Antibiotic susceptibility of clinical isolates of Staphylococcus intermedius and Staphylococcus aureus from dogs in Grenada.

Sixty-nine Staphylococcus intermedius isolates from various clinical conditions in dogs over a 3 year period were evaluated for in vitro susceptibility to 8 antibiotics using the disk diffusion method. The majority of the isolates originated from cases of pyoderma (30.4%), and otitis externa (29.0%). The least resistance was seen against cephalothin (1.6% of the 64 isolates, followed by chloramphenicol (3.9% of 26 isolates). Highest resistance was seen against ampicillin (34.4% of 32 isolates), followed by tertracycline (29.0% of 69 isolates). Resistance rates to other drugs were as follows: enrofloxacin 17.1%, gentamicin 7.3%, neomycin 6.1% and amoxicillin-clavulanic acid, 4.6%. Eleven isolates of S. aureus, showed no resistance to cephalothin. Overall the S. intermedius and S. aureus isolates were highly sensitive to cephalothin (98.7% susceptibility), suggesting that the first generation cephalosporins may be most useful drugs for treatment of Staphylococcus infection in dogs in Grenada.

Antibiotic susceptibility of clinical isolates of Staphylococcus intermedius and Staphylococcus aureus from dogs in Grenada.

Sixty-nine Staphylococcus intermedius isolates from various clinical conditions in dogs over a 3 year period were evaluated for in vitro susceptibility to 8 antibiotics using the disk diffusion method. The majority of the isolates originated from cases of pyoderma (30.4%), and otitis externa (29.0%). The least resistance was seen against cephalothin (1.6% of the 64 isolates, followed by chloramphenicol (3.9% of 26 isolates). Highest resistance was seen against ampicillin (34.4% of 32 isolates), followed by tertracycline (29.0% of 69 isolates). Resistance rates to other drugs were as follows: enrofloxacin 17.1%, gentamicin 7.3%, neomycin 6.1% and amoxicillin-clavulanic acid, 4.6%. Eleven isolates of S. aureus, showed no resistance to cephalothin. Overall the S. intermedius and S. aureus isolates were highly sensitive to cephalothin (98.7% susceptibility), suggesting that the first generation cephalosporins may be most useful drugs for treatment of Staphylococcus infection in dogs in Grenada.

Novel infectious bronchitis virus S1 genotypes in Mexico 1998-1999.

Seventeen infectious bronchitis virus (IBV) field isolates recovered from commercial broiler flocks in Mexico were identified by reverse transcription-polymerase chain reaction cycle sequencing of the S1 gene. The isolates were obtained from broilers on farms from the neighboring states of Queretaro and San Luis Potosi in 1998 and 1999. Flocks had an ongoing history of bacterial-complicated respiratory disease with mortality rates as high as 28% in spite of receiving live vaccinations for Massachusetts and Connecticut strains of IBV. Sequence analysis of the S1 gene identified two unique genotypes that have been described, as of this time, only in Mexico and thus appear to represent strains indigenous to the country. The Mex/1765/99 genotype was isolated from 64% (11/17) of the respiratory disease outbreaks. Three isolates (18%) were similar to the BL-56 genotype, a unique Mexican IBV strain observed initially in 1996. In addition to the two indigenous strains, three isolates (18%) were found to be the Connecticut genotype.

A dot immunoblotting assay (dot blot ELISA) for early detection of Newcastle disease antibodies in chickens.

An enzyme-linked immunosorbent assay using nitrocellulose blotting membrane (dot blot ELISA) was developed for the detection of antibodies against Newcastle disease virus (NDV) in chickens. In this method, a nitrocellulose blotting membrane was used as the solid phase carrier. NDV antigens were directly bound onto the nitrocellulose membrane that was set into a dot blot microfiltration apparatus. Efficiency of the assay was evaluated using known positive and negative NDV sera obtained from chickens. The ability of the assay to detect antibodies 2 days earlier than the standard hemagglutination-inhibition (HI) test was demonstrated on sera collected from chickens experimentally infected with NDV, La Sota strain.

Efficacy of combined killed-in-oil emulsion and live Newcastle disease vaccines in chickens.

