Longitudinal Serological Monitoring of Commercial Broiler Breeders for Fowl Adenoviruses (FAdVs)-Presence of Antibodies Is Linked with Virus Excretion.

Currently, the poultry industry worldwide is facing an emerging trend of fowl adenovirus (FAdV)-associated diseases with a significant economic impact, especially in meat-type chickens. Vertical transmission is an important feature of all FAdVs; hence, preventive measures mostly revolve around breeding stocks. However, knowledge about temporal development of FAdV infections in modern commercial settings is rare or even nonexistent. In the present study, longitudinal monitoring for FAdV was conducted in broiler breeder flocks located in a confined geographical region with intensive poultry production in Iran. For this, the antibody status of birds from 4 to 32 wk of age was monitored with a commercial FAdV-ELISA and virus neutralization test (VNT). In parallel, fecal shedding of FAdV was determined at the peak of egg production with real-time PCR and virus isolation. Overall, the commercial ELISA showed seroconversion of flocks before onset of production. VNT resolved in detail infection patterns of individual serotypes with a primordial FAdV-D (FAdV-2/-11) infection, frequently followed by FAdV-E (FAdV-8a, -8b) superinfection. FAdV-A (FAdV-1) was traced in half of the investigated flocks, while no evidence of infection with FAdV-C (FAdV-4, -10) was noted. Common serological profiles between different houses of the same farm indicate an overarching biosecurity. Serological profiles coupled with virological findings at the peak of egg production indicated that higher antibody levels, determined by ELISA, correlated with lower amounts of viral DNA in fecal excretion. Simultaneously, the number of isolated FAdVs belonging to distinct serotypes declined in accordance with a rise of neutralizing antibodies in birds, underlining the significance of serotype-specific antibodies in the epidemiology of FAdV in breeders. Investigations in breeders were complemented with screening of FAdV-associated diseases in local broilers over a 3-yr period; 26 cases of inclusion body hepatitis with dominant involvement of FAdV-11/FAdV-8b, one outbreak of adenoviral gizzard erosion related to FAdV-1, and no evidence of hepatitis-hydropericardium syndrome suggest that identical serotypes are maintained in the local poultry industry.

Artículo regular­Monitoreo serológico longitudinal en reproductores pesados comerciales para detectar adenovirus del pollo (FAdV): la presencia de anticuerpos está relacionada con la excreción de virus. Actualmente, la industria avícola en todo el mundo enfrenta a una tendencia emergente de enfermedades asociadas con adenovirus del pollo (FAdV) con un impacto económico significativo, especialmente en pollos de engorde. La transmisión vertical es una característica importante de los adenovirus del pollo, por lo que las medidas preventivas giran principalmente en torno a las poblaciones de reproductores. Sin embargo, el conocimiento sobre el desarrollo temporal de las infecciones por adenovirus del pollo en los entornos comerciales modernos es escaso o incluso inexistente. En el presente estudio, se llevó a cabo un seguimiento longitudinal de adenovirus del pollo en parvadas de reproductoras pesadas ubicadas en una región geográfica confinada con producción avícola intensiva en Irán. Para ello, se evaluó el estado de anticuerpos de las aves de 4 a 32 semanas de edad con una prueba comercial de ELISA para adenovirus del pollo y por la prueba de virus neutralización (VNT). En paralelo, se determinó la eliminación fecal de adenovirus del pollo en el pico de producción de huevos mediante un método de PCR en tiempo real y por aislamiento del virus. En general, la prueba de ELISA comercial mostró seroconversión de parvadas antes del inicio de la producción. La prueba de virus neutralización reveló en detalle los patrones de infección de los serotipos individuales con una infección primordialmente por FAdV-D (FAdV-2/-11), seguida frecuentemente por una superinfección por FAdV-E (FAdV-8a, - 8b). Se rastreó FAdV-A (FAdV-1) en la mitad de las parvadas investigadas, mientras que no se observó evidencia de infección por FAdV-C (FAdV-4, -10). Los perfiles serológicos comunes entre las diferentes casetas de la misma granja indican una bioseguridad generalizada. Los perfiles serológicos junto con los hallazgos virológicos en el pico de producción de huevo indicaron que los niveles más altos de anticuerpos, determinados por ELISA, se correlacionaron con cantidades más bajas de ADN viral en la excreción fecal. Simultáneamente, el número de adenovirus de pollo aislados pertenecientes a distintos serotipos disminuyó de acuerdo con un aumento de anticuerpos neutralizantes en aves, lo que subraya la importancia de los anticuerpos específicos de serotipo en la epidemiología del adenovirus del pollo en reproductores. Las investigaciones en reproductoras se complementaron con la detección de enfermedades asociadas a adenovirus en pollos de engorde locales durante un período de 3 años; 26 casos de hepatitis por cuerpos de inclusión con participación dominante de FAdV-11/FAdV-8b, un brote de erosión de molleja adenoviral relacionado con FAdV-1 y ninguna evidencia de síndrome de hepatitis-hidropericardio sugieren que se mantienen serotipos idénticos en la industria avícola local.

