Avian Reoviruses of the Same Genotype Induce Different Pathology in Chickens.

The emergence of avian reovirus variant strains has caused negative effects in the poultry industry worldwide. Regardless of the efforts in molecular characterization and classification of these variants, information about the pathogenicity, transmissibility, and immunosuppression in chickens is limited. The genomes of two variant strains (A and B) and a classic S1133 strain (C) belonging to the same sigma C genotype 1 were compared. Additionally, these strains were used in a challenge experiment to evaluate inoculated and indirectly exposed specific-pathogen-free chickens. The whole-genome sequence analysis of the three strains revealed nucleotide identity differences in the L3, M2, and S1 genes. Strains A and B also showed homology differences in the S4 gene, despite having high homologies in all other genes. The in vivo challenge experiments showed that, whereas variant A induced high viral loads in tendons, hearts, and duodena of inoculated chickens, variant B induced high viral loads in indirectly exposed chickens. Likewise, histopathology reflected differences in the pathologic effects induced by these strains. For instance, the B and C strains induced more severe microscopic lesions compared with the A strain. Lymphoid depletion was more severe in bursas than in thymi, and inoculated birds were more affected than exposed birds. In conclusion, different pathologic outcomes in chickens were observed depending on the strain and transmission route. This study provides insights onto the relationship between pathogenicity and genomic composition of avian reoviruses.

Reovirus aviares del mismo genotipo inducen diferentes patologías en pollos. La aparición de cepas variantes del reovirus aviar ha causado efectos negativos en la industria avícola en todo el mundo. Independientemente de los esfuerzos en la caracterización molecular y clasificación de estas variantes, la información sobre la patogenicidad, transmisibilidad e inmunodepresión en pollos es limitada. Se compararon los genomas de dos cepas variantes (A y B) y una cepa S1133 clásica (C) perteneciente al mismo genotipo 1 del gene sigma C. Además, estas cepas se utilizaron en un experimento de desafío para evaluar pollos libres de patógenos específicos inoculados y expuestos indirectamente. El análisis de la secuencia del genoma completo de las tres cepas reveló diferencias de identidad de nucleótidos en los genes L3, M2 y S1. Las cepas A y B también mostraron diferencias de homología en el gene S4, a pesar de tener altas similitudes en todos los demás genes. Los experimentos de exposición in vivo mostraron que, mientras que la variante A inducía altas cargas virales en tendones, corazones y duodeno en los pollos inoculados, la variante B inducía altas cargas virales en pollos expuestos indirectamente. Asimismo, la histopatología reflejó diferencias en los efectos patológicos inducidos por estas cepas. Por ejemplo, las cepas B y C indujeron lesiones microscópicas más graves en comparación con la cepa A. La despoblación linfoide fue más severa en las bolsas que en el timo, y las aves inoculadas resultaron más afectadas que las expuestas. En conclusión, se observaron diferentes resultados patológicos en pollos según la cepa y la vía de transmisión. Este estudio proporciona información sobre la relación entre la patogenicidad y la composición genómica de los reovirus aviares.

Infectious Bronchitis Virus Surveillance in Broilers in California (2012-20).

Infectious bronchitis virus (IBV) causes severe economic losses among chicken flocks worldwide. Although IBV molecular surveillance has been conducted in California broilers, seasonal and spatial-temporal trends in IBV prevalence are poorly defined. The goals of this study were to evaluate seasonal and spatial-temporal trends in IBV prevalence and to determine the predominant IBV genotypes obtained over the last 8 yr from a broiler company located in the California Central Valley. In total, 3439 broilers with a suspicion of IBV infection were submitted to the California Animal Health and Food Safety laboratories between January 2012 and February 2020. Swabs from tracheas, kidneys, and cecal tonsils from each submission were independently pooled and screened for IBV using reverse transcriptase quantitative PCR (RT-qPCR). Positive samples were submitted for virus isolation. Viral isolates were subject to a conventional RT-PCR targeting the S1 gene hypervariable region. Positive samples from this RT-PCR were sequenced, and phylogenetic analyses were performed. In total, 1243 pooled swab samples were positive for IBV. Positive results were more frequently detected in fall and winter months compared to spring. Spatial analyses revealed an IBV hot spot in the vicinity of Livingston, and two areas with a low prevalence (i.e., cold spots) around Riverdale. The IBV spatial-temporal distribution identified three significant clusters: one hot spot around Turlock from 2015 to 2016, a second hot spot around Merced from 2012 to 2016, and a cold spot around Fresno from 2017 to 2020. Predominant genotypes changed over time from IBV Cal 99, which was predominant between 2012 and 2014, to IBV 3099 in 2019. Vaccination efforts were initiated in 2018, and as a result, we detected an emerging variant with 92% similarity to CA 3099 in 2020. This work highlights the importance of ongoing surveillance in IBV prevention programs. Surveillance strategies are necessary to monitor trends in diseases such as infectious bronchitis, and the tools used for surveillance need to be sensitive enough to detect new variants and identify spatial-temporal trends.

