (MAS) by oral inoculation with several different combinations including intestinal homogenate, reovirus, and hemolytic Escherichia coli obtained from MAS-affected chickens and intestinal homogenate from healthy chickens (healthy homogenate) were performed in 1-day-old specific-pathogen-free (SPF) broilers. The MAS homogenate, serving as a positive control, induced weight gain depression and intestinal lesions such as cystic crypts of Lieberkühn, villus atrophy, and lymphoid and/or granulocytic infiltration. The healthy homogenate, the formalin-treated MAS homogenate, the formalin-treated healthy homogenate, and phosphate-buffered saline caused neither weight gain depression nor intestinal lesions. We were able to reproduce both weight gain depression and intestinal lesions by inoculation of reovirus either combined with the formalin-treated MAS homogenate or combined with healthy homogenate. Surprisingly, when hemolytic E. coli was added to the combination of reovirus with formalin-treated MAS homogenate, this did not cause weight gain depression although this combination caused the described intestinal lesions. Identical results were obtained with the combination of formalin-treated MAS homogenate with hemolytic E. coli or the combination of reovirus with hemolytic E. coli. The intestinal lesions were more severe and developed faster by combinations including reovirus and formalin-treated MAS homogenate. This study indicates that a combination of enteropathogenic reovirus with other agents or substances that are present in an intestinal homogenate from MAS-affected and healthy chickens can induce MAS in SPF broilers. Escherichia coli is not essential for induction of weight gain depression but can play a role in development of intestinal lesions. Furthermore, intestinal lesions alone will not always result in weight gain depression.

The Israeli vaccine strain of fowl poxvirus grows efficiently in chicken embryo fibroblasts but not in cell lines derived from monkey kidney or human fibroblasts. We developed two assays for the titration of the infectivity of this virus in secondary cultures of chicken embryo fibroblasts. The first is a focus assay, in which minimum essential medium and SeaKem ME agarose were used for the overlay media. Under these conditions, clear virus foci appeared after 5 days of incubation at 37 C. The second assay is a semiautomatic colorimetric test based on the ability of live cells in culture to reduce the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) to its formazan derivative. The reagent was added to infected chicken embryo fibroblasts in 96-well plates 10 days after infection. The formazan formed during 2 hr was extracted with dimethyl sulfoxide, and its absorbance was read by an automatic microplate spectrophotometer. A good correlation of the infectivity titers of the virus was obtained by the two methods.

Systemic Mycoplasma synoviae (MS) infection was induced experimentally in commercial turkeys with recent MS isolates (K4822D and K4774J) from turkey breeder flocks that exhibited no clinical signs typical of MS infection except for a low incidence of swollen footpads. The virulence of each strain was compared by evaluating gross and microscopic lesions, serologic responses, and MS isolation rates at 10 and 21 days postchallenge and by comparing these results with those obtained from a known virulent isolate (K1968), another previously characterized field isolate (K4463B), and unchallenged controls. All strains induced lesions typical of infectious synovitis but showed distinct differences in the extent of the gross and microscopic lesions and in the isolation rates from the tissues in turkeys. K1968 induced the most extensive lesions in hock and stifle joints and footpads, but strains K4822D, K4774J, and K4463B all induced synovitis and were similar in virulence for synovial tissues. Very mild respiratory lesions were induced by all of the strains studied. All strains yielded strong positive serologic responses. We concluded that these recent field isolates, although able to induce synovitis, are less virulent for turkeys than a known virulent strain. Nevertheless, under severe experimental challenge, these strains have the capability of causing lesions that may be incompatible with economical turkey production.

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.

