Nonspecific cellular defense of the avian respiratory system: a review.

The normal, steady-state, avian respiratory system has very low numbers of residing avian respiratory phagocytes (ARP). Birds must rely heavily on the influx of ARP to defend against infectious agents. The system is refractory to elicitation by inert stimulants, but responds efficiently to replicating bacteria, with very rapid influx of large numbers of activated ARP (polymorphonuclear neutrophils, heterophils, and macrophages) with increased phagocytic proportions and capacities. The numbers subside within a few a days. Activated ARP act in a non agent-specific manner: Pasteurella multocida-activated ARP can defend against a severe Escherichia coli airsacculitis. Parenteral routes of stimulation generally are not, respiratory routes are very, efficient in activating ARP. Heterophils are the most efficient in defensive reactions, such as oxidative burst, production of nitric oxide and killing of bacteria. Respiratory viruses may stimulate, but also may diminish some of the defensive functions of ARP. This is also true for attenuated, modified live virus vaccines. These vaccines must be used carefully in the presence of subclinical bacterial, mycoplasmal infections. Published literature on non-specific cellular defense of the avian respiratory system is very limited, particularly about interactions among multiple infectious agents and the system.

Analysis of bovine parainfluenza-3 virus replication in bovine embryonic lung cells by indirect fluorescent antibody and hemadsorption assays.

The temporal manifestation of bovine parainfluenza-3 virus (BPI3V) proteins in the cytoplasm and on the surface of bovine embryonic lung (BEL) cells was characterized by indirect fluorescent antibody (IFA) and hemadsorption (HAd) assays. Intracellular proteins appeared earliest at 0.5 h post inoculation (p.i.) and the infection spread to virtually all cells by 48 h p.i. Viral proteins on the surface of cells were seen first at 16 h p.i., and by 48 h p.i. the entire cell monolayer was IFA positive. Hundred-fold less virus in the inoculum delayed appearance of the intracellular as well as cell-surface viral proteins by 24 h and allowed the infection of only about 1/3 of the cells by 48 h p.i. Kinetics of the development in the proportion of HAd-positive cells correlated with those of the surface fluorescence-positive cells. Morphology of the manifestation of BPI3V proteins is characterized by microphotography.

Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 single-density, or by 1.077/1.119 double-density centrifugation, the 1.077/1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously.

Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs.

The objective of this study was to assess the risk of transmission of swine hepatitis E virus (swine HEV) to naïve pigs by inoculation with tissues or feces collected from pigs infected experimentally with swine HEV. Seventy-five, 3-week-old pigs were assigned randomly to 24 groups of 3-4 pigs and inoculated with homogenates of tissues (liver, heart, pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI). Each inoculum was prepared as a 10% suspension (w/v) in PBS buffer and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the swine bioassay. The inoculation route was intravenous for liver, heart and pancreas, and via stomach tube for skeletal muscle and fecal suspension. The liver homogenate inocula and feces collected at 3-7 and 14-20 DPI were positive for swine HEV RNA by RT-PCR. The pigs inoculated with liver homogenates collected at 3-7 and 14-20 DPI developed anti-HEV antibodies and swine HEV RNA was detected in their sera. Pigs inoculated with heart, pancreas, skeletal muscle homogenates or fecal suspensions failed to develop anti-HEV antibodies. These findings suggest that there is a potential risk of transmission of swine HEV via liver tissue from infected pigs in the early stages (3-20 DPI) of infection and the in vitro RT-PCR assay correlates well with the swine bioassay.

Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants.

In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system.

Proteolytic inactivation of simian-11 rotavirus: a pilot study.