Successful reproduction of adenoviral gizzard erosion in 20-week-old SPF layer-type chickens and efficacious prophylaxis due to live vaccination with an apathogenic fowl adenovirus serotype 1 strain (CELO).

Recently, outbreaks of adenoviral gizzard erosion (AGE) have been documented in pullets and layers housed free range and in enriched cage systems characterized by increased mortality and a negative impact on egg production. In the present study the pathogenicity of a fowl adenovirus serotype 1 (FAdV-1) field strain as well as the aetiological role of a FAdV-8a strain, both isolated from AGE affected pullets, were investigated in vivo in 20-week-old specific-pathogen-free (SPF) layer-type chickens. Furthermore, the efficacy of a single (week 17) and double (week 14 and 17) application of a live vaccine consisting of an apathogenic FAdV-1 (CELO strain) against challenge with virulent FAdV-1 was investigated. For the first time, AGE was successfully reproduced in adult birds after oral infection of 20-week-old SPF birds with a virulent FAdV-1 field isolate, characterized by pathological changes of the gizzard from 7 days post challenge onwards. In addition, a negative impact of the FAdV-1 infection on the development of the reproductive tract was observed. Thus, confirming the pathogenicity and aetiological role of FAdV-1 in the development of AGE and economic losses due to AGE in layers. In contrast, no pathological changes were observed in birds infected with FAdV-8a. Independent of a single or double application of the live FAdV-1 vaccine strain CELO, no gross pathological changes were observed in gizzards post challenge with the virulent FAdV-1, indicating that complete protection of layers against horizontal induction of AGE was achieved. Nonetheless, virulent FAdV-1 was detected in cloacal swabs and gizzards in both vaccinated groups post challenge determined by the application of an amplification refractory mutation system quantitative PCR used to differentiate between vaccine and challenge strains.

Fiber-based fluorescent microsphere immunoassay (FMIA) as a novel multiplex serodiagnostic tool for simultaneous detection and differentiation of all clinically relevant fowl adenovirus (FAdV) serotypes.

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test.

Development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes 1 and 4 in chickens.

Conventional serological methods for detection and differentiation of antibodies against fowl aviadenoviruses (FAdVs) are laborious and time-consuming, therefore ELISAs based upon recombinant proteins were developed in the present study to overcome this limitation for clinically relevant serotypes FAdV-1 and FAdV-4. In order to develop serotype-specific ELISAs, the two distinct fibers, fiber-1 (fib-1) and fiber-2 (fib-2), characteristically present only in FAdV-1 and FAdV-4, were applied separately as coating antigens. Sera raised against each recombinant fib-1 and fib-2 of FAdV-1 and FAdV-4 did not react with any of the heterologous fiber ELISAs, as anticipated by the low degree of amino acid identity between those FAdV fibers (23.1-41.2%), indicating that heterologous fibers do not share common epitopes. Testing of 172 monospecific sera, raised against all FAdV serotypes (1-8a and 8b-11), retrieved specificities between 99.3% and 100.0% for the ELISAs, further substantiating the serotype-specificity of fibers. Investigating sera from chickens experimentally inoculated with different FAdV-1 or FAdV-4 strains revealed that ELISAs were equally or more sensitive than the virus-neutralization (VN) test. Furthermore, strong correlations were demonstrated between fiber antibody titres and neutralization activity. Particularly, sera directed against live virus showed a pronounced fiber antibody response, which might be explained by an excessive production of fibers during infection. Application of the newly developed fiber ELISAs on field sera with heterogeneous serological status demonstrated high sensitivity and serotype-specificity of this test system, providing for the first time a diagnostic tool for mass screening of chicken flocks against FAdV serotypes, namely FAdV-1 and FAdV-4.

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture.

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.

Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E).

