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Clinical Update on Testing Modalities for Encephalitozoon cuniculi in Clinically Sick Rabbits
Topics in Medicine and Surgery
Clinical Update on Testing Modalities for Encephalitozoon cuniculi in Clinically Sick Rabbits Gregory Rich, DVM
Abstract This article describes the clinical presentation of rabbit patients infected with Encephalitozoon cuniculi that present to veterinary hospitals. The difficulty in obtaining a definitive diagnosis with this disease, even in animals with severe clinical signs, is frustrating to veterinarians and owners alike. A detailed review of current diagnostic testing methodologies and recent advances should help with the interpretation of E. cuniculi test results to communicate and inform owners on treatment and prevention of this devastating disease. Copyright 2010 Elsevier Inc. All rights reserved. Key words: Clinical signs; diagnostic test; Encephalitozoon cuniculi; neurologic; rabbit
E
ncephalitozoon cuniculi is a common disease of pet rabbits and is an internationally recognized disease affecting rabbit colonies.1-5 Encephalitozoon represents a genus of obligate intracellular parasites from the phylum Microsporidia, subphylum Sporozoa. The following species of Encephalitozoon have been identified and shown to be opportunistic pathogens for immunocompromised humans: E. intestinalis, E. hellum, E. bieniusi, and E. septata. In human patients with AIDS, E. cuniculi infections have been attributed to cases with interstitial pneumonitis, diarrhea, and muscle wasting.1 Infection of the host usually occurs after ingestion of food contaminated with rabbit urine that has E. cuniculi spores, although infection through inhalation of infectious spores has also been postulated. Vertical infection in utero from an infected dam is also possible. After ingestion or inhalation, monocytes infected with E. cuniculi spores transport the organism to primary colonization sites (e.g., kidneys, brain, lungs, liver).1-4 Spores can survive within the environment for up to 4 weeks at room temperature.3 Encephalitozoonosis is a problem in laboratory rabbits because its effects on the health of the animals can interfere with investigative results.3 A definitive antemortem diagnosis is difficult because histopathology is
226
required to demonstrate the presence of E. cuniculi. Serological testing is difficult to interpret because a positive titer may be indicative of exposure and not a true infection.6 Postmortem diagnosis can also be difficult because of the sparse number of organisms present in many chronically infected brain and/or renal tissues.1,7-10 Because of the inability of current diagnostic tests to provide adequate answers to the E. cuniculi status of a rabbit, there is ongoing investigative research being done to improve our understanding of how to interpret test results with parallel testing strategies and to improve overall test reliability and sensitivity/specificity.6-10
Clinical Signs The clinical picture of an E. cuniculi infected rabbit can vary from a subclinical carrier to acute death.1-5 NeuFrom West Esplanade Veterinary Clinic and Bird Hospital, Metairie, LA USA. Address correspondence to: Gregory Rich, DVM, West Esplanade Veterinary Clinic, 3640 West Esplanade, Metairie, LA 70002. E-mail: [email protected] © 2010 Elsevier Inc. All rights reserved. 1557-5063/10/1903-$30.00 doi:10.1053/j.jepm.2010.07.007
Journal of Exotic Pet Medicine, Vol 19, No 3 ( July), 2010: pp 226 –230
227
Clinical Testing for E. cuniculi in Symptomatic Rabbits
Figure 1. Disorientation and rolling behavior due to central nervous system manifestations of E. cuniculi infection.
rologic signs comprise the greatest percentage of patient presentations relative to a presumed encephalitozoonosis diagnosis. Vestibular disease and seizures are the most obvious clinical signs associated with a rabbit infected with E. cuniculi, although intermittent aggressive behavior, ataxia, seizures, and swaying may also be present.1 Posterior paresis and severe muscle wasting have been associated with late-stage disease presentations. Vestibular signs range from a mild head tilt to a severe inability for the patient to “right” itself, creating a constant rolling presentation.1,4,5 The neurological manifestations are presumed to occur as brain cells rupture and E. cuniculi organisms are released into surrounding tissues. Common terms used to describe the vestibular disease include wryneck, torticollis, head tilt, or labyrinthitis (Fig 1).1,5 Renal manifestations are equally quite varied.1-5 Subtle clinical signs such as urine scald in the perineal region and along the medial aspect of the
Figure 3. Cataract formation in an adult rabbit.
