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Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil
Author's Accepted Manuscript
Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil Suelen Berger Baldotto MS, DVM, Carolyn Cray PhD, Amália Turner Giannico DMV, Larissa Reifur PhD, DVM, Fabiano Montiani-Ferreira PhD, DVM
www.sasjournal.com
PII: DOI: Reference:
S1557-5063(15)00122-6 http://dx.doi.org/10.1053/j.jepm.2015.08.010 JEPM617
To appear in:
Journal of Exotic Pet Medicine
Cite this article as: Suelen Berger Baldotto MS, DVM, Carolyn Cray PhD, Amália Turner Giannico DMV, Larissa Reifur PhD, DVM, Fabiano Montiani-Ferreira PhD, DVM, Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil, Journal of Exotic Pet Medicine, http://dx.doi.org/10.1053/j.jepm.2015.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AEMV Forum
Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil Suelen Berger Baldotto, MS, DVM, Carolyn Cray, PhD, Amália Turner Giannico, DMV, Larissa Reifur, PhD, DVM, Fabiano Montiani-Ferreira, PhD, DVM From the Department of Veterinary Medicine, School of Veterinary Medicine, Federal University of Paraná, Brazil (Baldotto, Giannico, Reifur, Montiani-Ferreira), Division of Comparative Pathology, Miller School of Medicine, University of Miami, Florida, USA (Cray) Address correspondence to Fabiano Montiani-Ferreira, PhD, DVM, Laboratório de Oftalmologia Comparada, Federal University of Paraná, Rua dos Funcionários, 1540, Juvevê, CEP 80035-050, Curitiba City, Paraná, Brazil. Email address: [email protected]. Telephone: +55 41 3350-5616. Fax: +55 41 33505725.
Abstract Encephalitozoon cuniculi is an obligate intracellular parasite responsible for encephalitozoonosis, primarily an opportunistic infection of rabbits, but also other animals including humans which affects the nervous system, kidney, liver and eyes. This article reports the results of a research investigation that examined
the
seroepidemiological
data
from
rabbits
diagnosed
with
encephalitozoonosis in Brazil. A total of 186 pet rabbits (Oryctolagus cuniculus) either with subclinical infection or presenting with ophthalmic or neurological signs suggestive of encephalitozoonosis were included in the study. Using enzyme-linked immunosorbent assay (ELISA), antibodies to E. cuniculi were detected in 81.7% of the animals. Serological status did not correlate with place
of birth, age, gender, breed or fur color of the animals. Of the 152 seropositive rabbits, 89% (136 rabbits) were subclinical, supporting their role as E. cuniculi reservoirs. Of the 16 seropositive animals with clinical signs, 62.5% (5 rabbits) showed ophthalmic signs, 60% (6 rabbits) presented with neurological signs, and 62.5% (5 rabbits) had a combination of ophthalmic and neurologic signs. These results indicate a high prevalence of E. cuniculi infection in pet rabbits being cared for in Brazil, thus highlighting the importance of improving diagnostic and control measures. Further research studies are required to determine if the parasite strain is a significant factor for zoonotic transmission.
Key words: Encephalitozoon cuniculi; rabbit; zoonosis; serology; clinical signs; ELISA
Encephalitozoon cuniculi is a single-celled eukaryote belonging to the Microsporidia phylum within the Fungi kingdom.1 A wide range of mammals, including humans, can be infected with the E. cuniculi organism through the ingestion or inhalation of spores that can be shed in the feces, urine, or respiratory secretions of infected animals.1,2 Humans have been infected with the same E. cuniculi organism that has been isolated from dogs, cats, rabbits and birds, which suggest zoonotic transmission,3,4,5,6 which likely occurs through exposure to contaminated water, food, or air.1,7 The clinical course of encephalitozoonosis depends on the host’s immunological competence. Immune-competent hosts are usually subclinical carriers, show mild signs
of disease, or have reproductive problems, while immune-compromised individuals often present with a disseminated infection.7,8,9 The disease is of clinical significance in pet and laboratory rodents and rabbits. Rabbits can be chronically subclinical or show signs of renal insufficiency, including polyuria, polydipsia, pollakisuria and azotemia, and nervous system lesions, including head tilt, vestibular syndrome, and hind limb paralysis. The eye, which is immune privileged, can develop uveitis, secondary glaucoma, and cataracts.10,11,12 Immune-competent hosts are able to often control but not completely eliminate the infectious organisms. Treatment may contribute little to total resolution of disease, rather helping to reduce inflammation, secondary bacterial infections, and prevent spore formation.13 Conversely, immune-compromised hosts are considerably more susceptible to fatal infection. Despite the fact that E. cuniculi has been reported to infect animals and humans worldwide, only two prevalence studies have been conducted in Brazil, and these involved animals other than rabbits.9,14 The present work was conducted to provide more information regarding encephalitozoonosis in rabbits, specifically in Brazil, by determining the seroprevalence of antibodies to E. cuniculi in rabbits from southeastern and southern of the country.