The present study describes the development of a SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of all fowl adenovirus (FAdV) species. Primers were designed based on conserved nucleotide sequences within the 52K gene. Ten-fold serial dilutions of a vector DNA were used as standard for quantitation. The real-time PCR had an efficiency of 98%, a regression squared value of 0.999 and showed a range of 6.73-6.73×10(8) copies of FAdV DNA per reaction. The assay was highly specific for FAdVs and an exact quantitation of all 5 FAdV species (FAdV-A to FAdV-E) could be demonstrated. It was shown, that twelve FAdV serotypes (FAdV-1 to 8a, and 8b to 11) were detectable and quantifiable. Other viral genomes as well as uninfected chicken embryo liver (CEL) cells did not produce positive signal. Cloacal swabs were taken during the animal experiment, which was performed with all FAdV species. Shedding of FAdVs was investigated in cell culture, by conventional PCR and by the developed real-time PCR. The real-time PCR was found more sensitive than cell culture and conventional PCR. Detection and quantitation of FAdVs in different type of samples was possible by the new real-time PCR.

Trichomonas gallinae, in comparison to Tetratrichomonas gallinarum, induces distinctive cytopathogenic effects in tissue cultures.

In the present study the interaction of three genetically different clonal cultures of Trichomonas gallinae and Tetratrichomonas gallinarum with a permanent chicken liver (LMH) and a permanent quail fibroblast (QT35) cell culture was studied. Proliferation of T. gallinae cells was associated with a disintegration of the cell monolayer. The initial lesions on the LMH monolayer consisted of a progressive accumulation of the flagellate, forming clumps attached to the monolayer. A prolonged incubation time was characterized by appearance of holes in the cell monolayer with accumulation of trichomonads at their periphery. According to the severeness of the monolayer disruption differences among three tested T. gallinae clones were noticed. Furthermore, filtrates obtained either from axenic cultures of T. gallinae or from infected cell cultures produced a cytopathogenic effect similar to the protozoal cells, on both types of cell cultures. However, the destructive effect of the flagellates and their cell-free filtrates was much more pronounced on the LMH monolayer in comparison with the QT35 cells. Furthermore, freshly seeded LMH and QT35 cells suspended in cell-free filtrates of T. gallinae were unable to form a confluent monolayer. In comparison to T. gallinae, clonal cultures of T. gallinarum or their cell-free filtrates produced no effect on both types of monolayers. Interestingly, the cell-free filtrates obtained from both trichomonad species had an effect on the viability of both cell cultures. However, the cytotoxic effect of T. gallinarum filtrates was less severe than that recorded by T. gallinae. Consequently, for the first time a destruction of specified monolayers induced by T. gallinae-free filtrates could be demonstrated.

Two fiber genes of nearly equal lengths are a common and distinctive feature of Fowl adenovirus C members.

The complete genome or the genome region containing the two fiber genes of two reference strains and one field isolate representing both serotypes of Fowl adenovirus C were sequenced. Two fiber genes were revealed in the genomes of all three isolates. Fiber-1 and fiber-2 genes of several Fowl adenovirus C isolates were sequenced as well. Both serotypes 4 and 10 have two fiber genes. The genome region containing the fiber gene was also sequenced for the reference strain of Fowl adenovirus B. Just one fiber gene was revealed in this strain. Predicted amino acid sequences were compared to already published fiber sequences of different adenovirus isolates and one amino acid substitution within fiber-2 was detected in all of the Fowl adenovirus C isolates that were isolated from chickens with hepatitis-hydropericardium syndrome in comparison to apathogenic isolates. Phylogenetic analyses provided insights about the evolution of fiber genes in avian adenoviruses and their genetic relationships.

Distribution of nerve endings in the human dorsal radiocarpal ligament.

PURPOSE: The purpose of this investigation was to evaluate the nerve-ending apparatus populations within a large number of adult human dorsal radiocarpal (DRC) ligaments to test the hypothesis that the majority of nerve endings could be grouped into established classifications and that the nerve endings could be found in predictable locations within the substance of the ligament. METHODS: The DRC ligaments were harvested from 20 wrists of 10 fresh cadavers with an average age of 75.6 years within 12 to 18 hours of death. Before the tissues were harvested, radiographs were taken to exclude any arthritic conditions of the wrists. Tissues were fixed, sectioned with a cryostat, and serial sections were collected on glass slides. Slides were processed for fluorescence immunohistochemistry using antibody to protein gene product 9.5 and a secondary antibody conjugated to a fluorescent tag (Alexa Fluor 488). The sections were evaluated with an LSM-510 confocal laser microscope and a Kontron KS 400 image analyzer. Labeled nerve endings were counted, mapped, and reconstructed. RESULTS: The average number of nerve endings in each DRC ligament was 10.1+/-4.7. More than 76% of the nerve endings were found in the 2 ends of the ligament with 23.3% in the central third and approximately 80% distributed in the superficial layer. More than 80% of the nerve endings were discovered in the epiligamentous sheath rather than in the perifascicular spaces. CONCLUSIONS: The distribution of the nerve endings follows a consistent pattern. These results will provide a foundation of morphologic information useful in understanding normal and abnormal neural control of wrist joint mechanics.

