Mechanical transmission of turkey coronavirus by domestic houseflies (Musca domestica Linnaeaus)

Domestic houseflies (Musca domestica Linnaeaus) were examined for their ability to harbor and transmit turkey coronavirus (TCV). Laboratory-reared flies were experimentally exposed to TCV by allowing flies to imbibe an inoculum comprised of turkey embryo-propagated virus (NC95 strain). TCV was detected in dissected crops from exposed flies for up to 9 hr postexposure; no virus was detected in crops of sham-exposed flies. TCV was not detected in dissected intestinal tissues collected from exposed or sham-exposed flies at any time postexposure. The potential of the housefly to directly transmit TCV to live turkey poults was examined by placing 7-day-old turkey poults in contact with TCV-exposed houseflies 3 hr after flies consumed TCV inoculum. TCV infection was detected in turkeys placed in contact with TCV-exposed flies at densities as low as one fly/bird (TCV antigens detected at 3 days post fly contact in tissues of 3/12 turkeys); however, increased rates of infection were observed with higher fly densities (TCV antigens detected in 9/12 turkeys after contact with 10 flies/bird). This study demonstrates the potential of the housefly to serve as a mechanical vector of TCV.     

Hemorrhagic enteritis: virus distribution and sequential development of antibody in turkeys

Turkeys poults were inoculated intraperitoneally with hemorrhagic enteritis virus (HEV) at 4-1/2 weeks of age. Antibody response and sequential development of viral antigen in various tissues were monitored. An enzyme-linked immunosorbent assay (ELISA) was developed to study antibody production, and immunoperoxidase staining was used to determined sites of localization of the viral antigens in tissues. Results of ELISA and immunodiffusion tests were compared. ELISA detected antibody from day 3 post-infection (p.i.), and gel diffusion detected antibody from day 5 p.i. Peak ELISA antibody titer appeared from day 14 p.i. HEV antigen was detected from 2-6 days p.i. in the spleen, liver, intestine, kidney, and bone marrow; peak titers in the spleen were on day 3 p.i. Virus was not detected after day 6 p.i.     

Delayed replication of Marek's disease virus following in ovo inoculation during late stages of embryonal development

Several oncogenic and non-oncogenic isolates of Marek's disease virus (MDV) were inoculated into embryonated eggs on embryonation day (ED) 16 to 18, and embryos or chicks hatching from inoculated eggs were examined for infectious virus and viral internal antigen (VIA) in lymphoid organs. There was no evidence of extensive replication of MDV in any of the embryonic tissues examined. Levels of VIA peaked 4-5 days after chicks hatched. This indicated that MDV remained inactive during embryonation and did not initiate pathogenic events until chicks hatched. Because HVT replicated rapidly in the embryo but MDV did not, in ovo inoculation of HVT simultaneously with oncogenic MDV or several days after MDV resulted in significant protection (P less than 0.025) of hatched chicks against Marek's disease (MD). Little protection was obtained if HVT was given simultaneously with MDV or after MDV to chicks already hatched. The relative susceptibility of the embryo to extensive replication of the vaccine virus but not the challenge virus apparently accounted for protection against MD in chicks hatching from dually infected eggs.     

Pathogenesis of bovine viral diarrhoea-mucosal disease (BVD-MD) virus infection in tracheal organ cultures

Bovine foetal tracheal organ cultures were infected with two strains of BVD-MD virus and observed for 35 days. The effect of the virus was assessed by observing ciliary activity in gross tissue explants and histological changes in haematoxylin and eosin stained sections. Decrease in ciliary activity and mild epithelial degeneration were first observed at 4 days post infection (p.i.), the epithelial degeneration progressing to complete destruction by day 35 p.i. Viral titres in extracellular fluids rose sharply from day 4 p.i., reached peak levels (10⁵ TCID₅₀/ml) between 15 and 18 days p.i., and eventually declined but persisted at lower titres up to day 35 p.i., when observations were terminated.     

鸭源鸡杆菌对SPF雏鸡的感染特性研究

为研究鸭源鸡杆菌的流行病学特性,本研究采用从自然病例分离得到的鸭源鸡杆菌人工感染4日龄SPF雏鸡,通过形态学、PCR、荧光定量PCR和组织学等方法分别对感染后SPF雏鸡的组织脏器进行了细菌分离、检测和鉴定,用ELISA对其血清抗体水平进行检测.结果表明,人工感染鸡无临床症状,组织学检查感染鸡的肝脏、气管和肺脏组织均有损伤.人工感染后第3d和同居感染第2d鸡体内可分离出鸭源鸡杆菌,人工感染后第96d仍能够分离到鸡杆菌.ELISA方法证实人工感染后47 d和同居后32 d出现抗体高峰,持续2～3周,人工感染组和同居组抗体水平均高于对照组(p＜0.05);荧光定量PCR检测病料结果显示人工感染组和同居组的气管组织中细菌含量最高.本研究表明雏鸡在4日龄即可感染鸭源鸡杆菌,并能通过同居传播,长期带菌、排菌;感染后抗体产生缓慢、持续期短.本研究为鸭源鸡杆菌的流行病学、致病机理研究及防治措施的制定等提供了参考依据.     