Materials and Methods Animals, sample sources and collection In the present study, 186 pet rabbits (Oryctolagus cuniculus) were evaluated from three Brazilian states, including São Paulo state (Presidente Prudente city; 22° 07' 33" S 51° 23' 20" W), Paraná state (Curitiba city; 25° 25' 47" S 49° 16' 19" W), and Santa Catarina state (Federal Institute of Education and Pomerode’s Zoo, Pomerode city; 26° 44' 27" S 49° 10' 37" W). These cities are located in the center of the most developed, densely populated and industrialized region of Brazil (southeast and southern) (Fig. 1). The linear distances from Presidente Prudente to Curitiba is 600 km and from Curitiba to Pomerode is 200 km (Fig. 1). A clinical history including husbandry, age, gender, diet, and health status followed by an external physical examination including a neurological and ophthalmic exam was performed for each animal. During the physical examination, overt neurological signs (e.g. urinary incontinence, head tilt, ataxia, paralysis, circling, rolling, spontaneous nystagmus, tremors, opisthotonus), signs of renal insufficiency (e.g. polyuria, polydipsia, pollakisuria, azotemia) and/or ophthalmic abnormalities (e.g. cataracts, uveitis, glaucoma) were evaluated. Animals that exhibited one or more of the clinical signs often associated with rabbit encephalitozoonosis were considered clinically infected. Although hematology and biochemistry analyses were performed for a thorough assessment of general health, another objective of the study was to correlate possible changes in the blood results with seropositive E. cuniculi cases. Approximately 8 mL of blood was collected from the jugular vein from each animal for hematology (complete
blood count (CBC) and platelet count), serum biochemistry (serum alanine amino transferase, gamma glutamyl transpeptidase, alkaline phosphatase, urea and creatinine) and for disease specific serological tests. Complete blood counts were performed on an automated hematology analyzer BC-2800 Vet (Shenzhen Mindray BioMedical Eletronics Co, LTD, Shenzhen, China). Serum chemistries were measured using the chemistry analyzer BS-200 (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen City, China). Serum samples were frozen at -20oC prior to serology testing.
Enzyme-linked immunosorbent assay (ELISA) examination The frozen sera collected for this investigation was shipped overnight packed with dry ice to the Comparative Pathology Laboratory at the School of Medicine, at the University of Miami, Florida, for E. cuniculi testing. Commercially available ELISA plates (ECUN ELISA plate, Charles River Laboratories, Wilmington, Massachusetts) were coated with rabbit E. cuniculi (ATCC 50503, American Type Culture Collection, Manassas, Virginia) that had been grown in MRC-5 human lung fibroblasts cells. ELISA procedures were performed according to the Cray et al. (2009) methodology. 15 In brief; antigen was extracted via sodium dodecyl sulfate (SDS) treatment, heating, and centrifugation and adjusted to 3 μg/mL for plating. The ELISA plate was constructed to include two samples (ECUN antigen and a negative control antigen). Serum or plasma samples were serially diluted with phosphate-buffered saline (PBS) starting at a dilution of 1:32, and 50 μL of each dilution was added to each well. After incubation the plates were washed with 0.9% NaCl solution and polyoxyethylene sorbitan monolaurate 0.05%
(Tween 20, Sigma Chemical Corp, St. Louis, Missouri USA). Horseradish peroxidaseconjugated rabbit anti-IgG (Sigma Chemical Corp, St Louis, Missouri USA) was added and incubated for 30 min. The plate was washed and 50 µL of 2,2'-azino-bis-3ethylbenzothiazoline-6-sulphonic acid (Kirkegaard Perry Laboratories, Gaithersburg, Maryland USA) was added. The absorbance at 405 nm was measured with a microplate reader (SpectraMax 340PC, Molecular Devices, Sunnyvale, California USA). The index was calculated by dividing the absorbance from all samples by the negative control. Samples were considered positive when the ratio of sample to negative control was ≥ 1.5.
Statistical analysis Fisher's Exact Test was used to investigate the differences between ratios of rabbit groups and their characteristics (e.g. age, sex, health status, fur color, breed). P values <0.05 were considered statistically significant.
Results Of the 186 animals included in this study, 101 were female (54%) and 85 were male (46%). Ages of the subject rabbits ranged from 2.5 months to 4 years, weight 0.6 to 4.4 kg, and different breeds were represented, including 40.4% New Zealand, 12.3% chinchilla, 10.1% English spot, 17% rex, 4.2% lion head, 3.2% Californian, 1.6% Flemish giant and 11.2% unidentified breed. The most common coat colors were white (40%), black (10%), and black and white (11%).
A total of 152 (81.7%) rabbit serum samples were positive for antibodies to E. cuniculi the ELISA test previously described, using the 1:32 dilution as the cut-off. No significant differences were found comparing the number of seropositive and seronegative samples from the distinct Brazilian states investigated (P>0.05) (Table 1). There was no correlation between seropositive animals in relation to sex and age group (Table 2), breed or coat color. There was also not a significant correlation between seropositive and the hematology or serum chemistry profile results. Urea concentration (34.6 ± 12.2 mg/dL for seronegative and 41.2 ± 11.9 mg/dL for seropositive samples, P=0.055) and alkaline phosphatase (136.2 ± 90.4 U/L for seronegative and 191.4 ± 105.6 U/L for seropositive animals, P=0.06) were higher in seropositive animals; however, these serum chemistry correlations were not significant. The majority of the animals (160/186) did not show clinical signs of disease and among the subclinical animals, 85.0% were seropositive for E. cuniculi. The number of subclinical seropositive animals was significantly smaller (P=0.0001) than healthy seropositive animals (Table 2). Among 26 rabbits exhibiting overt signs of disease, 16 were seropositive (61.5%) and each had signs suggestive of encephalitozoonosis, including neurologic or ophthalmic signs, or a combination of both (Table 3). Clinical neurologic
and
ophthalmic
signs
that
were
considered
suspicious
for
encephalitozoonosis included urinary incontinence, hind limb paralysis (without any evidence and history of trauma), head tilt, cataracts, uveitis, glaucoma, and strabismus. The percentage of seronegative rabbits with neurological or ophthalmic signs (10/34) was significantly higher than seropositive rabbits with similar disease conditions (16/152) (P=0.02).