In an effort to develop alternate disinfectants for rotaviruses, pilot studies were conducted to determine if bacterial proteases could render simian-11 (SA-11) rotavirus non-infectious. SA-11 was found to be fairly temperature resistant, retaining a low-level of infectivity following 65 degrees C treatment for 2h at pH 8.5. It also resisted pH 8.5-5 at 45 degrees C for 2h. Alcalase, an alkaline protease, was the most effective of the various proteases (alcalase, durazym, neutrase, and savinase) tested. To analyze specific parameters for alcalase, SA-11 virus (10(5.5) median tissue culture infective dose/ml original titer) was treated at pH 6, 25 and 15 degrees C (simulated field conditions), with 0.1 and 1.0% alcalase. At pH 6.0 and 15 degrees C, 0.1% alcalase reduced SA-11 titer by 0. 75 log in 24h, and by 1.25 log in 120h. At 25 degrees C and pH 6.0, 0.1% alcalase reduced the titer by 2.25 log after 24h, and by 2.75 log in 120h. At pH 6.0 and 15 degrees C, 1% alcalase reduced SA-11 titer by 1.50 log in 24h and by 1.75 log in 120h. At the same enzyme concentration and pH, but at 25 degrees C the titer was reduced by 2. 75 log in the first 24h and by 3.25 log at 120h. These results show that the alkaline protease alcalase is capable of inactivating SA-11 virus to a certain degree depending on conditions. Further definition of operating parameters, demonstration of inactivation under field conditions and analysis whether the demonstrated degree of inactivation would decrease calf morbidity and mortality remain to be explored at this time.

Mice immune responses to Brucella abortus heat shock proteins. Use of baculovirus recombinant-expressing whole insect cells, purified Brucella abortus recombinant proteins, and a vaccinia virus recombinant as immunogens.

Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.

Stimulation of avian respiratory phagocytes by Pasteurella multocida: effects of the route of exposure, bacterial dosage and strain, and the age of chickens.

The effects of the route of exposure (intratracheal [IT], drinking water [DW] and aerosolization [AS]), the age of chickens, and the dose of two vaccine strains of Pasteurella multocida (CU and M-9) on the number, phagocytic proportion and capacity of macrophages, granulocytes and lymphocytes (collectively avian respiratory phagocytes [ARPs]) were analyzed. Administration of P. multocida via the DW even at very high dose failed to stimulate ARPs. In contrast, administration of both strains of P. multocida either IT or by AS resulted in rapid and highly significant increases in the numbers of ARPs. 10 x 10(9) colony forming units (cfu) of aerosolized P. multocidaCU strain activated ARPs maximally in both young (3-6 weeks of age) and old (4-6 months of age) chickens. Old chickens responded in dose dependent manner to 20 x 10(9), 8 x 10(9), and 4 x 10(9) cfu of aerosolized P. multocida CU strain. Young chickens responded significantly only to 8 x 10(9) CU organisms. The M-9 and CU strains had limited differences in inducing migration of ARPs into the respiratory system of chickens or elevating phagocytic proportions and capacity of ARPs. The results indicate that the analyzed factors influence the response of ARPs to P. multocida to various degrees.

Selective humoral immune response of Balb/C mice to Brucella abortus proteins expressed by vaccinia virus recombinants.

Genes encoding Brucella abortus Cu/Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54. Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the beta-gal reporter gene but no B. abortus DNA were also developed. Recombinants were analyzed in Western blotting using a polyclonal B. abortus immune serum. vUBSOD expressed a protein of apparent molecular weight of 28 kDa, composed of the 20 kDa B. abortus Cu/Zn-SOD and a protein approximately 8 kDa encoded by a portion of the vaccinia virus TK gene. vSB54 expressed a 54 kDa protein corresponding to the 54 kDa HtrA homologue. Recombinants vUSV-1 and vSC11 did not express B. abortus proteins. Groups of mice were inoculated intraperitoneally with 10(7) TCID50 of 1 of the 4 different recombinant vaccinia viruses and 5 weeks later their sera were analyzed for antibodies against vaccinia virus and B. abortus proteins. Each group of mice responded with antibodies to vaccinia virus. Sera of vSB54-inoculated mice recognized the 54 kDa HtrA homologue. vUBSOD did not induce a humoral immune response. These results represent the first report on the expression of B. abortus proteins by vaccinia virus recombinants and the first demonstrated immune response against a B. abortus protein expressed by such a recombinant.

