Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.

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.

Immune and physiological responses of turkeys with green-liver osteomyelitis complex.

A study of field turkeys was undertaken in order to determine the involvement of relative immunological differences in the etiology of turkey osteomyelitis complex (TOC). Lame and normal turkeys were sampled from commercial flocks just prior to processing in two separate trials. After testing for functions of both humoral and cellular immunity, the turkeys were necropsied and examined for lesions of TOC. There were significantly higher relative spleen and over weights and significantly lower body weights and relative bursal weights in birds with TOC. The birds with TOC had lower response to phytohemagglutinin-P in both in vivo and in vitro tests as well as lower circulating lymphocyte counts and higher monocyte, heterophil, and total white blood cell counts. There was a significantly higher antibody response to sheep red blood cells in turkeys with TOC, whereas antibody response to Salmonella pullorum antigen was not different. There were no significant differences in the percentages of mononuclear cells or heterophils able to phagocytize bacteria or latex particles, or kill bacteria; however, the heterophils from turkeys with TOC lesions did phagocytize significantly fewer latex particles per cell than did those of the healthy turkeys. Total serum protein, uric acid, and blood urea nitrogen levels were higher in birds with TOC, whereas hemoglobin, iron, alkaline phosphatase, and gamma-glutamyl-transferase levels were lower. Although many of the differences in birds with TOC could be caused by the normal host reaction to infection, further study may reveal innate differences that contribute to susceptibility to TOC.

Effect of the genetic selection of turkeys for increased body weight and egg production on immune and physiological responses.

Selection of poultry for fast growth rate is often accompanied by a reduction in specific immune responses or increased disease susceptibility. In this study, 17-wk-old male turkeys from each of four closed genetic lines, a randombred control (RBC) line and its subline (F) selected for increased 16-wk BW, and another RBC and its subline (E) selected for increased egg production, were tested for in vivo response to toe web inoculation with phytohemagglutinin-P (PHA-P), in vitro response of lymphocytes in whole blood to PHA-P and concanavalin A (Con A), hemolytic complement activity, differential white blood cell counts, hematology, and serum chemistry values. Fifteen male turkeys from each of two commercial lines, Com A and Com B, were also tested. The large-bodied F line birds had a lower toe web response to PHA-P, lower lymphocyte counts, and lower relative spleen weights than their smaller parent line. Body weights, total erythrocyte counts, blood urea nitrogen (BUN) levels, and in vitro mitogenic response to PHA-P and Con A were higher in the F line birds. Line E had lower hemolytic complement levels, lower relative spleen and relative bursal weights, and a higher in vitro mitogenic response to PHA-P than its parent line. The Com B line had a lower toe web response to PHA-P, and lower serum levels of gamma-glutamyltransferase and bilirubin than Com A. Line Com B had higher total RBC counts and higher levels of alanine aminotransferase (ALT) than Com A. These results support the concept that some changes in the cell-mediated immune response, as well as other physiological changes that may potentially affect immune response, appear to accompany selection for faster growth.

Reservoir competence of Alphitobius diaperinus (Coleoptera:Tenebrionidae) for Escherichia coli (Eubacteriales:Enterobacteriaceae).

Larval and adult lesser mealworms, Alphitobius diaperinus (Panzer), were found to harbor a Congo red-binding strain of Escherichia coli (Migula) Castellani & Chalmers both on the external surface of their body and internally for 12 d. Thereafter, E. coli was not detected, even though the beetles were exposed continuously to a food source inoculated with the bacteria. Lesser mealworm larvae and adults discharge E. coli bacteria in their feces for up to 6 and 10 d, respectively. However, bacteria were no longer detected in their feces after larvae underwent a single molt to the next larval stage. This indicated there was no transstadial transmission of this strain of E. coli. Consumed infected larvae were found to cause more 1-d-old chicks to have positive cloacal swabs for Congo red-binding E. coli than consumed infected adults. The data indicated that the lesser mealworm may play a role in the direct transmission of E. coli and contribute to the spread of this bacteria in broiler production systems. This may be achieved by beetles being directly consumed by chickens or indirectly by spread of the bacteria throughout the broiler house by lesser mealworm feces.

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.

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 concurrent infections of haemorrhagic enteritis virus or marble spleen disease virus and Eimeria meleagrimitis in turkeys.

Turkey poults given the combination of marble spleen disease virus and Eimeria meleagrimitis (EM) exhibited a greater pathogenic effect caused by the virus and illustrated by an increased spleen weight:gain index and antigen titre. Poults inoculated with both haemorrhagic enteritis virus (HEV) and EM exhibited an ameliorated pathogenic effect, in that the spleen weight:gain index and mean antigen titre were reduced. A reduction in the faecal score of the same birds was also noted when HEV was combined with EM. Poults receiving EM had lower numbers of circulating heterophils and a higher number of lymphocytes compared to the controls. These effects, however, were lost when either virus was simultaneously present with EM. Poults receiving both EM and HEV had the highest level of circulating monocytes.

