A comparison of methods of inducing lachrymation and tear collection in chickens for detection of virus-specific immuoglobulins after infection with infectious bronchitis virus

Abstract

Four-week-old specific-pathogen-free chickens were infected with virulent infectious bronchitis virus Massachusetts strain M41 via the ocular–nasal route. At weekly intervals up to 3 weeks post-infection, excess lachrymation was induced either by placing sodium chloride (salt) crystals on the eyes or by intramuscular injection of carbachol. Tears were collected using micropipettes or on filter paper. Levels of virus-specific immunoglobulin (Ig)A and IgG were similar, irrespective of the method of tear induction. When tears were collected using filter papers, IgG was detected in eluted samples but at significantly lower levels than in those collected by pipette. Collection of IgA in this way was even less productive, and only trace amounts were detected. Possible reasons for these discrepancies have been discussed.

Comparaison de méthodes induisant un larmoiement et permettant la récolte des larmes chez le poulet pour la détection des immunoglobulines spécifiques de virus après une infection par un virus de la bronchite infectieuse.

Des poulets, indemnes de microorganismes pathogènes spécifiés, ont été infectés par la souche virulente Massachusetts M41 du virus de la bronchite infectieuse via la voie oculonasale. Toutes les semaines jusqu’à la troisième après l'infection, un excès de larmes a été induit soit en plaçant des cristaux de chlorite de sodium sur les yeux ou en injectant par voie intramusculaire du carbachol. Les larmes ont été récoltées en utilisant des micropipettes ou des filtres à papier. Les niveaux d'IgA et d'IgG spécifiques de virus ont été similaires, quelle que soit la méthode d'induction des larmes. Pour les larmes récoltées sur papier filtre, les IgG ont été détectées dans des échantillons élués mais à des niveaux significativement inférieurs à celles récoltées à l'aide de pipettes. La récolte des IgA de cette façon a été encore moins productive et seules quelques traces ont été détectées. Les raisons possibles pour ces divergences sont discutées.

Ein Vergleich von Methoden zur Induktion der Tränensekretion und zur Tränensammlung bei Hühnern zum Nachweis virusspezifischer Immunglobuline nach Infektion mit dem Virus der infektiösem Bronchitis

Vierwöchige spezifisch-pathogen freie Hühner wurden mit virulentem Massachusetts-Stamm M41 des Virus der infektiösen Bronchitis (IBV) via okulonasaler Route infiziert. In wöchentlichen Intervallen bis zur dritten Woche post infectionem wurde eine übermäßige Tränensekretion entweder durch Auflegen von NaCl (Salz)-Kristallen auf die Augen oder durch intramuskuläre Injektion von Carbachol induziert. Die Tränen wurden mittels Mikropipetten oder Filterpapier gesammelt. Der Level virusspezifischer IgA und IgG war unabhängig von der Methode der Träneninduktion ähnlich. Bei der Tränensammlung mit Hilfe von Filterpapier wurde IgG in den gelösten Proben nachgewiesen, jedoch mit signifikant geringeren Gehalten als in den mit der Pipette gesammelten. Die Sammlung von IgA auf diese Art war sogar noch weniger ergiebig, so dass nur Spuren des Immunglobulins nachgewiesen wurden. Die möglichen Gründe für diese Diskrepanzen werden diskutiert.

Comparación de los métodos para inducir lagrimeo y recolección de lágrimas en pollos para detectar inmunoglobulinas virus-específicas tras la infección con virus de bronquitis infecciosa

Pollos libres de patógenos específicos de cuatro semanas de edad fueron infectados con la cepa Massachusetts M41 de virus de bronquitis infecciosa virulento (IBV) por vía oculonasal. A intervalos semanales hasta las 3 semanas postinfección, se indujo el exceso de lagrimeo mediante la introducción de cristales de cloruro sódico (sal) en los ojos o mediante inyección intramuscular de carbacol. Se recogieron lágrimas mediante micropipetas o en papel de filtro. Los niveles de IgA e IgG virus específicas fueron similares, independientemente del método de inducción al lagrimeo. Cuando las lágrimas fueron recogidas mediante papeles de filtro, las IgG fueron detectadas en muestras eluidas, pero a niveles mucho menores que las recogidas con pipetas. La recolección de IgA de esta forma fue incluso menos productiva, y únicamente se detectaron cantidades mínimas. Se discuten las posibles razones de estas discrepancias.

