Adherence of Ornithobacterium rhinotracheale to chicken embryo lung cells as a pathogenic mechanism

Ornithobacterium rhinotracheale is a bacterium that causes respiratory disease in birds and it has been isolated in countries with a large poultry production, including Mexico. The pathogenicity mechanisms of this bacterium have not been completely elucidated yet. The capacity of the bacterium to adhere to epithelial cells of chicken in vitro has been evidenced, and since this bacterium has been isolated from the lungs and air sacs of several avian species, the aim of this study was to determine if this bacterium can adhere to chicken lung cells. We used five O. rhinotracheale reference serovars (A–E) that were in contact with primary lung cells cultured from a 19-day-old chicken embryo. O. rhinotracheale adherence was evaluated through optical and transmission electron microscopies. The results revealed that O. rhinotracheale is capable of adhering to chicken embryo lung cells within 3?h of incubation with a diffuse adherence pattern. The adherence percentages of the chicken embryo lung cells were 51–96% according to the serovar of the bacterium. Relative adherence was from 4 to 8 bacteria per cell. Transmission electron microscope data revealed intracellular bacteria inside a vacuole in less than 3?h of incubation.     

Efficacy of gamithromycin against Ornithobacterium rhinotracheale in turkey poults pre-infected with avian metapneumovirus

Ornithobacterium rhinotracheale is an avian respiratory pathogen that affects turkeys. The objective of this study was to evaluate the clinical efficacy of gamithromycin (GAM) against O. rhinotracheale in turkeys. The birds were inoculated oculonasally with 10⁸ colony-forming units (cfu) of O. rhinotracheale, preceded by infection with avian metapneumovirus. In addition to a negative (CONTR?) and a positive control group (CONTR+) there were two treated groups administered GAM (6?mg/kg) either subcutaneously (GAM SC) or orally (GAM PO) by administration as a single bolus at one-day post-bacterial infection (p.b.i.). From the start of the avian metapneumovirus infection until the end of the experiment, the turkeys were examined clinically and scored daily. In addition, tracheal swabs were collected at several days p.b.i. Necropsy was performed at 4, 8 and 12 days p.b.i. to evaluate the presence of gross lesions, and to collect trachea and lung tissue samples and air sac swabs for O. rhinotracheale quantification. The clinical score of the GAM SC group showed slightly lower values and birds recovered earlier than those in the GAM PO and CONTR+ groups. O. rhinotracheale cfus were significantly reduced in tracheal swabs of the SC group between 2 and 4 days p.b.i. At necropsy, CONTR+ showed higher O. rhinotracheale cfu in lung tissues compared to the treated groups. Moreover, at 8 days p.b.i. only the lung samples of CONTR+ were positive. In conclusion, the efficacy of GAM against O. rhinotracheale was demonstrated, especially in the lung tissue. However, the PO bolus administration of the commercially available product was not as efficacious as the SC bolus.     

Localization of thein vivoexpression of P and F1 fimbriae in chickens experimentally inoculated with pathogenicEscherichia coli

Escherichia colicausing septicemia in poultry often possess F1 (type 1) and/or P fimbriae which may be involved in bacterial colonization and infection. To investigate the expression of these fimbriaein vivo, two pathogenicE. colistrains with different fimbrial profiles, TK3 (fim⁺/pap⁺) and MT78 (fim⁺/pap⁻), were administered to 2-week-old chickens by either the intratracheal or caudal thoracic air sac inoculation route. Antibodies specific for native F1 fimbriae were detected by ELISA and immunodot in the serum of chickens inoculated with either strain MT78 or strain TK3, irrespective of the route of inoculation. Antibodies specific for P fimbriae of serotype F11 were detected by ELISA and immunoblotting in the serum of chickens inoculated by either route with strain TK3. F1, but not P fimbriae, were expressed by bacteria colonizing the trachea of chickens inoculated by the air sac route with strain MT78 or TK3, as demonstrated by examination of frozen tissue sections using immunofluorescence. F1 fimbriae were also expressed by bacteria colonizing the air sacs and lungs, but not by bacteria in the blood or other internal organs, of chickens inoculated with either strain. P fimbriae were expressed by bacteria colonizing the air sacs, lungs, kidney, blood, and pericardial fluid, but not by bacteria colonizing the trachea, of chickens inoculated with strain TK3. Fimbriae-like structures were observed by electron microscopy on bacteria adhering to the epithelial cells of the air sacs of chickens inoculated with strain TK3. These results demonstrate that both strains MT78 and TK3 undergoin vivophase variation with respect to their fimbrial profiles and site of bacterial colonization in different organs of infected chickens and suggest that F1 fimbriae are important for initial bacterial colonization of the upper respiratory tract whereas P fimbriae are important for later stages of the infection.     