Vigilancia del virus de la bronquitis infecciosa en pollos de engorde en California (2012­2020). El virus de la bronquitis infecciosa (IBV) causa graves pérdidas económicas entre las parvadas de pollos en todo el mundo. Aunque la vigilancia molecular del virus de la bronquitis infecciosa se ha realizado en pollos de engorde en el estado de California, las tendencias estacionales y espacio-temporales sobre la prevalencia de este virus están mal definidas. Los objetivos de este estudio fueron evaluar las tendencias estacionales y espacio-temporales sobre la prevalencia del virus de la bronquitis infecciosa y determinar los genotipos predominantes de este virus obtenidos durante los últimos ocho años de una empresa de pollos de engorde ubicada en el Valle Central de California. En total, 3439 pollos de engorde con sospecha de infección por el virus de la bronquitis infecciosa se enviaron a los laboratorios de Salud Animal y Seguridad Alimentaria del estado de California entre enero del 2012 y febrero del 2020. Los hisopos de tráqueas, riñones y tonsilas cecales de cada caso se combinaron de forma independiente y se examinaron para detectar al virus de la bronquitis utilizando transcripción reversa y un método cuantitativo de PCR (RT-qPCR). Se enviaron muestras positivas para aislamiento del virus. Los aislados virales se sometieron a un método convencional de RT-PCR dirigido a la región hipervariable del gene S1. Se secuenciaron muestras positivas mediante la prueba RT-PCR y se realizaron análisis filogenéticos. Un total de 1243 muestras combinadas de hisopos dieron positivo para el virus de la bronquitis infecciosa. Los resultados positivos se detectaron con mayor frecuencia en los meses de otoño e invierno en comparación con la primavera. Los análisis espaciales revelaron un punto activo para el virus de la bronquitis infecciosa en las cercanías de Livingston y dos áreas con una baja prevalencia (es decir, puntos fríos) alrededor de Riverdale. La distribución espacio-temporal del virus de la bronquitis identificó tres grupos importantes: un punto activo alrededor de Turlock entre los años 2015 a 2016, un segundo punto activo alrededor de Merced entre los años 2012 a 2016 y un punto frío alrededor de Fresno entre los años 2017 a 2020. Los genotipos predominantes cambiaron con el tiempo, consideraron el subtipo IBV Cal 99, que fue predominante entre 2012 y 2014, a el tipo IBV 3099 en 2019. Los esfuerzos de vacunación se iniciaron en el 2018, y como resultado, detectamos una variante emergente con un 92% de similitud con el virus CA 3099 en 2020. Este trabajo destaca la importancia de la vigilancia continua en los programas de prevención para la bronquitis infecciosa. Las estrategias de vigilancia son necesarias para monitorear las tendencias en enfermedades como la bronquitis infecciosa, y las herramientas utilizadas para la vigilancia deben ser lo suficientemente sensibles como para detectar nuevas variantes e identificar tendencias espacio-temporales.

Infectious Coryza: Persistence, Genotyping, and Vaccine Testing.