A competitive enzyme immunoassay was developed to measure the changes in serum levels of ovotransferrin (OTF) during inflammation and infectious diseases in chickens. The assay is based on the competition of serum OTF with a fixed concentration of biotin-labeled OTF to bind to a rabbit anti-chicken transferrin antibody immobilized on microtiter wells. After several washing steps, the antibody-bound biotinylated OTF is probed with streptavidin–horseradish peroxidase conjugate (HRP) followed by a colorimetric detection of the HRP activity. The relative changes in the optical density of color are plotted against the competing concentrations of OTF with logarithmic regression to generate a standard curve that is used to determine the concentrations of OTF in unknown samples. Serum had no effect on the measurement of OTF. By this method, the time course changes of serum OTF levels in 4-wk-old male broiler chickens that were subjected to inflammation by croton oil injection were measured. The results showed croton oil–induced inflammation elevated serum OTF levels at 16 hr postinjection. OTF levels reached a peak by 72 hr, remained high through 120 hr, and returned to a basal level of olive oil–injected controls by 240 hr. There were no changes in serum OTF levels at any of the above time points in olive oil–injected control chickens. For studies with poultry diseases, specific-pathogen-free (SPF) male chickens were challenged with known bacterial and viral pathogens, and serum was collected at the height of the infection, i.e., 7 days after the challenge. Compared with uninjected controls, the SPF chickens challenged with Escherichia coli, fowl poxvirus, respiratory enteric orphan virus, infectious bursal disease virus, infectious bronchitis virus, or infectious laryngotracheitis virus had higher levels of OTF in serum. Inflammation-induced changes in serum OTF levels were also evident in the changes in the density of a 65-kD band protein corresponding to OTF. These results demonstrate that serum OTF may be a nonspecific clinical marker of inflammation associated with traumatic or infectious avian diseases.

Food poisoning by Staphylococcus aureus affects hundreds of thousands of people each year. Staphylococcus aureus also causes invasive diseases such as arthritis (in poultry) and septicemia (in poultry and humans). Foodborne disease is caused by the ingestion of a staphylococcal enterotoxin (SE). Enterotoxin has also been associated with other S. aureus illnesses in humans and domestic animals. In this study, polymerase chain reaction was used to detect the staphylococcal enterotoxin genes, SEA, SEB, SEC, SED, and SEE, in S. aureus isolates associated with invasive disease in poultry and humans. In the 34 poultry isolates, only one isolate was found to contain a SE gene, sec. In the 41 human isolates, over 51% tested positive for an SE gene with 12.2% positive for the gene for SEA, 2.4% for SEB, 22% for SEC, 24.4% for SED, and 0 for SEE. The disparity between the rates for SE gene(s) in poultry and human isolates suggests a lesser role for the enterotoxins in invasive poultry disease than in human disease.

Detection of infected poultry flocks is essential for controlling eggborne transmission of Salmonella enteritidis to humans. The present study evaluated the detection of antibodies in the sera of experimentally infected chickens by a fluorescence polarization assay with a tracer prepared from the O-polysaccharide of S. enteritidis and an enzyme-linked immunosorbent assay (ELISA) with an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with either 10⁶ or 10⁸ colony-forming units (CFU) of S. enteritidis (phage type 13a) or with 10⁸ CFU of Salmonella typhimurium. Serum samples were collected before inoculation and at five subsequent weekly intervals. Both assays successfully detected the majority of hens infected with S. enteritidis at either dose level, but they also identified a substantial number of hens infected with S. typhimurium as seropositive. The fluorescence polarization test detected S. enteritidis infection significantly more often and cross-reacted with sera from hens infected with S. typhimurium significantly less often than the ELISA. The fluorescence polarization assay also offered advantages in terms of speed and methodologic simplicity.

One hundred four Escherichia coli isolates were collected from internal tissues and the cloacae of broilers with colibacillosis or from the cloacae of healthy birds. The isolates were tested for the presence of DNA sequences for temperature-sensitive hemagglutinin (tsh), for P (pap) and F1 (pil) fimbriae, and for aerobactin synthesis (iuc) by DNA/DNA hybridization. The isolates were also tested for O1, O2, and O78 serogroups, serum and antibiotic resistance, and virulence in day-old chickens. The Tsh/Pil/Iuc was the major pathotype detected in 53.8% of isolates from internal tissues, as compared with only 28.8% of isolates from the cloacae. The Tsh/Pap/Iuc pathotype was detected at a lower frequency (15.4%) but only in isolates from internal tissues. Among the virulence-associated marker genes, tsh and iuc were detected in most of the isolates from internal tissues (90.4% and 92.3%), as compared with only 51.9% and 63.5% of isolates from the cloacae, respectively. pap was detected to a lesser extent, in 25% of isolates but only from internal tissues. In contrast to the pil gene, the tsh-, pap-, and iuc-DNA sequences were more frequently detected in isolates from internal tissues than in isolates from the cloacae. O-antigen typing revealed that 25% of isolates belonged to serogroups O1 (4.8%), O2 (9.6%), and O78 (10.6%). Although most isolates appeared to be resistant to serum, only isolates from internal tissues were virulent in day-old chickens in contrast to isolates from the cloacae. More than 10% of isolates were resistant to most of the antibiotics used for the study. However, less resistance to enrofloxacin and norfloxacin was observed. Our data suggest that the Tsh/Pil/Iuc and Tsh/Pap/Iuc pathotypes and Tsh and Iuc virulence-associated markers are important factors of avian pathogenic E. coli. Enrofloxacin appeared to be the best choice for treatment of the infection.

