Development of a lavage procedure to collect lung secretions from chickens for evaluating respiratory humoral immunity

Abstract

Mucosal immunology research has been hampered by the difficulty and labour-intensiveness of collecting samples. This is especially true for sites such as the lung, and the present paper describes a simple method for obtaining samples from this organ in chickens. Following sacrifice, the bird was placed on its back and the trachea was cut and exteriorized. Narrow-diameter tubing, to which a 30 ml syringe was attached, was threaded down the trachea to the bronchi and air was evacuated from the lung. Warm buffer was administered and the lung sample then aspirated, processed and frozen. In the current experiment this sampling system was tested on hens that were challenged with Salmonella Enteritidis. Elevated anti-Salmonella Enteritidis antibody levels in lung from infected hens were observed in significantly more infected hens than non-infected control hens in two trials. The simplicity and utility of this sampling system will make it a useful tool for those laboratories wishing to expand their humoral mucosal immunology capabilities, even for study of non-respiratory pathogens.

Développement d'une procédure de lavage pour récolter les secrétions pulmonaires des poulets permettant d'évaluer l'immunité humorale respiratoire

Les recherches sur l'immunologie muqueuse ont été freinées par la difficulté et la lourdeur du travail de récolte des échantillons. C'est principalement vrai pour certains organes tels les poumons et le présent article décrit une méthode simple pour obtenir des échantillons à partir de cet organe chez le poulet. Après euthanasie, l'animal est placé sur le dos et la trachée est coupée et extériorisée. Un tuyau de faible diamètre, auquel une seringue de 30 ml est attachée, est enfilé dans la trachée jusqu'aux bronches et l'air est évacué des poumons. Un tampon chaud est administré et les échantillons de poumons sont aspirés, traités et congelés. Cette méthode d'échantillonnage a été testée chez des pondeuses qui ont été éprouvées avec Salmonella Enteritidis (SE). Des niveaux élevés d'anticorps anti SE dans les poumons des pondeuses infectées ont été observés chez significativement plus de poules infectées que chez les témoins non infectés, dans deux essais. La simplicité et l'utilité de cette méthode d'échantillonnage en feront un outil utile pour les laboratoires désirant étendre leurs capacités en immunologie humorale muqueuse, voire même pour des études d'agents pathogènes non respiratoires.

Entwicklung einer Spültechnik zur Gewinnung von Lungensekret aus Hühnern für die Untersuchung der humoralen Immunität im Respirationstrakt

Die Untersuchung der Schleimhautimmunität ist bislang durch die Schwierigkeit und Arbeitsintensivität der Probenentnahmen erschwert worden. Dies galt insbesondere für ein Organsystem wie die Lunge. Die vorliegende Studie beschreibt eine einfache Methode zur Probengewinnung aus diesem Organ bei Hühnern. Unmittelbar nach der Tötung wurde das Tier auf den Rücken gelegt und die Trachea durchtrennt und vorgelagert. Ein Tubus mit engem Durchmesser, auf den eine 30-ml-Spritze aufgesetzt war, wurde in die Trachea bis zu den Bronchien eingeführt und die Luft aus der Lunge evakuiert. Anschließend wurde warme Pufferlösung eingegeben und dann die Lungenspülprobe aspiriert, bearbeitet und tiefgefroren. In dem beschriebenen Experiment wurde diese Spülmethode an Hennen getestet, die mit Salmonella Enteritidis (SE) infiziert worden waren. In zwei Versuchen wurden in den Lungen erhöhte Anti-SE-Antikörpertiter bei signifikant mehr infizierten als nicht infizierten Kontrollhennen festgestellt. Die Einfachheit und Anwendbarkeit dieser Probenentnahmemethode macht sie zu einem nützlichen Werkzeug für die Laboratorien, die ihre Fähigkeiten auf dem Gebiet der humoralen Schleimhautimmunologie auch für die Untersuchung nicht-respiratorischer Erreger erweitern möchten.

