High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus

Six-day-old turkeys were inoculated with turkey coronavirus (TCV) and an enteropathogenic Escherichia coli (EPEC) (isolate R98/5) that were isolated from poult enteritis and mortality syndrome (PEMS)-affected turkeys. Turkeys inoculated with only R98/5 did not develop clinically apparent disease, and only mild disease and moderate growth depression were observed in turkeys inoculated with only TCV. Turkeys dually inoculated with TCV and R98/5 developed severe enteritis with high mortality (38/48, 79%) and marked growth depression. R98/5 infection resulted in attaching/effacing (AE) intestinal lesions characteristic of EPEC: adherence of bacterial microcolonies to intestinal epithelium with degeneration and necrosis of epithelium at sites of bacterial attachment. AE lesions were more extensive and were detected for a prolonged duration in dually inoculated turkeys compared with turkeys inoculated with only R98/5. An apparent synergistic effect in dually inoculated turkeys was indicated by increased mortality, enhanced growth depression, and enhanced AE lesion development. The results suggest that TCV promoted intestinal colonization by R98/5; however, R98/5 did not appear to alter TCV infection. The present study provides a possible etiologic explanation for PEMS.     

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.     

Experimental inoculation of wild turkeys (Meleagris gallopavo) with Mycobacterium bovis

Although avian species are known to be susceptible to infection with Mycobacterium spp. organisms, much remains unknown about the susceptibility of birds to infection with M. bovis. The objective of this current study was to determine if wild turkeys (Meleagris gallopavo) can be infected with M. bovis when inoculated by the oral or intratracheal route. Six turkeys were orally inoculated and another six were inoculated via the trachea with a high dose of M. bovis, 1 x 10(5) CFU/ml. Six turkeys were sham-inoculated controls. Two turkeys from each treatment group were sacrificed on days 30, 60, and 90 postinoculation. There were no gross or microscopic lesions consistent with mycobacteriosis in the 23 inoculated turkeys over the 90-day duration of this study. Fecal cultures were also consistently negative for M. bovis when sampled before inoculation and on days 1, 30, and 60 postinoculation. Two intratracheally inoculated turkeys were positive for M. bovis in visceral tissues at 30 days postinoculation. However, this finding was only indicative of passive persistence of mycobacteria in the tissues and not of infection, as there were no attendant lesions or clinical compromise to support infection. Thus, it can be concluded that young wild turkeys are resistant to infection with M. bovis and, therefore, pose minimal threat as reservoir or spillover hosts for this organism.     

Pathogenicity of turkey coronavirus in turkeys and chickens

We designed this study to compare the replication potential of turkey coronavirus (TCV) and its effect in chickens and turkeys and to study the effect of singleand combined infection of turkey poults with TCV and astrovirus. We studied the pathogenicity of TCV in experimentally inoculated turkey poults and chickens by observing the dinical signs and gross lesions. Two trials were conducted with 1-day-old and 4-wk-old specific-pathogen-free turkey poults and chickens. One-day-old turkey poults developed diarrhea at 48 hr postinoculation. Poults euthanatized at 3, 5, and 7 days postinoculation had flaccid, pale, and thin-walled intestines with watery contents. The 4-wk-old turkeys had no clinical signs or gross lesions. One-day-old and 4-wk-old chicks developed no clinical signs or gross lesions although the TCV was detected in gut contents of the birds throughout the experimental period (14 days). In another experiment, mean plasma D-xylose concentrations in 3-day-old turkey poults inoculated with TCV, turkey astrovirus, or a combination of both viruses were significantly lower than in the uninoculated controls.     