Avian pneumovirus (APV) causes a respiratory disease in turkeys. The virus has been associated with morbidity and mortality due to secondary infections. Our objective was to determine if APV caused immunosuppression in the T-cell or B-cell compartments and to study the pathogenesis of the disease in APV maternal antibody–lacking 2-wk-old commercial turkeys. APV was administered by the eyedrop/intranasal route. Observations were made for gross lesions, viral genome, and T-cell mitogenesis and cytokine secretion at 3, 5, 7, 14, and 21 days postinoculation (DPI). During the acute phase of the disease that lasted for about 1 wk, the turkeys exposed to APV showed clinical signs characterized by nasal discharge and sinus swelling. Virus genome was detected by in situ hybridization in cells of turbinates and trachea at 3 and 5 DPI. At 3 and 5 DPI, spleen cells of the birds infected with APV markedly decreased proliferative response to concanavalin A (Con A). Con A and lipopolysaccharide stimulation of spleen cells from virus-exposed turkeys resulted in accumulation of nitric oxide–inducing factors (NOIF) in the culture fluid. NOIF were not detected in culture fluids of Con A-stimulated spleen cells of virus-free turkeys. APV did not compromise the antibody-producing ability of turkeys against several extraneous antigens such as Brucella abortus and tetanus toxoid.

A temperature-sensitive (Ts) mutant strain of Ornithobacterium rhinotracheale (ORT) was developed after exposure of the wild-type organism to N-methyl-N′-nitro-N-nitrosoguanidine. The Ts mutant strain grew at 31 C but had its growth inhibited at 41 C unlike wild-type parent strain. The Ts mutant and parent strains were characterized. Morphologic and biochemical properties of wild-type and mutant strains did not show any differences. The strains were also characterized by polymerase chain reaction (PCR)-based fingerprinting methods. Results showed similar patterns in repetitive sequences by repetitive PCR (enterobacterial repetitive intergenic consensus, highly conserved repeated DNA elements present in Streptococcus pneumoniae (BOX), repetitive extragenic palindromic, and Salmonella enteritidis repetitive element primers); however, random amplified polymorphic DNA fingerprinting was able to differentiate mutant and parent strains showing a unique pattern for each of the ORT strains. The rationale for the use of a Ts strain as a vaccine is based on the ability of the mutant to colonize the upper respiratory tract but not the lower respiratory tract and systemic system of the birds, where the wild-type strain causes severe lesions. In a preliminary evaluation, Ts strain of ORT was recovered from tracheas and choanae of Ts-treated turkeys for 13 days postadministration of the strain either in drinking water or by oculonasal instillation. Humoral immune response was detected in Ts-vaccinated but not in control group birds after 3 wk postadministration. Results suggest that Ts strain of ORT has promising potential use as a live vaccine for ORT.

The detection of either infectious virus, viral antigen, and/or viral RNA in different tissues of commercial broilers inoculated at 1 day of age with E/Del variant strain of infectious bursal disease virus (IBDV) was investigated at 2, 4, and 6 wk postinoculation (PI). Virus was readily isolated from homogenates of bursa, cecal tonsils, and bone marrow at 2 and 4 wk PI. Virus isolation coupled with immunoperoxidase assay or reverse transcription–polymerase chain reaction for IBDV-specific RNA extended the window of IBDV detection in the bursa of Fabricius to 6 wk PI. Serology indicated an active early virus infection; however, viral pathology was observed later and beginning at 4 wk PI. This study indicates that variant strains of IBDV may be present in commercial broilers longer than previously thought, and cecal tonsils and bone marrow may serve as nonbursal lymphoid tissues supporting virus replication at later time points PI.

The protection elicited by a temperature-sensitive (Ts) mutant of Ornithobacterium rhinotracheale (ORT) vaccine against challenge with pathogenic strain was investigated. In Experiment 1, specific serologic response to ORT was detected in 12%–19% of Ts-vaccinated birds at 3 wk postvaccination by either drinking water or oculo-nasal instillation. At 7 days postchallenge, 100% of Ts-vaccinated turkeys of all groups were able to respond with an ORT-specific antibody response, but the control group was not, suggesting the potential of Ts strain to evoke immune protection. The study also revealed a statistically significant ability of the Ts strain to protect vaccinated turkeys against gross lesions caused by the pathogenic strain of ORT in treated groups vs. control.