Introduction

Infectious bronchitis virus (IBV) induces a range of immune responses in chickens (Dhinakar Raj & Jones, 1997), with local immunity playing a major role in resisting infection, virus clearance and protection against challenge (Gomez & Raggi, 1974; Gillette, 1981; Hawkes et al., 1983; Dhinakar Raj & Jones, 1996). Hence, various researchers have studied the role of the Harderian gland in IBV-infected chickens and the presence of antibodies in the lachrymal fluid (Survashe et al., 1979; Davelaar et al., 1982; Cook et al., 1992; Toro et al., 1993; Dhinakar Raj & Jones, 1996; Toro et al., 1996). In order to do this, especially in young chicks, the induction of excess lachrymal fluid is essential and two main methods are commonly used. One is the application of a few crystals of sodium chloride to the eyes (Toro et al., 1993, 1996; Dhinakar Raj & Jones, 1996; Heckert et al., 2002) and the other requires the intramuscular injection of carbachol (Aitken et al., 1975; Cook et al., 1992). Salt application is cheap, simple to perform, excess lachrymation occurs almost immediately and the chicks recover from the initial irritation very quickly. Carbachol injection is more expensive, there is a delay of about 2 min before tears are produced and unwanted excess salivation also occurs. The two methods have never been compared directly to determine their relative efficiency in inducing amounts of lachrymal immunoglobulin (Ig)A or IgG. This paper describes an experiment to compare levels of virus-specific immunoglobulins in tears induced by these methods, using chicks experimentally infected with virulent IBV.

In poultry disease investigations by serology, sera are normally separated from whole blood collected from the brachial or jugular veins using needles and syringes. An alternative method of collection is to puncture the vein with a needle, and absorb the blood onto filter papers (Brugh & Beard, 1980; Ambali & Jones, 1991; Thangavelu et al., 2000). This approach is particularly convenient for multiple sampling under commercial conditions. In the laboratory, serum can be eluted from the filter papers and tested in the usual way. Tear collection in experimental chicks is usually done using micropipettes and requires care in execution. The filter paper method would seem to provide a simple and safe alternative, following induction of lachrymation by the aforementioned methods. In addition to experimental situations, it might also have application where local immune responses are being measured in field trials of vaccines. Thus, this paper also evaluates the filter paper technique for the collection of lachrymal fluid and subsequent measurement of virus-specific immunoglobulins after IBV infection.

Materials and Methods

Chicks

White Leghorn specific-pathogen-free chicken eggs (Lohmann Animal Health, Cuxhaven, Germany) were incubated and hatched at our laboratory. Chicks were housed in isolation rooms. Food and water were provided ad libitum.

Infectious bronchitis virus

IBV Massachusetts strain M41 was kindly supplied by Dr J.K.A. Cook (Houghton, UK) The virus underwent 10 passages in tracheal organ cultures and four passages in embryonated chicken eggs, after which the titre was 6.9 log₁₀ median embryo infective doses per millilitre

Experimental design

Thirty-six 4-week-old specific-pathogen-free chickens were separated into two isolation rooms, one for the uninfected controls and another for the infected group. There were two well-separated cages in each room, and in each was placed eight or 10 chickens (Table 1). Each bird in the infected group was inoculated with 100 μl IBV by the oculo-nasal route, and the two uninfected control groups were sham-inoculated with virus isolation medium (Eagle's serum-free minimum essential medium with glutamine, streptomycin [50 μg/ml] and penicillin [50 IU/ml]). At 7, 14 and 21 days post-infection, the following methods were used to induce lachrymation:

Table 1. Experiment design to study the levels of immunoglobulins in tears induced by salt or carbachol, and collected by pipettes or filter paper

			Eyes for lachrymal collection
Group	Designation^a	n	Right^b	Left^c
Uninfected
Salt	C salt	8	Pipette	Paper
Carbachol	C carba	8	Pipette	Paper
IBV-infected
Salt	IBV salt	10	Pipette	Paper
Carbachol	IBV carba	10	Pipette	Paper
^aUC salt, uinfected control chickens, tears induced using salt; C carba, uninfected control chickens, tears induced using carbachol; IBV salt, IBV-infected chickens, tears induced using salt; IBV carba, IBV-infected chickens, tears induced using carbachol.
^bTears collected using micropipette and tips.
^cTears collected using filter paper.