Discussion Encephalitizoon cuniculi is considered a parasite primarily associated with clinical disease in laboratory rabbits; however, the number of seropositive pet rabbits presenting with clinical disease signs has been increasing, thus increasing the risk to human health due to its zoonotic potential.16,17 The present study revealed that 81.7% (152/186) of rabbits from southern and southeastern Brazilian states tested by a disease specific ELISA were seropositive for E. cuniculi. In the group of subclinical animals, 85% (136/160) of were seropositive, and in overtly diseased rabbits, 61.5% (16/26) were seropositive, the reverse of what one would expect. The presence of neurologic or ophthalmic disease signs and seroprevalence for E. cuniculi have no statistical correlation in the population studied (Table 3). This high number of subclinical diseased animals could be biased as random sampling did not occur with this research study. However, the rabbits from this investigation were referred primarily by veterinarians and considering that pet rabbits in Brazil are not usually brought to veterinary clinics when healthy, the number of subclinical seropositive animals is considered significant. The authors believe that the high percentage of E. cuniculi infected rabbits observed in this survey may be explained by several factors: 1) Suitable environmental conditions for the dissemination of the parasite in Brazilian tropical and subtropical regions; 2) owners´ lack of knowledge of the disease and, consequently, its transmission; 3) poor general husbandry practices, which in most cases were less than ideal, especially in multiple rabbit households (i.e. rabbits having access to infected
urine and urine-contaminated food); 4) presence of several relatives living in the same household, resulting in the young having trans-placental exposure. This study performed in South America showed a high seroprevalence of E. cuniculi infection in rabbits when compared with other studies worldwide. Despite this discrepancy between the percentages of seropositive animals, both our study, as well as other investigations, showed an elevated number of subclinical seropositive rabbits. Results from E. cuniculi reports and seroepidemiological surveys in pet rabbits and other animals are available from several countries and also show a high percentage of subclinical seropositive animals. Two distinct surveys conducted in the United Kingdom revealed that antibodies to E. cuniculi were found in 23% and 52% of healthy pet rabbits18,19 while in Japan, the seroprevalence was 63.5%.10 In southern Italy, antibodies to E. cuniculi were identified in 67.2% (84/125) of the tested pet rabbits, 32% of them were subclinical20 and very similar results were found in pet rabbits examined in veterinary clinics located in the USA.15 In Germany, 43% (336/773) of the pet rabbits examined in an investigation were positive for E. cuniculi antibodies with 18% of the subclinical animals testing positive.21 Reports of E. cuniculi infecting a variety of animal species in several other countries, including dogs, goats, cows, horses, alpacas, lemurs and foxes, have been reported.14,22,23,24,25,26,27,28,29 Interestingly, wild rabbits appear to have no contact with E. cuniculi in Australia, England, and Scotland.30,31 The most common clinical signs observed in the animals in this study included urinary incontinence, hind limb paralysis, head tilt, cataract, uveitis, and glaucoma, all previously described as common signs in rabbits infected with E. cuniculi.12,15,16 Despite the fact that several rabbits in this study had typical neurological and ophthalmic signs
and were seropositive for E. cuniculi, there were also rabbits that exhibited similar clinical signs and tested seronegative. Although no disparity in prevalence between different geographic areas was observed in this study or in one investigation conducted in the United Kingdom, there was a significant distinct regional prevalence observed in Taiwan.18,32 In Taiwan the researchers suggest the inequality may be related to population density and the regional practice of allowing rabbits to graze outdoors versus rabbits raised completely indoors. Animals allowed to graze outdoors had a higher rate of infection than rabbits maintained inside a house. In the present study, such a trend was not detected possibly due to the small number of animals from distinct Brazilian states. In addition to geographic location, age, sex, health status, fur color, and breed showed no significant association with the prevalence of seropositive E. cuniculi animals. This could be explained by the fact that infection rates are more likely influenced by the degree of exposure to E. cuniculi rather than susceptibility.33 There was no association between seropositive rabbits and changes in hematological or biochemical values, except for a slight tendency towards an increase in urea and alkaline phosphatase levels; however, this difference was not statistically significant. Urea and creatinine have been shown to increase in the presence of kidney disease16 but the significance of slightly increased urea levels without concurrent increase in creatinine is unknown. Regarding liver function, researchers state that this disease can cause significant hepatic alterations,2 which may explain the slight changes in the alkaline phosphatase levels observed in this study; however, other hepatic enzymes were within normal limits. Alkaline phosphatase is non-specific, can change
with other disease processes, and may vary according to life stage. These findings strongly suggest that hematological and biochemical evaluations are not very useful as sole diagnostic tests for the diagnosis of E. cuniculi infection but can be useful as supporting evidence for other concurrent disease conditions. In conclusion, the present study determined the seroprevalence of E. cuniculi in rabbits from distinct geographic locations in Brazil. As previously mentioned, geographical surveys provide important epidemiological data relevant to human health, as E. cuniculi is a zoonotic organism.12 The high percentage of seropositive healthy rabbits detected in this study (143 of 171 = 83.6%) demonstrates the importance of performing routine testing and screening, especially when the owners belong to a high risk group, such as those suffering from immunosuppressive diseases. 12 This information may also be important, as E. cuniculi has been suggested as a cause of fatal canine neurological and renal disease and a cause of reproductive problems in other animals.8,9,23,34,35 Seropositive animals that exhibited clinical disease signs of encephalitozoonosis were associated with a slight, but not statistically significant increase in creatinine and alkaline phosphatase levels. Thus, for the animals presenting with clinical disease signs suggestive of encephalitozoonosis and increased alkaline phosphatase and creatinine, an initial serologic evaluation is recommended, and if negative, additional testing should occur 4 weeks later to assess for possible seroconversion.12
Acknowledgements The authors thank Dr. Gillian Shaw, Johns Hopkins University, Baltimore, Maryland, USA, for her invaluable help in the preparation of this manuscript.