In Experiment 2, seroconversion was detected by enzyme-linked immunosorbent assay in birds after they were given the Ts strain in drinking water in field conditions. The results of the field study showed mean scores of gross lesions of nonvaccinated/challenged groups to be up to seven times higher than those of the vaccinated/challenged group. In addition, reisolation rates and quantification of ORT colonies per gram of lung tissue were significantly lower for vaccinated/challenged than for nonvaccinated/challenged turkeys. In conclusion, results from laboratory and field experiments suggest that use of the Ts mutant strain of ORT as a live vaccine would be a suitable method to evoke protection against ORT infection in turkeys.

In order to know the effect of in vitro passages on the pathogenicity of the Salmonella gallinarum strain INTA 91, a lyophilized culture was compared with the same strain recently isolated from a sick bird. The mean lethal dose (LD₅₀) of the orally administered lyophilized culture was determined as 2.04 × 10⁸ colony-forming units (CFU)/chicken. There was no correlation between the LD₅₀ dose and the degree of disease produced; doses 10 or 100 times higher than the calculated LD₅₀ did not produce a more severe disease. In trial 1, chickens were challenged with 1.02 × 10⁹ CFU per chicken (5LD₅₀) of the lyophilized strain and reached 52.2% mortality at the end of the assay. In trial 2, three different groups of chickens were infected with a recent isolate of the same strain: 2.04 × 10⁸ CFU/chicken, 4.1 × 10⁸ CFU/chicken, and 2.1 × 10⁹ CFU/chicken (i.e., 1LD₅₀, 2LD₅₀, and 10LD₅₀ of the dose calculated for the lyophilized strain, respectively). These chicken groups presented higher mortality rates (90%, 100%, and 95%, respectively) than previous trials, showing that the S. gallinarum strain used here increased its virulence by in vivo infected chicken passage. In all assays, the disease started after an incubation period of around 5–6 days. To obtain reliable and reproducible results in future challenge experiments, a fixed limited number of in vitro passages of the S. gallinarum strain must be determined.

The objective of this study was to identify the causative agent of cellulitis in turkeys. Eighteen flocks from nine producers were sampled at the local processing plant, and 37 birds with cellulitis on legs or caudal thoracic area were obtained. None of the 37 birds with cellulitis had lesions in other organs. On gross examination, lesions were categorized into two groups: cellulitis with unopened skin lesions (type a) and cellulitis with opened skin lesions (type b). Histopathologically, cellulitis with unopened skin lesions had dermal necrosis with underlying fibrin and inflammatory exudate but cellulitis with open skin lesions had chronic granulomatous/granulation tissue-type reaction associated with foreign material. A complete bacteriologic study was conducted on 25 of 37 birds. Bacteria were isolated from 12 of the 25 birds with cellulitis lesions. No aerobic, microaerophilic, or anaerobic bacteria were isolated from the remaining 13 birds with cellulitis lesions. Escherichia coli was isolated in low numbers in mixed cultures with Proteus mirabilis, Lactobacillus spp., Klebsiella spp., and Staphylococcus spp. in 9 of 12 lesions. The remaining few cases yielded P. mirabilis in pure culture or in mixed culture with Pseudomonas aeruginosa. Types a and b cellulitis lesions in turkeys could be associated with primary contact dermatitis and skin abrasions, respectively. Their occurrence is likely associated with different management practices.

One-day-of-age broiler chickens were administered a commercial competitive exclusion (CE) product and then challenged by three different methods with an Escherichia coli O78:K80 that was pathogenic for poultry and resistant to six antibiotics. Three challenge methods were used on 2-day-old broilers: direct challenge, precolonized seeder, and instant seeder. Direct challenge was accomplished by administering the challenge E. coli per os. The precolonized seeder challenge had two chicks that had received the challenge E. coli 24 hr previously, whereas the instant seeder challenge had two chicks given the challenge E. coli per os with immediate placement with the experimental birds. One oral dose of the commercial CE product significantly reduced the colonization of the small intestine, large intestine, and ceca by the highly antimicrobial resistant poultry pathogenic E. coli O78:K80 at 7 and 14 days postchallenge by all three challenge methods. The overall mean reductions in colonization were 3.0 log₁₀ for the large intestine, 3.0 log₁₀ for the small intestine, and 4.0 log₁₀ for the cecum. The most severe challenge method, on the basis of the least amount of reduction of colonization of the challenge E. coli by the CE, was by the direct oral gavage at 2 days of age.

