Respiratory infection of turkeys with Listeria monocytogenes Scott A.

The pathogenesis of L. monocytogenes strain Scott A was studied by challenging day-old male turkey poults by air sac inoculation with tryptose phosphate broth containing 10(0) cfu (control), 10(4), 10(5), and 10(6) cfu (low challenge), or 10(7) and 10(8) cfu (high challenge) of the Scott A (serotype 4b) strain of L. monocytogenes. Mortality at 2 wk postinfection (PI) ranged from 25% for low challenge to 100% for high challenge (P= 0.0001). Gross and histopathological lesions were observed in heart, liver, spleen, lung, and bursa of Fabricius of mortalities at 4 days PI. Listeria monocytogenes challenge resulted in significantly decreased relative weight of the bursa of Fabricius and increased relative weight of the spleen, and L. monocytogenes was isolated by direct plating of liver, pericardium, brain, and both left and right stifle joint synovium (knee) cultures, as well as gall bladder, yolk sac, and cecal tonsil from transfer swabs onto Listeria-selective agar. Isolates were confirmed as positive using Gram stain, biochemical tests, and the Biolog system. High challenge resulted in confirmed L. monocytogenes isolation from 48% of left knee and 59% of right knee cultures. Low challenge resulted in isolation of L. monocytogenes from 11% of both left and right knee cultures. These results suggest that L. monocytogenes Scott A colonization of turkey knee synovial tissue can initiate in day-of-age poults and that L. monocytogenes Scott A can be invasive through air sac infection.

An outbreak of histomoniasis in turkeys infected with a moderate level of Ascaridia dissimilis but no Heterakis gallinarum.

Histomoniasis was diagnosed in a commercial turkey flock. All morbidity and mortality occurred in one house. Birds exhibited lesions characteristic for histomoniasis, and the diagnosis was confirmed by histopathologic examination. Affected turkeys were infected with moderate levels of Ascaridia dissimilis but not Heterakis gallinarum. Compression smears of hepatic tissues showed typical histotrophic phase Histomonas meleagridis, whereas cecal smears exhibited large numbers of Trichomonas gallinarum. A challenge experiment was conducted in which turkey poults were placed on contaminated litter. Although histomoniasis was not reproduced in the experiment, the birds did become infected with low numbers of A. dissimilis.

Identification and partial characterization of Arkansas isolates of chicken anemia virus.

Chickens from both broiler and broiler breeder pullet flocks experiencing symptoms of chicken anemia virus (CAV) infection were first observed at the Poultry Health Research Laboratory at the University of Arkansas in September 1992. Flocks had experienced higher than normal mortality with subcutaneous hemorrhages on the wings, neck, and thorax. Postmortem and histopathologic evaluation revealed thymus and bursal atrophy and lesions consistent with those reported for CAV infection. Because this infection had not previously been observed by Poultry Health Research Laboratory personnel in Arkansas-grown chickens, the establishment of a definitive diagnosis was deemed important. The presence of CAV was established by infecting MSB-1 cells with pooled liver homogenates from groups of 10 specific-pathogen-free chickens that had previously been inoculated in an attempt to experimentally reproduce the disease observed in the field. Cytopathic effects in the infected MSB-1 cells were first evident following the fifth passage. Indirect fluorescent antibody technique identified infected MSB-1 cells following at least five blind passages. To further confirm the presence of CAV, a polymerase chain reaction (PCR) technique was used to amplify a specific portion of the virus genome from infected MSB-1 cells and tissue extracts from several submitted chickens. Sequence analysis of a 186-bp PCR amplification product revealed that the Arkansas isolate was very similar to the Cuxhaven-1 isolate (99.5% sequence identity).

Histologic study of hepatic lesions in two turkey flocks.

Hepatic lesions were studied in two turkey flocks by euthanatizing 50 birds a week from the ages of 1 through 15 wk. Samples of liver that contained lesions and samples of duodenum, pancreas, ileum, and cecal tonsil were examined histologically. Lymphocytic infiltrations made up 82% and 75% of the hepatic lesions, and granulomas occurred in 18% and 25% of the livers. Nematode larvae were present in 12% and 15% of the hepatic lesions.

Broiler chicken with a duplicated lower intestinal tract.