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge.

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates.

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses.

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.

In vitro cytotoxic activity of equine lymphocytes on equine herpesvirus-1 infected allogenic fibroblasts.

The objectives of this study were to: (1) develop a technique to analyze the in vitro cytotoxic activity of lymphocytes from adult horses against equine herpesvirus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); (2) evaluate the ability of a 72-h in vitro incubation with interleukin-2 (IL-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; (3) compare the cytotoxic activity of lymphocytes isolated from pregnant mares and non-pregnant mares against EHV-1 infected EDF; (4) ascertain if any correlations existed between the percent cytotoxicity and percentage of lymphocytes phenotypically identified by five different mouse-anti-equine monoclonal antibodies; and (5) determine if any correlation existed between virus-neutralizing antibody titers and the percent cytotoxicity. Results of the study indicate that in vitro cytotoxic activity of equine lymphocytes against EHV-1 infected allogenic fibroblasts can be measured with a standard 4-h 51Cr release assay. This activity was enhanced by an in vitro incubation with IL-2. The cytolytic activity of freshly isolated lymphocytes was greater for non-pregnant than pregnant mares. However, after IL-2 stimulation the cytolytic activity was greater for lymphocytes from pregnant mares. A positive correlation was not detected between the percentage of phenotypically identified cells and the percent cytotoxicity, although several negative correlations were present. This suggests that the cytotoxic activity was either not mediated by any of the phenotypically identified cell populations or that the activity was

Precipitating and agglutinating activity in duck anti-soluble protein immune sera.

Ducks were inoculated parenterally, up to 4 times, with bovine serum albumin (BSA), B gamma globulin (BGG), or horse gamma globulin (HGG). Their sera were tested in immunodiffusion tests for precipitins. Only BSA and BGG induced precipitins. Only 7 of 443 sera tested, obtained from 3 of 62 inoculated ducks, developed a precipitin line with homologous antigen. All 7 sera were obtained from the earliest bleeding (7 days) after inoculation. Sera were also tested for agglutinins in direct passive hemagglutination (DPHA) and direct red-cell-linked antigen tests (DRCLAT) and for nonagglutinating antibodies in indirect red-cell-linked antigen tests (IRCLAT). No duck had a passive-hemagglutination-demonstrable primary immune response. Demonstrable DPHA titers after subsequent inoculations were very low compared with responses to these antigens of other species noted by other workers. Duck immune response was greatly diversified: all inoculation regimens that induced agglutinins in some ducks left others completely unstimulated. Precipitins and agglutinins of the same ducks correlated well: those 7 sera that had precipitins also had the highest DPHA titers for the 3 donor ducks. However, hemagglutinating titer and presence of precipitins in sera of different ducks correlated poorly. Increasing the age of the ducks at the first inoculation from 6 to 10 weeks increased the number of responders and DPHA titers of their sera. Non-agglutinating antibodies were demonstrated in anti-BSA sera: in IRCLAT, the sera had titers 2-to-256-fold higher than the sera in DPHA or DRCLAT had. Duck immunoglobulins were deficient in those immunological reactions (precipitation and agglutination) that require functional bivalency.

Immune response of the duck to particulate (red blood cell) antigens.

