Are anti-ganglioside antibodies associated with proventricular dilatation disease in birds?

Experimental design

Two experiments were performed. The first experiment aimed to analyze whether or not the inoculation of gangliosides in an avian species (chickens) could lead to the development of neurological or gastrointestinal signs that could resemble PDD. Only histologic examination was performed in this experiment. A second experiment was performed in order to determine if the inoculation of crude nervous tissue or purified gangliosides in a psittacine species (quaker parrots) could lead to neurological or gastrointestinal disease, resembling those seen in cases of PDD. In this experiment, blood samples were collected in order to evaluate anti-ganglioside antibody and fibrinogen levels. Samples were also collected for histologic examination.

Inoculum preparation and purification

Gangliosides were extracted from brain, spinal cord, sciatic and brachial nerves, and nerve of Remak from ten healthy chickens, and three healthy PaBV-free quaker parrots, using a modified technique (Svennerholm & Fredman, 1980) adapted for this purpose by Pesaro (Pesaro, 2008).

Thin layer chromatography (TLC)

The isolation of brain gangliosides was confirmed using a thin-layer chromatography (Scandroglio et al., 2009). Given the lack of commercially available avian gangliosides, a bovine ganglioside mixture (Millipore, Billerica, MA, USA) was used as the standard for the TLC analysis. The solvent system utilized for this purpose was composed of 50:42:11 volume of chloroform/methanol/0.2% aqueous CaCl₂. After separation, gangliosides were stained using Ehrlich’s reagent, prepared with 6 g of p-dimethylaminobenzaldehyde in 50 ml of 37% hydrochloric acid and 50 ml of 95% ethanol.

Experimental animals

The experiments described here were performed under animal use protocol number IACUC 2014-0006, approved by the Texas A&M University Institutional Animal Care and Use Committee. To determine if the inoculation of brain gangliosides into an avian species could induce neurologic disease, we performed a preliminary study using 17 healthy female, white, leghorn chickens, between 10 and 15 weeks old, and vaccinated for Marek’s disease originated from Texas A&M University Poultry Science Center, which were divided into two groups: the first group was comprised of 11 chickens (C1–C11), inoculated with 1 ml of purified gangliosides and FCA in the pectoral muscle. The second group consisted of six chickens (C12–C17) inoculated only with phosphate-buffered saline (PBS) and served as controls. All chickens were inoculated twice with 31 days between each inoculation, as performed before by Pesaro (2008). The chickens that did not present clinical signs were euthanized 62 days post inoculation (dpi).

The second experiment included 5 healthy adult quaker parrots (Myiopsitta monachus) selected from a colony tested for PaBV every three months by RT-PCR and Western blot, as previously described (Guo et al., 2014), and were consistently negative at all times. The quaker parrots were divided into 3 groups and a solution was injected in the pectoral muscle, as follows: group 1, one quaker parrot (QP1) was inoculated with 0.5 ml of FCA (negative control); group 2, two quaker parrots (QP2 and QP3) were inoculated with 0.5 ml of an emulsion of FCA and purified quaker brain gangliosides; and group 3 (QP4 and QP5) consisted of two quaker parrots inoculated with 0.5 ml of an emulsion of FCA and crude nervous extracts (mixture of brain, spinal cord, sciatic, and brachial nerves, and nerve of Remak). All quaker parrots were inoculated three times, with 24 days interval between each inoculation and euthanized at 83 dpi (Fig. 1). In contrast to the chickens, an additional inoculation was performed in the quaker parrots in order to stimulate the production of antibodies. All animals in this experiment were anesthetized with Isofluorane and then euthanized with carbon dioxide (CO₂). All birds were clinically evaluated daily and any abnormal signs (particularly neurological or gastrointestinal signs) or behavior were documented. Blood samples from quaker parrots were collected at different time points for Western blot, ELISA and fibrinogen determination (Fig. 1).

RT-PCR

The quaker parrots used in this experiment were further tested for PaBV by RT-PCR. Cloacal and choanal swabs were collected for RT-PCR 3 days before inoculation and 25 dpi. Samples from brain, spinal cord and nerves were also collected during necropsy at 83 dpi and tested by RT-PCR. Briefly, RNA was extracted from samples using a QIAamp^® Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) and reverse-transcribed and amplified using primers targeting the PaBV matrix gene, as previously described (Guo et al., 2014). Positive and negative samples from other psittacine birds not related to this study were used as positive and negative controls. The amplification products were analyzed by a 1.5% agarose gel electrophoresis. The nervous tissues used for the preparation of the inoculum were also tested by RT-PCR for PaBV prior to inoculation into the quaker parrots.

Fibrinogen assessment

Plasma samples from quaker parrots were submitted to the Clinical Pathology laboratory at the Veterinary Medicine Teaching Hospital at Texas A&M University for measurement of fibrinogen concentrations using the heat precipitation method as previously described (Millar, Simpson & Stalker, 1971). Samples were collected at 1 dpi, 25 dpi, 48 dpi and 49 dpi (Fig. 1).

Pathology

A systematic necropsy was performed on all birds and representative tissues of CNS, PNS, gastrointestinal tract, skin, musculoskeletal, endocrine and genitourinary systems were collected and processed for routine histological examination.