The reemergence of infectious coryza (IC) caused by Avibacterium paragallinarum (AP) as an acute and occasionally chronic respiratory disease in domestic poultry has caused severe losses in several U.S. states. The disease is also associated with decreased egg production in layers and increased condemnations from air sac infections in broilers. A series of applied experiments were performed to elucidate the persistence of AP in infected broiler flocks, to genotype AP strains isolated from field cases, and to evaluate commercial and autogenous vaccine protection in commercial and specific-pathogen-free (SPF) chickens. Experimental evaluation of environmental persistence suggests that AP did not persist more than 12 hr in a hypothetically contaminated environment. Additionally, other detected potential pathogens such as Gallibacterium anatis and infectious bronchitis virus caused mild respiratory signs in the exposed birds. The HMTp210 and HagA genes of four IC field strains were sequenced and compared with published sequences of HMTp210 and HagA. The HMTp210 phylogeny showed a marginally imperfect clustering of the sequences in genogroups A, B, and C. Although not definitive, this phylogeny provided evidence that the four field strains aligned with previously characterized serovar C strains. Moreover, the base pair homology of the four strains was 100% identical to serovar C reference strains (H-18 and Modesto). HagA phylogeny was unclear, but interestingly, the IC field strains were 100% homologous to C-1 strains reported from Mexico and Ecuador. Finally, vaccine protection studies in commercial hens indicate that clinical signs are induced by a combination of IC and other concomitant pathogens infecting commercial birds. Additionally, vaccine protection experiments performed in SPF hens indicated that protection provided by the two commercial vaccines tested provided a reduction in clinical signs and bacterial shedding after two applications.

Coriza infecciosa: Persistencia, genotipificación y pruebas para vacunas. El resurgimiento de la coriza infecciosa (CI) causada por Avibacterium paragallinarum (AP) como una enfermedad respiratoria aguda y ocasionalmente crónica en aves domésticas ha causado graves pérdidas en varios estados de los Estados Unidos. La enfermedad también se asocia con una disminución en la producción de huevo en gallinas de postura y al incremento de decomisos por infecciones de los sacos aéreos en pollos de engorde. Se realizó una serie de experimentos aplicados para dilucidar la persistencia de A. paragillanarum en parvadas de pollos de engorde infectados, para genotipificar las cepas de A. paragallinarum aisladas de casos de campo y para evaluar la protección de vacunas comerciales y autógenas en pollos comerciales y en aves libres de patógenos específicos (SPF). La evaluación experimental de la persistencia ambiental sugiere que A. paragallinarum no persistió más de doce horas en un ambiente hipotéticamente contaminado. Además, otros patógenos potenciales detectados como Gallibacterium anatis y el virus de la bronquitis infecciosa causaron signos respiratorios leves en las aves expuestas. Los genes HMTp210 y HagA de cuatro cepas de campo de coriza infecciosa se secuenciaron y compararon con las secuencias publicadas de HMTp210 y HagA. La filogenia de HMTp210 mostró una agrupación marginalmente imperfecta de las secuencias en los genogrupos A, B y C. Aunque no es definitiva, esta filogenia proporcionó evidencia de que las cuatro cepas de campo se alinearon con cepas del serovar C previamente caracterizadas. Además, la homología de pares de bases de las cuatro cepas fue 100% idéntica a las cepas de referencia del serovar C (H-18 y Modesto). La filogenia de HagA no fue clara, pero curiosamente, las cepas de campo de coriza infecciosa fueron 100% similares con las cepas C-1 reportadas en México y Ecuador. Finalmente, los estudios de protección de vacunas en gallinas comerciales indican que los signos clínicos son inducidos por una combinación de coriza infecciosa y otros patógenos concomitantes que infectan a las aves comerciales. Además, los experimentos de protección de vacunas realizados en aves libres de patógenos específicos indicaron que la protección proporcionada por las dos vacunas comerciales analizadas proporcionó una reducción en los signos clínicos y en la eliminación bacteriana después de dos aplicaciones.

Intervention strategies for laryngotracheitis: impact of extended downtime and enhanced biosecurity auditing.

