Journal of Veterinary Diagnostic Investigation Vol. 19 Issue 3, 317-322
Copyright © 2007 by the American Association of Veterinary Laboratory Diagnosticians
Infections Caused by Pathogenic Free-living Amebas (Balamuthia Mandrillaris and Acanthamoeba Sp.) In Horses
Hailu Kinde1,
Deryck H. Read,
Barbara M. Daft,
Michael Manzer,
Robert W. Nordhausen,
Daryl J. Kelly,
Paul A. Fuerst,
Gregory Booton and
Govinda S. Visvesvara
Correspondence: 1Corresponding Author: Hailu Kinde, CAHFS San Bernardino Branch, 105 West Central Avenue, San Bernardino, CA 92408, e-mail: hkinde{at}ucdavis.edu
 |
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.
Key Words: Acanthamoeba culbertsoni • Balamuthia mandrillaris • granulomatous amebic encephalitis
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.
View larger version (127K):
[in this window]
[in a new window]
|
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.
|
|
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 systemb 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 co-author'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.
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 colleagues6 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.
|
Acknowledgments
|
|---|
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).
|
Sources and manufacturers
|
|---|
From the California Animal Health and Food Safety Laboratory System (CAHFS), San Bernardino Branch, 105 West Central Ave, San Bernardino, CA 92408 (Kinde, Read, Daft), CAHFS—Davis, School of Veterinary Medicine, University of California, Davis, CA 95616 (Manzer,Nordhausen), College of Biological Sciences,Ohio State University, Columbus, OH43210 (Kelly, Fuerst, Booton)., andDivision of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341 (Visvesvara). 
a. Qiagen Inc., Valencia, CA.
b. Applied Biosystems, Foster City, CA.
|
References
|
|---|
- Anderson M.P., Oosterhuis J.E., Kennedy S., Benirschke K.: 1986, Pneumonia and meningoencephalitis due to ameba in lowland gorilla. J Zoo An Med 17:87–91.
- Booton G.C., Kelly D.J., Chu Y-W.D.V., Seal D.V., Houang E., Lam D.S., Byers TJ, Fuerst P.A.: 2002, 18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong. J Clin Microbiol 40:1621–1625.[Abstract/Free Full Text]
- Culbertson C.G.: 1961, Pathogenic Acanthamoeba (Hartmannella). Am J Clin Pathol 35:195–202.[Medline]
- Davis C.R., Barr B.C., Pascoe J.R., Olander H.J., Dubey J.P.: 1999, Hepatic sarcocystosis in a horse. J Parasitol 85:965–968.[Medline]
- Fuentealba I.C., Wikse S.E., Read W.K., Edwards J.F., Visvesvara G.S.: 1992, Amebic meningoencephalitis in a sheep. J Am Vet Med Assoc 200:363–365.[Medline]
- Kinde H., Visvesvara G.S., Barr B.C., Nordhausen R.W., Chiu P.H.: 1998, Amebic meningoencephalitis caused by Balamuthia mandrillaris, (leptomyxid ameba) in a horse. J Vet Diagn Invest 10:378–381.[Free Full Text]
- Lozano-Alarcon F.G., Bradley A., Houser B.S., Visvesvara G.S.: 1997, Primary amebic meningencephalitis due to Naegleria fowleri in a South American tapir. Vet Pathol 34:339–243.
- Ma P., Visvesvara G.S.: 1990, Naegleria and Acanthamoeba infections: review. Rev Infect Dis 12:490–513.[Medline]
- Martinez A.J., Visvesvara G.S.: 1997, Free-living, amphizoic and opportunistic amebas. Brain Pathol 7:583–598.[Medline]
- Martinez A.J., Visvesvara G.S.: 1991, Laboratory diagnosis of pathogenic free-living amebas: Naegleria, Acanthamoeba, and Leptomyxid. Clin Lab Med 11:861–872.[Medline]
- Naginton J.P., Watson G., Playfair T.J., McGill J., Jones B.R., Steele A.D.: 1974, Amoebic infection of the eye. Lancet 2:1537–1540.[Medline]
- Pearce J.R., Powel H.S., Chandler F.W., Visvesvara G.S.: 1985, Amebic meningoencephalitis caused by Acanthamoeba castellanii in a dog. J Am Vet Med Assoc 187:951–952.[Medline]
- Schroeder J.M., Booton G.C., Hay J., Niszl I.A., Seal D.V., Markus M.B., Fuerst P.A., Byers T.J.: 2001, Use of subgenic 18S rDNA PCR and sequencing for generic and genotypic identification of Acanthamoeba from human cases of keratitis and from sewage sludge. J Clin Microbiol 39:1903–1911.[Abstract/Free Full Text]
- Stothard D.R., Schroeder-Diedrich J.M., Awwad M.H., Gast R.J., Ledee D.R., Rodriguez-Zaragoza S., Dean C.L., Fuerst P.A., Byers T.J.: 1998, The evolutionary history of the genus Acanthamoeba and the identification of eight new 18s rRNA gene sequence types. J Eukaryot Microbiol 45:45–54.[Medline]
- Visvesvara G.S., Stehr-Green J.K.: 1990, Epidemiology of free living ameba infections. J Protozool 37:25–33.
- Visvesvara G.S., Schuster F., Martinez A.J.: 1993, Balamuthia mandrillaris N.G. N. Sp. agent of amebic meningoencephalitis in humans and other animals. J Eukaryot Microbiol 40:504–514.[Medline]
TOP
Abstract
References