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log₁₀ antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose–response study, there was a significant difference in the mean log₁₀ antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4⁺ and CD8⁺ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA⁺ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA⁺ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.     

Time-course investigation of infection with a low virulent Pasteurella multocida strain in normal and immune-suppressed 12-week-old free-range chickens

Twelve-week-old indigenous chickens, either immune-suppressed using dexamethasone (IS) or non-immune-suppressed (NIS), were challenged with a low virulent strain, Pasteurella multocida strain NCTC 10322^T, and developed clinical signs and pathological lesions typical of chronic fowl cholera. NIS birds demonstrated much more severe signs of fowl cholera than IS birds. With few exceptions, signs recorded in IS and NIS birds were of the same types, but significantly milder in the IS birds, indicating that immune suppression does not change the course of infection but rather the severity of signs in fowl cholera. P. multocida signals by fluorescent in situ hybridization (FISH) were observed between 1 h and 14 days in the lungs, trachea, air sacs, liver, spleen, bursa of Fabricius and caecal tonsils, while signals from other organs mostly were observed after 24 h. More organs had FISH signals in NIS birds than in IS birds and at higher frequency per organ. Many organs were positive by FISH even 14 days post infection, and it is suggested that these organs may be likely places for long-term carriage of P. multocida following infection. The present study has demonstrated the spread of P. multocida in different tissues in chickens and distribution of lesions associated with chronic fowl cholera, and pointed to a decrease of pathology in IS birds. Since dexamethasone mostly affects heterophils, the study suggests that these cells play a role in the development of lesions associated with chronic fowl cholera in chickens.     

Synergy between avian pneumovirus and Ornithobacterium rhinotracheale in turkeys

The purpose of this study was to assess the possible synergism between Ornithobacterium rhinotracheale (ORT) and avian pneumovirus (APV), inoculated into turkeys via the natural route, for the reproduction of respiratory disease. Three-week-old specific pathogen free turkeys were inoculated oculonasally with either APV subtype A, ORT or both agents using two different time intervals (3 and 5 days) between APV and ORT. The birds were observed clinically on a daily basis and swabbed intratracheally at short, regular intervals. They were killed at 1, 3, 5, 8 and 15 days post single or dual inoculation and examined for gross lesions at necropsy. Samples of the turbinates, trachea, lungs, air sacs, heart, pericardium and liver were taken for bacteriological and/or histological examination. Combined APV/ORT infections resulted in overt clinical signs and a longer persistence of ORT in the respiratory tract and aggravated the macroscopic and histological lesions in comparison with the groups given single infections. In all ORT-challenged turkeys, ORT was isolated from the turbinates, trachea and lungs, but in turkeys infected with both agents ORT was frequently found in the air sacs and on a single occasion in the heart and pericardium. The time interval between APV and ORT inoculation did not have a significant effect on the outcome of the dual infection. A conspicuous important feature was the attachment of ORT to the cilia of the epithelium of the turbinates and trachea of both ORT-infected and APV/ORT-infected birds. In conclusion, the results show that ORT is able to adhere to and colonize the respiratory tract but, under the circumstances used in this study, is not capable of inducing respiratory disease without viral priming.     