Oral challenge of turkey poults with Mycoplasma iowae

Day-old poults were inoculated orally each day for 7 days with 0.2 ml of Mycoplasma iowae, strain D112, 10(8) colony-forming units/ml. Cloacal swabs were taken from each poult during the inoculation period and at selected intervals until 21 days after the last inoculation. Most poults shed mycoplasmas persistently after inoculation. Cloacal swabs from eight out of ten poults were positive at 21 days after the last inoculation. Feces of poults in the infected group were normal, and there was no significant rise in cloacal temperature. At necropsy, mycoplasmas were recovered from tissues of the respiratory tract, gastrointestinal tract, spleen, and kidney. In the gastrointestinal tract, the most frequent recoveries were from the wall of the distal portion of the small intestine, cecum, and large intestine. Recovery of M. iowae from these organs and tissues indicated infection following oral challenge.     

The location of primary replication of the herpesvirus of bovine malignant catarrhal fever in rabbits

The herpesvirus of malignant catarrhal fever (MCFV) was isolated from the spleen of $\text{2}\text{3}$ rabbits 4 days after the intravenous inoculation of infectious lymph node suspension, while no virus could be isolated at 2 and 6 days post inoculation (p.i.). Indirect immunofluorescence identified the antigen-positive cells at 4 days p.i. as medium sized lymphocytes lying in the venous sinuses of the spleen, lower numbers of fluorescing cells being seen in the paracortical areas of lymph nodes and in the thymus. Scanty fluorescence, without isolation of virus, was evident in the spleen at 6 days p.i. but by day 8 p.i. virus could be isolated irregularly from spleen and lymph nodes and at 12 days p.i. was found in all lymphoid tissues in those rabbits with lymphadenitis and pyrexia. The primary replication in the spleen at 4 days is compared with other herpes induced lymphoproliferative disorders.     

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.     

Experimental bovine coronavirus in turkey poults and young chickens

The DB2 calf strain of bovine coronavirus (BCV) was used to inoculate 1-day-old specific-pathogen-free (SPF) turkey poults in three trials. In all trials, the birds developed clinical signs of enteritis at 48-72 hr postinoculation. Birds euthanatized at 3, 5, and 7 days postinoculation (DPI) had flaccid, pale intestines with watery contents, and the ceca were markedly enlarged with frothy contents. Coronavirus particles were detected by immune electron microscopy with BCV antibodies from the intestinal contents of birds killed at 3, 5, 7, and 12 DPI. Body weights of inoculated poults killed at 3, 5, and 7 DPI were significantly reduced as compared with controls. Hemagglutinating antibodies were detected in sera of convalescent birds at 12 DPI. However, experimental inoculation of 1-day-old SPF chicks in two trials with the same virus resulted in no clinical signs or macroscopic or microscopic lesions. No coronaviruses were detected from intestinal contents, and there were no significant differences in body weights of inoculated and noninoculated control chicks.     

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.     

Observations on the pathogenicity of Alcaligenes faecalis in chickens

A series of trials was conducted in which specific-pathogen-free (SPF) leghorn chicks were exposed to various isolates of Alcaligenes faecalis. Chicks were inoculated with A. faecalis alone or in combination with Newcastle disease/infectious bronchitis (Nc/Br) vaccine, laryngotracheitis vaccine, infectious bursal disease virus, or Mycoplasma gallisepticum. The response was evaluated by morbidity, mortality, airsacculitis, reisolation of A. faecalis, and histopathological lesions of tracheas. Although A. faecalis was recovered up to 42 days postinoculation in some cases, no clinical signs were directly attributed to simple A. faecalis infection. None of the other agents significantly increased the severity of A. faecalis signs or lesions, except that A. faecalis-infected chicks that were given Nc/Br vaccine had prolonged microscopic tracheal lesions. In another trial, the effects of A. faecalis in young SPF leghorns, non-SPF broilers, and turkeys were compared. Broiler-type chicks were more susceptible than leghorns and less susceptible than poults. Consequently, the use of leghorns as a model for studying this infection is questioned.     

