Infections Caused by Pathogenic Free-Living Amebas ( Balamuthia Mandrillaris and Acanthamoeba sp.) in Horses

Abstract

This article describes amebic infections in 4 horses: granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba culbertsoni and systemic infections caused by Acanthamoeba sp. The former infection occurred in 1 of 4 horses spontaneously without any underlying conditions; the latter amebic infection was perhaps “opportunistic” considering the visceral involvement by this protozoan in association with Aspergillus sp. and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, culture, polymerase chain reactions, and electron microscopy are presented.

Table 1.

Infections with Acanthamoeba sp. and Balamuthia mandrillaris in 4 horses: clinicopathologic findings.

	Case 1	Case 2	Case 3	Case 4
Animal description	20-yr-old Warmblood gelding	28-yr-old Quarter Horse mare	22-yr-old Thoroughbred gelding	1-yr-old Thoroughbred filly
Clinical signs	Ataxia, incoordination, leaning to 1 side over 3 mo, unresponsive to antibiotics and anti-inflammatory drugs; later developed fever and became progressively ataxic; animal was euthanized	Respiratory disease over 1 wk and then developed diarrhea and endotoxemia; condition progressively worsened; animal was euthanized	Slight depression and decreased feed intake over 5 days; showed progressive difficulty to rise, ataxia, whole-body trembling, violence falling and recumbency; animal was euthanized.	Hind leg ataxia and anorexia over weeks; prolapsed rectum and colitis; unresponsive to antibiotics and anti-inflammatory drugs; progressive ataxia, dribbling of urine, and recumbency; found dead overnight
Gross pathology	Bilateral, multiple, grey, firm pulmonary nodules (1.5–2 cm in diameter) with smooth grey “meaty” appearance on cut surface; malacic area (5 mm in diameter) on frontal cerebral cortex	Peritoneal cavity contained approximately 1 liter of straw-colored clear watery fluid; large colon and cecum were filled with grey-green, watery fluid; lungs had randomly distributed grey firm nodules, pale tan appearance on cut section (1 cm in diameter), and thrombosis of pulmonary vessels; kidneys were slightly enlarged and cortices appeared pale pink in color; uterus was mildly enlarged and lumen contained thin, white fluid	Left thalamic region of brain was swollen (approximately 4 cm across), yellow, and wet; with central spherical pink, palpably firm mass (5 mm across); hyperemic duramater of left olfactory fossa	Both lungs were markedly enlarged and had disseminated palpably firm, miliary light-grey foci (3-4 mm in diameter); red and markedly enlarged tracheobronchial lymph nodes; thickened gastric glandular mucosal wall (1.3 cm) and multiple circular ulcers up to 4 mm in diameter; kidneys had multiple cortical pale grey foci (1-2 μm); spinal cord had focal hemorrhages at the level of second thoracic segment on dorsolateral aspect
Histopathology	Severe subacute bronchointerstitial pneumonia with predominant neutrophilic infiltrates; multifocal to coalescing necrosis of parenchyma, and fibrin thrombosis of vessels; perivascular inflammation consisted of infiltration of lymphocytes, plasma cells, and neutrophils; numerous intralesional amebic trophozoites and cysts (15-20 μm) with basophilic, granular cytoplasm, centrally located nucleus, and densely staining nucleolus (Fig. 1A); cerebral cortex was focally	Chronic active pneumonia with necrosis, abscessation, pleuritis, vascular thrombosis, and acute infarction; intralesional amebic trophozoites (15-30 μm) and cysts (15-20 μm) with finely granular basophilic cytoplasm and single nucleus containing centrally located densely staining nucleolus (Fig. 1F); mucosa of ventral colon contained hyaline proteinaceous material; other findings were multifocal chronic glomerulonephritis with glomerular	Granulomatous encephalitis (confined to thalamus) characterized by presence of foreign-body-type giant cells in neuropil with intralesional amebic trophozoites; amebas were approximately spherical and 12–15 μm in diameter, with slightly basophilic granular cytoplasm and eccentrically placed nuclei (Fig. 1D); these findings were accompanied by thick mononuclear leukocytic perivascular cuffs, necrosis of neuropil, fibrinoid necrosis of	Chronic mild ulcerative enteritis and colitis; subacute to chronic suppurative necrotic gastritis; moderate multifocal granulomatous tubulonephritis with intralesional ameboid bodies; granulomatous tracheobronchial lymphadenitis with intralesional ameboid bodies similar to those described in Cases 1 and 2; thymic atrophy; severe multifocal to coalescing necrotizing
Histopathology (continued)	necrotic and had marked cellular infiltration of macrophages, lymphocytes, and few neutrophils; amebic organisms similar to those in lung were present adjacent to blood vessels or within necrotic foci surrounded by mixed inflammatory cell infiltrates (Fig. 1C)	obsolescence, mild suppurative endometritis, and mild multifocal myocardial necrosis with fibrosis Brain was not examined	small blood vessels, and vasogenic edema; in other areas of the brain there was extensive chronic nonsuppurative encephalitis (from medulla to basal ganglia) consisting of perivascular cuffing by large mononuclear leukocytes	granulomatous pneumonia with intralesional amebas (Fig. 1F); multifocal granulomatous hemorrhagic myelitis and meningitis involving
dorsolateral tracts of first and second thoracic segments of spinal cord
Identification and characterization of amebas	Ameba was cultured from lung and further characterized as Acanthamoeba culbertsoni by IIFA, IHC, and PCR*	Acanthamoeba sp. was identified by IIFA and IHC*	Ameba was cultured from brain and characterized as Balamuthia mandrillaris by IIFA, IHC, and PCR*	Acanthamoeba sp. was identified by IIFA and IHC*
Bacteriology/mycology	No bacterial pathogens detected	Pseudomonas sp., Escherichia coli, and Aspergillus sp. were detected in lungs	No bacterial pathogens detected	Salmonella enterica serovar Eastbourne detected in stomach wall