Ornithobacterium rhinotracheale: A review

Ornithobacterium rhinotracheale is a relatively recently discovered bacterium of the rRNA superfamily V. It is of worldwide distribution in commercial poultry, in which it is associated with respiratory diseases, and it is also found in wild birds. Airsacculitis and pneumonia are the most common features of infection with O. rhinotracheale. These signs can be induced by aerosol in intra-tracheal or intra-thoracic administration of the organism, and can be aggravated by other factors, such as respiratory viruses, bacteria or climatic conditions. Osteitis, meningitis and joint-infections, which can be induced by intravenous application, have been associated with O. rhinotracheale, but it remains uncertain whether the organism should be regarded as a primary pathogen. The infection can be transmitted horizontally by aerosol, as well as vertically through eggs, which probably accounts for its rapid and worldwide spread. Although O. rhinotracheale is difficult to identify, some commercial identification systems have been found to be suitable, although the media used in such systems will not always support its growth. A PCR assay was also found to be suitable for identification purposes. Twelve serotypes can be distinguished within the species O. rhinotracheale, of which serotype A is the most prevalent. Genetic investigation has revealed that more species or subspecies probably exist within the genus Ornithobacterium. Therapeutic treatment of the disease can be difficult because acquired resistance against the regular antibiotics is very common within the genus. Vaccination with autogenous inactivated vaccines has been successful in reducing clinical signs, but success depends on the adjuvant used. Only potent oil adjuvants are effective in young birds with maternal antibodies, but the use of these adjuvants is known to induce some local reactions. Live vaccination is feasible, but up to now, no avirulent strains of O. rhinotracheale have been found. Vaccination of broiler breeders induced protection against experimental infection of the progeny to at least 3 weeks of age.     

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A–E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.     

Microcomputer-assisted morphometric analysis of airsacculitis caused by Mycoplasma gallisepticum in immunised and nonimmunised chickens

Inflammation caused by inoculation of Mycoplasma gallisepticum (MG) into the air sacs was compared in chickens previously exposed by intramuscular injection of MG with that produced in nonimmunised chickens. Air sacs from chickens inoculated with Frey's media, from those receiving only intramuscular injections of MG, and from nonimmunised non-challenged birds were included in the evaluations. The airsacculitis produced in immunised chickens was more severe at 48 hours post-challenge infection than that produced in nonimmune chickens. At 96 hours after challenge, the airsacculitis in nonimmunised chickens was significantly more severe than that produced in chickens of any other experimental group. The protective effect of immunisation at 96 hours was demonstrated clearly by using the techniques of ring-stabilisation for collection, processing, and embedding of air sacs and microcomputer-assisted morpho-metric analysis. Air sac wall thickness and area measurements correlated positively with subjective impressions on the degree of airsacculitis and with determinations made by point count analysis of the relative percentage of selected components of inflammation.     

Respiratory tract infection caused by Mycobacterium bovis in a black swan (Cygnus atratus)

A 3-year-old male black swan (Cygnus atratus), belonging to a private collection, died suddenly and was subjected to post mortem examination. At necropsy, caseous exudate was observed in the lungs and air sacs; granulomatous lesions characterized by epithelioid macrophages and abundant mycobacteria were observed microscopically. Avian tuberculosis associated with Mycobacterium bovis was confirmed by bacteriologic isolation, biochemical tests and molecular methods. The organism was identified as spoligotype SB0140, which is frequently found in cattle and people in North America. In this case, interspecies transmission could have been the source of infection because the swan cohabited with cattle.     

Antibiotic sensitivity and resistance in Ornithobacterium rhinotracheale strains from Belgian broiler chickens

Establishing the antibiotic sensitivity of the avian respiratory pathogen Ornithobacterium rhinotracheale is difficult because of the organism's complex growth requirements and the unusually frequent occurrence of resistance. The minimal inhibitory concentrations of 10 antibiotics were determined for 45 strains of O. rhinotracheale from Belgian broiler chickens collected from 45 farms between 1995 and 1998. They were compared with the type strain, which was isolated from a turkey, and a strain isolated from a rook. All the broiler strains were resistant to lincomycin and to the β -lactams ampicillin and ceftiofur. Less than 10% of the strains were sensitive to the macrolides tylosin and spiramycin, tilmicosin and flumequine. A few strains were sensitive to enrofloxacin and doxycycline. All strains were sensitive to tiamulin.     