The pathology and pathogenesis of Salmonella stanley infection in experimental chicks

Experimental infection with Salmonella stanley was produced by oral, intravenous and intramuscular routes in day-old chicks. The earliest evidence of the presence of the organisms was in duodenal mucosa six hours after oral infection. Following oral infection the organisms were detected in the duodenum from six hours to five days, in the caecum from 12 hours to nine days, liver, spleen and blood from 24 hours to seven days. The resistance to infection was found to be significant after 10 days old, but not up to six days old. The work confirmed that the survival time of birds given S stanley by the intravenous or intramuscular routes was inversely proportional to the dose up to a maximum beyond which the survival time was not further decreased by dose increase. The presence of S stanley in tissues and blood was detected by isolation and by the fluorescent antibody technique.     

A comparison of the virulence of three strains of infectious bovine rhinotracheitis virus

The virulence of three strains of infectious bovine rhinotracheitis (IBR) virus was compared in six-month-old Ayrshire-cross calves. The strains were an isolate from a recent severe outbreak of IBR in Scotland (Strichen strain), the prototype British strain (Oxford strain) and a North American isolate (Colorado strain). The Colorado and Strichen strains produced the characteristic clinical signs and pathological lesions of severe IBR three to four days post infection (p.i.). The Strichen strain was slightly more virulent, possibly as a result of its having been passaged fewer times in tissue culture. In contrast, the Oxford strain produced a mild clinical response with minimal pathological lesions. Virus was recovered from nasal swabs for a longer period from the calves infected with the Strichen strain (up to 13 days p.i.) and Colorado strain (up to 12 days p.i.) than from the animals infected with the Oxford strain (up to 10 days p.i.). These findings support the suggestion that the recent epidemic of severe IBR in Britain had resulted from the importation of a “new” strain of virus.     

Demonstration in live chickens of the carrier state in infectious laryngotracheitis

Two separate groups of nine-week-old specific pathogen free cockerels maintained in isolation were infected with a field strain of infectious laryngotracheitis (ILT) virus, either by intratracheal or combined intranasal and supraconjunctival inoculation. Birds were monitored for virus shedding from five sites on alternate days during the acute phase and three times weekly until week 17. They were then treated with cyclophosphamide on three consecutive days and thereafter swabbed daily. During the acute phase clinical signs were observed and virus was recovered from ocular and nasal sites for up to six to eight days. Initially after the acute phase no virus could be detected. However, from the seventh week after infection intermittent, apparently spontaneous shedding was detected in four of five birds in each group. There was no clear effect of cyclophosphamide treatment on re-excretion patterns, possibly because of the high levels of virus shedding already occurring. Thus, a carrier state for ILT virus has been demonstrated experimentally in live clinically recovered birds.     

An enzyme-linked immunosorbent assay for detection of Bordetella avium infection in turkey flocks: sensitivity, specificity, and reproducibility.

An enzyme-linked immunosorbent assay (ELISA) for detection of Bordetella avium infection in turkey poults was developed. One-week-old poults challenged intratracheally with 10(12) colony-forming units of B. avium had detectable titers (greater than or equal to 11), with an average of 13.6% positive samples when the birds were 6 to 11 weeks old. The method was sensitive enough to detect maternal antibodies to B. avium in poults up to 3 weeks of age. The same poults challenged at 1 week of age had 100% tracheal infection up to 3 weeks of age, which dropped to 0% by 6 weeks. The method resulted in no false-positive samples (titer = 0) from birds not infected with B. avium and tested weekly between 4 and 11 weeks of age. Antibodies in turkey flocks infected with Newcastle disease virus, hemorrhagic enteritis virus, and Mycoplasma meleagridis, and birds infected with Escherichia coli had no apparent cross-reactivity to the B. avium antigens used in the ELISA. The percentages of B. avium-positive serum samples collected from different turkey flocks did not significantly differ (P greater than 0.05) when samples were tested by the developed ELISA at different times, an indication of the reproducibility of the method.     