To study effects of experimental cryptosporidiosis, broiler chickens were infected per os with 5 × 10⁵ oocysts of Cryptosporidium baileyi and Cryptosporidium meleagridis. In the first experiment, chickens were infected with oocysts of C. baileyi at the age of 7, 14, and 21 days. In the second experiment, chickens were infected with oocysts of C. baileyi, C. meleagridis, or both cryptosporidial species at the age of 7 days. Although clinical signs of infection were apparent, neither final live weight nor mortality was significanty influenced in chickens infected with a single Cryptosporidium species. In chickens infected with C. meleagridis, the growth retardation was observed in the 2-wk period after infection. The compensatory growth, however, started when the oocyst shedding had ceased. The number of oocysts in excreta specimens of chickens infected with C. meleagridis was two to three times lower than in excreta of chickens infected with C. baileyi. Chickens infected with both C. baileyi and C. meleagridis (5 × 10⁵ oocysts of each) had significantly lower final live weight and worse feed efficiency than chickens of other groups. Concurrent infection did not influence individual C. baileyi or C. meleagridis oocyst shedding.

Mycoplasma gallisepticum infection results in numerous clinical signs including a reduction in egg production in laying chickens. Attempts to prevent mycoplasmosis have included vaccination with both killed and attenuated live M. gallisepticum strains. Live vaccines provide reduction in clinical signs and have been shown to replace indigenous strains when used in a consistent program for several placements. Antibiotic therapy is another option for controlling losses associated with mycoplasmosis. Therapeutic antibiotics with activity against mycoplasma approved for use in poultry include tetracyclines and tylosin. These drugs also are approved for feed efficiency when administered in the feed at levels below the therapeutic index for mycoplasma. The data presented here suggest that birds vaccinated with the live 6/85 strain of M. gallisepticum and then fed tylosin, at the approved level for feed efficiency, exhibit a serologic vaccine response similar to that of unmedicated birds but show improved feed efficiency.

Minimum inhibitory concentrations (MICs) were determined in vitro for 7 antibiotics (aivlosin, enrofloxacine, tylosin, tiamulin, kitasamycin, chlortetracycline, and oxytetracycline) against eight recent local Argentinean isolates and two standard strains of Mycoplasma synoviae. Aivlosin (3-acetyl-4"-isovaleryl tylosin tartrate), tylosin, and tiamulin showed the lowest MICs with MIC₉₀s of 0.006, 0.012, and 0.05 µg/ml, respectively. Except one strain that showed resistant values to chlortetracycline (≥12.5 µg/ml), all the analyzed strains were susceptible in different degrees to all the antibiotics tested. In this study, the improved activity of the tylosin-derived drug, aivlosin, was confirmed because it showed, in most strains, MIC values half those for tylosin.

A rapid, sensitive, and specific polymerase chain reaction (PCR) assay was developed for the direct detection of Campylobacter in environmental samples from hatcheries. PCR, with a set of primers specific for the Campylobacter flaA short variable region (SVR), detected the presence of Campylobacter in both fluff and eggshell samples; however, a determination of whether the organism was living or dead could not be made. Conventional cultural methods detected no Campylobacter from the same samples. An additional benefit of the direct PCR assay is it allows for the production of a product that can be sequenced to provide further epidemiologic information.

In the present study, the influence of stress from handling and transport on some frequently examined blood parameters of racing pigeons was evaluated. After 3 hr, there was a highly significant (P < 0.01) increase in the number as well as in the percentage of heterophils and decrease of lymphocytes. In clinical chemistries, increases of creatine kinase and glucose and a decrease of uric acid were observed. There was a mean decrease of the total white blood count of >15% that was less significant (P < 0.05). Changes in lactate dehydrogenase, basophils, and monocytes did not prove to be significant; eosinophils, aspartate aminotransferase, total protein, and the packed cell volume were not influenced by stress.