References 1. Didier ES, Stovall ME, Green LC, et al: Epidemiology of microsporidiosis: sources and modes of transmission. Vet Parasitol 126:145-166, 2004 2. Cox JC, Hamilton RC, Attwood HD: An investigation of the route and progression of Encephalitozoon cuniculi infection in adult rabbits. J Protozool 26:260-265, 1979 3. Mathis A, Michel M, Kuster H, et al: Two Encephalitozoon cuniculi strains of human origin are infectious to rabbits. Parasitology 114:29-35, 1997 4. Mathis A, Weber R, Deplazes P: Zoonotic potential of the microsporidia. Clin Microbiol Rev 18:423-445, 2005 5. Kasickova D, Sak B, Kvac M, et al: Sources of potentially infectious human microsporidia: molecular characterisation of microsporidia isolates from exotic birds in the Czech Republic, prevalence study and importance of birds in epidemiology of the human microsporidial infections. Vet Parasitol 165:125-130, 2009 6. Benz P, Maass G, Csokai J, et al: Detection of Encephalitozoon cuniculi in the feline cataractous lens. Vet Ophthalmol 1:37-47, 2011
7. Didier ES, Didier PJ, Snowden KF, et al: Microsporidiosis in mammals. Microbes Infect 2: 709-720, 2000 8. Fournier S, Liguory O, Sarfati C, et al: Disseminated infection due to Encephalitozoon cuniculi in a patient with AIDS: case report and review. HIV Med 1:155-161, 2000 9. Goodwin D, Gennari SM, Howe DK, et al: Prevalence of antibodies to Encephalitozoon cuniculi in horses from Brazil. Vet Parasitol 142:380-382, 2006 10. Igarashi M, Oohashi E, Dautu G, et al: High seroprevalence of Encephalitozoon cuniculi in pet rabbits in Japan. J Vet Med Sci 70:1301-1304, 2008 11. Kunzel F, Gruber A, Tichy A, et al: Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits. Vet Parasitol 151:115-124, 2008 12. Santaniello A, Dipineto L, Rinaldi L, et al: Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy. Res Vet Sci 87:67-69, 2009 13. Sieg J, Hein J, Jass A, et al: Clinical evaluation of therapeutic success in rabbits with suspected encephalitozoonosis. Vet Parasitol 187:328-332, 2012 14. Lindsay DS, Goodwin DG, Zajac AM, et al: Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia. J Parasitol 95:760-763, 2009 15. Cray C, Arcia G, Schneider R, et al: Evaluation of the usefulness of an ELISA and protein electrophoresis in the diagnosis of Encephalitozoon cuniculi infection in rabbits. Am J Vet Res 70:478-482, 2009
16. Kunzel F, Joachim A: Encephalitozoonosis in rabbits. Parasitol Res 106:299-309, 2010 17. Varga M: Questions around Encephalitozoon cuniculi in rabbits. Vet Rec 174:347348, 2014 18. Harcourt-Brown FM, Holloway HK: Encephalitozoon cuniculi in pet rabbits. Vet Rec 152: 427-431, 2003 19. Keeble EJ, Shaw DJ: Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in the United Kingdom. Vet Rec 158:539-544, 2006 20. Dipineto L, Rinaldi L, Santaniello A, et al: Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy. Zoonoses Public Health 55:173-175, 2008 21. Hein J, Flock U, Sauter-Louis C, et al: Encephalitozoon cuniculi in rabbits in Germany: prevalence and sensitivity of antibody testing. Vet Rec 174:350, 2014 22. Cislakova L, Literak I, Balent P, et al: Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders. Ann Agric Environ Med 8:289-291, 2001 23. Halanova M, Letkova V, Macak V, et al: The first finding of antibodies to Encephalitozoon cuniculi in cows in Slovakia. Vet Parasitol 82:167-171, 1999
24. Levkutova M, Hipikova V, Faitelzon S, et al: Prevalence of antibodies to Encephalitozoon cuniculi in horses in the Israel. Ann Agric Environ Med 11:265-267, 2004 25. Malcekova B, Halanova M, Sulinova Z, et al: Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals. Res Vet Sci 89:358-361, 2010 26. Murphy TM, Walochnik J, Hassl A, et al: Study on the prevalence of Toxoplasma gondii and Neospora caninum and molecular evidence of Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis infections in red foxes (Vulpes vulpes) in rural Ireland. Vet Parasitol 146:227-234, 2007 27. Wasson K, Peper RL: Mammalian microsporidiosis. Vet Pathol 37:113-128, 2000 28. Webster JD, Miller MA, Vemulapalli R: Encephalitozoon cuniculi-associated placentitis and perinatal death in an alpaca (Lama pacos). Vet Pathol 45:255-258, 2008 29. Yabsley MJ, Jordan CN, Mitchell SM, et al: Seroprevalence of Toxoplasma gondii, Sarcocystis neurona, and Encephalitozoon cuniculi in three species of lemurs from St. Catherines Island, GA, USA. Vet Parasitol 144:28-32, 2007 30. Cox JC, Pye D, Edmonds JW, et al: An investigation of Encephalitozoon cuniculi in the wild rabbit Oryctolagus cuniculus in Victoria, Australia. J Hyg (Lond) 84:295-300, 1980
31. Cox JC, Ross J: A serological survey of Encephalitozoon cuniculi infection in the wild rabbit in England and Scotland. Res Vet Sci 28:396, 1980 32. Tee KY, Kao JP, Chiu HY, et al: Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan. Vet Parasitol 183:68-71, 2011 33. Kunstyr I, Naumann S: Head tilt in rabbits caused by pasteurellosis and encephalitozoonosis. Lab Anim 19:208-213, 1985 34. Snowden KF, Lewis BC, Hoffman J, et al: Encephalitozoon cuniculi infections in dogs: a case series. J Am Anim Hosp Assoc 45:225-231, 2009 35. Tosoni A, Nebuloni M, Ferri A, et al: Disseminated microsporidiosis caused by Encephalitozoon cuniculi III (dog type) in an Italian AIDS patient: a retrospective study. Mod Pathol 15:577-583, 2002 Figure legend Fig. 1. Map of Brazil demonstrating the different states and regions. Dots indicate cities in the three states in which a population of rabbits (Oryctolagus cuniculus) was investigated: Red dot Presidente Prudente (São Paulo); Black dot - Curitiba (Paraná); Yelow dot - Pomerode (Santa Catarina).
