Isolation and serial propagation of turkey rotaviruses in a fetal rhesus monkey kidney (MA104) cell line

Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.     

Development of protein A-gold immunoelectron microscopy for detection of bovine coronavirus in calves: comparison with ELISA and direct immunofluorescence of nasal epithelial cells

A protein A-colloidal gold immunoelectron microscopy (PAG-IEM) technique was developed for the detection of bovine coronavirus (BCV) in the feces and nasal secretions of infected calves. Feces or nasal swab fluids were incubated sequentially with hyperimmune bovine anti-bovine coronavirus serum and protein A-gold, negatively stained, applied to formvar-coated copper grids and viewed using an electron microscope. The PAG-IEM method specifically identified BCV particles and possible subviral particles in feces and nasal-swab fluids from infected calves. The PAG-IEM method did not label other enveloped enteric viruses or morphologically similar fringed particles commonly found in feces. Detection of BCV using PAG-IEM was compared with ELISA and direct immunofluorescence (IF) of nasal epithelial cells by monitoring fecal and respiratory tract shedding of BCV from two experimentally infected and two naturally infected calves from birth to 3 weeks of age. PAG-IEM and ELISA detected shedding of BCV in fecal (4/4 animals) and nasal (3/4 animals) samples for an average of 5.25 days each. The observed agreement of BCV detection by PAG-IEM and ELISA was 85%. PAG-IEM may be a more sensitive immunoassay for the detection of BCV in diagnostic specimens from infected neonatal calves than ELISA. BCV infection of nasal epithelial cells was detected by immunofluorescence in 4/4 calves, persisted for the duration of the study in 2/4 calves and was sporadic in the other two animals. The observed agreement of BCV detection by PAG-IEM and IF was 57%.     

Further characterization of the agent causing coryza in turkeys

A total of 128 isolates of Alcaligenes faecalis, from the respiratory tract of turkeys and chickens, were identified and divided into two types designated type I and type II. Type I isolates were pathogenic in poults, hemagglutinated guinea pig red blood cells (RBCs), and did not grow on minimal essential medium (MEM) agar, and most did not grow in 6.5% NaCl broth. Type II isolates were nonpathogenic and nonhemagglutinating and grew on MEM agar, and most grew in 6.5% NaCl broth. Hemagglutination of guinea pig RBCs was a reliable characteristic for distinguishing type I from type II isolates, and it correlated with pathogenicity. In serological studies using 62 type I and 21 type II isolates, cross-reactions were observed when type I but not type II antigens were used to test antisera in the microagglutination test. Eleven bacterial isolates, different from type I and type II isolates, were urease-positive. Although frequently isolated from turkeys with coryza, these isolates were nonpathogenic and were always found in association with type I A. faecalis. Urease-positive isolates and type I and type II A. faecalis isolates were stable following 50 in vitro passages. Bordetella avium sp. nov. (the nomenclature suggested in Europe for A. faecalis) was pathogenic in poults. The colonial morphology, biochemical characteristics, and hemagglutinating activity of B. avium sp. nov. were the same as those of type I A. faecalis isolates. Based on the results of these studies, it was concluded that type I A. faecalis is the etiologic agent of turkey coryza.     

Preliminary Studies on Stocking Density and Production of Hamoor Epinephelus tauvina in PVC-Lined Raceways

The results of two stocking density trials on the nursery and grow-out stages of Epinephelus tauvina (Family: Serranidae), in PVC-lined raceways are presented.
At the nursery stage, fry of 17.1 g initial mean weight showed no significant differences in growth rate, survival rate and condition factor when stocked at densities of 200 and 400 fish/m³ over a period of 52 days. Fish grew to mean weights of 61.7 and 63.7 g, giving growth rates of 0.86 and 0.90 g/fish/day and final biomasses of 12.1 and 2S.2 kg/m³ for densities of 200 and 400 fish/m³, respectively. Survival rates were excellent for both treatments at 98percnt; or greater. Food conversion efficiency was slightly improved at the higher density.
At the grow-out stage, E. tauvina of mean weights ranging from 150-170 g cultured for a period of 215 days grew better at a density of 5 fish/m³ than at densities of 20 and 60 fish/m³ (final size: 770, 560 and 450 g with growth rates of 2.8, 1.8 and 1.4 g/fish/day, respectively). Survival rates were higher at the two lower densities. Overall, total biomass increased with stocking density (3.9, 11.1 and 23.4 kg/m³, for 5, 20 and 60 fish/m³, respectively). These results indicate that hamoor has potential to be successfully cultured in raceways.     