Young ducklings were inoculated intravenously (IV) twice with either chicken or sheep red blood cells (CRBC, SRBC). Breeder ducks were inoculated IV twice with an increased dose of CRBC. Sera of these actively immunized ducks and sera of day-old ducklings from the CRBC-inoculated breeder ducks were tested for hemagglutinins in direct-hemagglutination (DHA) tests. The young ducklings and the breeders responded with very low DHA titers to CRBC and SRBC. Although ducklings inoculated with CRBC developed a true secondary response to a second CRBC inoculation, ducklings inoculated with SRBC failed to develop elevated titers after a second of SRBC inoculation. Half the breeders failed to develop higher titers to a second CRBC inoculation. Sera from the majority of day-old ducklings hatched from eggs laid after one inoculation of the breeders had no or only minimal DHA activity. Sera of ducklings hatched from eggs laid after the second inoculation had somewhat higher DHA titers, and in greater proportion. Still, many of those duckling sera had no or minimal DHA titers. Results with Sephadex G-200 fractions indicated that hemagglutinins after one inoculation resided primarily in duck immunoglobulin M (DIgM). Sera of ducks inoculated twice with CRBC had hemagglutinating activity in DIgG, too. Hemagglutinating activity was demonstrated in both 2-mercaptoethanol-sensitive (DIgM) and -resistant (DIgG) antibodies. The conspicuously low titers in all DHA-positive sera agree with results of previous studies, suggesting that DIg's are deficient in immunological reactions requiring functional bivalency.

Humoral immune response of the duck to duck hepatitis virus: virus-neutralizing vs. virus-precipitating antibodies.

Ducks were induced to develop high-level duck hepatitis virus (DHV)-neutralizing antibodies by inculation with a chicken-embryo-adapted DHV via subcutaneous, intramuscular, and intratracheal routes. Administration of the DHV orally in a gelatin capsule failed to stimulate immune response in the ducks. Contact controls of these ducks also remained negative for anti-DHV antibodies. These observations indicated that the DHV administered orally, in gelatin capsule, failed to infect the ducks. None of numerous duck anti-DHV immune sera, with virus-neutralizing activity in the range of 1.8 to 5.57 log10 median- embryo-infective-dose (EID50) neutralization index, developed precipitin lines against a variety of DHV preparations tested in low- and high-ionic-strength agar. The results suggest that the agar-gel immunodiffusion test is unsuitable for serologic testing of duck sera for anti-DHV antibody activity. Virus-neutralizing activity was revealed in both immunoglobulin M (IgM) and IgG classes of sera of actively immunized ducks. Immunodiffusion tests of Sephadex G-200 fractions of 1-day-old duckling sera with monospecific rabbit anti-duck IgM (DIgM) serum failed to detect DIgM. These results demonstrated that the IgM is not being transferred from the dam to the newly hatched ducklings. Seven- and 14-day-old ducks had DIgM in their sera. However, this IgM had no DHV-neutralizing activity, indicating that it was newly developed by the ducklings, which had no active DHV immune response, not having been exposed to DHV.

Immunoelectrophoresis of duck sera and immunoglobulins.

Duck whole sera (DWS) and immunoglobulins (DIg) were studied in immunoelectrophoresis (IE) and immunodiffusion (ID) tests by rabbit (R) anti-DWS and R anti-DIg sera, as well as by R antisera monospecific for DIgM and DIgG classes. Immunoelectrophoresis of adult DWS against R anti-DWS illustrated the presence of DIgM and two subclasses of DIgG. These observations were verified by the use of monospecific R anti-DIgM and R anti-DIgG sera. It appeared that DIgM is an electrophoretically heterogeneous protein with components that migrate more slowly than IgM of other species. The cathodal tip of the DIgM extended into the gamma2 migration zone almost as far as the DigG arc. Changes in the IE pattern of DWS from 1-to-14-day-old ducklings were observed that reflected a reduction in DIgG content from the age of 1 to 7 days followed by a gradual change toward the IE appearance of adult sera. Immunoelectrophoresis and ID failed to detect DIgM in sera of newly hatched ducklings. This class of DIg appeared in sera of 7-day-old duckling and seemed to be more concentrated in sera of 14-day-old ducklings.