An outbreak of vaccinal infectious laryngotracheitis (LT) began in 2005 involving 57 ranches of two broiler companies in California. Standard biosecurity, and cleaning and disinfection programs along with vaccination, did not stop the outbreak. Due to the close proximity and number of birds in the same geographic area, the decision was made by both companies to attempt a joint regional and zonal depopulation strategy. The strategy involved extended downtime between flock placements on ranches located within close proximity to one another. This extended downtime on each ranch ranged from 30 to 91 days. An extensive biosecurity audit, with more than 70 items, was implemented. Briefly, this included heating all houses to 37 C for 100 hr, removing the litter, cleaning and disinfecting everything on the ranches, then again heating the houses to 37 C for 100 hr. Used litter was spread on crops away from poultry, or was sent to a litter processor for pasteurization. Extensive surveillance for LT at 28, 35, and 42 days of age was performed on the initial flocks, which had been placed immediately after the extended downtime. Since completion of this plan in early 2008, LT was diagnosed on only two of the previously 57 affected ranches. Those two ranches, and those within close proximity, went through the extended downtime program and biosecurity audit a second time. Currently, both companies are free of LT. This program lends credence to the importance of cooperation between companies to consider all the ranches within close proximity as the population at risk. In the control of LT in broilers, the program also highlights the necessity for extended downtime and enhanced biosecurity auditing of all flocks.

Partial Molecular Characterization and Pathogenicity Study of an Avian Reovirus Causing Tenosynovitis in Commercial Broilers.

This study describes the molecular characterization of avian reoviruses (ARVs) isolated during an outbreak in commercial chickens between 2015 and 2016. In addition, a pathogenicity study of a selected ARV strain isolated from a field case of viral tenosynovitis in commercial broiler chickens was performed. On the basis of phylogenetic analysis of a 1088-bp fragment of the ARV S1 gene, the investigated sequences were differentiated into five distinct genotypic clusters (GCs), namely GC1, GC2, GC3, GC4, and GC6. Specific-pathogen-free (SPF) and commercial broiler chickens were challenged with the GC1 genetic type MK247011, at 14 days of age via the interdigital toe web. No significant effects in body weight gain and feed conversion were detected in both chicken types. The Δ interdigital web thickness was most severe at 4 days postchallenge (DPC) in both the SPF and broiler subgroups. The inflammation in SPF birds was slightly more severe compared with broilers. Neither mortality nor clinical signs occurred in the infected groups for the duration of the experiment, despite the presence of significant microscopic lesions in challenged birds. Microscopic changes of tenosynovitis became evident at 3 DPC, with the highest incidence and severity detected at 14 and 21 DPC, respectively. Seroconversion against ARV occurred 3 wk postchallenge, and the microscopic lesions detected in tendon and heart sections were highly compatible with those described in the field. Increased severity of tenosynovitis and epicarditis lesions were noted in the ARV-challenged groups compared with the control groups. Although SPF and broiler chickens showed comparable responses to the challenge with an ARV genetic variant, detected lesions were subclinical, denoting the limitations of our challenge approach. The age selected in this experiment possibly influenced the course of the infection. Data from this study highlight the genotypic diversity of isolates in California, and the outcome of the pathogenicity study can be used as a basis to improve protocols for pathogenicity studies to characterize ARV variants causing clinical disease in the field.