TABLE 1: Seroprevalence of Encephalitozoon cuniculi in rabbits from three Brazilian states, based on enzymelinked immunosorbent assay (ELISA). State No. tested No. positive % Paraná 157 129 82.2 Santa Catarina 18 13 72.2 São Paulo 11 10 90.9 186 152 81.7 Total
TABLE 2: Number of rabbits according to sex, age, and clinical status that were seropositive for Encephalitozoon cuniculi based on enzyme-linked immunosorbent assay (ELISA) and P value of the comparisons between the groups. Rabbit data No. tested No. positive % of positive P* Sex Male 85 70 82.3 0.91 Female 101 82 81.2 Age 2.5 to 6 months 124 103 83.1 6 months to 2 years 59 47 79.7 0.85 2 years to 4 years 3 2 66.7 Clinical status Symptomatic 26 16 61.5 0.0001 Asymptomatic 160 136 85.0 Total 186 152 81.7 * Fisher’s Exact Test.
TABLE 3: Number of rabbits with and without neurologic and ophthalmic clinical signs that were seropositive for Encephalitozoon cuniculi based on enzymelinked immunosorbent assay (ELISA). Clinical signs No. tested No. seropositive % of positive Neurological 10 6 60.0 Ophthalmic 8 5 62.5 Neurological and ophthalmic 8 5 62.5 Total 26 16 61.5
Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil Suelen Berger Baldotto MS, DVM, Carolyn Cray PhD, Amália Turner Giannico DMV, Larissa Reifur PhD, DVM, Fabiano Montiani-Ferreira PhD, DVM
www.sasjournal.com
PII: DOI: Reference:
S1557-5063(15)00122-6 http://dx.doi.org/10.1053/j.jepm.2015.08.010 JEPM617
To appear in:
Journal of Exotic Pet Medicine
Cite this article as: Suelen Berger Baldotto MS, DVM, Carolyn Cray PhD, Amália Turner Giannico DMV, Larissa Reifur PhD, DVM, Fabiano Montiani-Ferreira PhD, DVM, Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil, Journal of Exotic Pet Medicine, http://dx.doi.org/10.1053/j.jepm.2015.08.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AEMV Forum
Seroprevalence of Encephalitozoon cuniculi Infection in Pet Rabbits in Brazil Suelen Berger Baldotto, MS, DVM, Carolyn Cray, PhD, Amália Turner Giannico, DMV, Larissa Reifur, PhD, DVM, Fabiano Montiani-Ferreira, PhD, DVM From the Department of Veterinary Medicine, School of Veterinary Medicine, Federal University of Paraná, Brazil (Baldotto, Giannico, Reifur, Montiani-Ferreira), Division of Comparative Pathology, Miller School of Medicine, University of Miami, Florida, USA (Cray) Address correspondence to Fabiano Montiani-Ferreira, PhD, DVM, Laboratório de Oftalmologia Comparada, Federal University of Paraná, Rua dos Funcionários, 1540, Juvevê, CEP 80035-050, Curitiba City, Paraná, Brazil. Email address: [email protected]. Telephone: +55 41 3350-5616. Fax: +55 41 33505725.
Abstract Encephalitozoon cuniculi is an obligate intracellular parasite responsible for encephalitozoonosis, primarily an opportunistic infection of rabbits, but also other animals including humans which affects the nervous system, kidney, liver and eyes. This article reports the results of a research investigation that examined
the
seroepidemiological
data
from
rabbits
diagnosed
with
encephalitozoonosis in Brazil. A total of 186 pet rabbits (Oryctolagus cuniculus) either with subclinical infection or presenting with ophthalmic or neurological signs suggestive of encephalitozoonosis were included in the study. Using enzyme-linked immunosorbent assay (ELISA), antibodies to E. cuniculi were detected in 81.7% of the animals. Serological status did not correlate with place
of birth, age, gender, breed or fur color of the animals. Of the 152 seropositive rabbits, 89% (136 rabbits) were subclinical, supporting their role as E. cuniculi reservoirs. Of the 16 seropositive animals with clinical signs, 62.5% (5 rabbits) showed ophthalmic signs, 60% (6 rabbits) presented with neurological signs, and 62.5% (5 rabbits) had a combination of ophthalmic and neurologic signs. These results indicate a high prevalence of E. cuniculi infection in pet rabbits being cared for in Brazil, thus highlighting the importance of improving diagnostic and control measures. Further research studies are required to determine if the parasite strain is a significant factor for zoonotic transmission.
Key words: Encephalitozoon cuniculi; rabbit; zoonosis; serology; clinical signs; ELISA
Encephalitozoon cuniculi is a single-celled eukaryote belonging to the Microsporidia phylum within the Fungi kingdom.1 A wide range of mammals, including humans, can be infected with the E. cuniculi organism through the ingestion or inhalation of spores that can be shed in the feces, urine, or respiratory secretions of infected animals.1,2 Humans have been infected with the same E. cuniculi organism that has been isolated from dogs, cats, rabbits and birds, which suggest zoonotic transmission,3,4,5,6 which likely occurs through exposure to contaminated water, food, or air.1,7 The clinical course of encephalitozoonosis depends on the host’s immunological competence. Immune-competent hosts are usually subclinical carriers, show mild signs
of disease, or have reproductive problems, while immune-compromised individuals often present with a disseminated infection.7,8,9 The disease is of clinical significance in pet and laboratory rodents and rabbits. Rabbits can be chronically subclinical or show signs of renal insufficiency, including polyuria, polydipsia, pollakisuria and azotemia, and nervous system lesions, including head tilt, vestibular syndrome, and hind limb paralysis. The eye, which is immune privileged, can develop uveitis, secondary glaucoma, and cataracts.10,11,12 Immune-competent hosts are able to often control but not completely eliminate the infectious organisms. Treatment may contribute little to total resolution of disease, rather helping to reduce inflammation, secondary bacterial infections, and prevent spore formation.13 Conversely, immune-compromised hosts are considerably more susceptible to fatal infection. Despite the fact that E. cuniculi has been reported to infect animals and humans worldwide, only two prevalence studies have been conducted in Brazil, and these involved animals other than rabbits.9,14 The present work was conducted to provide more information regarding encephalitozoonosis in rabbits, specifically in Brazil, by determining the seroprevalence of antibodies to E. cuniculi in rabbits from southeastern and southern of the country.