Carbachol

Chickens in one control group and one IBV-infected group were injected with carbachol (Sigma-Aldrich, St Louis, MO, USA) at a dose of 0.4 mg/kg (Cook et al., 1992) into the pectoral muscle. Approximately 2 min later, excess tear secretion was induced.

Salt

Approximately 0.003 g fine sodium chloride crystals were sprinkled onto one eye while keeping the eyelids held open (Dhinakar Raj & Jones, 1996), and within 1 min excess lachrymation occurred.

At each sampling time tears were collected from at least four birds using the following two methods.

Micropipette (fluid)

Tears were allowed to accumulate before being carefully collected by a pipette. The fluid was immediately placed in 1.5 ml microfuge tubes, and stored at −70°C.

Filter paper

Accumulated tears were absorbed onto a Whatman number 1 filter paper and the absorbed area was marked with a pencil. Thereafter the papers were treated in a similar manner as for the blood-spot filter papers described previously (Brugh & Beard, 1980; Ambali & Jones, 1991). The papers were dried at 37°C for 2 h, and were then placed in airtight plastic bags, which were sealed and stored at 4°C until used (Brugh & Beard, 1980; Ambali & Jones, 1991).

Detection of antibodies

Tears collected by pipettes or on filter paper were processed as described in the following for testing by enzyme-linked immunosorbent assay (ELISA).

Pipette (fluid)

Samples were thawed prior to the assay and diluted 1:10 in phosphate-buffered saline (Dhinakar Raj & Jones, 1996), and 50 μl was used in the ELISA.

Filter papers

These were prepared as described by Thangavelu et al. (2000). Briefly, after absorbing tears, 5 mm diameter discs were cut from the filter paper using a hand punch. Two discs from each sample were placed in a well of a flat-bottomed 48-well plate, and eluted with 100 μl phosphate-buffered saline (pH 7.2). For elution, the plate was placed on a shaker for 2 h at room temperature and thereafter left static at 4°C overnight. ELISAs were performed the next day using 50 μl eluted supernatant of each sample.

IBV-specific IgA and IgG in tracheal washes were assayed using an indirect ELISA (Dhinakar Raj & Jones, 1996), except that the plates were coated with partially purified IBV M41 antigen. This was done by ultracentrifugation at 20 000×g for 90 min of previously clarified allantoic fluid from eggs inoculated with IBV 48 h previously. The resulting pellet was washed and resuspended in phosphate-buffered saline (pH 7.2). The virus suspension was overlaid onto 25% sucrose and ultracentrifuged at 20 000×g for 3 h. The pellet was then washed and resuspended in phosphate-buffered saline, aliquoted and stored at −70°C until use. Monoclonal antibodies to the immunoglobulins were kindly provided by Dr T.F. Davison (Institute for Animal Health, Compton, Newbury, UK). Corrected optical density values were calculated by deducting the optical density values of non-antigen-coated wells from those of the test wells (Fournier-Caruana et al., 2003). The mean antibody corrected optical density values were compared using a Students t test.

Results

Observations on methods of induction and collection of tears

Excess lachrymation was successfully induced by carbachol injection and salt application, but the latter usually elicited a greater volume of tears within a shorter period of time. Approximately 200 to 400 μl and 100 to 200 μl could be collected using the salt and carbachol methods, respectively. In addition, if needed, more tears could be induced with further application of salt, but this was not possible with carbachol as excessive salivation resulted in difficulty in breathing. Induction of hypersalivation made tear collection more difficult in view of possible contamination of the tears due to frequent head-shaking. Furthermore, as carbachol need to be injected intramuscularly, it stimulated a systemic effect, so excessive lachrymation was concurrently produced in both eyes. This was inconvenient, as it was difficult to collect tears from both eyes at the same time. This was not a problem with salt, as tears were produced only with local application to each eye.

