Clinical observations and pathology of the reproductive tract associated with natural infection of an avian influenza (AI) virus in an AI vaccinated layer flock

Published on: 6/9/2017
Author/s : Magdalena Escorcia 1, Juan Carlos Del Río 2, Daniel Marrufo 1, Félix Sánchez Godoy 1, Seneca Comonfort-Díaz 1, Beatriz Gómez 3, Elizabeth Castillo-Villanueva 3. / 1 Departamento de Medicina y Zootecnia de Aves. Universidad Nacional Autónoma de México, México, DF, México; 2 Unidad de Investigación Multidisciplinaria, Universidad Nacional Autónoma de México, Cuautitlán Izcalli, Edo de México, México; 3 Departamento de Microbiología y Parasitología. Universidad Nacional Autónoma de México. México, DF, México.

This paper describes the clinical findings, gross and microscopic lesions of the reproductive tract, and the negative effects on productive parameters (egg drop, decrease of egg quality), observed in a flock of vaccinated commercial layers affected by the highly pathogenic avian influenza H7N3 in Mexico during the outbreak that occurred from Summer 2012 to Summer 2013. The viral infection was demonstrated by immunochemistry and immunofluorescence assays. The morphopathological diagnosis included severe, necrotic, and serofibrinous transmural salpingitis, with necrotic vasculitis and multifocal thrombi. According to the findings described in this study, the application of the vaccine derived from the AIV isolate A/cinnamon teal/Mexico/2817/2006H7N3, applied only once, with a dose of 512 hemagglutination units (HAU) did not elicit effective protection against the disease, as opposed to the observations under experimentally controlled conditions that have been published. It is possible that several factors in the field may have an impact on the vaccine’s efficacy.


Key Words: Avian Influenza, reproductive lesions, vaccine dose, vaccination schedule.


In June 2012, a highly pathogenic avian influenza (HPAI) virus, subtype H7N3, was identified as the cause of a severe disease outbreak in commercial layer farms in the Western State of Jalisco. Approximately 22.4 million birds died due to the infection or to the preventive stamping out of neighboring farms.6 On July 26, 2012, a national vaccination program was established using an inactivated H7N3 avian influenza (AI) vaccine in commercial layer farms, inside the area under control, in conjunction with other measures to limit the spread of the disease.7 The HPAI viruses involved in this outbreak shared a genetic identity of 97% with other viruses isolated from wild birds in North America.4


The suspected infection was reported in July 2012 by the veterinarian in charge of the farm. The farm was located in a zone where previous cases had been reported and confirmed.9 Since the official report from the Mexican animal health authorities, there was an increase in clinical surveillance, and there was a coordinated effort to evaluate egg production curves of at-risk farms. During the outbreak, clinical histories were monitored for clinical signs and lesions in the reproductive tract, as well for egg production records.


The layer flock described in this report was a commercial 60-week-old Hy Line W-36 flock located in Acatic, Jalisco. This flock had been previously vaccinated at 51 weeks of age with one dose (0.5 ml) of an inactivated, oil emulsion avian influenza vaccine, subtype H7N3, elaborated with the virus A/cinnamon teal/Mexico/2817/2006H7N3, with a dose of 512 hemagglutination units (HAU) per bird. The mortality rate remained constant at 0.1% per day, prior to the onset of clinical signs (between weeks 57 and 57.4). Based on clinical observations, it was estimated that the natural challenge to the HPAI virus occurred around 57 weeks of age. At 57.5 weeks of age, a steady and continuous increase in the mortality rate was observed with a peak at 58.4 weeks (6 days after the onset of increased mortality) with 6.7 per day. After peak, mortality dropped to 3.9% per day for the next few days and dropped to 1.6% per day two days later. Mortality continued dropping until it reached 0.1% per day, approximately 13 days later. At 57 weeks of age, the hen-day egg production (EHD) was 90.8% and declined according to a normal egg production curve. However, with the onset of mortality, the EHD also dropped. The lowest EHD percentage (31.8%) did not coincide in time with the peak of mortality. The EHD reached its lowest level seven days after the peak of mortality, and although egg production started an apparent recovery, it remained very low.


At 60 weeks of age, when birds apparently had recovered from the infection as determined by a normal mortality rate, the flock continued to lay eggs that were different sizes, soft shelled, wrinkled, white speckled, too porous, and sometimes shell-less. Furthermore, some eggs with small yolks and the lack of thick albumin were also observed. A lack of differentiation between true and false layers was also observed.


Post-mortem examinations revealed in most birds congested follicles, the presence of caseous exudates in oviducts, internal laying, and peritonitis. Furthermore, in some of the hens examined, atrophic ovaries and oviducts were observed.


Complete oviduct samples were obtained, fixed in formalin solution, and routinely processed for histopathology; then they were stained with hematoxylin and eosin. In the oviduct lumen, there were extensive zones of necrosis characterized by abundant cell debris mixed with fibrillary, eosinophilic material indicative of fibrin, eosinophilic, spherical deposits of yolk and inflammatory cells consisting of heterophils and a few macrophages. Extensive necrosis zones regarding the lining of epithelial cells and glandular epithelium were observed; some of these glands were dilated and had large amounts of cellular debris. Submucosa, muscularis, and serosa layers were also expanded due to the presence of abundant homogenous eosinophilic material mixed with fibrin. Blood vessels were surrounded with aggregates of inflammatory cells, including lymphocytes, a few plasma cells, and macrophages. The wall in some of these blood vessels was thickened and intensely eosinophilic (fibrinoid vasculitis), and in some areas, thrombi were noted (Figure 1). Severe acute, necrotic, and serofibrinous transmural salpingitis with necrotic vasculitis and multifocal thrombi was issued as morphological diagnosis. The same lesions were observed in the different portions of the oviduct.