Following the introduction of routine vaccination regimes with different types of Newcastle disease (ND) vaccines, the incidence of velogenic viscerotropic Newcastle disease (VVND) in commercial poultry worldwide has declined dramatically. Unfortunately, these vaccination regimes are not feasible in free-range and backyard systems of poultry production practiced in many developing countries. In this study, we sought to develop a single vaccination regime in chickens with ND vaccines to elicit a long-lasting high level of ND virus (NDV) antibodies adequate to protect chickens against ND. The level of antibody response, as measured by the hemagglutination-inhibition (HI) test, and the degree of protection against the virulent strain of NDV were studied in chickens immunized with different vaccines. The vaccines used were: killed-in-oil emulsion (subcutaneous; s.c.) plus live virus (oculanasal; o.n.), given concurrently; experimental vaccine (s.c.) plus live virus (o.n.), given concurrently; killed-in-oil (s.c.); experimental vaccine prepared by homogenizing commercial live vaccine and oil emulsion (s.c.); and live virus (o.n.). The results obtained in this study indicate that concurrent administration of oil emulsion and live NDV vaccines induced the best antibody response, but there was no significant difference in protection among the vaccinated groups.

Active immunization against vasoactive intestinal peptide prevents photo-induced prolactin secretion in turkeys.

Photostimulation initiates and maintains the rise in circulating prolactin (PRL) observed during the reproductive cycle of the female turkey. Vasoactive intestinal peptide (VIP) is the principal PRL-releasing factor. This study tested the hypothesis that gonadal stimulatory photoperiods stimulate PRL secretion by releasing hypothalamic VIP. Therefore, an experiment was designed to determine if VIP immunoneutralization altered photo-induced PRL secretion. Reproductively quiescent female turkeys were divided into two groups comprising turkeys actively immunized with synthetic VIP conjugated to keyhole limpet hemocyanin (VIP-KLH; immunized; n = 48) or KLH alone (control; n = 48). The first immunization was administered 6 weeks before photostimulation. Blood samples were collected at frequent intervals prior to and following photostimulation, and plasma PRL concentrations were determined. Vasoactive intestinal peptide antibody titer was estimated from the percentage of 125I-labeled VIP bound to plasma diluted 1:1000. At the onset of photostimulation (Day 0), plasma PRL levels were similar for immunized and control turkeys (9.1 +/- 0.3 versus 8.9 +/- 0.3 ng/ml, respectively). Plasma PRL of control birds increased (P < 0.05) by Day 16 of photostimulation, reaching a peak value of 724.9 +/- 90.1 ng/ml on Day 84. In contrast, plasma PRL remained essentially unchanged in immunized birds. Titer of anti-VIP antibodies expressed as 125I-VIP bound by plasma in immunized birds was 10.9 +/- 1.5% on the day of photostimulation. Incubation behavior was blocked in immunized birds, whereas 75% of controls exhibited incubation behavior. The control group laid 1.83 eggs/ week/hen compared to 3.40 eggs/week/hen in immunized hens. These findings suggest that photoperiodic modulation of PRL secretion in the turkey is influenced by hypothalamic VIP neuronal system.

A dot immunobinding assay (dot-ELISA) for the rapid serodiagnosis of Salmonella enteritidis infection in chickens.

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

A type-specific avian influenza virus subunit vaccine for turkeys: induction of protective immunity to challenge infection.

The fraction NP/HA (nucleoprotein/haemagglutinin) obtained from n-octyl-beta-D-glucopyranoside-treated influenza A H5N2 virus was highly enriched for NP with residual haemagglutinin. This preparation was incorporated in ISCOMs. This potent 'immunostimulating complex' induced the production of high antibody titres in turkeys. The NP/HA ISCOMs preparation was found to protect turkeys from both homologous and heterologous challenge infection as shown by reduced viral titres in the lung and trachea of vaccinated turkeys. Clearance of the virus from trachea and lungs was seen at late stages of infection. The vaccine also induced a cellular immune response as measured by T-cell proliferation and a delayed-type hypersensitivity response. The results reported in this study demonstrate that the NP/HA ISCOM vaccine is capable of inducing type-specific immunity and that it has potential utility as a vaccine in turkeys.

Adjuvanted subunit vaccines for the control of Salmonella enteritidis infection in turkeys.

Liposomes and immunostimulating complexes (ISCOM) are adjuvants that have been known to potentiate the immune response to membrane proteins. Adjuvanted outer membrane proteins (OMP) from Salmonella enteritidis were evaluated for their protective efficacy against S enteritidis infection in turkeys. The adjuvanted vaccines prepared for evaluation were: positive or negatively charged liposomes, lipid-conjugated ISCOM, and mineral oil vaccines. These preparations were compared with that of a whole cell bacterin and protein alone. After vaccination, turkeys were challenge-exposed with a nalidixic acid-resistant strain of S enteritidis. They were monitored for clinical signs of disease, antibody response, bacterial shedding pattern, and clearance of the challenge S enteritidis from internal organs. Results indicated a significantly (P < 0.05) higher antibody response to the positively charged liposomal OMP vaccine, compared with the whole cell bacterin. The antibody response to positively charged liposomal OMP vaccine was greater when a booster dose of this preparation was given. Shedding of S enteritidis was decreased in all vaccinated and challenge-exposed turkeys (P < 0.001). The tissues from a high percentage (90 to 100%) of birds that received a booster vaccination of the liposomal (+ or -) and ISCOM vaccine were culture-negative for S enteritidis.