*

IHC = immunohistochemistry; IIFA = indirect immunofluorescence assay; PCR = polymerase chain reaction.

Amebic encephalitis in humans and animals is caused by several species of free-living amebas belonging to the genera Acanthamoeba, Naegleria, and Balamuthia. 1,5,6,8,12,15,16 In humans free-living amebas cause 3 well-defined diseases: 1) primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri, 2) granulomatous amebic encephalitis (GAE) caused by Acanthamoeba spp. and Balamuthia mandrillaris, and 3) chronic amebic keratitis (AK) caused by species of Acanthamoeba. Both PAM and chronic AK occur in healthy individuals while GAE is often associated with patients having acquired immunodeficiency. 8 , 14 , 15 The occurrence of GAE in animals with immunodeficiency has not been documented. The clinicopathologic findings of the 4 affected horses are described in Table 1.

Formalin-fixed and paraffin-embedded brain (Cases 1 and 3) and lung (Cases 1, 2, and 4) tissue sections were deparaffinized, and indirect immunofluorescence (IIF) and/or immunohistochemistry (IHC) assays were performed as described previously. 6 Acanthamoeba culbertsoni (Fig. 1B), B. mandrillaris (Fig. 1E), and Acanthamoeba sp. (Fig. 1G) were detected.

Attempts were made to isolate the organisms from frozen-thawed unfixed brain (Cases 1 and 3) and lung tissues (Case 1). The brain and lung specimens had been stored at −70°C for 60 days and were transferred on dry ice to the Centers for Disease Control (CDC), Atlanta, Georgia, where cultivation was attempted as previously described. 16 The cultured amebas were further characterized by IIF as A. culbertsoni (from the lung of Case 1) and B. mandrillaris (from the brain of Case 3). In addition, the amebas were processed for transmission electron microscopy and examined in either a Zeiss 10 C or a Zeiss (LEO) model 906E transmission electron microscope at 60 kv accelerating voltage as previously described. 4 Ultrastructural characteristics of the amebas are shown in Fig. 1H, I, and J.

Confirmation of the identity of the amebas and genotyping was performed using polymerase chain reaction (PCR). Centrifuged pellets of amebas (CDC V409C) and CDC V433) were suspended separately in 200 μl of UNSET buffer, and DNA was extracted. 2 , 13 Approximately 100 to 200 ng of genomic DNA from CDC V409C and CDC V433 was used for PCR amplifications. Nuclear 18S rDNA amplification was performed with primers CRN5 (5′-TGGTTGATCCTGCCAGTAG-3′)and SSU2 (5′-CCGCGGCCGCGGATCCTGATCCCTCCGCAGGTTCAC-3′), which span approximately 99% of the gene. PCR products of nuclear 18S rDNA were prepared for automated sequencing using the QIAquick PCR purification kit. a These products were then directly sequenced using automated fluorescent sequencing protocols on an Applied Biosystems automated sequencing system b using primers previously used in Acanthamoeba studies. 14 The 18S rDNA sequence of B. mandrillaris V433 is available under GenBank accession number AF477021, and the 18S rDNA sequence of A. culbertsoni V409C is available under accession number DQ499151. The mitochondrial 16S rRNA gene from A. culbertsoni (CDC V409C) was amplified using primers previously used in one of the coauthor's laboratories (G. B.) to amplify this region in Acanthamoeba sp. 7 The A. culbertsoni (CDC V409C) 16S rDNA sequence is available in GenBank under accession number AF479542. The mitochondrial 16S rRNA gene from B. mandrillaris (CDC V433) was amplified by PCR using primers that specifically amplify this target in B. mandrillaris. 2 The 16S rDNA sequence is available in GenBank under accession number AF477017. The nuclear 18S and mitochondrial 16S rDNA sequences of A. culbertsoni V409C and B. mandrillaris V433 were aligned in the sequence alignment editor ESEE and compared to other sequences in our database to determine their identification. 3 PCR sequence comparison of both the nuclear and mitochondrial sequences from CDC V409C (from Case 1) and CDC V433 (from Case 3) determined the identity of these isolates as A. culbertsoni (Acanthamoeba genotype T10) and B. mandrillaris, respectively.