A case is described of a duplicated lower intestinal tract in a broiler chicken. The ileum branched to form two lower intestinal tracts. The ileum of the malformed branch was swollen and attached to paired ceca that were grossly enlarged, and the rectum was a swollen blind sac, disconnected from the exterior of the bird.

Pathogenicity studies of an Arkansas variant infectious bursal disease virus.

A variant infectious bursal disease virus (IBDV), IBDV-s977, was blind passaged in cell culture, plaque purified, and attenuated by serial passage at a high multiplicity of infection (MOI) in chick embryo fibroblasts (CEF). Cell culture passages of virus caused less bursal atrophy and splenomegaly than did the original isolate and retained immunogenicity; however, virus tended to persist for a longer time in the bursa and spleen of birds infected with the highest CEF passages. Antibody to both low MOI and high MOI passages of IBDV-s977 poorly neutralized virus that was isolated from bursal tissue 28 days postinfection (PI). The spleens of chickens infected with the eighteenth CEF passage were negative for virus at 3 and 7 days PI but had high titers of virus at 14 and 28 days PI. There was also more virus in the bursa of birds infected with the fifteenth and eighteenth CEF passages at 28 days PI than at 7 or 14 days PI. Defective interference (DI) was demonstrated when cell cultures were coinfected with a constant amount of low MOI virus and serial dilutions of high MOI virus. There was an increase in interference score with increased passage number in CEF, and there was more interference in virus passaged at a high MOI. There was an inverse relationship between interference score and bursal lesion score and splenomegaly at 7 days PI, indicating that DI particles may be involved in virus attenuation. There was a positive relationship between interference and viral persistence in the bursa and spleen at 28 days PI. Antiserum to s977 was shown to enhance the nonlytic replication of s977 in CEF, presumably within macrophages, providing a possible mechanism for the pathotypic variation seen in emerging strains of IBDV.

Antigenic characterization of an Arkansas isolate of infectious bursal disease virus.

The s977 strain of infectious bursal disease virus (IBDV) was isolated in northwest Arkansas in 1977 from the bursae of young broilers with high maternal antibody titers to the Moulthrop strain of IBDV (BursaVac). The comparison of a plaque-purified isolate of s977 with other IBDV serotype 1 and serotype 2 strains using virus neutralization indicates that s977 is a subtype of serotype 1 vaccine viruses and the MD variant strain of IBDV and has no relatedness to the Delaware Variant A (VarA) virus. In vivo cross-protection studies in specific-pathogen-free white leghorn chickens showed that an inactivated vaccine using s977 antigen was 2.5 times more protective against challenge with s977 than was an inactivated IBDV Variant E (VarE) vaccine. The vaccination of maternally immune broiler chicks with live s977 did not provide protection against subsequent challenge, indicating that s977 does not have enough antigenic difference to break through maternal immunity. Analysis of denatured viral polypeptides using polyacrylamide gel electrophoresis showed that s977 and two reported variant strains, 51 and VarE, share three protein bands, 90 kD (VP1), 40 kD (VP2), and 31 kD (VP3), that were not observed in BursaVac. BursaVac and s977 shared a 74 kD precursor band that was absent or very faint in the VarE and 51 strains. The most unique characteristic of s977 was the relative abundance of a wide, 56-63 kD band that contained two distinct immunoreactive bands when blotted with antiserum to s977. BursaVac contained a 56 kD band that failed to react with s977 antiserum. Analysis of polypeptide bands using laser densitometry indicated the presence of a number of bands between 20 kD and 25 kD in the s977, 51, and VarE preparations but only a 25 kD band in BursaVac. The number of bands decreased with the degree of relatedness to standard vaccine strains. It appears that, antigenically, S977 may hold an intermediate position between the classic virus strains and the more recently reported serotype 1 variants.

A survey of sixty turkey flocks exhibiting hepatic foci taken at time of processing.