E.tenella初次感染雏鸡外周血T淋巴细胞亚群及增殖能力变化的动态研究

应用流式细胞术（ FCM）和MTS比色法检测雏鸡初次感染柔嫩艾美尔球虫（ E． tenella）后外周血中CD3＋CD4＋、CD3＋CD8＋T淋巴细胞及其增殖能力的动态变化。数据显示,E． tenella初次感染雏鸡后7～20 d CD3＋CD4＋、CD3＋CD8＋T 淋巴细胞比例明显高于对照组雏鸡（ P＜0．05和P＜0．01）,并且均于12 d时达到峰值。初次感染后7～20 d雏鸡外周血中CD4＋／CD8＋T淋巴细胞亚群的比值明显升高,于感染后16 d时达到1．78。 E． tenella初次感染雏鸡后12～20 d外周血淋巴细胞增殖能力明显高于对照组雏鸡（ P＜0．05）,并于16 d达到峰值。试验表明E． tenella初次感染雏鸡能激活淋巴细胞产生增殖反应,CD3＋CD4＋、CD3＋CD8＋T淋巴细胞在抵抗E． tenella初次感染过程中有重要作用。     

A novel "small round virus" inducing poult enteritis and mortality syndrome and associated immune alterations

The role of a novel "small round virus" (SRV) isolated from poult enteritis and mortality syndrome (PEMS) cases in inducing PEMS and associated immune alterations was examined in this study. Specific-pathogen-free and conventional poults were orally challenged with SRV and/or turkey coronavirus and monitored for clinical signs. Intestines, thymus, bursa, and spleens were examined for SRV antigen at various days postinoculation (DPI). Peripheral blood lymphocytes (PBLs), thymocytes, and splenic lymphocytes from inoculated poults or lymphocytes isolated from healthy poults after incubation with SRV in vitro were examined for lymphoproliferative potential against concanavalin A (Con A). The incidence of lymphocyte subpopulations in the peripheral blood and thymic lymphocytes of SRV-challenged poults was examined by flow cytometry. The results of these studies showed that the SRV challenge induced diarrhea, growth suppression, and atrophy of thymus and bursa resembling those of PEMS in field and/or experimental infections. The SRV antigen was detected in intestinal tissues soon after infection (i.e., at 2 and 4 DPI), whereas lymphoid tissues such as thymus, bursa, and spleen were positive for SRV antigen starting at 4 DPI until 8 DPI, suggesting virus translocation to lymphoid organs. The responsiveness of PBLs to Con A at 2 DPI was significantly reduced in all virus challenge groups (e.g., 28% and 22% in the SRV-alone group in studies 1 and 2, respectively) below the uninfected group. However, this suppressed response was no longer evident in the SRV group by 7 DPI. The SRV incubation with normal thymocytes and splenocytes in vitro resulted in significantly reduced lymphoproliferative response against Con A (41.2% and 10.49% reductions at 1:50 SRV dilution vs. controls in thymocytes and splenocytes, respectively). Flow cytometry analysis revealed a sudden decline at 2 DPI in the numbers of CD4- CD8+ lymphocyte subset in PBLs of SRV-infected poults. However, by 8 DPI, SRV-challenged poults had relatively higher CD4- CD8+ lymphocytes in PBLs. On the contrary, thymocytes had higher percentages of CD4- CD8+ lymphocytes at 2 and 4 DPI and reached comparable levels at 8 DPI in controls and SRV-infected poults. No differences were observed in CD4+ CD8- lymphocyte numbers in controls vs. SRV-infected poults. The findings of these studies imply that SRV may be a promising primary etiologic agent of PEMS. Furthermore, the SRV infection may compromise the lymphocyte-mediated immune defenses by reducing lymphoproliferation and the CD4- CD8+ (presumably T-cytotoxic cells) lymphocytes during the acute stage of SRV infection.     

Hemorrhagic enteritis of turkeys: influence of maternal antibody and age at exposure

The effect of maternal antibody (MAB) to hemorrhagic enteritis (HE) on the response of turkeys to infection with virulent and avirulent strains of HE virus (HEV) was examined. The influence of age at exposure and treatment with HEV antibody on development of clinical HE also was studied. MAB protected poults from clinical HE for up to 6 weeks of age. MAB also interfered with vaccination against the disease for at least 5 weeks after hatching, as indicated by absence of HEV antigen in spleens and by poor seroconversion at 6 days and at 3 weeks post-vaccination, respectively. The incidence of clinical HE in MAB-negative poults was significantly higher in poults inoculated with virus at 15 days of age or older than in poults inoculated at 1-13 days of age. Further, MAB-negative poults embryonally inoculated with virulent or avirulent strains of HEV did not develop disease; these poults developed antibody and resisted challenge with virulent virus at 6 weeks of age. Poults treated with HE antibody within 1 hour of challenge or at 1, 3, or 5 weeks before challenge with virulent virus were protected against lesions and mortality induced by HEV. These results suggest that MAB may influence susceptibility of turkeys to infection with HEV for at least 5 to 6 weeks after hatching, unlike the case with most other viral infections of poultry. The results confirm that early age resistance to clinical HE is independent of MAB and suggest that such resistance persists for up to 13 days of age. The data also suggest that turkeys lacking MAB can be immunized against HE by embryo vaccination.