Rapid detection and quantification of Cryptosporidium baileyi oocysts in feces and organs of chickens using a microscopic slide flotation method

A simple semiquantitative microscopic slide flotation (MSF) method using modified Sheather's sugar solution (MSSS) is presented for the rapid detection and quantification of Cryptosporidium baileyi oocysts in the feces and mucosal and/or organ scrapings of chickens. Oocyst shedding was evaluated by examination of the surface of coverslips, and the average quantitative score (0–5) recorded for 10 microscopic fields (magnification ×250) is reported. The equivalence between these scores and the actual number of oocysts counted per gram of feces was assessed (r _s=0.89; P < 0.001). The applicability of this method was tested by comparison of the kinetics of oocyst shedding in feces of inoculated chickens with those reported by other authors working under similar conditions. In organs the MSF method was compared to histology. Fewer false-negative results were obtained using MSF versus the histology method. The MSF method was particularly more efficient in tracheae with low levels of infection and in the lungs, regardless of the level of infection. The MSF method was also very efficient in detecting oocysts in air sacs from chickens with aerosacculitis. It provides a specific and sufficiently sensitive, simple, rapid, reliable, and low-cost means of diagnosing C. baileyi in the feces and organs of chickens. This method can be used in the routine diagnosis of cryptosporidia in chickens, and it could be extended to other avian species and used in epidemiology studies to evaluate the prevalence of cryptosporidiosis in fowl. Received: 31 May 1999 / Accepted: 21 July 1999     

Detection of proteins and nucleic acids of Newcastle disease virus in Eimeria acervulina

Ten-day-old specific pathogen free (SPF) chickens were inoculated simultaneously with Eimeria acervulina and Newcastle disease virus (NDV). By employing immunofluorescent staining and in situ hybridization techniques, we detected NDV proteins and nucleic acids in different life stages of E. acervulina. However, no NDV particle was found within E. acervulina by electron microscopy. Oocysts from E. acervulina that contained NDV proteins and nucleic acids could elicit antibodies against NDV after repeated inoculation into SPF chickens. Moreover, the proportion of oocysts from chickens infected with E. acervulina and NDV which could be induced to sporulate in vitro was lower than those from chickens infected with E. acervulina alone. These results indicate that nucleic acids and proteins of NDV can exist within E. acervulina, and stimulate an immune response against NDV in chickens, and that NDV may also interfere with the sporulation of oocysts.     

Isolation and Characterization of Small-Colony Variants of Ornithobacterium rhinotracheale

Ornithobacterium rhinotracheale is a Gram-negative bacterium associated with respiratory diseases in many avian species, with worldwide distribution, and it causes significant economic loss to the poultry industry. In this study, the isolation and characterization of O. rhinotracheale small-colony variants (SCVs) are described for the first time. O. rhinotracheale isolates (n = 27) were recovered from tracheal samples (n = 321) collected from different avian species with clinical signs of respiratory disease. Of the 27 O. rhinotracheale isolates, 21 (77.8%) showed SCVs in their primary cultures. Five O. rhinotracheale SCV isolates showed high levels of stability and were chosen for further characterization with their wild-type (WT) isolates. Stable O. rhinotracheale SCVs were oxidase negative, while their WT isolates were positive. Growth curves for stable O. rhinotracheale SCVs indicated lower growth rates and longer lag phases than for their WT isolates. Furthermore, it was possible to increase the efficacy of the broth medium in supporting the growth of O. rhinotracheale WT isolates by supplementing it with 5% fetal bovine serum (FBS) and 2% IsoVitaleX Enrichment. Antibiotic susceptibility tests showed that O. rhinotracheale SCVs had higher MIC values than their WT isolates. This study suggests that successful antibiotic treatment of respiratory diseases associated with O. rhinotracheale must take into consideration the resistance patterns of O. rhinotracheale SCVs. Intracellular persistence in murine RAW 264.7 macrophages revealed that O. rhinotracheale SCV28 had higher survival rates than its WT isolate. Finally, small-colony variants may be important contributors to the pathogenesis of O. rhinotracheale.     

