Campylobacter colonization is not associated with proventricular dilatation disease in psittacines.

Psittacine proventricular dilatation disease (PDD) is a neurological disease caused by parrot bornaviruses. A competing theory suggests that intestinal colonization by Campylobacter species may also be a potential cause of PDD or that their presence may be required for disease development. This theory proposes that PDD results from the activities of antiganglioside antibodies on enteric neurons in a manner similar to the pathogenesis of Guillain-Barré syndrome in humans. We therefore cultured feces from domestic chickens as well as from multiple parrot species to determine whether Campylobacter spp. could be detected in the latter. We failed to detect Campylobacter in a flock of cockatiels known to be highly susceptible to experimental parrot bornavirus-induced PDD. Even in naturally infected psittacines suffering from clinical PDD, no Campylobacter species were detected. Conversely, Campylobacter was readily cultured from domestic poultry samples and confirmed by using matrix-associated laser desorption ionization mass spectroscopy/real-time polymerase chain reaction. We conclude that not only are Campylobacter infections of psittacines uncommon, but also that infection by Campylobacter species is not related to the etiology of PDD.

Introduction

Proventricular dilatation disease (PDD) is a neurologic disease of parrots. Clinically, it presents as gastrointestinal dysfunction characterized by anorexia, crop stasis, and a massively dilated proventriculus. These effects appear to be a consequence of the destruction of enteric neurons.1–3 The sole confirmed causative agent of psittacine PDD is parrot bornavirus (PaBV). Proventricular dilatation attributable to other bornaviruses has been observed in geese and canaries.3,4 PDD does not develop in the absence of PaBV, and Koch’s postulates have been repeatedly fulfilled linking PaBV to the development of PDD.5,6 Thus, PDD is readily and consistently induced in cockatiels (Nymphicus hollandicus) by intramuscular challenge with tissue cultured PaBV.

There is a competing theory that attributes PDD to the development of autoantibodies against gangliosides and suggests that PDD is a form of Gullain–Barré syndrome (GBS).7,8

Many cases of GBS in humans appear to be induced by the development of cross-reacting antibodies to enteric Campylobacter jejuni. The proposed pathophysiology suggests that PDD is mediated by immune responses to the lipo-oligosaccharide of C. jejuni. These exhibit molecular mimicry to gangliosides expressed on neurons, especially at the nodes of Ranvier.9–11 As a result, antibodies directed against Campylobacter antigens also target gangliosides within the myelin sheath, and these antibodies will then cause demyelination and impaired nerve function. This impairment, it is suggested, would result in the gastrointestinal symptoms associated with PDD.

This study was therefore designed to determine whether Campylobacter was present in the intestinal microbiota of healthy PDD-susceptible and resistant psittacines as well as birds suffering from clinical PDD.

Members of the Campylobacter genus are enteric, helical bacteria that are microaerophilic, resistant to cephalosporin, oxidase positive, and, for the most part, catalase positive. C. jejuni has a natural reservoir in chickens, and some passerine species. It is widespread in wild birds, especially feral pigeons, crows, and waterfowl.12–17 It is a significant cause of human food poisoning due to its high prevalence in commercial poultry. C. jejuni prevalence ranges from 45% at the flock level and 93% at the farm level.18 It can thus be considered a component of the normal chicken microbiota. Chicken fecal samples therefore served as positive controls in the search for Campylobacter.

Materials and methods

Sixty three avian fecal samples from 52 birds were cultured to test for the presence of Campylobacter spp. Of these, 26 samples were from chickens and 27 from psittacines. Aviary birds that were tested included 6 Monk parakeets (Myiopsitta monachus), 5 cockatiels (Nymphicus hollandicus), 12 macaw (Ara sp) species (six twice), six from African gray parrots (Psittacus erithracus) (three twice), two from Amazon parrots sp (one twice), and two from a blue-headed parrot (Pionus menstruus). All these birds were housed in the Texas A&M Avian Health Complex. Four single parrot samples were obtained from birds admitted to the Texas A&M Veterinary Medical Teaching Hospital for diagnosis and treatment of suspected PDD. These included one each of a macaw, African gray, Amazon sp, and sulfur-crested cockatoo (Cacatua galerita). This study was performed under a protocol approved by Texas A&M University Animal Use and Care Committee: IACUC 2014-0169.

