Kakambi V. Nagaraja

Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys.

Antibodies to avian pneumovirus (APV) were first detected in Minnesota turkeys in 1997. Virus isolation was attempted on 32 samples (28 tracheal swabs, 4 pools of trachea and turbinates) that were positive for APV by reverse transcriptase polymerase chain reaction (RT-PCR). The cell cultures used were chicken embryo fibroblast (CEF), Vero cells, and QT-35 cells. Five virus isolates were obtained from these samples, and the identity of the isolates was confirmed by RT-PCR. Four isolates were obtained by inoculation of CEF cells, and 1 isolate was obtained in QT-35 cells after 3–7 blind passages in cell cultures. Vero cells did not yield any isolate on primary isolation; however, all 5 isolates could be adapted to grow in Vero cells following primary isolation in CEF or QT-35 cells. This is the first report of isolation of APV in Minnesota and also the first report of primary isolation of APV in QT-35 cells.

Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys

Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production. Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells. In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells. In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin. Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V. By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V. None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.

Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar Enteritidis infection of chickens.

ABSTRACT Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), andfimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14− strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.

Effect of time and temperature on growth of Salmonella enteritidis in experimentally inoculated eggs.

Influence of time and temperature on Salmonella enteritidis multiplication in experimentally injected eggs was examined. There was an increase in the number of S. enteritidis with the increase in temperature of egg storage. There was less increase of S. enteritidis in eggs stored at 4 degrees C than in eggs held at temperatures higher than 4 degrees C (P less than 0.05). These results suggest a possible method for monitoring commercial eggs for the presence of S. enteritidis. It was concluded that the chances of recovery of S. enteritidis can be increased 10(6)-fold or more by holding the eggs at temperatures of 21 or 27 degrees C for more than 20 days and culturing their contents.

Molecular Epidemiology of Subgroup C Avian Pneumoviruses Isolated in the United States and Comparison with Subgroup A and B Viruses

ABSTRACT The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

Ornithobacterium rhinotracheale Infection in Turkeys: Experimental Reproduction of the Disease

This report details the first experimental production of clinical disease, mortality, and pathology resembling that of field infections by using Ornithobacterium rhinotracheale alone. Twenty-two-week-old male turkeys were exposed to O. rhinotracheale or lung homogenate from O. rhinotracheale-infected turkeys. Within 24 hr after inoculation, turkeys given O. rhinotracheale or lung homogenate intratracheally were depressed and coughing and had decreased feed intake. By 48 hr, several birds were coughing blood and ultimately died. Grossly, the lungs were reddened, wet, and heavy, failed to collapse, and were covered by tenacious tan-to-white exudate. Microscopically, the parabronchi and air capillaries were filled with fibrin, heterophils, macrophages, and small numbers of gram-negative bacteria. The pleura was often covered by a thick layer of fibrin, heterophils, and macrophages. Turkeys that survived to day 7 postinoculation had severe, subacute pneumonia. Ornithobacterium rhinotracheale was recovered from the lungs of most birds with pneumonia and was also cultured from the air sacs, sinuses, tracheas, spleens, and livers. All turkeys inoculated with O. rhinotracheale developed antibodies to O. rhinotracheale detectable by the serum plate agglutination test.

Feasibility of Using Proteins from Salmonella gallinarum vs. 9R Live Vaccine for the Prevention of Fowl Typhoid in Chickens

Proteins from a field strain of Salmonella gallinarum MSG1 were compared with 9R live vaccine strain for their protection against experimental fowl typhoid in chickens. Proteins from S. gallinarum gave better protection than the 9R live vaccine as measured by clearance of challenge organism from internal organs. Proteins given twice with an adjuvant at 200 micrograms/100 g body weight resulted in 95% protection, compared with 60% protection with 9R given orally. The 9R live vaccine produced more hepatic and splenic lesions and, when administered orally as a single dose, was the least protective (60%). In the group vaccinated subcutaneously with a single dose of 9R without an adjuvant, both the challenge strain and the 9R vaccine strain were isolated from the ovaries of some birds. All chickens vaccinated with 9R strain or with proteins developed antibodies detectable by microagglutination test, and in some vaccinated groups as many as 100% of the birds developed antibody levels detected by seroagglutination.

Isolation and identification of Ornithobacterium rhinotracheale from commercial turkey flocks in the upper midwest

Increased death loss was seen in a flock of 22-wk-old tom turkeys. The predominant postmortem lesion was fibrinopurulent pneumonia and pleuritis. Within 5 wk, turkey flocks on 17 other farms developed similar problems. All affected flocks during the 5-wk period were between 14 and 22 wk of age, and the severity of clinical signs and the degree of mortality increased with age. Ornithobacterium rhinotracheale was isolated in pure culture from affected lungs. Further investigation by tracheal swab culture of 261 flocks between 5 and 7 wk of age resulted in detection of O. rhinotracheale in 43% of the flocks.

Pathogenesis of Avian Pneumovirus Infection in Turkeys

Avian pneumovirus (APV) is the cause of a respiratory disease of turkeys characterized by coughing, ocular and nasal discharge, and swelling of the infraorbital sinuses. Sixty turkey poults were reared in isolation conditions. At 3 weeks of age, serum samples were collected and determined to be free of antibodies against APV, avian influenza, hemorrhagic enteritis, Newcastle disease, Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, Ornithobacterium rhinotracheale, and Bordetella avium. When the poults were 4 weeks old, they were inoculated with cell culture–propagated APV (APV/Minnesota/turkey/2a/97) via the conjunctival spaces and nostrils. After inoculation, four poults were euthanatized every 2 days for 14 days, and blood, swabs, and tissues were collected. Clinical signs consisting of nasal discharge, swelling of the infraorbital sinuses, and frothy ocular discharge were evident by 2 days postinoculation (PI) and persisted until day 12 PI. Mild inflammation of the mucosa of the nasal turbinates and infraorbital sinuses was present between days 2 and 10 PI. Mild inflammatory changes were seen in tracheas of poults euthanatized between days 4 and 10 PI. Antibody to APV was detected by day 7 PI. The virus was detected in tissue preparations and swabs of nasal turbinates and infraorbital sinuses by reverse transcription polymerase chain reaction, virus isolation, and immunohistochemical staining methods between days 2 and 10 PI. Virus was detected in tracheal tissue and swabs between days 2 and 6 PI using the same methods. In this experiment, turkey poults inoculated with tissue culture-propagated APV developed clinical signs similar to those seen in field cases associated with infection with this virus.