rear legs and “bladder sludge” have been noted in cases testing positive for E. cuniculi. Dysuria is thought to be secondary to the neurologic manifestations of the disease or from muscle wasting associated with the patient’s general illness. Mild azotemia and elevations in serum/plasma creatinine are occasionally noted, but these abnormal findings are not reliable indicators for E. cuniculi infections (Fig 2). Intraocular disease, such as cataract formation, hypopyon, and/or uveitis, is commonly diagnosed in pet rabbits.1-5 A large percentage of rabbit cases with intraocular disease have tested positive for E. cuniculi using serologic, histopathologic, or polymerase chain reaction (PCR) testing.1-5 Separate studies by Kunzel and Csokai revealed that a high percentage of cases with phacoclastic uveitis secondary to lens rupture tested positive for E. cuniculi on PCR analysis of lens material (Fig 3).7,9 Nonspecific disease presentations of general malaise, weight loss of unknown origin, anorexia, sore hocks, and diarrhea are common complaints of owners with sick rabbits. Diagnostic testing for E. cuniculi in these cases may identify this organism as a factor in these nonspecific disease processes, but should not preclude a complete medical workup.
Diagnostic Features
Figure 2. Urine scald caused by renal disease and dysuria from E. cuniculi infection.
Encephalitozoon cuniculi causes a wide range of disease manifestations in pet rabbits. Subclinical carriers are a primary source of disease exposure in multiple rabbit households, pet stores, and breeder colonies.1 A susceptible rabbit can become infected by ingesting food contaminated with urine. There has been verification
228 that young rabbits can be infected by a diseased parent during their first few days of life.1 As mentioned earlier, vertical transmission in utero may also occur. Natural infection in young rabbits results in seroconversion at 8 to 10 weeks of age.1 Under normal circumstances, natural infection with E. cuniculi produces an immunoglobulin M seroconversion. High antibody levels can be detected for up to 6 to 9 weeks postinfection, whereas spores can be detected in an infected rabbit’s urine for up to 3 months. The presence of increased antibody levels precedes the demonstration of intracellular organisms in renal tissue by 2 weeks, histologic changes in renal tissue by 4 weeks, and histologic changes in brain tissue by 8 weeks.1 Similar to other chronic infectious diseases (e.g., feline infectious peritonitis) and obligate intracellular pathogens (e.g., avian chlamydiosis), a positive antibody titer to E. cuniculi only confirms exposure. It has been shown that a considerable percentage of rabbits have high titers even in the absence of clinical signs.7 Numerous attempts to use current DNA-probe diagnostic testing have been performed to allow quicker, more accurate confirmation of the disease process. PCR analysis of rabbit encephalitozoonosis cases have been studied7-10; however, both urine and cerebrospinal fluid samples tested with PCR analysis were found to be unreliable for detection of E. cuniculi in either subclinical patients or those showing overt disease signs.9 PCR testing of phacoemulsified lens material from rabbits with phacoclastic uveitis did prove to be a sensitive sample source. Scientific investigations have shown that even postmortem identification of E. cuniculi in infected rabbits may be challenging.10 In a study by Csokai and coworkers,10 the authors could identify very few spores in confirmed encephalitozoonosis cases with severe cerebral histologic lesions using Ziehl-Neelsen and acid-fast trichrome stains. In renal tissue, organisms may often be difficult to identify because of the severe fibrotic or granulomatous reaction caused by the infected cells. E. cuniculi infection should be considered in the differential for rabbit cases in which granulomatous inflammatory lesions are present in renal or brain tissue samples.1,3,7
Serology Using Enzyme-linked Immunosorbent Assay (ELISA) and Protein Electrophoresis Using laboratory submissions to the University of Miami, Division of Comparative Pathology, Miller
Rich