Isolation of infectious bursal disease virus from the lesser mealworm, Alphitobius diaperinus (Panzer).

Infectious bursal disease virus (IBDV) was isolated from adult lesser mealworms, Alphitobius diaperinus (Panzer), up to 14 d after exposure, but isolation of the virus was erratic over this period of time. The virus was undetected after 24 h in beetle larvae. Virus was isolated from the adult beetle's mouth parts, foregut, midgut, hindgut, and blood 24 h after they fed on feed inoculated with IBDV. Ten days after exposure, virus was isolated from the foregut but not the blood, mouth parts, or remaining digestive tract of the adult beetles. The adult lesser mealworm is capable of serving as a reservoir for IBDV, rather than a fomite, between broiler growouts.

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.

Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae).

The reservoir competence of the lesser mealworm, Alphitobius diaperinus (Panzer) is reported for Salmonella typhimurium (Loeffler) relative to broiler chicken production. Salmonella typhimurium was isolated from feces of the adult lesser mealworm at least 28 d after feeding for 24 h on 1 g of chicken feed inoculated with 3 x 10(8) bacteria/ml. All larvae fed S. typhimurium ceased voiding the bacteria in their feces before pupal molt, except one. One beetle continued to void S. typhimurium after it emerged as an adult, providing evidence that transstadial transmission of S. typhimurium may occur. The bacteria were found both on the external body surface and inside the body of surface-sterilized adults and larvae during 16 d of exposure. Salmonella-positive cloacal swabs were obtained from 1-day-old broiler chicks within 24 h after eating one infected lesser mealworm adult or larva.

Evaluation of the humoral immune response to different antigens in Arkansas Regressor and Progressor chickens.

Arkansas Regressor and Progressor chickens were re-evaluated for their immune response to different antigens. Chickens received i.v. injection of either SRBC (10 birds per line) or Salmonella pullorum (SP; 10 birds per line) at 7 wk of age, and sera were collected at 6, 13, and 20 d postimmunization. A third group of birds (10 birds per line) received and i.m. injection of GAT emulsion at 7 and 12 wk of age, and sera were collected at 10 and 14 wk of age. There were significant differences between the two lines in their humoral immunity to SRBC, SP, and GAT. Such results suggest genetic control of humoral immunity to these antigens in these lines. It is unknown whether humoral immunity to these antigens is correlated to regression of tumors induced by Rous sarcoma virus.

Antibody detection in matched chicken sera and egg-yolk samples by commercial enzyme-linked immunosorbent assay kits for Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, and avian reovirus.

ELISA kits have been used to detect antibody in egg yolk. The major advantage eggs offer over blood samples is the ability to collect samples without compromising flock biosecurity. A disadvantage to using egg yolk over sera concerns the method of preparing yolk for antibody testing. The technique used in this study involved a simple dilution method with no mixing or extraction. To determine the adequacy of yolk samples to replace serum samples, a serum sample and the first six eggs were obtained from each of 50 commercial leghorn hens. Mean titers were consistently larger for serum than for yolk, but the size of the difference varied with the virus. The variation of mean egg titer was comparable to that of the serum titer. Correlations between a hen's serum titer and the mean titer from hen eggs were only moderate, ranging from 0.35 to 0.85 across viruses and systems. The ability to predict the serum titer of a single hen by the mean titer from hen eggs may be inadequate.

Evaluation of the interaction of Eimeria meleagrimitis with hemorrhagic enteritis virus or marble spleen disease virus in turkeys.

The interaction of Eimeria meleagrimitis with hemorrhagic enteritis virus (HEV) or marble spleen disease virus (MSDV) was studied in 4-week-old female turkeys. Birds given either virus in combination with the coccidia showed greater weight gain than did birds given HEV alone. A combination of MSDV and E. meleagrimitis resulted in significantly lower oocyst production when oocysts were counted from individual birds. Levels of serum glucose, serum albumin, and total protein were reduced in birds given HEV either alone or in combination with E. meleagrimitis. Birds receiving E. meleagrimitis alone or in combination with either MSDV or HEV exhibited higher blood urea nitrogen (BUN) levels than birds in all other treatments. Birds receiving HEV or the combination of E. meleagrimitis and either HEV or MSDV had significantly lower serum triglycerides and cholesterol. Serum amylase was lower in poults receiving HEV alone or combined with E. meleagrimitis, and serum alkaline phosphatase was lower in the HEV-only treatment.

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.