Characterization of an Infectious Bronchitis Virus Isolated from Commercial Layers Suffering from False Layer Syndrome.

Infectious bronchitis virus (IBV) is a gammacoronavirus that primarily induces an upper respiratory disease in chickens, also affecting the urogenital tract and occasionally leading to a condition called false layer syndrome (FLS), where sexually mature hens ovulate normally but are unable to lay eggs. Here, we describe an outbreak of FLS in Arizona from which an IBV variant that is nearly 90% homologous to DMV/1639 using the Spike subunit 1 gene, named AZ/FLS/17, was isolated and used in challenge experiments. Three-day-old specific-pathogen-free chicks were challenged with AZ/FLS/17 or M41 in high and low doses, and the disease outcomes were compared. Overall, no differences in microscopic lesions or viral loads in the reproductive tract were detected between AZ/FLS/17- and M41-infected birds. To minimize the losses linked to FLS in the problematic flocks, an updated live-attenuated IBV vaccine protocol including the use of the Ma5 strain at the hatchery was implemented, resulting in a drastic reduction of false layers in the subsequent flocks. To monitor the circulation of wild-type and vaccine strains in this population, a molecular surveillance study was performed. Samples were collected at 1, 7, 14, and 21 days of age, and from laying hens at 30 and 36 wk. In older birds, the IBV strains detected were more diverse than at 1 and 7 days of age. Nevertheless, live vaccine combinations are still widely used to decrease the losses caused by FLS in commercial egg laying flocks worldwide.

Caracterización de un virus de la bronquitis infecciosa aislado de aves de postura comerciales que padecían el síndrome de la falsa ponedora. El virus de la bronquitis infecciosa (IBV) es un gammacoronavirus que induce principalmente una enfermedad de las vías respiratorias superiores en los pollos, que también afecta el tracto urogenital y ocasionalmente conduce a una condición llamada síndrome de la falsa ponedora (FLS), donde las gallinas sexualmente maduras ovulan normalmente pero no pueden producir huevos. En este estudio se describe un brote del síndrome de la falsa ponedora en Arizona a partir del cual se aisló una variante del virus de bronquitis que es casi 90% similar a la variante DMV/1639 usando el gene S1, la cual se denominó AZ/FLS/17, y se usó en experimentos de desafío. Pollos de tres días de edad libres de patógenos específicos (SPF) fueron desafiados con el virus AZ/FLS/17 o con el virus M41 en dosis altas y bajas, y se compararon los resultados de la enfermedad. En general, no se detectaron diferencias en las lesiones microscópicas o en las cargas virales en el tracto reproductivo entre las aves infectadas con el virus AZ/FLS/17 y el virus M41. Para minimizar las pérdidas relacionadas con el síndrome de la falsa ponedora en las parvadas problemáticas, se implementó un protocolo actualizado con vacuna viva atenuada contra la bronquitis infecciosa que incluía el uso de la cepa Ma5 en la incubadora, lo que resultó en una reducción drástica de las gallinas falsas ponedoras en las parvadas subsecuentes. Para monitorear la circulación de cepas de tipo silvestre y vacunales en esta población, se realizó un estudio de vigilancia molecular. Se recolectaron muestras a los 1, 7, 14 y 21 días de edad y de gallinas de postura a las 30 y 36 semanas. En aves mayores, las cepas del virus de bronquitis detectadas fueron más diversas que a los días 1 y 7 de edad. Sin embargo, las combinaciones de vacunas vivas todavía se utilizan ampliamente para disminuir las pérdidas causadas por el síndrome de la falsa ponedora en las parvadas de postura de huevo comercial en todo el mundo.

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.

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.

Retrospective Study of Histomoniasis (Blackhead) in California Turkey Flocks, 2000-2014.

The flagellate parasite Histomonas meleagridis causes a severe disease in turkeys. Since nitarsone, the last antiflagellate feed additive, was withdrawn from the market, H. meleagridis has gained increasing clinical and economic importance, and frequently entire turkey flocks are lost to the disease. Even before the antiflagellate market withdrawal, H. meleagridis has been recognized as a noteworthy disease in commercial turkeys. The aim of this study was to compile and analyze data on histomoniasis outbreaks diagnosed by the California Animal Health and Food Safety System in the years 2000 through 2014 while nitarsone was still available. Sixty-two cases were included in the study. In all cases, the parasite was detected by histopathology. Five cases were from breeder flocks, 44 from commercial meat turkey flocks, and 13 from other flocks such as backyard flocks or pet turkeys. There were between one and eight cases each year with no clear trend over time. Most cases occurred in the warmer months between April and October with a clear peak in August. Turkeys in the flocks with the disease were aged between 2 wk and 15 mo, with a median age of 9 wk. In cases for which mortality was reported, daily mortality ranged between 0.04% and 2% with an average of 0.34% and a median of 0.15%. Five-week mortality in infected houses on three infected ranches was between 1.3% and 68%. In 12 cases, the parasite was observed in organs other than ceca and liver. These included spleen, kidney, bursa of Fabricius, proventriculus, lung, pancreas, and crop. In 58 cases, histomoniasis was considered the most significant and primary diagnosis; in four cases histomoniasis was regarded as an incidental finding. In 14 other outbreaks, detailed information was available. In 11 cases, more than one house on the ranch was affected; in five cases the disease spread to all houses. Total mortality in turkeys from affected flocks ranged between 12% and 65%. In eight flocks, the birds were marketed with a weight that was considered too light. In conclusion, the present study shows that even with nitarsone available as antiflagellate feed, additive histomoniasis was a serious disease of turkeys in the United States with an epidemiology similar to that observed in Europe.