Effect of avian influenza virus infection on the phagocytic function of systemic phagocytes and pulmonary macrophages of turkeys.

The effects of avian influenza virus (AIV) infection on systemic phagocytes and pulmonary macrophages of turkeys were studied. There was a significant increase (P < 0.0001) in oxidative burst in systemic phagocytes of AIV-inoculated turkeys on 2, 4, 6, and 8 days postinoculation (PI), as measured by chemiluminescence. There was also a significant increase (P < 0.02) in oxidative burst in pulmonary macrophages on day 4 PI. The chemiluminescence response was depressed on 6, 8, and 10 days PI in AIV-inoculated turkeys compared with controls. The increase in oxidative response in both systemic phagocytes and pulmonary macrophages correlated with the peak virus titer in the lungs and trachea of AIV-inoculated inoculated turkeys. Bacterial killing by pulmonary macrophages from AIV-inoculated turkeys was reduced on days 6 and 10 PI compared with uninoculated controls. Histopathological changes in trachea were more pronounced on day 6 PI in AIV-inoculated turkeys; no significant changes were detected in the lungs. These data indicate that compromised functional capacity of pulmonary macrophages predisposes turkeys to secondary bacterial infections.

Effect of intravenous inoculation of avian influenza virus on reproduction and growth in mallard ducks.

An avian influenza virus isolate, A/Mallard/Ohio/184/86 (H5N1), was evaluated for its effects on reproduction in isolation-reared adult mallard ducks (Anas platyrhynchos) and growth rate in juvenile mallards after intravenous inoculation. There was a significant decrease in egg production in the experimental group during the first week after inoculation, but it returned to the normal production level during the second week. No effect was seen on egg weight, shape, or fertility. Ducklings receiving this influenza virus isolate did not differ from controls in their rate of growth.

Antigen-capture enzyme immunoassay for detection of avian influenza virus in turkeys.

A double-antibody sandwich ELISA (DAS-ELISA) was developed for detection of avian influenza virus (AIV) antigen. A monoclonal antibody to the viral nucleoprotein (NP) was used to coat the ELISA plates. A direct DAS-ELISA and an indirect DAS-ELISA were evaluated. In the direct DAS-ELISA, monoclonal antibody to the AIV NP conjugated with horseradish peroxidase was used. The direct DAS-ELISA was evaluated for its sensitivity to detect purified NP; this procedure detected as little as 0.1 ng. In the indirect DAS-ELISA, rabbit NP antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect DAS-ELISA was evaluated for its ability to detect the AIV antigen in tracheal and cloacal specimens from turkeys inoculated with AIV. Results of indirect DAS-ELISA were compared with those of conventional virus isolation. Percentage agreement between indirect DAS-ELISA and virus isolation in AIV-positive samples was found to be 76.1% and, in AIV-negative samples, it was found to be 82.1%. These results indicate that the DAS-ELISA might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

Effect of temperature on the stability of avian influenza virus antigens under different storage conditions.

The combined effect of time and temperature on the stability of two avian influenza virus (AIV) isolates concentrated with polyethylene glycol (PEG), stored at different temperatures, and used in the preparation of avian influenza vaccine was evaluated in turkeys at 24 hr and at 12, 24, 30, 36, and 42 months of storage. The differences detected between antibodies raised in turkeys by vaccines made from isolates under different storage conditions, times, and temperatures were not significant (P > 0.05), especially with vaccines prepared from one isolate. Virus recovery rates following challenge studies of vaccinated birds were similar. However, birds that were vaccinated twice had lower rates of virus recovery from the trachea, lungs, pancreas, and fecal samples following challenge infection. The results suggest that if stable isolates of AIV can be identified, such isolates can be rapidly concentrated with PEG and stored at -20 C or -196 C for at least 42 months without any loss of potency in the vaccine prepared from these isolates. This would reduce the costs associated with vaccine storage and subsequent expiration dates.

Comparative evaluation of tissue trophism characteristics in turkeys and mallard ducks after intravenous inoculation of type A influenza viruses.

Ten avian type A influenza viruses consisting of seven waterfowl-origin, one pheasant-origin, and two turkey-origin viruses were evaluated for their pathogenicity potential after intravenous inoculation into domestic turkeys and mallard ducks (Anas platyrhynchos). The replicative abilities and tissue trophism properties of each virus isolate were examined in both species. The overall virus-isolation rate and histopathological lesion score were greater in the turkeys than in the ducks. The waterfowl-origin viruses caused more tissue damage in turkeys than in ducks but had a narrower tissue distribution range. The pheasant isolate was extremely pathogenic in turkeys but had limited distribution and little effect in ducks. The turkey isolates were more pathogenic in turkeys than in ducks. The pancreas was the most severely affected organ in turkeys, followed by kidney and liver. The spleen and bursa were the most commonly affected organs in ducks.

