Abstract

Tyzzer's disease was diagnosed as the cause of mortality in free-ranging hatch-year passerine birds in Southern California, US. Birds presented with ataxia, tremors, and opisthotonos and died or were euthanized. Postmortem examination of four birds identified encephalitis on microscopy with characteristic stacked, slender bacterial rods visible with special stains. Infection with Clostridium piliforme was confirmed by real-time PCR.

Tyzzer's disease, caused by the bacterium Clostridium piliforme, has been reported to cause mortality in laboratory (Rights et al. 1947) and domestic (Uzal et al. 2015) animals as well as wild mammals in captivity, such as white-tailed deer fawns (Odocoileus virginianus; Brooks et al. 2006), and in some free-ranging wildlife like the muskrat (Ondatra zibethicus; Wobeser 2001). While neonates or juveniles are typically susceptible to natural infection, poor hygiene, stressors, or corticosteroid therapy leading to immunosuppression is associated with clinical disease in individuals of all ages. Overcrowding has been established as an important factor for disease in laboratory mice (Rights et al. 1947).

Clostridium piliforme enters the body through the distal intestines where it reaches the liver via portal circulation and disseminates to the myocardium, classically known as the intestine–liver–heart triad. Spontaneous encephalitis is a rare manifestation of Tyzzer's disease, reported only in gerbils (Veazey et al. 1992) and in a juvenile passerine in captivity (Mete et al. 2011). We describe an outbreak of C. piliforme encephalitis in free-ranging passerines.

Between June and August 2013, increased admissions of hatch-year woodpeckers and jays were reported to the Wildlife Investigations Laboratory (Rancho Cordova, California, USA) by the Big Bear Alpine Zoo, San Bernardino County (Big Bear Lake, California, USA). The birds presented with neurological symptoms including ataxia, tremors, and torticollis, and either died shortly after admission or were euthanized. Twelve carcasses were submitted frozen to Wildlife Investigations Laboratory, and six birds in the freshest condition were selected for postmortem exam at the California Animal Health and Food Safety Laboratory (Davis, California, USA). Three Acorn Woodpeckers (Melanerpes formicivorus) were submitted in July 2013 and identified as A, B, and C, and one Northern Flicker (Colaptes auratus), identified as D, and two California Scrub Jays (Aphelocoma californica), identified as E and F, were submitted in August 2013.

At necropsy, all birds were in good nutritional condition with no significant gross findings. Brain, skeletal muscle, peripheral nerves, trachea, lungs, liver, heart, kidneys, spleen, bursa when present, esophagus, proventriculus, ventriculus, and intestines were collected in 10% buffered formalin for histopathology. A fecal pool from each group, A–C and D–F, were submitted for Salmonella spp. PCR, and aerobic culture was performed on swabs from liver and lung of each bird. Virology included PCR for avian influenza virus and avian paramyxovirus-1 on oropharyngeal swabs, and for West Nile virus on kidney. Toxicology included liver tissue screening for carbamate and organophosphate insecticides, selenium, and heavy metals (lead, manganese, mercury, cadmium, copper, iron, zinc, arsenic, and molybdenum) and brain for cholinesterase activity. All test results were negative or within normal ranges except for positive Salmonella spp. PCR on the flicker and jay fecal pool (D–F). This was considered incidental due to lack of lesions and inability to recover the organism from other organs that would suggest a systemic infection.

Significant findings detected on histopathology included inflammatory lesions in the brain of four birds (A, C, D, E): regionally extensive cellularity composed of heterophils, necrotic debris and karyorrhexis with neuronal degeneration (Fig. 1) present in the cerebral cortex of birds A, D, and E, while C had lesions in the brain stem. Steiner's silver stain highlighted the “haystack” pattern of argyrophilic slender rods (Fig. 2) compatible with C. piliforme in the brain lesions of A and D. Real-time PCR for C. piliforme using a FAM-TAMRA–labeled probe (Borchers et al. 2006) on DNA extracted from formalin-fixed, paraffin-embedded sections of brain from the four birds positively identified the bacterium. Due to the lack of histological brain lesions, PCR was not performed on birds B and F, and the cause of neurologic disease in these two birds was undetermined.