Structural proteins of classic and variant strains of infectious bursal disease viruses.

Structural polypeptides of six tissue-culture-origin (BGM-70 continuous cell line) infectious bursal disease viruses representing classic and variant strains of serotype 1 and one serotype 2 strain were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, two of the variant strains were propagated in vivo in bursa of Fabricius and compared with those grown in cell culture. Differences among the structural proteins of serotype 1 viruses were minor and probably of no value in differentiating these viruses. However, distinct differences were observed between serotype 1 and 2 viruses. The bursa-derived viruses were different from those propagated in cell culture in molecular weights and in proportions of the proteins. The bursa-derived strains had protein migration patterns similar to those described for tissue-culture-incomplete virus particles.     

Induction of functional defects in macrophages by a poult enteritis and mortality syndrome-associated turkey astrovirus.

The interaction of a poult enteritis and mortality syndrome (PEMS)-turkey astrovirus-Ohio State University (TAst-OSU) with the mononuclear phagocytic system cells, namely macrophages, was examined after in vitro and in vivo exposure. In vitro exposures were performed by incubating adherent turkey macrophages with various volumes of 10(6) 50% embryo infective dose (EID50)/ml TAst-OSU stock, whereas for in vivo challenge, poults were given a 200 microl inoculum of 10(6) EID50/ml TAst-OSU stock at 7 days of age. Results show that TAst-OSU in vitro exposure reduced macrophage viability relative to controls (P < 0.05) and decreased phagocytosis (P < 0.05) and intracytoplasmic killing of Escherichia coli (P < 0.05) after a 42-48-hr exposure. Poults challenged with TAst-OSU in vivo recruited almost 50% fewer Sephadex-elicited inflammatory cells in the abdominal cavity (P < 0.05) as compared with the sham controls. Similar to in vitro exposure, macrophages isolated from in vivo TAst-OSU-challenged poults exhibited reduced percentage of phagocytic macrophages (P < 0.05) as well as fewer intracytoplasmic E. coli per phagocytic macrophage (P < 0.05). TAst-OSU-challenged poults had a greater number of viable E. coli in their spleens (P < 0.05) after an intravenous E. coli challenge as compared with the non-TAst-OSU-challenged control poults. Macrophage-mediated cytokines and metabolites were also examined during this study. Both in vitro and in vivo TAst-OSU challenge resulted in reduced interleukin (IL)-1 and IL-6 activity. On the contrary, nitrite levels in macrophage culture supernatant fraction of TAst-OSU-challenged macrophages were significantly higher (P < or = 0.05). The findings of these studies indicated that TAst-OSU challenge induced defects in macrophage effector functions, implying that PEMS-turkey astrovirus can potentially impair the immune response of turkeys, thereby leading to enhanced susceptibility of turkeys to secondary, perhaps even fatal, bacterial infections.     

Immunogenicity and antigenicity of infectious bursal disease virus serotypes I and II in chickens

Chickens were inoculated with infectious bursal disease virus serotype I or serotype II to determine if their immune system can distinguish between the two serotypes. Chickens had neutralizing antibodies to only serotype I viruses following exposure to serotype I viruses, and chickens had antibodies to only serotype II viruses following exposure to serotype II viruses. No cross-reactions were observed between antisera prepared to each of these two serotypes using a cross-virus-neutralization assay. Signs of disease were detected only in birds exposed to a virulent serotype I isolate. Chicks exposed to the serotype II viruses were not protected from challenge with a virulent serotype I isolate. In one experiment, antibodies to a serotype II isolate, which were detected before challenge, did not protect chicks from challenge with a virulent serotype I isolate.