Typing of Haemophilus paragallinarum strains by using enterobacterial repetitive intergenic consensus-based polymerase chain reaction.

The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum.

Approach for selection of individual enteric bacteria for competitive exclusion in turkey poults.

The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.

Changes in serum ovotransferrin levels in chickens with experimentally induced inflammation and diseases.

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 OTE 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.

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.

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.

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.

Effects of different floor types and levels of washing of waterers on broiler performance and bacteria count of drinking water.

The objective of the present experiment was to determine the effect of different flooring materials and washing of waterers on broiler performance. The floor treatments were 1) black, plastic-coated expanded metal, relatively rigid (B); 2) white plastic, semi-rigid, with rectangular openings (WR); 3) white plastic, semi-rigid, with square openings (WS); and 4) 3 cm of rice hull litter (C). One hanging waterer was placed in each pen. Wash treatments were 1) trough and bell washed every Monday, Wednesday, and Friday (AW); 2) wash trough only on Monday, Wednesday, and Friday (TW); and 3) the waterers were never washed after the 2nd wk (NW). Broilers reared on C has significantly lower BW than those broilers on B floors. Broilers reared on the B and WS floors had significantly higher breast blister scores and percentage of birds with blisters than broilers reared on C floors. Broilers reared on C had lower enlarged feather follicle scores than those reared on all raised floors and a lower percentage of enlarged feather follicles than those broilers reared on WS or WR floors. Broilers reared on WS+TW had significantly better feed conversion than WS+AW, B+TW, and B+AW treatments. Broilers reared on WR+TW treatment were significantly higher in breast blister score than broilers reared on WR+AW, C+TW, and C+AW treatments. Broilers reared on C+TW and C+AW treatments were significantly lower in breast blister score except for broilers reared on C+NW, WR+AW, and WS+AW treatments. Broilers reared on C+NW treatment were significantly lower in enlarged feather follicle score than those broilers reared on B+TW, WR+AW, and WS+NW treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Action spectrum of antiviral factor from chicken sera.

Rous sarcoma virus infections of regressor line chickens stimulate the transient production of antiviral factors in the serum. Earlier the present authors reported that a viral neutralization factor (VNF) inactivated Rous sarcoma virus during a 3-h incubation. The VNF is likely to have a broad antiviral and antimicrobial spectrum because it is active against several unrelated pathogenic poultry viruses. The present study measured the activity of VNF against Newcastle disease virus, infectious bursal disease virus, and infectious bronchitis virus. The VNF is active in immunologically incompetent systems and must be preincubated with the virus in order to inhibit it. Based upon the current experiments, it is proposed that VNF is not an immunomodulator but directly inactivates the virus. The VNF agent appears to be one of a newly identified class of nonspecific antiviral agents produced in vivo in chickens in response to a viral infection.

Manganese dipyridoxyl diphosphate. Effect of dose, time, and pulse sequence on hepatic enhancement in rats.

We used an animal model to investigate the hepatic enhancement characteristics of manganese dipyridoxyl diphosphate (MnDPDP) related to time, dose, and pulse sequence. The contrast doses selected were in the human tolerance range. Using an SE 300/15 pulse sequence, maximum mean hepatic enhancement of 45% (8 mumols/kg) and 58% (12 mumols/kg) over baseline was seen during a plateau maintained between 5 and 50 minutes postinjection in the 8 mumols/kg group, and between 10 and 90 minutes in the 12 mumols/kg group. This plateau was followed by a very gradual decline in hepatic enhancement. Using either 4 or 8 mumols/kg, there was a significant increase in postcontrast hepatic intensity on all relatively T1-weighted pulse sequences (spin echo [SE] 300/15, inversion recovery [IR] 1400/20/400, gradient echo [GE] 47/13/80 degrees, and GE 60/20/30 degrees) except GE 47/13/80 degrees at 4 mumols/kg. At 8 mumols/kg there was superior enhancement, with IR 1400/20/400 and SE 300/15, but at 4 mumols/kg there was no consistently superior sequence. None of the relatively T2-weighted pulse sequences (SE 2000/50, SE 2000/100, or GE 100/30/20 degrees) demonstrated a significant change in hepatic intensity using either dose of contrast. The data suggest that the best combination of dose, pulse sequence, and time for hepatic imaging with MnDPDP is 8 mumols/kg using heavily T1-weighted sequences 5 to 60 minutes following contrast administration.

Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability.

Three experiments were conducted to characterize the variation in enzyme-linked immunosorbent assay (ELISA) kits for infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). Expt. 1 was carried out to determine the variation in assay results when the same pools of low-, medium-, and high-titered serum were assayed. Significant variation occurred among separate lots and among test plates within the same lots for the IBV and IBDV assays. In most cases, variability between days and among technicians was not significant. Coefficients of variation were larger than is acceptable for immune-type assays. In the IBDV assay with high-titered serum, most of the wells in the plates reached maximum absorbance and were not capable of detecting titers above 1:8000-1:9000. Expt. 2 was conducted to determine the effects of varying the length of the ortho-phenylene-diamine (OPD) incubation time upon assay results. Either 7-, 12-, or 15-minute OPD incubation times were used. Incubation time significantly affected mean titer at all combinations of assay types and times, except determinations on the low-titered IBV samples. Expt. 3 was conducted to determine the effects of three different dilution methods on observed IBDV titer. The use of non-standard dilutions had significant effects on observed titer. In the medium- and high-titered samples, the use of two different dilution methods at 1:5000 rather than 1:500 resulted in titers that were three to four times those observed at the 1:500 dilution.