Figure 1

Histology of Clostridium piliforme encephalitis in a hatch-year Northern Flicker (Colaptes auratus) is characterized by marked, regionally extensive cellularity due to infiltrations of heterophils with neuronal degeneration and necrosis. Bar=20 μm. Similar lesions were observed in four birds including this flicker, as the cause of increased incidence in neurologic disease in wild passerines in San Bernardino County, California, USA.

Figure 1

Histology of Clostridium piliforme encephalitis in a hatch-year Northern Flicker (Colaptes auratus) is characterized by marked, regionally extensive cellularity due to infiltrations of heterophils with neuronal degeneration and necrosis. Bar=20 μm. Similar lesions were observed in four birds including this flicker, as the cause of increased incidence in neurologic disease in wild passerines in San Bernardino County, California, USA.

Figure 2

Silver stain (Steiner) section of the brain lesions in the hatch-year Northern Flicker (Colaptes auratus) demonstrates the argyrophilic stacks of characteristic long curvilinear Clostridium piliforme (arrows), occurring mostly in neurons and among heterophils and necrotic debris. Bar=10 μm.

Figure 2

Silver stain (Steiner) section of the brain lesions in the hatch-year Northern Flicker (Colaptes auratus) demonstrates the argyrophilic stacks of characteristic long curvilinear Clostridium piliforme (arrows), occurring mostly in neurons and among heterophils and necrotic debris. Bar=10 μm.

Although Tyzzer's disease has previously been described in individual, captive birds, this report documents the first outbreak involving multiple, free-ranging wild birds. Similar to the captive cases, which include a lorikeet (Trichoglossus haematodus; Raymond et al. 2001), a cockatiel (Nymphicus hollandicus; Saunders et al. 1993), and a Taveta Golden Weaver (Ploceus castaneiceps; Mete et al. 2011), the outbreak we describe involved neonatal and juvenile birds. Transmission of C. piliforme is fecal–oral by ingestion of spores in a contaminated environment (Wobeser 2001). As such, free-ranging mammals, and possibly healthy adult birds acting as carriers, may have been the source of infection in the hatch-year woodpeckers and scrub jay. Since overcrowding and other stressors are often cited as predisposing factors for infection in captivity (Rights et al. 1947), anthropomorphic modifications to the local environment may have contributed to the outbreak while also aiding in detection. Big Bear Lake is a 16.4 km2 community bordered by a mountain lake and the San Bernardino National Forest. This local area of urbanization may have functioned to bring wild birds and mammalian hosts into closer contact through the presence of artificial food, water and shelter, altered microclimate, and/or decreased predation risk (Bradley and Altizer 2007).

Similar to the case in the weaver, encephalitis was the main histological finding in the two woodpeckers, the Northern Flicker, and one scrub jay described here. This contrasts to C. piliforme infection in mammals, which usually consists of the enteritis-hepatitis-myocarditis triad. Liver necrosis and organisms were observed in the two psittacines, though there was an absence of intestinal lesions. Necrotizing hepatitis or enteritis was not a feature of Tyzzer's disease in the weaver or the passerines reported here, with the exception of one woodpecker (B) that had acute random necrosis in the liver without inflammation or organisms and the cause was not identified. Studies in mice have revealed that significant intestinal damage is a prerequisite for infection of organs other than the liver (Tsuchitani et al. 1983). Given that the carcasses in this case were frozen until submission and that intestines are prone to autolyze rapidly, especially in small-bodied birds, we could not adequately rule out enteritis. However, natural infection by C. piliforme causing encephalitis seems to be a more consistent finding in passerines. In these avian species, the neurologic clinical signs, the unusual microscopic hypercellularity of regions of the brain due to infiltrating leukocytes, and the silver stain highlighting the characteristic slender rods prompts the diagnosis of Tyzzer's disease. Nevertheless, the advanced post-mortem changes often present in wildlife can be immense, and PCR has been a highly useful tool for confirming the diagnosis.

We thank D. Richardson with the Big Bear Alpine Zoo for bringing these cases to our attention.

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