Samples were collected using sterile swabs and refrigerated for transfer to the laboratory. Samples were plated shortly thereafter. Two types of selective plate media were used for culturing the fecal samples: Campy BBL w/10% sheep blood (Becton Dickinson, # 221728, Franklin Lakes, NJ, USA) and Campy–Cefex (Becton Dickinson, #292487). The fecal samples were swabbed directly onto the agar plates and streaked with a heat-sterilized metal loop. Phosphate-buffered saline was added to the fecal collection tubes to create a fecal slurry which was then used to inoculate Bolton Broth media. If bacterial growth was insufficient on direct fecal plating, the Bolton Broth culture was inoculated on to the selective plate media. A strain of C. jejuni (ATCC 33291), was cultured at the same time to serve as a positive control.

The plates were incubated in one of two containers: hard, air-fast canisters for more than two plates, with an Anaeropack-MicroAero pack (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan), or in groups of two plates in BioBag Type Cfj bags (Becton Dickinson, #261212); incubation was done at a temperature of 40°C for 2 days under microaerophilic conditions, after which the cultures were removed, plates described, and suspect colonies transferred to new agar plates. Preliminary confirmation for Campylobacter spp. was achieved with a combination of Gram stain, catalase, and oxidase tests, while real-time polymerase chain or matrix-associated laser desorption ionization mass spectrometry (MALDI-TOF, Bruker Bioytper Compass, Billerica, MA, USA) analysis were used for definitive confirmation of cultured Campylobacter and their species.19 For the samples analyzed by matrix-associated laser desorption ionization mass spectroscopy, a log score was generated based on the peaks obtained from the sample and the comparison of the obtained data against a library of bacterial standards. A positive result was reported if the log score was greater than or equal to 2.000 (on a scale of 0.000–3.000) – indicative of an acceptable probability of sample classification at the species level.

Results and discussion

Of the 26 chicken fecal samples, eight (30%) were determined to contain C. jejuni. All 27 psittacine-derived samples were negative. These negative birds included an African Gray (Psittacus erythracus) (ID# R2786) and a Great Green Macaw (Ara ambigua)(ID#. 229721), both of which were previously diagnosed as suffering from PDD and were shedding PaBV in their droppings at the time of sampling. Additionally, none of the four parrots admitted to the Texas A&M veterinary clinic on suspicion of PDD were Campylobacter positive. (One macaw, one African gray, one Amazona sp, and a sulfur-crested cockatoo). Additionally, it should be pointed out that the cockatiels housed in the aviary belonged to a flock that had been shown repeatedly to be consistently susceptible to developing typical PDD upon challenge with PaBV-1, genotype 2.

Campylobacter is present in many wild birds including pigeons, waterfowl, starlings, and corvids.13,14,20,21 There has been but a single report of Campylobacter in parrots; during a disease outbreak in Peru.22 We have investigated the normal gut microbiota of wild and captive scarlet macaws and failed to detect Campylobacter.23 Likewise, Campylobacter has not been detected in the normal microbiota of the cockatiel.24 Conversely, Campylobacter species, especially C. jejuni, are pervasive in chicken gut flora.18 Our poultry sample contained Campylobacter as expected, and thus served as a positive control. The absence of Campylobacter in psittacine samples suggests that colonization by this bacterium may infrequent in these species.

Rossi et al8 have postulated that PDD is not exclusively caused by PaBV infection and that some cases may be induced by Campylobacter, particularly C. jejuni. The number of confirmed PDD cases analyzed here is insufficient to rule out Campylobacter infection as a cause or contributing factor to PDD. However, we can induce PDD at-will in cockatiels by inoculation of PaBV-1 genotype 2, and as described above, our cockatiel flock appears to be free of Campylobacter. GBS can be induced in poultry but its manifestations in no way resemble PDD. They are primarily abnormalities in motor behavior.25 It has long been known that Borna disease in rodents is mediated by activated T-cells directed against infected neurons expressing bornaviral N protein.26 It is probable that the situation in birds is the same, and so we suggest that C. jejuni does not play a significant role in the pathogenesis of PDD.