Cellular defense of the avian respiratory system: dose-response relationship and duration of response in intratracheal stimulation of avian respiratory phagocytes by a Pasteurella multocida bacterin.

In experiments analyzing dose-response, intratracheal inoculation of chickens with 10(8) and 10(9) avirulent Pasteurella multocida organisms induced the migration within 24 hr of large numbers of respiratory lavage cells (RLC) with increased phagocytic proportions and phagocytic capacity. Doses from 10(4) to 10(7) organisms per bird resulted in elevated numbers of RLCs that were not significantly higher (P > 0.05) than values of uninoculated or mock-inoculated control chickens. When analyzing the duration of response, we found that inoculation with 10(9) organisms resulted in significantly higher (P < 0.05) numbers of RLCs for 63 to 89 hr without significant elevation in phagocytic proportion and capacity. The numbers of RLCs were elevated, although not significantly (P > 0.05), up to 11 days after inoculation. These results indicate that RLCs migrate to the respiratory tract only in response to a relatively high number of stimulating bacterial organisms and that the duration of response is relatively short. Although there were elevated numbers of RLCs beyond 89 hours after stimulation, the question remains as to whether these cells would enhance nonspecific defense of the respiratory system of chickens.

Cellular defense of the avian respiratory tract: paucity of free-residing macrophages in the normal chicken.

Avian respiratory macrophages (ARM) were obtained from lungs and air sacs of 122 White Plymouth Rock chickens, ranging from 376 to 3800 g in weight. Procedures involved lavaging through the surgically prepared trachea with either a 15-g cannula or French #8 pediatric urinary catheters. Factors, in different combinations, investigated for their effects on the ARM yield, were: lavage fluids (0.85% physiologic saline, 0.1 M phosphate-buffered saline, Ca-Mg-free Hanks' solution, Eagle's minimum essential medium); additives (10 U heparin/ml, 0.1% EDTA, 12 mM lidocaine); lavage repetitions (from 3 to 10); fluid temperature (room and 41 C); and lavage time (fluid retention up to 35 min). None of the lavage methods emerged clearly as the best, with phosphate-buffered saline and 0.85% physiologic saline alone as good as when combined with additives. Although 10 lavages yielded more ARM, it appeared that the majority of ARM washed off into the early lavages. Chickens from a line selected for large body size had more ARM than those from a line selected for small body weight. Regardless of genetic line, however, the chickens yielded a very low number of ARM compared with mammalian species of the same or smaller weight. Most of the birds yielded only 200,000 to 300,000 ARM, with minimum yields being less than 10,000, the maximum being 2 million ARM. Either these results point to a deficiency in the defense system of the chicken's respiratory tract against bacteria, mycoplasma, fungi, and viruses, or mechanisms other than macrophages are primary in resistance to pathogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular defense of the avian respiratory system. Influx of phagocytes: elicitation versus activation.

We studied various means of inducing avian phagocytes to migrate to the respiratory tract. No significant and consistent increases in the number of avian respiratory phagocytes (ARP) were elicited by intravenous inoculation with Escherichia coli lipopolysaccharide (LPS), Saccharomyces cerevisiae glucan (G), and Freund's incomplete adjuvant (FIA) in a water-in-oil-in-water emulsion; subcutaneous inoculation with the LPS-G-FIA homogenate; or aerosolized exposure to LPS-G-FIA, thioglycolate, and proteose-peptone. Intravenous inoculation with heat-killed Corynebacterium parvum resulted in a significant increase in the number of ARP by day 6 after inoculation; intratracheal inoculation of C. parvum effected a more rapid and higher level of phagocyte migration to the respiratory tract. Intratracheally administered E. coli induced significant migration of phagocytes to the respiratory system so that by 24 hours postinoculation, the group average number of ARP was about 50-100 times as high as the number in unstimulated control birds. None of the birds yielding high numbers of phagocytes from their respiratory tract had signs of respiratory disease.