Materials and Methods Animals, sample sources and collection In the present study, 186 pet rabbits (Oryctolagus cuniculus) were evaluated from three Brazilian states, including São Paulo state (Presidente Prudente city; 22° 07' 33" S 51° 23' 20" W), Paraná state (Curitiba city; 25° 25' 47" S 49° 16' 19" W), and Santa Catarina state (Federal Institute of Education and Pomerode’s Zoo, Pomerode city; 26° 44' 27" S 49° 10' 37" W). These cities are located in the center of the most developed, densely populated and industrialized region of Brazil (southeast and southern) (Fig. 1). The linear distances from Presidente Prudente to Curitiba is 600 km and from Curitiba to Pomerode is 200 km (Fig. 1). A clinical history including husbandry, age, gender, diet, and health status followed by an external physical examination including a neurological and ophthalmic exam was performed for each animal. During the physical examination, overt neurological signs (e.g. urinary incontinence, head tilt, ataxia, paralysis, circling, rolling, spontaneous nystagmus, tremors, opisthotonus), signs of renal insufficiency (e.g. polyuria, polydipsia, pollakisuria, azotemia) and/or ophthalmic abnormalities (e.g. cataracts, uveitis, glaucoma) were evaluated. Animals that exhibited one or more of the clinical signs often associated with rabbit encephalitozoonosis were considered clinically infected. Although hematology and biochemistry analyses were performed for a thorough assessment of general health, another objective of the study was to correlate possible changes in the blood results with seropositive E. cuniculi cases. Approximately 8 mL of blood was collected from the jugular vein from each animal for hematology (complete
blood count (CBC) and platelet count), serum biochemistry (serum alanine amino transferase, gamma glutamyl transpeptidase, alkaline phosphatase, urea and creatinine) and for disease specific serological tests. Complete blood counts were performed on an automated hematology analyzer BC-2800 Vet (Shenzhen Mindray BioMedical Eletronics Co, LTD, Shenzhen, China). Serum chemistries were measured using the chemistry analyzer BS-200 (Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen City, China). Serum samples were frozen at -20oC prior to serology testing.
Enzyme-linked immunosorbent assay (ELISA) examination The frozen sera collected for this investigation was shipped overnight packed with dry ice to the Comparative Pathology Laboratory at the School of Medicine, at the University of Miami, Florida, for E. cuniculi testing. Commercially available ELISA plates (ECUN ELISA plate, Charles River Laboratories, Wilmington, Massachusetts) were coated with rabbit E. cuniculi (ATCC 50503, American Type Culture Collection, Manassas, Virginia) that had been grown in MRC-5 human lung fibroblasts cells. ELISA procedures were performed according to the Cray et al. (2009) methodology. 15 In brief; antigen was extracted via sodium dodecyl sulfate (SDS) treatment, heating, and centrifugation and adjusted to 3 μg/mL for plating. The ELISA plate was constructed to include two samples (ECUN antigen and a negative control antigen). Serum or plasma samples were serially diluted with phosphate-buffered saline (PBS) starting at a dilution of 1:32, and 50 μL of each dilution was added to each well. After incubation the plates were washed with 0.9% NaCl solution and polyoxyethylene sorbitan monolaurate 0.05%
(Tween 20, Sigma Chemical Corp, St. Louis, Missouri USA). Horseradish peroxidaseconjugated rabbit anti-IgG (Sigma Chemical Corp, St Louis, Missouri USA) was added and incubated for 30 min. The plate was washed and 50 µL of 2,2'-azino-bis-3ethylbenzothiazoline-6-sulphonic acid (Kirkegaard Perry Laboratories, Gaithersburg, Maryland USA) was added. The absorbance at 405 nm was measured with a microplate reader (SpectraMax 340PC, Molecular Devices, Sunnyvale, California USA). The index was calculated by dividing the absorbance from all samples by the negative control. Samples were considered positive when the ratio of sample to negative control was ≥ 1.5.
Statistical analysis Fisher's Exact Test was used to investigate the differences between ratios of rabbit groups and their characteristics (e.g. age, sex, health status, fur color, breed). P values <0.05 were considered statistically significant.
Results Of the 186 animals included in this study, 101 were female (54%) and 85 were male (46%). Ages of the subject rabbits ranged from 2.5 months to 4 years, weight 0.6 to 4.4 kg, and different breeds were represented, including 40.4% New Zealand, 12.3% chinchilla, 10.1% English spot, 17% rex, 4.2% lion head, 3.2% Californian, 1.6% Flemish giant and 11.2% unidentified breed. The most common coat colors were white (40%), black (10%), and black and white (11%).
A total of 152 (81.7%) rabbit serum samples were positive for antibodies to E. cuniculi the ELISA test previously described, using the 1:32 dilution as the cut-off. No significant differences were found comparing the number of seropositive and seronegative samples from the distinct Brazilian states investigated (P>0.05) (Table 1). There was no correlation between seropositive animals in relation to sex and age group (Table 2), breed or coat color. There was also not a significant correlation between seropositive and the hematology or serum chemistry profile results. Urea concentration (34.6 ± 12.2 mg/dL for seronegative and 41.2 ± 11.9 mg/dL for seropositive samples, P=0.055) and alkaline phosphatase (136.2 ± 90.4 U/L for seronegative and 191.4 ± 105.6 U/L for seropositive animals, P=0.06) were higher in seropositive animals; however, these serum chemistry correlations were not significant. The majority of the animals (160/186) did not show clinical signs of disease and among the subclinical animals, 85.0% were seropositive for E. cuniculi. The number of subclinical seropositive animals was significantly smaller (P=0.0001) than healthy seropositive animals (Table 2). Among 26 rabbits exhibiting overt signs of disease, 16 were seropositive (61.5%) and each had signs suggestive of encephalitozoonosis, including neurologic or ophthalmic signs, or a combination of both (Table 3). Clinical neurologic
and
ophthalmic
signs
that
were
considered
suspicious
for
encephalitozoonosis included urinary incontinence, hind limb paralysis (without any evidence and history of trauma), head tilt, cataracts, uveitis, glaucoma, and strabismus. The percentage of seronegative rabbits with neurological or ophthalmic signs (10/34) was significantly higher than seropositive rabbits with similar disease conditions (16/152) (P=0.02).