Fifty turkey flocks including 24 16-week-old male flocks and 26 20-week-old male flocks were sampled at time of processing. Hepatic foci were cultured for aerobic and anaerobic bacteria. The majority of these did not have any bacteria recovered from the lesions. Of the bacteria that were recovered, most were facultative anaerobes, with Escherichia coli and Salmonella sp. comprising the most common isolates. All of the birds examined (300 total) for parasites were infected with varying levels of Ascaridia dissimilis. The highest average worm burden was found in the 20-week-old flocks. Heterakis gallinarum were found in only a few of the younger turkeys (16 weeks old) and not in any of the older birds. An analysis of the spatial distribution of the hepatic foci performed in an additional 10 turkey flocks (500 birds) revealed that, although present on the surface of all regions of the liver, 56.12% of the lesions were found on the left hepatic lobe and 43.88% were found on the right hepatic lobe.

The effect of dietary copper sulfate on infectious proventriculitis.

Oral inoculation of day-old broiler chicks with a crude homogenate of affected proventricular tissue, or the same homogenate filtered through a .2 micron filter caused proventricular lesions similar to those responsible for carcass contamination of broilers at processing. Dietary copper sulfate (CUS) has also been shown to produce similar lesions. In this study, we investigated the interaction between crude proventriculus homogenate or filtered proventriculus homogenate and 1 g/kg CUS added to a standard chicken diet. Cobb x Cobb female broiler chicks were distributed into six groups with four replicate battery pens per group. Birds were fed either a standard broiler starter diet or the same diet with 1 g/kg CUS. Each dietary treatment was inoculated per os with 1 mL of either sterile saline, unfiltered homogenate, or filtered homogenate. Both crude and filtered homogenates had a much stronger affect on proventriculus score than did Cu by itself, resulting in no interaction between either homogenate or filtrate and CUS. There was a significant and possibly antagonistic interaction on proventriculus relative weights in the CUS by filtrate group during Week 1 and a synergistic interaction in the CUS by homogenate group during Week 4. Body weights were decreased in birds fed homogenate or CUS, but not in birds fed filtrate. There was a protective effect shown by filtrate on body weight of birds fed both filtrate and CUS only during Week 1. There was a synergistic decrease in body weight of birds fed homogenate and CUS during Week 2. Overall feed conversion efficiency was significantly decreased in the homogenate treatment (P = .04) and decreased in the birds fed CUS (P = .1). There was a (4.2 vs 2.3) (P = .1) decrease in feed conversion efficiency in birds fed both homogenate and CUS. Natural exposure to low levels of the infectious agent present in the homogenates may interact with excess dietary CUS, resulting in increased proventriculus size and decrease in body weight and feed conversion efficiency.

Experimental reproduction of proventriculitis using homogenates of proventricular tissue.

Proventriculitis is a problem affecting the processing of broiler carcasses, particularly those processed at 4 to 5 wk of age. The proventriculus and the gastric isthmus connecting the proventriculus to the gizzard are enlarged and swollen and often rupture during processing, causing carcass contamination. This study suggested that a filterable agent found in homogenated proventriculi can cause lesions similar to those seen in field cases. Proventriculitis was produced independently of an effect on growth, and only unfiltered homogenate caused stunting. Field birds with severe proventriculitis were shown to have increased body weights compared with birds without proventriculitis or with milder lesions. Intestinal weakness was not associated with proventriculitis and field birds with the most severe proventriculitis had stronger intestines. Although infectious proventriculitis has generally been reported as one of the lesions associated with stunting syndrome, these data suggest that it may have an independent etiology.

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV.

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.

The effect of microaerosolized hydrogen peroxide on bacterial and viral poultry pathogens.

The effect of microaerosolized H2O2 on bacterial and viral poultry pathogens was investigated. Bacterial cultures and viruses were dried on sterile glass Petri dishes and subjected to direct and indirect 5% (H2O2) microaerosol mist. In the trials using Escherichia coli and Staphylococcus aureus, there was complete inactivation following exposure to H2O2. Using Salmonella typhimurium, indirect exposure resulted in only partial inactivation whereas direct exposure to H2O2 gave complete inactivation. For the viruses studied, 5% H2O2 microaerosol mist completely inactivated infectious laryngotracheitis virus. Newcastle disease virus, infectious bronchitis virus, and avian influenza virus showed reduced infectivity but were not completely inactivated. Avian reovirus susceptibility varied with the method of exposure and infectious bursal disease virus was highly resistant. The use of 10% H2O2 mist, however, resulted in total inactivation of infectious bursal disease virus. The effect of 10% H2O2 on equipment and selected materials representative of a hatcher or poultry house was investigated. A solar cell calculator, a thermostat containing a microswitch, and samples of uncoated steel, galvanized steel, and uncoated aluminum were subjected to 10 fumigation cycles. No damage was detected in the calculator and the thermostat. Both the uncoated steel and the galvanized steel showed signs of oxidation. The aluminum did not show signs of oxidation.