Changes in lymphoid organs and blood lymphocytes induced by vitamin A deficiency and Newcastle disease virus infection in chickens

The effect of vitamin A deficiency in the presence or absence of Newcastle disease virus infection (NDV, La Sota strain) on weight of lymphoid organs and on the number and type of circulating white blood cells (WBC) was investigated in chickens. Day-old chickens with limited vitamin A reserves were fed purified diets containing either marginal (ad libitum) or adequate (pair-fed) levels of vitamin A and at 21–28 days of age; half the chickens in each group were infected with NDV. Vitamin A deficiency resulted only in significantly lower absolute and relative weights of bursa of Fabricius and after infection both weights of bursa and thymus were significantly lower. Relative weight of spleen was significantly higher after infection irrespective of vitamin A status. Liver weights were not affected by vitamin A status and/or NDV infection. Both vitamin A deficiency and NDV infection resulted in lymphopenia, while the lowest number of WBC were observed in vitamin A-deficient chickens during the acute phase of NDV (5 days after infection). Subsequent to lymphopenia due to NDV infection, a marked lymphocytosis was observed in controls and to a lesser extent in vitamin A-deficient birds. These results indicate that vitamin A deficiency, which is aggravated by concomitant NDV infection, affects lymphoid cell systems.     

Respiratory (especially pulmonary) and urinary infections of Cryptosporidium in layer chickens

Respiratory (especially pulmonary) and urinary infections of Crypto-sporidium were diagnosed in four of 26 chickens from a layer flock. Macroscopically, the lungs had various degrees of yellowish white pneumonic foci. Occasionally caseous exudates were present in the lungs and air sacs. Histologically, there was marked epithelial hyperplasia in the air capillaries, atria, parabronchi, secondary bronchi and trachea. Cryptosporidia were detected on the epithelial cell surface or in the lumens of them in the lungs. Macrophages containing cryptosporidia were frequently seen in the lumens and walls of air capillaries and atria and in the cavities of parabronchi and secondary bronchi. Cellular infiltration was observed in the interstitium. Aspergillosis or bacterial infection was also present in the lungs and air sacs. The ureters and collecting ducts, collecting tubules and distal convoluted tubules of the kidneys demonstrated marked hyperplasia of epithelial cells associated with cellular infiltration. Cryptosporidial attachment on the epithelial cell surface of them were noted. Ultrastructurally, trophozoites, schizonts and macrogametocytes of Crypto sporidium were recognised in the lungs, kidneys and ureters. This study suggests that Cryptosporidium may induce bronchopneumonia with epithelial hyperplasia and may invade the urinary tract of chickens that are infected with other infectious agents.     

Assessment of Mycoplasma gallisepticum vaccine efficacy in a co-infection challenge model with QX-like infectious bronchitis virus

Mycoplasma gallisepticum (MG) is the primary cause of chronic respiratory disease in poultry. We investigated the protective efficacy of the live-attenuated ts-11 and 6/85 MG vaccines against a local MG strain and, in order to enhance signs and mimic a typical field situation, we co-infected birds with a virulent strain of QX-like infectious bronchitis virus (IBV). Both vaccines showed similar ability to protect infected chickens from clinical signs, although ts-11 performed slightly better. Despite the lower protection against clinical disease, 6/85-vaccinated birds had significantly (P?≤?0.05) lower tracheal lesion scores and mucosal thickness at day 28 post-vaccination (7 days post-challenge [dpc] with MG, 2 dpc IBV) and day 31 post-vaccination (10 dpc MG challenge, 5 dpc IBV) compared to ts-11 vaccinated birds, but these difference was not significant at day 33 (12 dpc MG, 7 dpc IBV). Pathogen infection and replication was assessed by qPCR, and the 6/85 vaccine produced a more significant (P?≤?0.05) reduction in MG replication in the lungs, kidneys and livers but enhanced late replication in bursae and caecal tonsils. In contrast, the ts-11 vaccine had a more pronounced reductive effect on replication in tracheas, air sacs, bursae and heart at days 28 and 31, yet increased replication in lungs. Interestingly, both vaccines provided non-specific protection against IBV challenge. The co-challenge model provided useful data on vaccine efficacy, especially on days 31 and 33, and tracheas, lungs, air sacs, kidneys, liver and caecal tonsils were the best organs to assess.     

Cellular defense of the avian respiratory system: influx and nonopsonic phagocytosis by respiratory phagocytes activated by Pasteurella multocida.