Discussion Encephalitizoon cuniculi is considered a parasite primarily associated with clinical disease in laboratory rabbits; however, the number of seropositive pet rabbits presenting with clinical disease signs has been increasing, thus increasing the risk to human health due to its zoonotic potential.16,17 The present study revealed that 81.7% (152/186) of rabbits from southern and southeastern Brazilian states tested by a disease specific ELISA were seropositive for E. cuniculi. In the group of subclinical animals, 85% (136/160) of were seropositive, and in overtly diseased rabbits, 61.5% (16/26) were seropositive, the reverse of what one would expect. The presence of neurologic or ophthalmic disease signs and seroprevalence for E. cuniculi have no statistical correlation in the population studied (Table 3). This high number of subclinical diseased animals could be biased as random sampling did not occur with this research study. However, the rabbits from this investigation were referred primarily by veterinarians and considering that pet rabbits in Brazil are not usually brought to veterinary clinics when healthy, the number of subclinical seropositive animals is considered significant. The authors believe that the high percentage of E. cuniculi infected rabbits observed in this survey may be explained by several factors: 1) Suitable environmental conditions for the dissemination of the parasite in Brazilian tropical and subtropical regions; 2) owners´ lack of knowledge of the disease and, consequently, its transmission; 3) poor general husbandry practices, which in most cases were less than ideal, especially in multiple rabbit households (i.e. rabbits having access to infected
urine and urine-contaminated food); 4) presence of several relatives living in the same household, resulting in the young having trans-placental exposure. This study performed in South America showed a high seroprevalence of E. cuniculi infection in rabbits when compared with other studies worldwide. Despite this discrepancy between the percentages of seropositive animals, both our study, as well as other investigations, showed an elevated number of subclinical seropositive rabbits. Results from E. cuniculi reports and seroepidemiological surveys in pet rabbits and other animals are available from several countries and also show a high percentage of subclinical seropositive animals. Two distinct surveys conducted in the United Kingdom revealed that antibodies to E. cuniculi were found in 23% and 52% of healthy pet rabbits18,19 while in Japan, the seroprevalence was 63.5%.10 In southern Italy, antibodies to E. cuniculi were identified in 67.2% (84/125) of the tested pet rabbits, 32% of them were subclinical20 and very similar results were found in pet rabbits examined in veterinary clinics located in the USA.15 In Germany, 43% (336/773) of the pet rabbits examined in an investigation were positive for E. cuniculi antibodies with 18% of the subclinical animals testing positive.21 Reports of E. cuniculi infecting a variety of animal species in several other countries, including dogs, goats, cows, horses, alpacas, lemurs and foxes, have been reported.14,22,23,24,25,26,27,28,29 Interestingly, wild rabbits appear to have no contact with E. cuniculi in Australia, England, and Scotland.30,31 The most common clinical signs observed in the animals in this study included urinary incontinence, hind limb paralysis, head tilt, cataract, uveitis, and glaucoma, all previously described as common signs in rabbits infected with E. cuniculi.12,15,16 Despite the fact that several rabbits in this study had typical neurological and ophthalmic signs
and were seropositive for E. cuniculi, there were also rabbits that exhibited similar clinical signs and tested seronegative. Although no disparity in prevalence between different geographic areas was observed in this study or in one investigation conducted in the United Kingdom, there was a significant distinct regional prevalence observed in Taiwan.18,32 In Taiwan the researchers suggest the inequality may be related to population density and the regional practice of allowing rabbits to graze outdoors versus rabbits raised completely indoors. Animals allowed to graze outdoors had a higher rate of infection than rabbits maintained inside a house. In the present study, such a trend was not detected possibly due to the small number of animals from distinct Brazilian states. In addition to geographic location, age, sex, health status, fur color, and breed showed no significant association with the prevalence of seropositive E. cuniculi animals. This could be explained by the fact that infection rates are more likely influenced by the degree of exposure to E. cuniculi rather than susceptibility.33 There was no association between seropositive rabbits and changes in hematological or biochemical values, except for a slight tendency towards an increase in urea and alkaline phosphatase levels; however, this difference was not statistically significant. Urea and creatinine have been shown to increase in the presence of kidney disease16 but the significance of slightly increased urea levels without concurrent increase in creatinine is unknown. Regarding liver function, researchers state that this disease can cause significant hepatic alterations,2 which may explain the slight changes in the alkaline phosphatase levels observed in this study; however, other hepatic enzymes were within normal limits. Alkaline phosphatase is non-specific, can change
with other disease processes, and may vary according to life stage. These findings strongly suggest that hematological and biochemical evaluations are not very useful as sole diagnostic tests for the diagnosis of E. cuniculi infection but can be useful as supporting evidence for other concurrent disease conditions. In conclusion, the present study determined the seroprevalence of E. cuniculi in rabbits from distinct geographic locations in Brazil. As previously mentioned, geographical surveys provide important epidemiological data relevant to human health, as E. cuniculi is a zoonotic organism.12 The high percentage of seropositive healthy rabbits detected in this study (143 of 171 = 83.6%) demonstrates the importance of performing routine testing and screening, especially when the owners belong to a high risk group, such as those suffering from immunosuppressive diseases. 12 This information may also be important, as E. cuniculi has been suggested as a cause of fatal canine neurological and renal disease and a cause of reproductive problems in other animals.8,9,23,34,35 Seropositive animals that exhibited clinical disease signs of encephalitozoonosis were associated with a slight, but not statistically significant increase in creatinine and alkaline phosphatase levels. Thus, for the animals presenting with clinical disease signs suggestive of encephalitozoonosis and increased alkaline phosphatase and creatinine, an initial serologic evaluation is recommended, and if negative, additional testing should occur 4 weeks later to assess for possible seroconversion.12
Acknowledgements The authors thank Dr. Gillian Shaw, Johns Hopkins University, Baltimore, Maryland, USA, for her invaluable help in the preparation of this manuscript.