Endobronchial inoculation of various doses of Haemophilus (Actinobacillus) pleuropneumoniae in pigs

Twelve-week-old specific-pathogen-free pigs were inoculated deep in the bronchi with Haemophilus (Actinobacillus) pleuropneumoniae strain 13261 in doses ranging from 8 x 10(1) to 9 x 10(7) colony-forming units (CFU). Pigs that survived infection were euthanatized and examined 48 hours after inoculation. The relationship between dose and severity of disease was evaluated clinically and the weight of pneumonic lesions was compared. The relationship between infection dose and weight of pneumonic lesions proved to be unimodal and not linear. Inoculation of 10(4) CFU of strain 13261 resulted in severe pneumonic lesions and mortality of 29%. In contrast, death was not observed after inoculation with 10(6) CFU of strain 13261 and pneumonic lesions were less severe (P less than 0.05). An infective dose of 10(3) CFU induced pneumonic lesions that tended (not statistically significant) to be less severe than those induced by a dose of 10(4) CFU. The peak fever response in all infected pigs was observed from 6 to 12 hours after inoculation. Leukocytosis developed within 12 hours after inoculation, because of an increase of neutrophilic granulocytes. Thereafter, WBC count decreased owing to lymphopenia. Serum iron concentration decreased 80% after inoculation, and zinc concentration decreased 54%.     

Experimental and serologic observations on avian pneumovirus (APV/turkey/Colorado/97) infection in turkeys

An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States.     

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.     

Shedding of Mycobacterium bovis in the nasal mucus of cattle infected experimentally with tuberculosis by the intranasal and intratracheal routes

Four groups of six calves were infected experimentally with either a low dose of approximately 10(4) colony-forming units (cfu) or a high dose of approximately 10(6) cfu of Mycobacterium bovis. Each dose was delivered by the intranasal and intratracheal routes. More severe disease was observed in the groups inoculated with the high dose. Visible lesions were identified in 21 of the 24 animals, all of which also gave positive skin tests and interferon-gamma (IFN-gamma) responses. Nasal shedding was detected in 15 of the 24 animals and the frequency of shedding was influenced by both the route and the dose of infection; no shedding was observed in the group infected intratracheally with the low dose. Two of the 15 confirmed shedders had no visible lesions at postmortem examination; both of these calves gave IFN-gamma responses but only one was skin test positive.     

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.     

Infections with enteropathogenic Escherichia coli (EPEC) in cattle. 1. Comparison of different animal models and a cell culture system for the establishment of a detection system for "attaching and effacing" (AE) lesions]

In several animals species, enteropathogenic Escherichia coli (EPEC) were described as agents causing diarrhea. The histopathogenic pattern of EPEC is due to a typical adherence to enterocytes, called "attaching and effacing" (AE). This lesions are characterized by the formation of pedestals, cups and a marked loss of microvilli on enterocytes. In view of using an "in vitro system" (HeLa-cell culture) to test the adherence of EPEC, we first tested bovine EPEC in several laboratory animals. Various strains of mice, one day chicks (peroral) and a three day old calf (ligated intestinal loops) were inoculated with a bovine pathogenic EPEC (S 102-9). The adherence of EPEC "in vivo" was histologically, electron microscopically and bacteriologically investigated and compared to adherence to HeLa cell cultures. AE-lesions were found on calf enterocytes as well as on HeLa-cells, no lesions were seen in mice and chicks. The ligated intestinal loop test seems to be a useful model to compare "in vivo" to "in vitro" adherence.     

Pathogenesis of Newcastle disease in commercial and specific pathogen-free turkeys experimentally infected with isolates of different virulence

The pathogenesis of five different Newcastle disease virus (NDV) isolates representing all pathotypes was examined in commercial and specific pathogen-free (SPF) turkeys. Experimentally-infected birds were monitored clinically and euthanatized, with subsequent tissue collection, for examination by histopathology, by immunohistochemistry for the presence of NDV nucleoprotein, and by in situ hybridization for the presence of replicating virus. Clinically, the lentogenic pathotype did not cause overt clinical signs in either commercial or SPF turkeys. Mesogenic viruses caused depression in some birds. Turkeys infected with velogenic neurotropic and velogenic viscerotropic isolates showed severe depression, and neurologic signs. Histologic appearances for all strains had many similarities to lesions observed in chickens inoculated with the various isolates; that is, lesions were present predominantly in lymphoid, intestinal, and central nervous tissues. However, in general, disease among turkeys was less severe than in chickens, and turkeys could be considered a subclinical carrier for some of the isolates.     