Poultry have a very limited number of resident macrophages in the normal steady-state respiratory tract. Thus, poultry must rely heavily on active migration of phagocytic cells to the lungs and air sacs in defending against respiratory pathogens. Intratracheal administration of a live, apathogenic Pasteurella multocida vaccine (Choloral; Clemson University strain) increased the number of avian respiratory phagocytes (ARP; obtained by lavage of lungs and air sacs) within 24 h by 3 orders of magnitude compared with the number of ARP obtained from mock-inoculated controls and from nonreacting chickens. Chickens yielding a high number of ARP did not show any sign of respiratory disease. Flow cytometric analysis of ARP that were exposed to 20 nonopsonized fluorescent microspheres per ARP for 30 min at 37 degrees C demonstrated a fivefold increase in the percentage of actively phagocytic cells in the ARP populations of stimulated chickens compared with the percentage of phagocytic ARP for mock-inoculated control birds. The phagocytic capacity (relative number of engulfed microspheres) of ARP from stimulated birds doubled during the same time. The flow cytometric observations were confirmed by fluorescence microscopy. These results indicate that activation by avirulent replicating agents of phagocytic cells of chicken to migrate to the respiratory tract may be a means of defending poultry against air sacculitis and pneumonia.     

Clinical, serological and histopathological signs of toxoplasmosis in broiler chickens (Gallus domesticus) after experimental infection

To identify the features of experimental toxoplasmosis in broiler chickens (Gallus domesticus), a total of 48 birds aged 25?days were randomly assigned to one of four groups of 12 birds each. Tachyzoites of Toxoplasma gondii were injected intraperitoneally at doses of 5?×?10⁵ (group A), 1?×?10⁶ (group B) and 1.5?×?10⁶ (group C), and chickens in group D were treated with an injection of saline only (control). Before and after experimental infection, serum samples from all chickens were tested for antibodies against T. gondii with the Sabin–Feldman reaction. After infection, the clinical signs in all the chickens were recorded daily, and blood smears were prepared to determine parasitemia. Paraffin-embedded tissue sections were used for semi-nested polymerase chain reaction (PCR) to detect the T. gondii B1 gene. Half of the chickens in each group were killed 25?days and half were killed 35?days after infection. All serum samples from chickens in groups A, B and C contained titers of T. gondii antibodies. However, there were no clinical signs suggesting toxoplasmosis. On day 15, the protozoan was observed in blood smears in groups A, B, and C. Analysis by PCR was negative. Microscopic lesions were observed in the brain, heart, liver, pancreas, kidney, spleen, skeletal muscle, proventriculus and lungs, but not in the eyes. Although chickens in group A were exposed to the lowest dose of T. gondii tachyzoites, lesions in this group were relatively more severe than those observed in groups B and C, which were exposed to higher doses of tachyzoites. Group B showed acute signs of toxoplasmosis with few microscopic lesions, whereas group C showed no lesions. Although no stages of the parasite were found in histopathological sections of skeletal muscle, the potential risks of infected chicken meat for public health cannot be disregarded.     

Co-infection of turkeys with Escherichia coli (O78) and H6N1 avian influenza virus

Respiratory diseases are responsible for major economic losses in poultry farms. While in most cases a single pathogen is not alone responsible for the clinical outcome, the impact of co-infections is not well known, especially in turkeys. The purpose of this study was to assess the possible synergism between Escherichia coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1), in the turkey model. Four-week-old commercial turkeys were inoculated with either H6N1, O78 or both agents simultaneously or three days apart. We have established an experimental infection model of turkeys using aerosolization that better mimics field infections. Birds were observed clinically and swabbed on a daily basis. Necropsies were performed at 4 and 14 days post single or dual inoculation and followed by histological and immunohistochemical analyses. Combined LPAIV/E. coli infections resulted in more severe clinical signs, were associated with higher mortality and respiratory organ lesions (mucous or fibrinous exudative material in lungs and air sacs), in comparison with the groups given single infections (P?E. coli inoculation (none or three days) did not have a significant effect on the outcome of the dual infection and disease although slightly greater (P?>?0.05) respiratory signs were observed in turkeys of the E. coli followed by H6N1 inoculated group. Microscopic lesions and immunohistochemical staining supported clinical and macroscopic findings. Efficient virus and bacteria replication was observed in all inoculated groups. E. coli and H6N1 thus exercise an additive or synergistic pathogenic effect in the reproduction of respiratory disease.