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16. Kunzel F, Joachim A: Encephalitozoonosis in rabbits. Parasitol Res 106:299-309, 2010 17. Varga M: Questions around Encephalitozoon cuniculi in rabbits. Vet Rec 174:347348, 2014 18. Harcourt-Brown FM, Holloway HK: Encephalitozoon cuniculi in pet rabbits. Vet Rec 152: 427-431, 2003 19. Keeble EJ, Shaw DJ: Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in the United Kingdom. Vet Rec 158:539-544, 2006 20. Dipineto L, Rinaldi L, Santaniello A, et al: Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy. Zoonoses Public Health 55:173-175, 2008 21. Hein J, Flock U, Sauter-Louis C, et al: Encephalitozoon cuniculi in rabbits in Germany: prevalence and sensitivity of antibody testing. Vet Rec 174:350, 2014 22. Cislakova L, Literak I, Balent P, et al: Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders. Ann Agric Environ Med 8:289-291, 2001 23. Halanova M, Letkova V, Macak V, et al: The first finding of antibodies to Encephalitozoon cuniculi in cows in Slovakia. Vet Parasitol 82:167-171, 1999
24. Levkutova M, Hipikova V, Faitelzon S, et al: Prevalence of antibodies to Encephalitozoon cuniculi in horses in the Israel. Ann Agric Environ Med 11:265-267, 2004 25. Malcekova B, Halanova M, Sulinova Z, et al: Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals. Res Vet Sci 89:358-361, 2010 26. Murphy TM, Walochnik J, Hassl A, et al: Study on the prevalence of Toxoplasma gondii and Neospora caninum and molecular evidence of Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis infections in red foxes (Vulpes vulpes) in rural Ireland. Vet Parasitol 146:227-234, 2007 27. Wasson K, Peper RL: Mammalian microsporidiosis. Vet Pathol 37:113-128, 2000 28. Webster JD, Miller MA, Vemulapalli R: Encephalitozoon cuniculi-associated placentitis and perinatal death in an alpaca (Lama pacos). Vet Pathol 45:255-258, 2008 29. Yabsley MJ, Jordan CN, Mitchell SM, et al: Seroprevalence of Toxoplasma gondii, Sarcocystis neurona, and Encephalitozoon cuniculi in three species of lemurs from St. Catherines Island, GA, USA. Vet Parasitol 144:28-32, 2007 30. Cox JC, Pye D, Edmonds JW, et al: An investigation of Encephalitozoon cuniculi in the wild rabbit Oryctolagus cuniculus in Victoria, Australia. J Hyg (Lond) 84:295-300, 1980
31. Cox JC, Ross J: A serological survey of Encephalitozoon cuniculi infection in the wild rabbit in England and Scotland. Res Vet Sci 28:396, 1980 32. Tee KY, Kao JP, Chiu HY, et al: Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan. Vet Parasitol 183:68-71, 2011 33. Kunstyr I, Naumann S: Head tilt in rabbits caused by pasteurellosis and encephalitozoonosis. Lab Anim 19:208-213, 1985 34. Snowden KF, Lewis BC, Hoffman J, et al: Encephalitozoon cuniculi infections in dogs: a case series. J Am Anim Hosp Assoc 45:225-231, 2009 35. Tosoni A, Nebuloni M, Ferri A, et al: Disseminated microsporidiosis caused by Encephalitozoon cuniculi III (dog type) in an Italian AIDS patient: a retrospective study. Mod Pathol 15:577-583, 2002 Figure legend Fig. 1. Map of Brazil demonstrating the different states and regions. Dots indicate cities in the three states in which a population of rabbits (Oryctolagus cuniculus) was investigated: Red dot Presidente Prudente (São Paulo); Black dot - Curitiba (Paraná); Yelow dot - Pomerode (Santa Catarina).
TABLE 1: Seroprevalence of Encephalitozoon cuniculi in rabbits from three Brazilian states, based on enzymelinked immunosorbent assay (ELISA). State No. tested No. positive % Paraná 157 129 82.2 Santa Catarina 18 13 72.2 São Paulo 11 10 90.9 186 152 81.7 Total
TABLE 2: Number of rabbits according to sex, age, and clinical status that were seropositive for Encephalitozoon cuniculi based on enzyme-linked immunosorbent assay (ELISA) and P value of the comparisons between the groups. Rabbit data No. tested No. positive % of positive P* Sex Male 85 70 82.3 0.91 Female 101 82 81.2 Age 2.5 to 6 months 124 103 83.1 6 months to 2 years 59 47 79.7 0.85 2 years to 4 years 3 2 66.7 Clinical status Symptomatic 26 16 61.5 0.0001 Asymptomatic 160 136 85.0 Total 186 152 81.7 * Fisher’s Exact Test.
TABLE 3: Number of rabbits with and without neurologic and ophthalmic clinical signs that were seropositive for Encephalitozoon cuniculi based on enzymelinked immunosorbent assay (ELISA). Clinical signs No. tested No. seropositive % of positive Neurological 10 6 60.0 Ophthalmic 8 5 62.5 Neurological and ophthalmic 8 5 62.5 Total 26 16 61.5