A longitudinal study of green-liver osteomyelitis complex in commercial turkeys.

Two flocks of Nicholas tom turkeys from separate farms with histories of above-average condemnations for turkey green-liver osteomyelitis complex (TOC) were studied throughout a 16-week growout. Fifty birds from each farm were necropsied each week for 15 weeks, and birds that had green livers, osteomyelitis in the proximal tibia, or swollen joints were cultured for aerobic bacteria along with an equal number of control birds. At processing, TOC lesions and green livers were obtained for bacterial culture and histopathology. Green-liver-associated TOC was not observed until the turkeys were 9 or 10 weeks of age. The incidence of TOC was higher on one farm, which also had a higher incidence of airsacculitis, higher early and weekly mortality, seroconversion to Newcastle disease virus and Mycoplasma meleagridis, and significantly higher average body weights, relative spleen weights, and relative liver weights. Both farms had a high incidence of intestinal lesions and infestation with Ascaridia dissimilis. Histological evaluation of green livers revealed hyperplasia of bile ducts, dilation of sinusoids, and pigment-containing Kupffer's cells, some of which stained positive for iron. The bacterial isolates most frequently cultured from bones and livers were pleomorphic gram-variable coccobacilli, which grew visible colonies only after a series of subcultures and extended incubation.

A survey of two commercial turkey farms experiencing high levels of liver foci.

Two turkey farms that had previously experienced high levels of liver condemnations at slaughter were monitored through one complete growout cycle. Liver foci appeared at both farms by week 2. More than 80% of the liver foci sampled did not have any aerobic or facultative bacteria isolated from the lesions. Low numbers of Ascaridia dissimilis larvae were found on both farms by week 3 in the growout. The patterns for the ascaridiasis at both farms were similar, although one of the farms had a higher number of ascarids earlier than the other. Neither farm had high levels of adult ascarids present, although the average larval burden was high. Piperazine was administered at both farms on multiple occasions, but there were no significant decreases in the level of adult ascarids following administration. There was no apparent development of immunity, since all stages of the life cycle remained stable, even late in the growout. The simultaneous appearance of the liver foci and the A. dissimilis indicate that the ascarids may be responsible for the hepatic pathology.

Age-related changes in the incidence of tibial dyschondroplasia in turkeys.

Age-related changes in the incidence and severity of tibial dyschondroplasia (TD) and its relationship to body weight and other bone pathologies were studied in turkeys from ages 2 through 15 wk. A steady increase in the incidence and severity of TD was observed from 11 wk onwards. Whereas there was no correlation between the body weight and TD in early ages, during Weeks 14 and 15 the turkeys with severe TD had significantly higher body weight relative to those without or with mild lesions. Starting from Week 13, hemorrhage was evident in the base of TD-affected growth plates of many turkeys, which increased linearly during the following weeks. Histological examination of these areas revealed mild necrosis with erosion in TD tissues accompanied by occasional bone formations, fibrosis, and the presence of multinucleate chondroclasts and large number of erythrocytes adjacent to lesions. Sporadic rod-like bacterial colonies were discernible in TD tissues and cartilage canals. These studies show 1) whereas the onset of TD may have an independent etiology, the severity of the disease may be influenced by a higher body weight; and 2) subepiphyseal hemorrhages possibly facilitate a healing-type response that leads to replacement of TD tissue by way of resorption, bone formation, and fibrosis, but simultaneously makes the tissue accessible to bacteria.

Development of PCR method specific for Marek's disease virus.

A rapid polymerase chain reaction (PCR) assay specific for Marek's Disease Virus (MDV) was developed. This assay was able to detect MDV in inoculated chick kidney cells at dilutions of 10(-5). Negative PCR results were obtained using uninoculated chick cells, Marek's Disease Vaccine (SB), Herpesvirus of Turkeys (HVT) and Fowl Laryngotracheitis Vaccine (LT). Bursae, feathers and kidneys from MDV infected chickens were positive in the PCR assay. The same tissues from normal chickens were negative. This method required only 0.5 h for sample preparatory, 3 h for PCR application and 1 h for electrophoresis. Internal probe hybridization confirmed that the PCR products are from MDV, but this hybridization will not be necessary for future MDV detection.