Desarrollo de un procedimiento de lavado para recoger secreciones pulmonares en pollos para evaluar la inmunidad humoral respiratoria

La investigación en la inmunidad de mucosas ha encontrado dificultades debido al hecho que la recolección de muestras es laboriosa. Esto es especialmente cierto para muestras como el pulmón y el presente trabajo describe un método simple para obtener muestras de este órgano en pollos. Tras el sacrificio, el ave se colocaba sobre la espalda y se cortaba y exteriorizaba la tráquea. Se intubaba mediante un tubo de diámetro estrecho al cual se insertó una jeringa de 30 ml, y se colocaba por la tráquea, hasta los bronquios, para evacuar el aire de los pulmones. Una solución tampón caliente era administrada a la muestra de pulmón y después era aspirada, procesada y congelada. En el presente experimento, este tipo de muestreo fue utilizado para testar gallinas que habían sido inoculadas con Salmonella Enteritidis (SE). Se detectaron niveles elevados de anticuerpos anti-SE en pulmones de gallinas y las diferencias de estos anticuerpos en gallinas infectadas y no infectadas fueron significativas en dos experimentos. La simplicidad y la utilidad de este sistema de muestreo lo hará una herramienta útil para aquellos laboratorios a los cuales les interese expandir su capacidad para el estudio de la inmunidad humoral de mucosas, incluso para el estudio de patógenos no respiratorios.

Introduction

Most infections are initiated at one of the many mucosal surfaces in the body, and to counter these attacks the individual develops an immune response at these sites. The immunity can be either cellular or humoral, and both serve important roles in the defence against microbial invasion. Even in the absence of a direct challenge, the current concept of the common mucosal immune system (McGhee et al., 1992) would dictate that infection in one site, such as the alimentary tract, would result in an immune response at distant mucosal surfaces such as the lung. Our group has been developing sampling procedures for different mucosal surfaces in the chicken to enable us to study the interplay of these sites in the evolution of the overall mucosal immune response. The avian lung is the target of infection by a variety of pathogens and a distinct immune response can be detected in this organ. Detecting the respiratory humoral immune response requires the collection of lung lavage samples. Previous sampling procedures used multiple syringe catheter set-ups, stopcocks and blow-up cuffs (Holt, 1979; Fulton et al., 1990; Toth et al., 1987) that were very labour intensive and not suitable for incorporation into the normal immunology laboratory.

In the current manuscript, we describe a simple single-syringe/tubing lavage procedure that allows for the rapid collection of lung lavage samples suitable for use in antibody studies. The system was used on hens challenged orally with Salmonella Enteritidis (SE), an enteric organism not ordinarily known to colonize the lung, to test how readily immunity generated against an intestinal infection can be found in the respiratory tract.

Materials and Methods

Chickens

Groups of 17 and 16 Single Comb White Leghorn chickens (>60 weeks old) were obtained from the specific pathogen free flock maintained at the Southeast Poultry Research Laboratory for trials 1 and 2, respectively. The hens were housed in climate-controlled biocontainment buildings with full access to antibiotic-free feed and water. In trial 1, nine hens were challenged orally with 1 ml of 1×10⁷ SE/ml nalidixic acid-resistant SE while eight hens were left unchallenged in a separate building. At 4 weeks post challenge, the hens were bled from the wing vein and crop lavage samples were obtained as described by Holt et al. (2002).

The hens were then sacrificed and lung lavage samples taken as described in the following. The blood samples were allowed to coagulate and then the sera collected and frozen until analysis. The crop flushes were centrifuged for 5 min at 12 000×g and the supernatants frozen at −20°C until the time for assay. In trial 2, eight hens were challenged as already described with 1 ml of 1×10⁹ SE/ml while eight hens were left unchallenged in a separate room. At 5 weeks post challenge, the hens were sacrificed and lung lavage samples were obtained as described in the following.

Lung lavage protocol

The hens were euthanized by CO₂ inhalation. The chickens were placed on their backs, the skin was opened above the trachea and the trachea was cut. The lung lavage device consisted of 15 cm of 1/16 in internal diameter, 1/8 in outer diameter TygonJ tubing (Fisher Scientific, Norcross, Georgia, USA) inserted 1 cm into a second 15 cm TygonJ tubing (1/8 in internal diameter, 3/16 in outer diameter) that was attached to a syringe containing 10 ml lavage solution (1 M Tris/glycine buffer with 0.25% Tween 20, pH 7 to 8). The narrow diameter tubing was inserted down the trachea to the syrinx (Figure 1a). The large tubing sealed the trachea. Air was withdrawn from the lungs through the syringe and the lavage fluid was administered slowly into the lung and then withdrawn (Figure 1b). Blood-tinged samples were routinely discarded. Processing of the lung lavage sample entailed centrifugation for 5 min at 12 000×g and freezing the supernatants at −20°C until the time for assay.