Immunofluorescent studies on localization of secretory immunoglobulins in the intestines of turkeys recovered from turkey coronaviral enteritis

The present study concerns the production of specific secretory antibodies in turkeys orally inoculated with turkey coronavirus (TCV). Tissue localization of secretory antibodies in the intestines of recovered birds 4 and 5 months later was demonstrated by immunofluorescence technique. Intestinal sections were stained by sandwich method, using concentrated TCV antigen and anti-TCV conjugate. It was suggested that local synthesis of antibody might be responsible for life-long immunity seen in these birds after recovery from infection.     

Colonisation of conventional weaned pigs by enteropathogenic Escherichia coli (EPEC) and its hazard potential for human health

Enteropathogenic Escherichia coli (EPEC) bacteria frequently cause severe enteric diseases primarily in children and in young rabbits. Their pathogenicity for pigs has been tested by oral infection of colostrum-deprived newborn, and of severely immunosuppressed weaned pigs, but colonisation of conventional weaned pigs by porcine EPEC has not been experimentally studied. EPEC show similarities to enterohaemorrhagic E. coli (EHEC) additionally carrying shiga toxin genes integrated into the chromosome by lambdoid phages. We have demonstrated earlier that the porcine EPEC prototype strain P86-1390 (O45) could be transduced in vivo (in ligated loops of weaned pigs), by Stx2 phage derived from a human EHEC. Thus, the ability of this porcine EPEC strain to colonise conventional weaned pigs under farming conditions became a question of relevance to human health. To clarify this question, four intragastric infection experiments were performed on a total of 95 conventional weaned pigs. The EPEC P86-1390 and other well-characterised porcine EPEC strains were applied to 54 pigs, leaving 41 weaned pigs as negative controls. In three experiments moderate predispositions were applied: coinfections with enterotoxigenic E. coli (ETEC) or with low-virulence TGE coronavirus, application of fumonisin B1 with a normal therapeutic dose of dexamethasone, and the increase of soybean protein concentration in the feed. A total of 41 weaned pigs served as negative controls inoculated with a commensal porcine E. coli. Housing conditions simulated the farm environment. As an overall result, ileal segments of 18.5% of infected pigs were shown to be colonised by EPEC, while no EPEC were detected in the ilea of controls. Among predisposing factors occurring on farms, feed protein content increased by 20% (26.3% crude protein, provided by 48% soybean meal) seemed to enhance EPEC colonisation and resulted in the mobilisation of spontaneous latent EPEC/ETEC infection. The results indicate that under normal farm conditions porcine EPEC may colonise conventional weaned pigs by inducing ileal attaching effacing (AE) lesions with reasonable frequency, without clinical signs. The results also suggest that conventional weaned pigs may represent undetected reservoirs of porcine EPEC, potentially giving rise to the emergence of new types of EHEC due to natural transduction by Stx phages.     

Transmission and immunopathology of the avian influenza virus A/Anhui/1/2013 (H7N9) human isolate in three commonly commercialized avian species

H7N9 virus infection is a global concern, given that it can cause severe infection and mortality in humans. However, the understanding of H7N9 epidemiology, animal reservoir species and zoonotic risk remains limited. This work evaluates the pathogenicity, transmissibility and local innate immune response of three avian species harbouring different respiratory distribution of α2,6 and α2,3 SA receptors. Muscovy ducks, European quails and SPF chickens were intranasally inoculated with 10⁵ embryo infectious dose (EID)₅₀ of the human H7N9 (A/Anhui/1/2013) influenza isolate. None of the avian species showed clinical signs or macroscopic lesions, and only mild microscopic lesions were observed in the upper respiratory tract of quail and chickens. Quail presented more severe histopathologic lesions and avian influenza virus (AIV) positivity by immunohistochemistry (IHC), which correlated with higher IL‐6 responses. In contrast, Muscovy ducks were resistant to disease and presented higher IFNα and TLR7 response. In all species, viral shedding was higher in the respiratory than in the digestive tract. Higher viral shedding was observed in quail, followed by chicken and ducks, which presented similar viral titres. Efficient transmission was observed in all contact quail and half of the Muscovy ducks, while no transmission was observed between chicken. All avian species showed viral shedding in drinking water throughout infection.     