Biological and molecular characterization of H13N2 influenza type A viruses isolated from turkeys and surface water.

The pathogenicity potential of two H13N2 influenza viruses, one isolated from turkeys and the other isolated from surface water, was evaluated in turkeys, chickens, and mallard ducks (Anas platyrhynchos) after intracranial and oculonasal inoculation. Both isolates replicated in turkey poults, causing depressed weight gain, morbidity and mortality; both also caused histopathological lesions, such as mild to severe pancreatitis, hepatitis, and nephritis in turkeys. These isolates replicated in mallard ducklings but not in chickens. There was depressed weight gain in ducklings given the H13N2 isolate from water. Neither isolate caused morbidity or mortality in ducklings or chicks after inoculation.

Effect of an H5N1 avian influenza virus infection on the immune system of mallard ducks.

Avian influenza virus (AIV) of waterfowl origin, A/Mallard/Ohio/184/86 (H5N1), was used to evaluate the effect of AIV infection on the functional capabilities of the immune system in mallard ducks. The three main arms of the immune system--humoral, cell-mediated, and cellular--were evaluated. The integrity of the humoral immune system after AIV infection was evaluated by measuring total immunoglobulin and IgG antibody production to sheep erythrocytes and Brucella abortus antigen using hemagglutination and microagglutination assays, respectively. Cell-mediated immunity was evaluated using mitogen/antigen stimulation assays, and by measuring the cutaneous basophilic hypersensitivity response to intradermal phytohemagglutinin-P inoculation. The cellular component of the immune response was evaluated using whole-blood chemiluminescence and bacterial clearance assays. Results showed that infection with this AIV isolate suppressed T-cell function and enhanced macrophage phagocytic activity.

A lipid-conjugated immunostimulating complex subunit vaccine against Salmonella infection in turkeys.

Immunostaining complexes (ISCOMs) are multimeric particles and have been used successfully for presentation of membrane proteins. In this study, outer-membrane proteins (OMPs) from Salmonella heidelberg were incorporated into lipid-conjugated ISCOM particles and evaluated for their use in a vaccine for turkeys against homologous and heterologous Salmonella challenge. Two types of lipid-conjugated ISCOMs were examined: ISCOM-phospholipid and ISCOM-sphingolipid preparations. The turkeys were challenged with one of the three Salmonella serotypes: S. heidelberg, S. reading, or S. enteritidis. The turkeys were monitored for clinical signs, shedding pattern post-challenge, and clearance of the challenge Salmonella from selected internal organs. Vaccines containing OMP with either lipid-conjugated ISCOM preparation produced significantly greater (P < 0.01) immune response than OMP alone. Cloacal swabs from turkeys given OMP along with ISCOM-phospholipid and challenged with a homologous serotype were completely negative for Salmonella. A certain degree of cross-protection against heterologous Salmonella was afforded by both OMP-ISCOM vaccines. The isolation rate of Salmonella from internal organs was significantly lower (P < 0.0001) in vaccinated turkeys than in unvaccinated controls.

Influenza in commercial broiler breeders.

Influenza was detected in a flock of broiler breeders during routine serological monitoring. Although there were no clinical signs, egg production may have been affected in hens on one story of a two-story breeder house. Intensive measures were taken to avoid transmission to other farms. Two months after the flock was found to be serologically positive, sentinel hens were placed in the flock, and they became serologically positive 1 month later. In spite of this evidence for virus being present in the flock, no detectable transmission to any other farm occurred.

Enhancement of antibody response of turkeys to trivalent avian influenza vaccine by positively charged liposomal avridine adjuvant.

Trivalent avian influenza (AIV) antigens (H4N8, H5N2 and H7N3), mixed with positively charged, negatively charged and neutral avridine-containing liposomes, and oil-emulsion were subcutaneously administered to 6-week-old turkeys. Charged liposomal avridine adjuvant, either positive or negative, produced a better antibody response than uncharged liposomal avridine or oil-emulsion adjuvants when used in a trivalent avian influenza vaccine. The antibody response to the different antigens was generally greater to the positively charged adjuvanted vaccine compared with the negatively or neutral charged or oil-emulsion adjuvanted vaccines and these differences were significant (P less than 0.05) with the three antigens. The results suggest that the positively charged liposomal avridine plays a significant role as adjuvant to the AIV antigens.