Pathology and Tissue Distribution of an LPAI H5N8 of North American Lineage Isolated from an Outbreak in Commercial Japanese Quail (Coturnix c. japonica) in the Central Valley of California.

This report describes the pathology and tissue distribution of avian influenza (AI) antigens by immunohistochemistry (IHC) in the tissues of commercial layer quail from a natural outbreak of low pathogenic avian influenza (LPAI) H5N8. LPAI virus H5N8 of North American lineage was diagnosed in commercial Japanese quail hens ( Coturnix coturnix japonica) in California based on serology, reverse-transcriptase real-time polymerase chain reaction, virus isolation, and sequencing. The sudden increase in mortality in a flock of laying quail hens had prompted the submission of 15 live and 5 dead, 10- to 15-wk-old quail to the California Animal Health and Food Safety Laboratory System, Turlock branch in the beginning of April 2014. There was mild bilateral swelling of the eyelids and greenish diarrhea in 4/15 live quail submitted. On postmortem examination, there were severe, extensive hemorrhages and multifocal, confluent pale foci in the pancreas in 10/20 birds. Liver gross lesions in five birds ranged from a few pale areas to numerous disseminated foci. Histology revealed moderate to severe necrosis of acinar cells in the pancreas with little or no inflammation in most of the birds. Livers had acute multifocal coagulative necrosis of hepatocytes with fibrin exudation and infiltration of few to large numbers of heterophils and lymphocytes randomly scattered throughout. The AI virus was detected in the nucleus and cytoplasm of pancreatic acinar cells and hepatocytes by IHC targeting the nucleoprotein of the AI virus. A few birds had AI antigen in the reticuloendothelial cells of the spleen, endothelial cells of the lungs, epithelium of the respiratory mucosa, and lamina propria of the intestine. The severity of the lesions observed in this natural outbreak of LPAI in quail was higher than that expected for the pathotypic presentation in this species.

Trichodinosis associated with pathology of the reproductive tract in waterfowl.

Trichodinid ciliophorans are opportunistic parasites of many species of fish, amphibians, and molluscs, but yet never reported in association with lesions in birds. Postmortem and histopathological evaluation of a commercial adult Toulouse gander and female goose, and a wild Mallard drake revealed the presence of severe pathological parasitic colonization of their reproductive tracts. Histopathological findings included moderate to severe granulocytic inflammation, acanthosis, accentuation of the rete pegs, and proliferative hyperplastic squamous metaplasia of the mucosa of the ejaculatory ducts and groove, sulcus spermaticus, glandular part of the phallus (cavum penis), and oviduct in association with large numbers of ciliated protozoa anchored to the tissues or free in the lumen. These protozoa had characteristic morphological features analogous to the family of Trichodinidae. The source of this parasitism could not be determined. To our knowledge, this is the first report of trichodinosis associated with pathology in birds.

Respiratory tract trichomoniasis in breeder squab candidates in Northern California.

Breeder squab candidates between the ages of 6 and 16 wk were submitted to the California Animal Health and Food Safety Laboratory, Turlock branch, as a result of respiratory distress and increased mortality. These cases were submitted from one Northern California commercial squab operation on three separate occasions occurring between December 2007 and March 2008. Severe trichomoniasis was identified, primarily in the tracheal epithelium and lung of squabs, with few or no lesions in the oral cavity, crop, esophagus, and livers, where the organism commonly infiltrates. Infiltration of the trachea and lung sections with trichomonads was associated with a severe inflammatory response in the surrounding tissue. Diagnosis was confirmed with the use of histopathology and an immunoperoxidase special stain. Oxytetracycline supportive antibiotic therapy to prevent secondary bacterial infections was administered to remaining squabs on the farm, but no specific treatment regimen was instituted. This novel respiratory presentation of trichomoniasis continued over a period of 3 mo, until mortality gradually returned to normal.

Pre-processed bovine milk quality in Trinidad: Prevalence and characteristics of bacterial pathogens and occurrence of antimicrobial residues in milk from collection centres

The study was conducted to assess the impact of the changes in the milk collection system in Trinidad (from twice daily collection to once, introduction of chilling facilities to the collection centres and transportation of milk to the processing plant in insulated truck instead of in metal churns at ambient temperature) on the microbial load and antimicrobial residue quality of the milk as well as the temperature and pH of milk, using standard methods. The presence of antimicrobial residues was detected using the Delvo test kit. Of a total of 266 milk samples from churns, the mean ñ sd temperature and log10 ñ sd TAPC per ml was 20.36 ñ 7.91 øC and 6.3 ñ 1.09 respectively. For 20 milk samples from the chillers, the mean temperature and log10 ñ sd TAPC per ml was 15.10 ñ 2.73 øC and 7.04 ñ 0.33 respectively compared with corresponding values for 36 samples collected from the truck, 11.64 ñ 4.22 øC and 7.11 ñ 0.62 respectively (P < 0.05; X2). The mean TAPC, staphylococcal and E. coli counts per ml of milk from churns were significantly (P < 0.05; X2) higher for milk at low temperature (0–20 øC) compared with milk at high temperature (>30 øC). Eight (4.2 per cent) of 192 milk samples tested contained antimicrobial residues. Of 168 S. aureus isolates tested, 24 (14.3 per cent) were enterotoxigenic while 53 (45.3 per cent) of 117 isolates tested exhibited resistance to various antimicrobial agents while of 386 isolates of E. coli tested, 3 (0.8 per cent) were O157 strain and 129 (64.5 per cent) of 200 isolates exhibited resistance to antimicrobial agents. It was concluded that despite the changes, the microbial load of milk was still high suggesting poor sanitary practices at the farm level. The detection of antimicrobial residues agents coupled with toxigenic S. aureus and E. coli isolates could pose health hazards to consumers.