Infection of juvenile falcons (Falco spp.) with intestinal Lawsonia intracellularis

Abstract Intestinal infection of many host species with Lawsonia intracellularis are widely reported. Analyses of infections among carnivorous falcons have not previously been reported. Fifty juvenile captive falcons (Falco spp.) with or without Lawsonia infection were investigated in the United Arab Emirates, including clinical laboratory methods. Fresh intestinal biopsy samples were analysed by microbiological techniques for Lawsonia and other bacteria and by standard parasitological and pathological methods. Lawsonia intracellularis infection was diagnosed by microbiological examination and qPCR in 10 of 50 juvenile falcons at case examination. Seven of these 10 falcons were of normal clinical appearance, and the other three had other contributing factors to ill‐thrift. A range of other conditions were noted in 40 case control falcons. This first report of Lawsonia infection in falcons suggests that the agent may have a limited contribution to clinical disease in these birds, including ill‐thrift syndromes. This lack of clinical disease association mimics that noted among Lawsonia infections recorded in other avian families.


INTRODUCTION
Birds in the order Falconidae (falcons) have narrow pointed wings, keen eyesight, and a tomial tooth beak formation. Falcons are carnivorous and chase small live prey, such as rodents, and kill them using their beaks (Mindell et al., 2018). Among the 40 falcon species, peregrine falcons (Falco peregrinus), saker falcons (Falco cherrug), gyrfalcons (Falco rusticolus), and their various hybrids are sometimes raised and conditioned in captivity for exhibition or sporting purposes (Fleming et al., 2011). Falcons bred and raised in captivity are typically fed small rabbits or rodents, such as mice, hamsters, or rats and/or small birds such as young quail, ducks, or chickens (Fleming et al., 2011) nile and adult falcons can suffer a range of clinical conditions, such as ill-thrift and diarrhoea, due to campylobacteriosis, mycobacteriosis, dietary mis-management, or coccidiosis (Caryospora sp.) (Kubiak & Forbes, 2011;Samour, 2006).
Lawsonia intracellularis is an obligate intracellular bacterium identified as the causative agent in intestinal lesions of proliferative enteropathy in a wide range of animal species (McOrist & Gebhart, 2005). In some mammalian hosts (pig, horse, hamster, rabbit), the infection is both commonly reported and an important cause of clinical ill-thrift syndromes, whereas in many other hosts, it appears to be a relatively rare and unusual clinical event. Lawsonia intracellularis is a single-strain bacterium; its entry mechanisms and intracytoplasmic life  The purpose of this study was to investigate juvenile falcons presented for veterinary attention, including ill-thrift, at avian medicine clinics in the United Arab Emirates. L. intracellularis, conducted using methods described previously (Love et al., 1977), semi-quantitative PCR specific of these samples (100 mg per bird) for L. intracellularis, conducted using preparation methods and oligo-primers as described previously (Nathues et al., 2009).

Besides
Among pathology investigations, lower intestinal tract biopsy samples from three birds (# 1, 6 and 7; selected opportunely) were routinely processed for histopathology, sectioned at 3 µm and stained by haematoxylin and eosin, or Warthin-Starry silver impregnation. Cohort biopsy samples from these birds were also fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer. After routine processing and staining with uranyl acetate/lead citrate, ultra-thin sections were examined via transmission electron microscopy.

RESULTS
The results of case investigations are summarized in Table 1. The falcon species presented to avian clinics in the United Arab Emirates for this study: gyr falcon (F. rusticolis), peregrine falcon (F. peregrinus) or cross breed (gyr/peregrine) were considered typical of falcons held in local captive bird facilities. In 10 juvenile falcons (# 1 to # 10),

F I G U R E 1 Intestinal biopsy of falcon intestinal epithelial cells.
Numerous intracytoplasmic curved bacteria (arrow) in immature epithelial cell. Romanowsky stain.
bacterial examination and qPCR results were consistent with intestinal L. intracellularis infection (see Table 1). Seven of these 10 falcons were presented for veterinary examination in the absence of clinical signs, and only three were presented for investigation of clinical illthrift. Other identified causes of ill-thrift and/or poor performance in the latter three falcons and in un-infected case controls included husbandry mis-management, aspergillosis, bacterial (Campylobacter spp.), or coccidial (Caryospora spp.) enteritis (see Table 1). Segmented filamentous bacterial forms were visualized in association with the intestinal epithelium in 10 cases (data not shown) and were considered commensal bacteria (Hedblom et al., 2018).
Staining of lower intestinal tract biopsy samples in cases # 1 to # 10 typically showed curved, intracellular Gram-negative bacteria within immature intestinal epithelial cells (see Figure 1). Identical staining of samples from other cases was consistently negative for such bacteria.
Transmission electron microscopy of biopsy samples from selected cases with bacterial examination and qPCR evidence of L. intracellularis infection confirmed immature intestinal epithelial cells containing intracytoplasmic bacteria. These bacteria were curved, vibrioid, and Gram-negative (tri-laminar envelope), measuring 1.25-1.75 µm long and 0.25-0.4 µm wide (see Figure 2). The organisms were consistent with Lawsonia phenotype, consistently located free in the cytoplasm and occasionally noted in apposition to cell mitochondria (see Figure 2).
Histopathology examination of limited cohort biopsy samples from these selected cases indicated normal crypt/villus architecture, with no distinct intracellular elements.

Infection of immature intestinal cells with L. intracellularis has been
reported in numerous animal groups, and we report it here for the first time in falcons (Falco spp.  et al., 1988;Kubiak & Forbes, 2011;Samour, 2006 Lawsonia intracellularis forms a homogenous genus and species within the Desulfovibrio family, with no genetic differences between isolates collected from different animal hosts (Bengtsson et al., 2020).
This lack of speciation indicates that the bacterium is of recent evolutionary lineage (Schmitz-Esser et al., 2008). Lawsonia requires intracellular mitochondrial sources of energy and appears to be capable of an induced phagocytosis towards intestinal epithelial cells of many avian and mammalian hosts (Schmitz-Esser et al., 2008). This process was also evident in the infected falcons in this study (see Figure 2).
Lawsonia is considered capable of interrupting normal crypt cell differentiation into mature villus cells, thereby causing the distinctive lesions of proliferative enteropathy noted in many host animal species (McOrist et al., 1996). While in some hosts (pigs, horses, hamsters, rabbits), Lawsonia infections are clearly associated with significant lesions of proliferative enteropathy, in many other groups of animals, it occurs as rare and irregular recorded events (e.g., dogs, rats, foxes, monkeys) (Cooper & Gebhart, 1998). However, in avian hosts, members of both the Passeriformes and Galliformes appear to be relatively resistant to Lawsonia infection, with very low infection rates and repeated failure to demonstrate susceptibility upon challenge exposure (Collins et al., 1999;McOrist et al., 2003;Ohta et al., 2017;Viott et al., 2013). It is naturally likely that juvenile falcons have a higher rate of exposure to Lawsonia due to the carnivorous consumption of rodents or rabbits in their diet. While advisable, these potential sources of infection are not routinely tested. It is also possible that, in common with the other aforementioned bird groups, the gastrointestinal tract of falcons