Caracterización molecular parcial y estudio de patogenicidad de un reovirus aviar que causa tenosinovitis en pollos de engorde comerciales. Este estudio describe la caracterización molecular de reovirus aviares (ARV) aislados durante un brote en pollos comerciales entre los años 2015 y 2016. Además, se realizó un estudio de patogenicidad de una cepa de reovirus seleccionada que fue aislada de un caso de campo de tenosinovitis viral en pollos de engorde comerciales. Con base en el análisis filogenético de un fragmento de 1088 pb del gene S1 de reovirus, las secuencias investigadas se diferenciaron en cinco grupos genotípicos distintos (GCs), denominados, GC1, GC2, GC3, GC4 y GC6. Aves libres de patógenos específicos (SPF) y pollos de engorde comerciales se desafiaron con el tipo genético GC1 MK247011 a los 14 días de edad a través de la membrana interdigital. No se detectaron efectos significativos en el aumento de peso corporal ni en la conversión de alimento en ambos tipos de aves. El grosor de la banda interdigital diferencial fue más severa a los cuatro días posteriores al desafío en las aves libres de patógenos específicos y en los pollos de engorde. La inflamación en las aves libres de patógenos específicos fue ligeramente más severa en comparación con los pollos de engorde. No se presentó mortalidad ni signos clínicos en los grupos infectados durante la duración del experimento, a pesar de la presencia de lesiones microscópicas significativas en las aves desafiadas. Los cambios microscópicos de la tenosinovitis se hicieron evidentes a los tres días postinoculación, con la mayor incidencia y severidad detectadas a los 14 y 21días postinoculación, respectivamente. La seroconversión para reovirus ocurrió tres semanas después del desafío, y las lesiones microscópicas detectadas en secciones de tendón y corazón fueron altamente compatibles con las descritas en el campo. El aumento en la severidad de las lesiones de tenosinovitis y epicarditis se observó en los grupos expuestos a reovirus aviar en comparación con los grupos de control. Aunque las aves libres de patógenos específicos y los pollos de engorde mostraron respuestas comparables ante el desafío con una variante genética de reovirus, las lesiones detectadas fueron subclínicas, lo que denota las limitaciones de nuestro enfoque de desafío. La edad seleccionada en este experimento posiblemente influyó en el curso de la infección. Los datos de este estudio resaltan la diversidad genotípica de los aislamientos en California y el resultado del estudio de patogenicidad se puede usar como base para mejorar los protocolos de los estudios de patogenicidad para caracterizar las variantes de reovirus que causan enfermedades clínicas en el campo.

Genotypic Characterization of Emerging Avian Reovirus Genetic Variants in California.

This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

Turkey breeder hen age affects growth and systemic and intestinal inflammatory responses in female poults examined at different ages posthatch.

This trial examined the effect of 2 turkey breeder hen ages (33 or 55 wk of age) on performance, intestinal histology, and inflammatory immune response of female turkey poults grown to market weight. Using a randomized design, female poults were separated by breeder flock age (n = 8 floor pens/breeder flock age; n = 26 poults/pen; 0.195 m(2)/bird), fed identical commercial diets (9 phases), and grown to market weight (approximately 11.4 kg/ bird). At young ages, poults from the older breeder flock tended to have higher BW (P < 0.01 for d 7, P < 0.09 for d 63), although feed consumed was not significantly different due to breeder flock age (P > 0.20 for all ages). After approximately 63 d posthatch, no difference in BW was observed, suggesting that poults from the younger breeder flock were eventually able to compensate for initial reductions in performance. In addition to growth measurements on d 10, 24, and 65 posthatch, poults were vaccinated with lipopolysaccharide (LPS, from Salmonella Typhimurium; 0.5 mg/kg of BW intraabdominally) or not vaccinated (control), and intestinal histology and plasma haptoglobin were assessed at 24 h postadministration. In control birds, intestinal villus length was greater for poults from the older breeder flock (P < 0.05), as was crypt depth (P < 0.05 for d 11 and 25). Plasma haptoglobin levels did not change in 11-d-old poults after LPS administration, but they increased with LPS at d 25 and 66 posthatch (P < 0.05 for each). At d 66 posthatch, poults from the younger flock had increased haptoglobin levels post-LPS compared with those from the older breeder flock (P < 0.05). In general, LPS administration increased villus width in the jejunum and ileum (P < 0.05 for each), increased lamina propria width in the duodenum and ileum (P < 0.05 for each), and decreased ileum crypt depth (P < 0.05). Overall, poults from the older breeder flock had reduced inflammatory responses, even at 9 to 10 wk posthatch, even though performance was similar in poults from the 2 flocks by this age.

Immunoglobulin receptors used in avian Mycoplasma identification.

Among 77 isolates of avian Mycoplasma examined, IgG receptors were demonstrated on six of the nine species represented. Species identified with receptors were: Mycoplasma gallisepticum (two receptors of approximately 84,000 and 75,000 molecular weight [MW]), M. gallinarum (one receptor of approximately 135,000 MW), M. pullorum (one receptor of approximately 130,000 MW), M. gallinaceum (one receptor of approximately 125,000 MW), M. iowae (two receptors of approximately 50,000 and 38,000 MW), and M. synoviae (two receptors of approximately 91,000 and 81,000 MW). The IgG receptors demonstrated in Western blots consistently identified each of the six species. No IgG receptors were detected on M. iners, M. gallopavonis, or M. meleagridis.