School of Medicine, along with input from several private practitioners, a testing protocol for antemortem diagnosis of E. cuniculi using commercially available ELISA plates and standard agar gel electrophoresis was established.4 An adaptation to the existing qualitative “positive or negative” reporting format was used by determining a titer through serial dilutions. Titers were reported as the last sample dilution that resulted in a positive reaction on the ELISA plate for that patient. As has been the process for case follow-up while the patient is being treated and after therapy has ceased for a variety of other infectious diseases, titers hopefully allow the veterinary practitioner to observe a drop in antibody titers in relation to a positive treatment response. The basis of titer analysis is that a high initial titer correlates to the severity of the infectious disease state of the patient on presentation, and a drop in titer during and after treatment is indicative of an improved health status owing to a diminished immune response because of a reduced population of organisms present in the patient’s body.4 The use of serum/plasma protein electrophoresis to monitor disease states has been in place for more than 2 decades. As far back as 1977, it has been possible to separate cat sera into 10 fractions.11 Today, more than 200 plasma proteins have been described and quantified in man and animals. Proteins, being charged particles, move across a gradient in an electrically charged field. The following are the general bands produced in most animals on an electrophoretic screening: albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin. Some avian species produce a prealbumin fraction. For rabbits, the plasma proteins of interest include albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin.4 The albumin to globulin (A/G) ratio is also a useful parameter to measure in suspected E. cuniculi cases. In a recent article it was noted that E. cuniculi suspect cases had a lower A/G ratio than clinically normal rabbits and higher gamma globulin levels than clinically normal rabbits.4 These findings were not unexpected because albumin is a “negative acute phase protein,” meaning albumin is expected to decrease in the acute phase of certain inflammatory processes. In addition, even though E. cuniculi infections may appear clinically acute to the owner, they are classically a chronic inflammatory disease process; hence a rise in gamma globulins is expected in active cases. Using the combination of E. cuniculi antibody titer testing and protein electrophoresis,
229
Clinical Testing for E. cuniculi in Symptomatic Rabbits
a veterinary clinician may be able to determine the severity of the case at presentation and provide some prognostic evaluation for the patient and owner. Sequential serum/plasma analysis during or at the end of a treatment protocol may allow the practitioner the ability to determine if antibody levels and gamma globulin levels have dropped to a point where treatment can be discontinued and the clinical aspect of the case considered to be resolved. In a recent article by Dr. Carolyn Cray submitted to Exotic DVM as a Special Report, the following tables were included to further enhance the benefit of using specific antibody titers with protein electrophoresis (Tables 1 and 2).4,12 The conclusions drawn from these data support that an antemortem diagnosis of an active E. cuniculi infection could be made when the patient has a high E. cuniculi titer, a decreased A/G ratio, and a normal beta globulin level.4,12
Conclusion Continued research on antemortem diagnostic profiles, treatment protocols, and histological analysis of both treated and untreated E. cuniculi– infected animals will be critical to further our understanding of the total clinical picture of the affects of this organism on the overall health of rabbits. There is much more information that needs to be determined concerning posttreatment infectivity of rabbits that test positive for E. cuniculi. Necropsy analysis of renal and brain tissue from rabbits that either died exhibiting clinical signs consistent with encephalitozoonosis or old age, as well as those euthanized because of a severe disease state, will be essential to develop a true understanding of the epidemiological results of
Table 2. Protein electrophoresis changes by fraction in ECUN suspect and non-ECUN clinically abnormal clinical groups
Clinical Group
Titer
Clinically normal Non-ECUN clinically abnormal ECUN suspect Neurologic (n ⫽ 54) Ocular (n ⫽ 19) Renal (n ⫽ 12)
794.0 ⫾ 171.9 742.3 ⫾ 245.0 1324.0 ⫾ 138.0 1679.0 ⫾ 218.5 344.5 ⫾ 382.7 628.6 ⫾ 560.2
ECUN, Encephalitozoon cuniculi; ELISA, enzyme-linked immunosorbent assay.
ECUN Suspect
Non-ECUN Clinically Abnormal
Total protein A/G ratio Albumin Alpha-1 Alpha-2 Beta Gamma
— Decreased Decreased
— Decreased Decreased
Decreased — Increased
Decreased Increased Increased
ECUN, Encephalitozoon cuniculi; EPH, protein electrophoresis; A/G, albumin to globulin.
our current and future testing and treatment regimens.
Acknowledgments I wish to thank Drs. Carolyn Cray and Susan Kelleher for their support and the use of their library materials in accumulating the data needed for this article.
References 1. 2. 3. 4.
Table 1. Mean ⴞ SEM antibody titer results of ELISA by clinical group
EPH Value
5.
6. 7. 8.