Figure 1.  Procedure for obtaining a lavage sample from chickens. 1a: Tubing is inserted into the trachea down to the syrinx. 1b: Lavage fluid is injected into the lung and then aspirated back into the syringe.

Serum, crop and lung response to SE infection

For assay, the lung lavage and crop samples were thawed and serial two-fold dilutions using phosphate-buffered saline containing 0.05% Tween 20 (Sigma Chemical Co., St Louis, Missouri, USA) were made. For the serum samples, an initial 1:250 dilution of the serum was made and then serial two-fold dilutions were made thereafter. The dilutions were added to enzyme-linked immunosorbent assay trays to which 10 µg/ml SE lipopolysaccharide (LPS) (Sigma) had been adsorbed and were assayed as previously described (Holt et al., 1999). Titres were determined as the last dilution giving an OD₄₀₅ reading 1.5 times that of the negative control obtained previously from an uninfected chicken. Significant differences between the percentage of response positives in the lung, crops and serum from non-infected birds versus infected birds were determined via Students t test (Shott, 1992).

Results and Discussion

The described lung lavage procedure permits simple, rapid collection of lung samples with minimal labour, enabling a broader group of individuals to conduct lung immunity research. Salmonella infections are not routinely thought to include the respiratory tract and, as a result, little attention has been accorded this region with respect to immune responses. In the current study, hens were orally infected with SE and the progression of immunity in the serum, crop and lung were evaluated. In trial 1, eight of nine hens 4 weeks post oral SE challenge showed elevated serum IgG anti-SE antibodies (P<0.05) with average titres of 500, while one of eight of the non-challenged hens was positive (Figure 2a). In the alimentary tract, an IgA anti-SE response was observed in nine of nine crop samples from challenged hens (P<0.005) with average titres of 640 compared with two of eight of the non-challenged hens (Figure 2b), one having a titre of 10 and the second of 160. These results indicate that a good alimentary immune response resulted from the SE infection. In the lung, a specific although much more modest response was observed. Specific IgA anti-SE antibodies were detected in eight of nine lung lavage samples obtained from the infected hens, although the bulk of the titres were 20 with one individual each having a titre of 40, 80, or 360. Three of eight non-challenged hens tested positive with titres of 10. As no SE could be cultured from these non-challenged controls, the positive responses probably reflect LPS determinants shared between members of the Enterobacteriaceae generating cross-reactive antibodies detectable by the LPS enzyme-linked immunosorbent assay. In trial 2, eight of eight lung lavage samples were positive compared with one of eight of the non-challenged controls (P<0.001). Antibody responses in the lung were much more robust in this trial with the challenged hens exhibiting an average titre of 168 while the average titre in non-challenged hens was 2.5.

Figure 2.  2a: Plot of the percentage of positive IgG anti-SE samples in serum and IgA anti-SE samples in the crop and lung flushes from non-infected hens (filled bar) versus infected hens (open bar) in trial 1. Numbers in parentheses represent the mean titres for each group. * Significantly different from non-infected controls (P<0.05). 2b: Plot of the percentage of positive IgA anti-SE sample lung flushes from non-infected hens (filled bar) versus infected hens (open bar) in trial 2. Numbers in parentheses represent the mean titres for each group. * Significantly different from non-infected controls (P<0.05).

The possibility of respiratory immunity due to SE entering and infecting the lung, either through aspiration during challenge or through airborne transmission, could not be ruled out. Chickens can be infected in the respiratory tract with Salmonella spp. (Baskerville et al., 1992; Cooper et al., 1996; Lever & Williams, 1996; Nakamura et al., 1997; Gast et al., 1998; Holt et al., 1998; Leach et al., 1999). However, during administration of the challenge organism, the hens exhibited no respiratory signs indicative of aspiration of the inoculum and, in previous studies of airborne transmission, lung colonization was shown to be low (Gast et al., 1998). The results more probably indicate that an infection in the bowel can result in immunity at other mucosal sites such as the lung.

Translations of the abstract in French, Germany and Spanish are available on the Avian Pathology website.