For lachrymal fluid collection, the use of filter paper was simple and convenient. More careful restraining of the birds and its eyelids are required when accumulated tears are withdrawn into pipette tips.

Detection of antibodies

Pipettes

For IBV-infected chickens, levels of virus-specific IgA and IgG in tears induced using either salt or carbachol were identical irrespective of the methods used (Figures 1 and 2).

Figure 1. IBV-specific IgA in tears induced after salt application or carbachol injection, and collected using pipette tips. No significant differences between the groups.

Figure 2. IBV-specific IgG in tears induced after salt application or carbachol injection, and collected using pipette tips. No significant differences between the groups.

Filter paper disc

Only trace amounts of virus-specific IgA were detected after either method of tear induction (data not shown). However, for IgG similar levels were detected after both methods of collection (Figure 3). At 21 days post-infection, levels of IgG in tears obtained using filter papers were significantly (P<0.001) lower than those collected using pipettes (Figures 2 and 3), while at 14 days post-infection significantly (P<0.003) lower levels of IgG were found only between the carbachol groups (Figures 2 and 3).

Figure 3. IBV-specific IgG in tears induced after salt application or carbachol injection, and collected using filter papers. No significant differences between the groups.

Discussion

Excess lachrymation was induced using either salt or carbachol, and it appears that levels of IgA and IgG in tears of IBV-infected chickens were similar, irrespective of the method of induction. However, in our experience, the use of salt is preferred to carbachol, as it is cheap, convenient to use and a sufficient volume of tears can be produced within a short period of time. In contrast, carbachol needs to be injected intramuscularly and it takes longer to induce tear secretion, which is sometimes insufficient in volume. Furthermore, it also causes hypersalivation, resulting in head-shaking, which increases the risk of contaminating the tears. In such circumstances, birds must be carefully restrained. One advantage of carbachol is that it does not irritate the eyes, unlike the application of the salt. However, after salt application, the eyes appear normal after 10 to 15 min.

In this experiment, tears collected using pipette tips were stored at −70°C but those collected on filter papers were air-dried and stored at 4°C as described for blood-spot collection (Brugh & Beard, 1980; Ambali & Jones, 1991). After such storage, only trace amounts of IgA were detected in the eluted supernatants. In addition, levels of IgG in the same samples were significantly lower than in those collected using tips and stored at −70°C. There are several factors that could have been responsible for this. First, it is possible that the IgA could have been lost due to the adsorption onto the wells of the plastic plates used for elution from the discs. In a small ancillary study in which wells used for elution were tested for residual IgA by an ELISA, using anti-immunoglobulins monoclonal antibodies, it was shown that substantial amounts of each immunoglobulin were indeed retained on the plastic wells (Ganapathy & Jones, unpublished). Second, the temperature and duration of storage of the discs could have been important. In the current study, the filter papers were tested after 4 to 7 weeks at 4°C, and this is likely to have contributed to the poor detection rates. Working with human tears collected using capillary tubes, Sitaramamma et al. (1998) reported that protein concentrations of immunoglobulins remained stable for only 1 week when stored at 4°C but for up to 4 months at −70°C. Finally, the tears may have been over-diluted, since the elution was done according to a protocol established for blood (Thangavelu et al., 2000). This is probable, as the amounts of IgG in the tears, mostly due to transudation (Toro et al., 1993), are likely to be less concentrated than the blood.

In conclusion, both methods induced excess tear production but salt was more convenient and simpler to use. Furthermore, it appears that the salt concentration in the collected lachrymal fluids did not compromise the sensitivity of immunoglobulin detection, as similar levels of IBV-specific IgA and IgG were detected irrespective of lachrymal induction methods. For tear collection, both methods could be used but the filter paper technique needs further work before it can be recommended. In particular, the problems associated with tear dilution and storage need to be addressed.

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