Figure 1.

Histologic sections of equine lung and brain and ultrastructures of amebas with amebic infections. A, photomicrograph of lung (Case 1) showing alveolar spaces flooded with pink proteinaceous fluid with predominant neutrophilic response and intralesional ameboid bodies (arrows); hematoxylin and eosin (HE); bar = 10 μm; B (insert), IHC staining with anti-Acanthamoeba sp. serum of lung section showing amebic organisms; C, photomicrograph of brain (Case 1) showing that the neuropil is focally infiltrated by leukocytes, predominantly neutrophils with intralesional amebic bodies (arrow); HE; bar = 10 μm; D, photomicrograph of brain (Case 3) showing granulomatous encephalitis with an amebic cyst (arrow); note that the neuropil is replaced by “gitter cells” and mononuclear leukocytes; E (insert), IHC staining using anti-B. mandrillaris serum (Case 3); bar = 10 μm; F, photomicrograph of lung (Case 4) showing necrotizing pneumonia with numerous ameboid bodies (arrows); HE; bar = 10 μm; G (insert), indirect immunofluorescence staining with anti-Acanthamoeba serum in lung (Case 4); bar = 10 μm; H, electron micrograph of 4 Acanthamoeba cysts from case 1; note wrinkled ectocyst (ect), well-defined endocyst (en), and nucleus (N) in 2 cysts at top; bar = 5 μm; I (insert), electron micrograph of Acanthamoeba cyst, culture filtrate from lung (Case 1) contains lipid droplet (l), nucleolus (n), and plasma membrane (Pm); bar = 1 μm; J, electron micrograph of trophozoite of B. mandrillaris (culture aspirate of lung, Case 3); note dense cytoplasm contains lipid droplet (L), mitochondrion (M), and N with double n; bar = 2.6 μm.

Amebic infection in horses is very rare, and it is noteworthy to mention that all cases have originated in southern California between 1998 and 2001. Kinde and colleagues 6 reported meningoencephalitis in another horse that was also indigenous to southern California. The reason for this cluster of cases in southern California is not clear. Amebas can be easily missed in histologic sections because of their resemblance to macrophages; even when the disease is suspected, confirmation is often not possible because of lack of awareness of the disease and the scarcity of reagents. The authors believe that perhaps there are more cases in horses of amebic infections, “out there,” but not all neurologic cases are submitted to diagnostic laboratories for necropsy examination.

In human patients GAE due to Acanthamoeba species (Acanthamoeba castellanii, A. culbertsoni, and possibly other amebas) has been reported in chronically ill and debilitated or immunologically impaired individuals with severe derangements of host defenses. 8 In Case 4, the horse may have been immunocompromised as it showed thymic atrophy and systemic amebic infection and salmonellosis due to Salmonella enterica serovar Eastbourne. Case 2 had infections with Aspergillus sp., Pseudomonas sp., and Escherichia coli, suggesting that the horse may have already had a weakened immune system and became prone to opportunistic amebic infection. For cases 1 and 3 there was no known predisposing factor or detectable underlying disease and the clinicopathologic findings were due to primary amebic infection. The route of invasion and penetration into the central nervous system in human cases of GAE is hematogenous, probably from a primary site in the lower respiratory tract. 10 GAE has been reported with chronic ulceration of the skin resulting with terminal hematogenous spread to the brain. 8 In all 3 horses with Acanthamoeba infection the lung was highly likely to be the primary site of infection; from there, the organism may have spread hematogenously to other organs including the brain. In Case 3 no evidence of skin lesions, pneumonia, or microscopic lesions in the cribiform plate, olfactory fossae, and ethmoid bones was noted. In general in horses, the pathogenesis of amebic infection is not known but it is speculated that perhaps it is similar to that of humans. Although amebic encephalitis appears to be rare in horses, it should be included in the differential diagnosis with other neurologic conditions.

Acknowledgements. We thank S. Kwiek, E. J. Hurley, and R. Sriram for their technical assistance and Drs. D. Vrono, K. Valko, and T. Brauer (veterinary practitioners) for submitting the cases. Part of the work of P. A. F., D. J. K., and G. C. B. (co-authors) was supported by a grant from the National Institutes of Health (grant EY09073).