Figure 1. 1A.Photomicrograph of magnum, with large areas of necrosis of the tubular glands of the mucosa (arrows), copious amounts of serofibrinous exudate in the submucosa and muscular organ (asterisk), aggregates of cells around blood vessels (head arrow), and fibrinoid necrosis of the vessel wall (silhouetted arrowhead). Likewise, the serosa exhibits extensive areas of necrosis (rectangle). Hematoxylin and eosin. Bar=200 µm. 1B. Photomicrograph of a blood vessel in the submucosa of the magnum. The wall has segmental fibrinoid necrosis interspersed with inflammatory cells (arrow) and perivascular lymphocyte (silhouetted arrow). Hematoxylin and eosin. Bar=50 µm. 1C. Photomicrograph of magnum. The muscle layer is expanded by copious amounts of exudate serofibrinous (asterisks) interspersed with heterophiles (arrows). Hematoxylin and eosin. Bar=50 µm. 1D. Photomicrograph of the magnum showing intense immunopositivity in the superficial and deep areas of the tubular glands (arrows). Immunohistochemistry Technique. Bar=500 µm.


Infected cells were identified by a granular yellowish-brown staining and by fluorescent cells (FITC) observed in the uterus lumen as well as in necrotic ciliated and glandular cells (Figures 2). Furthermore, non-necrotic cells of tubular glands showed marked positivity in the nucleus, cytoplasm, and basal membrane. Immunopositivity was also observed in macrophages, endothelial cells, and muscle fibers.


Figure 2. Localization of the viral NS1 protein in hen’s uteruses. Representative micrographs of hens’ uteruses showing the distribution of the NS1 viral protein in the infected tissue. A) Magnification X10 and B) Magnification X20.


This avian influenza outbreak marks the second time that Mexico has faced a challenge from a highly pathogenic strain of avian influenza virus; the first one occurred in 1994 with a subtype H5N2. It was rapidly controlled and eradicated.11 This second outbreak was caused by a subtype H7N3, and its control has been taking longer. Previously published research3,10 has demonstrated the importance of antigen concentrations in vaccine preparation. However, there is not enough information on the number of vaccine doses, frequency of application, or serological titers to avoid the infection and dissemination of the virus in commercial laying hens. 3,10


In agreement with Ziegler et al. in 199911 and Kinde et al. in 20035, who demonstrated damage in the reproductive tract from AIVs of low pathogenicity, we also observed similar lesions in the oviduct and reproductive tract (salpingitis).


The isolated virus involved in the described report was characterized by the Mexican animal health authorities through molecular techniques, and was confirm to be regarded as highly pathogenic avian influenza subtype H7N3.6


A particular characteristic of this outbreak in commercial layers is that, in recovered birds, there was a return to normal appetite and mortality; however, egg quality and production rates were not recovered. These observations suggest that the tissue damage originated by the infection on the mucosal oviduct, resulting in scarring and not tissue regeneration that correlated with the loss in egg quality and production.


It is important to emphasize the need to make public all knowledge generated about this agent. We cannot accept the possibility of living with this virus; our work must be directed towards its eradication. The only way to achieve this is through improved knowledge.1



The authors thank Maria J. Gomora and Rodriguez-Mata V for their technical assistance in the histological process and Dr. Armando Mirande and Alejandro Banda Castro for helping with the English editing of this manuscript.



The present work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT; Grant #126619 and #78862), by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica-UNAM (PAPIIT-UNAM; Grant # IN-201110 and IN-204007) and by the School of Medicine, UNAM.



The handling and sampling of animals was performed as indicated in the Mexican Official Code (Norma Oficial Mexicana) 062-ZOO-1999, Technical Specifications for the Production, Care, and Use of Laboratory Animals, Chapter 9: Euthanasia. The disposal of remains was performed according to the National System of Animal Health Emergencies under the terms of Article 78 of the Federal Law on Animal Health, to diagnose, prevent, control, and eradicate the virus Avian Influenza A, subtype H7N3.2



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2. FMVZ: Facultad de Medicina Veterinaria y Zootecnia. Norma Oficial Mexicana. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio: 2014

3. García A et al. Efficacy of inactivated H5N2 influenza vaccines against lethal A/Chicken/Queretaro/19/95 infection. Avian Dis. 1998; 42 (2): 248–56

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9. SENASICA: Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria. Julio 2012: Acuerdo mediante el cual se activa, integra y opera el dispositivo nacional de emergencia en salud animal, en los términos del articulo 78 de la Ley Federal de Sanidad Animal, con objeto de diagnosticar, prevenir, controlar y erradicar el virus de la Influenza Aviar tipo A, subtipo H7N3:

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12. Ziegler AF et al. Characteristics of H7N2 (nonpathogenic) avian influenza virus infections in commercial layers in Pennsylvania, 1997-98. Avian Dis. 1999; 43: 142–149

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