Interaction of reovirus and Cryptosporidium baileyi in experimentally infected chickens

No clinical signs, gross lesions, or increased mortality were observed in specific-pathogen-free chickens orally inoculated at 5 days of age with Cryptosporidium baileyi, reovirus 2035, reovirus 2408, or combinations of these agents. Weight gain of chickens inoculated with only reovirus 2408 was depressed 0-8 days postinoculation (PI) (P less than 0.01) but not for the 21-day period PI. Weight gain of chickens inoculated with only reovirus 2035 was not affected. Cryptosporidium baileyi infection significantly depressed weight gain 8-14 days PI but not for the entire 21-day period PI. Weight gain of chickens infected with both C. baileyi and reovirus 2035 was significantly depressed 0-14 days PI and for the entire 21-day period PI. Dual infection with C. baileyi and either reovirus appeared to promote shedding of both agents. Cryptosporidia were found principally in the rectum 2-10 days PI and in the bursa of Fabricius 6-10 days PI. Reovirus infection did not cause any microscopic lesions and did not modify lesions caused by C. baileyi 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.     

Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specific-pathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.     

Shedding patterns of dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis

Although substantial fecal shedding is expected to start years after initial infection with Mycobacterium avium subspecies paratuberculosis (MAP), the potential for shedding by calves and therefore calf-to-calf transmission is underestimated in current Johne’s disease (JD) control programs. Shedding patterns were determined in this study in experimentally infected calves. Fifty calves were challenged at 2 weeks or at 3, 6, 9 or 12 months of age (6 calves served as a control group). In each age group, 5 calves were inoculated with a low and 5 with a high dose of MAP. Fecal culture was performed monthly until necropsy at 17 months of age. Overall, 61% of inoculated calves, representing all age and dose groups, shed MAP in their feces at least once during the follow-up period. Although most calves shed sporadically, 4 calves in the 2-week and 3-month high dose groups shed at every sampling. In general, shedding peaked 2 months after inoculation. Calves inoculated at 2 weeks or 3 months with a high dose of MAP shed more frequently than those inoculated with a low dose. Calves shedding frequently had more culture-positive tissue locations and more severe gross and histological lesions at necropsy. In conclusion, calves inoculated up to 1 year of age shed MAP in their feces shortly after inoculation. Consequently, there is potential for MAP transfer between calves (especially if they are group housed) and therefore, JD control programs should consider young calves as a source of infection.     

Specific mucosal IgA immunity in turkey poults infected with turkey coronavirus

The objective of this study was to elucidate the kinetics and magnitudes of specific IgA antibody responses in intestines of turkey poults infected with turkey coronavirus (TCV). Turkey poults were orally inoculated with TCV at 10 days of age. Intestinal segment cultures were administered for duodenum, jejunum, and ileum and the IgA antibody responses were analyzed at 1, 2, 3, 4, 6, or 9 weeks post-infection (PI) in two different experiments. The kinetics of virus-specific IgA antibody responses in duodenum, jejunum, and ileum were similar: gradually increased from 1 week PI, reached the peak at 3 or 4 weeks PI, and declined afterward. The virus-specific IgA antibody responses in duodenum, jejunum, and ileum showed negative correlation with duration of TCV antigen in the corresponding locations of intestine with Spearman's correlation coefficient of -0.85 (p=0.034), -0.74 (p=0.096), and -0.75 (p=0.084), respectively. Moreover, the virus-specific IgA antibody responses in serum were positively correlated with that of duodenum (coefficient=0.829, p=0.042), jejunum (coefficient=0.829, p=0.042), and ileum (coefficient=0.771, p=0.072) segment cultures, suggesting that the induction of specific IgA response in serum was predictive of an IgA response in intestine. The results indicate that intestinal mucosal IgA antibodies to TCV are elicited in turkeys following infection with TCV. The local mucosal antibodies may provide protective immunity for infected turkeys to recover from TCV infection.