Mapping lubricin in canine musculoskeletal tissues.

Lubricin, also known as superficial zone protein or PRG4, has many distinct biological functions, including lubrication, antiadhesion, and as a regulator of cell growth. This study investigated lubricin in canine musculoskeletal tissues using RT-PCR and immunohistochemistry. One or more variants were noted in canine flexor digitorum profundus (FDP) tendon, Achilles tendon, patellar tendon, A2 pulley, anterior cruciate ligament (ACL), knee lateral collateral ligament (LCL), articular cartilage, meniscus, muscle, and skin. We found 6 N-terminal lubricin splicing variants. The variants with larger sizes were identified in FDP tendon, ACL, LCL, A2 pulley, and cartilage. Lubricin was distributed both on the tissue surfaces and at the interface of fiber bundles within tissues, but this distribution varied by tissue type. We conclude that lubricin is present in many tissues; variations in splicing and physical distribution suggest that the variants of lubricin may play different roles in different locations.

Expression and mapping of lubricin in canine flexor tendon.

Matrix composition and the biomechanical environment are intimately interdependent in most connective tissues. Lubricin has many distinct biological functions, including lubrication, antiadhesion, and cytoprotection in cartilage, tendons, and other tissues. This study investigated the distribution of lubricin in the canine flexor digitorum profundus (FDP) tendon by immunohistochemistry. Lubricin was found both on the tendon surface and at the interface of collagen fiber bundles within the tendon, where the cells are subjected to shear force in addition to tension and compression. The expression of lubricin in regions of the canine flexor tendon that differ in mechanical or nutritional environment was also investigated using RT-PCR. Six N-terminal splicing variants were identified from six distinct anatomical regions of flexor tendon. The variants with larger sizes were noted in regions subjected to significant shear and compressive forces. Lubricin is ubiquitous in the FDP tendon, with variations in distribution and splicing that appear to correspond to discrete anatomic locations that differ by mechanical or nutritional environment.

Nerve endings of the wrist joint: a preliminary report of the dorsal radiocarpal ligament.

As part of an investigation of the articular nerve ending populations in the wrist joint capsule associated with the anterior and posterior interosseous nerves, this study addresses the nerve ending population in the dorsal radiocarpal ligament. The ligaments were harvested from four wrists of two fresh cadavers within 12 h of death. Tissues were fixed, cryostat sectioned, and processed for fluorescence immunohistochemistry using antibody to protein gene product 9.5 (PGP 9.5), a general or pan neuronal marker, and a secondary antibody conjugated to a fluorescent tag (Alexa Fluor 488). The sections were evaluated with a confocal laser microscope and an image analyzer. Labeled nerve endings were mapped, measured, and categorized. Type I (Ruffini-like ending), Type III (Golgi-like tendon organ) and Type IV (noncorpuscular) nerve endings could be identified in all four DRC ligaments, with Types I and IV dominating. These receptors were distributed primarily over the superficial two thirds of the ligament (>80%), and near the bony attachments (>70%). The dorsal radiocarpal ligament has a rich sensory innervation from the posterior interosseous nerve terminating in nerve endings located in the superficial two-thirds of the ligaments, primarily near bony attachment sites.

Fluorescence immunohistochemistry and confocal scanning laser microscopy: a protocol for studies of joint innervation.

We have developed a reliable technique for labeling and examining neural structures in soft tissues associated with articular joints and have tested it in human wrist joints under various specimen-related conditions. The labeling protocol employs an immunohistochemical process with a panneuronal marker (PGP 9.5) as the primary antibody and Alexa Fluor 488 as the fluorescing secondary antibody. Imaging was done using a confocal laser scanning microscope, which produced exceptionally detailed three-dimensional images of nerve endings and transiting nerve fibers from thick sections of wrist joint ligaments obtained from human cadavers. The protocol provided a practical postmortem window for specimen acquisition and processing without significant apparent worsening of image quality. The images produced are resistant to fading with repeated exposure to a fluorescent light source, which gives many opportunities for observation. Background staining is minimal, producing high contrast labeling of target tissues, which, in turn, enhances image analysis.