Serology

Quaker parrot sera were evaluated for reactivity against the crude and purified extracts used for inoculation by Western blot. This technique was performed based on previously described methods (Guo et al., 2014; Towbin et al., 1979). Briefly, crude and purified extracts were mixed with gel loading buffer and electrophoresed on 10% polyacrylamide gels. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane in transfer buffer at 100 mA for 2 h. Membranes were incubated in blocking solution at room temperature for 2 h and then cut into strips and incubated for 2 h with each individual bird serum diluted 1:1,000 in blocking solution and acting as the primary antibody. The membrane stripes were then incubated with alkaline phosphatase labeled goat anti-macaw IgY antibody diluted 1:5,000 in blocking buffer for 1 h. Membranes were rinsed and stained with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) (Sigma-Aldrich, St.Louis, MO, USA).

Measurement of anti-gangliosides antibodies was performed using enzyme-linked immunosorbent assay as previously described (Alaedini et al., 2002), with minor modifications for use with bird serum. Sera from 1 dpi, 25 dpi and 48 dpi from each quaker parrot was analyzed by ELISA and a healthy macaw serum was also used as negative control. Microtiter plates were coated with 100 µl per well of the crude or purified extracts in triplicate, and incubated at 4 °C overnight. Following incubation, wells were blocked with 5% fat-free dried milk in PBST for 1 h. Serum from each bird was diluted 1:5,000 and added to the wells and incubated for 1 h at room temperature. After three washes of PBST, the plates were incubated for 1 h with 100 µl of anti-macaw IgY antibody. TMB (3,3′, 5,5;-tetramethylbenzidine) substrate (Invitrogen, Carlsbad, CA) was added and incubated for 15 min. Sulfuric acid (H₂SO₄) was used to stop the reaction and optical density (OD) was measured at 450 nm. Sera with absorbance values higher than 0.1 were considered positive.

Thin Layer Chromatography (TLC)

Using a solvent system comprised by chloroform/methanol/0.2% aqueous CaCl₂ 50:42:11 volume, we were able to separate the aqueous phase of the inoculum demonstrating the presence of gangliosides when compared to the standard bovine ganglioside mixture. Using Ehrlich’s reagent, gangliosides reacted as grey spots on the TLC plate.

Clinical signs

Chicken C1 presented with mild walking difficulty and weakness at 14 dpi and was euthanized. All of the other chickens remained healthy and were euthanized at the end of the experimental period at 62 dpi. None of the quaker parrots presented clinical signs that resembled PDD (i.e., undigested seeds in the feces, regurgitation, neurological signs) or a peripheral neuropathy that could resemble GBS. However, nonspecific signs such as mild depression (QP2 and QP5) and weight loss (QP4) were observed.

PaBV-RT-PCR

All quaker parrots tested negative for PaBV on cloacal and choanal swabs by RT-PCR 3 days before inoculation and 25 dpi. Samples collected from the brain, spinal cord and nerves from all quaker parrots at time of euthanasia, as well as the inoculum used to inject the birds, were negative for PaBV by RT-PCR.

Fibrinogen analysis

Fibrinogen levels, an important inflammatory marker in birds, were increased in QP5, 24 h after each inoculation, reaching levels up to 400 mg/dL (Table 1). QP2, QP3, QP4 had variable fibrinogen levels, with the highest levels at 49 dpi. The fibrinogen levels for control QP1 were less than 100 mg/dL throughout the study.

Serology

The Western blot using the crude extract showed fewer bands from the sera from the quaker parrots inoculated with the purified ganglioside extract (QP2 and QP3) and several nonspecific bands for the birds inoculated with the crude extract (QP4 and QP5) (Fig. 2). The Western blot from control QP1 did not present any bands. The blots loaded with the purified extract only reacted with the sera of the birds inoculated with the purified extract, showing fewer faint bands when compared to the crude extract blots. A 45 kDa band was observed in the blots from QP2 and QP3, which were treated with purified gangliosides. This is the molecular weight of ganglioside binding proteins (Riggott & Matthew, 1996).

All inoculated quaker parrots (QP2–QP5) presented a marked increase in the absorbance values on ELISA (Fig. 3) using the purified and crude ganglioside extracts when comparing the first timepoint to the second and third timepoints. Due to the lack of a positive control originated from a quaker parrot with proved anti-ganglioside antibodies, the ELISA evaluation was performed based on the exposure of QP1 serum to the crude and purified extracts and also the serum from a healthy macaw exposed to these extracts. For the plates coated with the purified extract, QP2 had absorbance values, almost two times higher than sera from QP1, when comparing the second and third timepoints. The plates coated with crude extract, QP4 had absorbance values from the second and third timepoints almost three times higher when compared to QP1 (control). The sera from QP1 and the healthy macaw sera had constant low values at all 3 timepoints for plates coated with purified and crude extracts.

Pathology

Chicken C1 (euthanized at 14 dpi) had an intense inflammatory reaction in the site of the inoculation in the pectoral muscle. No gross lesions were observed in any of the quaker parrots. Histologically, C1, presented with a mild perivascular and perineural lymphocytic infiltrate in the proventriculus, while C5, and C6 presented with minimal to mild, focal, lymphocytic aggregates in the cerebrum. QP2 had a mild, multifocal, lymphoplasmacytic encephalitis in the optic tectum (Fig. 4A). QP4 had a mild, multifocal lymphoplasmacytic and histiocytic myelitis in the thoracic and lumbar segments (Fig. 4B).