During 1998, hydropericardium syndrome was observed among 3-to-6-wk-old broilers in 45 different flocks of Haryana, India, with mortality ranging between 10% and 30%. Fowl adenovirus (FAV) was isolated from one of the affected flocks by chicken embryo liver cell culture. Serum neutralization test and polymerase chain reaction assay coupled with restriction enzyme analysis confirmed that the isolated virus belonged to FAV serotype 4. The disease was reproduced in 28-day-old broilers by subcutaneous and oral inoculation of isolated FAV4 alone. Typical hydropericardium and basophilic intranuclear inclusions in hepatocytes were observed in experimental birds by day 4 postinoculation.

This case report examines an Arbor Acres female × Peterson male crossbred 6-wk-old female broiler chicken with four legs, two cloacae, and three ceca. The intestines occupied the caudodorsal portion of the abdominal cavity with three ceca attached to the terminal end of the ileum. The left lateral cecum was larger and had a divided distal end that terminated into 2-cm-long blind sacs. The rectum was dilated and divided into two cloacae that each opened on the dorsolateral aspect of both the right and left cloacal elevator muscles. The two extra legs were attached to the pygostyle by cartilage and skin tissue. Both femoral heads of the extraneous legs were fused together. The extra legs were smaller in size (approximately 17 cm in length) compared with the normal legs (approximately 22 cm). The femoral heads of the two normal legs were disarticulated from the acetabulum.

Eleven market turkey flocks developed a respiratory disease characterized by coughing, swollen sinuses and nasal discharge. These symptoms first appeared between 3 and 16 days of age. Avian pneumovirus (APV) RNA was detected by reverse transcriptase (RT)–polymerase chain reaction (PCR) in six of six flocks tested. APV was detected by immunohistochemistry in turbinates of three of three affected flocks tested. Virus isolation attempts were negative. Ten of 11 flocks became seropositive on the APV enzyme-linked immunosorbent assay. Five weeks prior to hatch of these affected market turkeys, several breeder flocks in one geographic area had developed clinical signs and experienced decline in egg production typical of APV infection. In two breeder flocks, acute and convalescent sera indicated APV infection during the period of declining egg production. Attempts to detect APV RNA by RT-PCR from choanal cleft swabs of newly hatched poults were successful. Attempts to isolate the virus from these PCR-positive samples were negative.

An increase in leg condemnations was experienced in several commercial meat flocks of male and female turkeys. Affected legs had hematomas around and above the tibiotarsus–tarsometatarsus (hock) joint. Avulsion of the common retinaculum from the external condyle was observed when the skin and muscular fascia were removed. A farm investigation was performed and similar changes were observed in turkeys between 10 and 19 wk of age. Interestingly, birds with avulsion of this fibrinous bridge were not lame. Body weights were normal, no infectious agent was detected, and mineral contents of bones and tendons were within normal limits. Avulsion of the retinaculum might not have been diagnosed in the past because the birds are not lame and the lesion is tightly covered by the skin and muscular fascia.

This report chronicles recurring outbreaks of Haemoproteus lophortyx infection in captive bobwhite quail. Clinically, the signs of infection included reluctance to move, ruffled appearance, prostration, and death. These signs were associated with parasitemia, anemia, and the presence of large megaloschizonts in skeletal muscles, particularly those of the thighs and back. The average cumulative mortality for flocks experiencing outbreaks was over 20%. In a typical outbreak, mortality rose when the birds were 5–6 wk of age, peaked in 8-to-10-wk-old quail, and declined rapidly when the quail were 9–11 wk old. Outbreaks occurred exclusively between the months of May and October, and warm weather was determined to be a risk factor for H. lophortyx mortality. This protozoan most likely overwinters in native California quail in the area and is transmitted to quail on the ranch by an insect vector that emerges in warm weather. Infection of the large population of naïve bobwhite quail on the ranch leads to amplification of H. lophortyx, resulting in epidemics in successive flocks.

A leiomyosarcoma was found in the gizzard of a 57-day-old female broiler chicken weighing 1.8 kg. Grossly, the tumor mass, 13.0 × 8.5 × 10.0 cm, enveloped the gizzard and had a gelatinous appearance due to the rich production of mucin. Miliary metastatic tumors were noted in the liver. Histopathologically, there was marked production of mucus throughout the tumor tissue, and densely or loosely arranged long spindle-shaped leiomyosarcoma cells proliferated. The tumor cells had a low rate of mitosis, showed slight cellular atypia, and, immunohistochemically, were positive for actin, α-smooth muscle actin, and desmin. Electron microscopically, various amounts of microfibrils with focal densities, dense patches, and basal plates were observed.