Use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys.

Three hundred fifty 1-day-old large white turkeys were reared in brooding batteries to 10 days of age, after which they were moved to floor pens on litter. At 7 weeks of age, poults were allotted into four treatment groups as follows: 1) virulent hemorrhagic enteritis virus (HEV) alone (100 turkeys), 2) Escherichia coli alone (100 turkeys), 3) HEV + E. coli (100 turkeys), and 4) negative controls (50 turkeys). HEV was given orally at 7 weeks of age, followed by E. coli challenge in the drinking water 2 days later for 10 consecutive days. All groups were observed daily for mortality, both during and after challenge. Turkeys that died or were moribund were necropsied, and cultures were taken from the liver and bone marrow for bacterial isolation. Total mortality rates were 23% in the HEV + E. coli group, 10% in the HEV-only group, 3% in the E. coli-only group, and 0% in the negative control group. Cumulative mortality values were significantly different from those of the negative controls (P < or = 0.05) for HEV only and the HEV + E. coli group. E. coli was isolated from the liver and bone marrow of almost all turkeys that died.

Effect of age of maternal flock and strain on the incidence of tibial dyschondroplasia in growing chicks.

Two experiments were conducted to study the effects of age of the maternal flock (27 vs. 57 weeks of age at time the eggs were set) and strain of broiler chicks (Cobb 500, Ross, Arbor Acres, and Avian) on the incidence of tibial dyschondroplasia (TD) in broiler cockerels grown to 3 weeks of age. The chicks were fed a corn/soybean meal-based diet containing 3145 kcal metabolizable energy corrected for nitrogen retention (MEn)/kg, 24% protein, and 0.50% available phosphorus. Each treatment was replicated four times with eight chicks per pen. In Expt. 1, chicks from the old parent flock had significantly higher body weights (P < or = 0.05) than those from the young parent flock. There were no significant differences in feed efficiency or incidence of TD. In Expt. 2, body weights, feed efficiency, and TD were unaffected by strain.

High mortality of domestic turkeys associated with Ascaridia dissimilis.

Third- and fourth-stage Ascaridia dissimilis larvae were isolated from commercial white turkey intestinal scrapings from two farms that were experiencing high mortality. Lesions consisted of a necrotic-like enteritis that was most severe in the jejunum. Subsequent bacteriological isolation yielded heavy growth of Escherichia coli and Clostridium perfringens. The rate of mortality declined rapidly when the turkeys were administered 18 ppm fenbendazole for 7 days.

Use of an enzyme-linked immunosorbent assay to measure antibody levels in turkey breeder hens, eggs, and progeny following natural infection or immunization with a commercial Bordetella avium bacterin.

Twelve large white turkey hens were immunized with a commercially available Bordetella avium bacterin. Hens and eggs were tested using an enzyme-linked immunosorbent assay (ELISA) to determine the response to the bacterin. Three hundred poults were then obtained from two commercial flocks, the hens of one flock having been immunized with the same bacterin used on the group of 12 turkeys. Titers of the poults were monitored for 7 weeks, and poults were challenged by exposure to infected poults at 1, 7, 14, and 21 days post-hatch. Hens produced an antibody response following immunization, with a parallel antibody response being detected in eggs. Maternal antibodies were present in poults from immunized hens. Poult titers declined to near the level of poults from unimmunized hens by 14 days of age. Poults from immunized hens challenged at 1 and 7 days were resistant to development of clinical disease and gross lesions, whereas all poults from unimmunized hens exhibited clinical signs and gross lesions. After 14 days, the resistance of both groups to development of clinical disease, became near equal, neither group being affected as severely as the unimmunized hens challenged at days 1 and 7. Six commercial turkey breeding flocks and their progeny that had not been vaccinated for B. avium and had no history of B. avium infection were evaluated with the B. avium ELISA. There were variations between the flocks, with poult titers reflecting those found in the hens.