Harcourt-Brown FM: Textbook of Rabbit Medicine. Oxford, UK, Butterworth-Heinemann, 2002 Harcourt-Brown FM: Encephalitozoon cuniculi infection in rabbits. J Exotic Pet Med 13:86-93, 2004 Harcourt-Brown FM, Holloway HK: Encephalitozoon cuniculi in pet rabbits. Vet Rec 152:427-431, 2003 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 Deeb BJ, Carpenter JW: Neurologic and musculoskeletal diseases, in Quesenberry KE, Carpenter JW (eds): Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery (ed 2). St. Louis, MO, Elsevier/ Saunders, pp 203-211, 2004 Keeble EJ, Shaw DJ: Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in the United Kingdom. Vet Rec 158:539-544, 2006 Csokai J, Joachim A, Gruber A, et al: Diagnostic markers for encephalitozoonosis in pet rabbits. Vet Parasitol 163:18-26, 2009 Dipineto L, Rinaldi L, Santaniello A, et al: Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy. Zoon Public Health 55:173-175, 2008
230 9. 10.
Rich Kunzel F, Gruber A, Tichy A, et al: Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits. Vet Parasitol 151:115-124, 2008 Csokai J, Gruber A, Kunzel F, et al: Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation. Parasitol Res 104:629-635, 2009
11.
12.
Kaneko JJ: Serum proteins and the dysproteinemias, in Kaneko JJ, Harvey JW, Bruss ML (eds): Clinical Biochemistry of Domestic Animals (ed 5). San Diego, CA, Academic Press, pp 117-138, 1997 Cray C: New testing option for the diagnosis of Encephalitozoon cuniculi in rabbits. Exotic DVM 11:27-28, 2009
Clinical Update on Testing Modalities for Encephalitozoon cuniculi in Clinically Sick Rabbits Gregory Rich, DVM
Abstract This article describes the clinical presentation of rabbit patients infected with Encephalitozoon cuniculi that present to veterinary hospitals. The difficulty in obtaining a definitive diagnosis with this disease, even in animals with severe clinical signs, is frustrating to veterinarians and owners alike. A detailed review of current diagnostic testing methodologies and recent advances should help with the interpretation of E. cuniculi test results to communicate and inform owners on treatment and prevention of this devastating disease. Copyright 2010 Elsevier Inc. All rights reserved. Key words: Clinical signs; diagnostic test; Encephalitozoon cuniculi; neurologic; rabbit
E
ncephalitozoon cuniculi is a common disease of pet rabbits and is an internationally recognized disease affecting rabbit colonies.1-5 Encephalitozoon represents a genus of obligate intracellular parasites from the phylum Microsporidia, subphylum Sporozoa. The following species of Encephalitozoon have been identified and shown to be opportunistic pathogens for immunocompromised humans: E. intestinalis, E. hellum, E. bieniusi, and E. septata. In human patients with AIDS, E. cuniculi infections have been attributed to cases with interstitial pneumonitis, diarrhea, and muscle wasting.1 Infection of the host usually occurs after ingestion of food contaminated with rabbit urine that has E. cuniculi spores, although infection through inhalation of infectious spores has also been postulated. Vertical infection in utero from an infected dam is also possible. After ingestion or inhalation, monocytes infected with E. cuniculi spores transport the organism to primary colonization sites (e.g., kidneys, brain, lungs, liver).1-4 Spores can survive within the environment for up to 4 weeks at room temperature.3 Encephalitozoonosis is a problem in laboratory rabbits because its effects on the health of the animals can interfere with investigative results.3 A definitive antemortem diagnosis is difficult because histopathology is
226
required to demonstrate the presence of E. cuniculi. Serological testing is difficult to interpret because a positive titer may be indicative of exposure and not a true infection.6 Postmortem diagnosis can also be difficult because of the sparse number of organisms present in many chronically infected brain and/or renal tissues.1,7-10 Because of the inability of current diagnostic tests to provide adequate answers to the E. cuniculi status of a rabbit, there is ongoing investigative research being done to improve our understanding of how to interpret test results with parallel testing strategies and to improve overall test reliability and sensitivity/specificity.6-10
Clinical Signs The clinical picture of an E. cuniculi infected rabbit can vary from a subclinical carrier to acute death.1-5 NeuFrom West Esplanade Veterinary Clinic and Bird Hospital, Metairie, LA USA. Address correspondence to: Gregory Rich, DVM, West Esplanade Veterinary Clinic, 3640 West Esplanade, Metairie, LA 70002. E-mail: [email protected] © 2010 Elsevier Inc. All rights reserved. 1557-5063/10/1903-$30.00 doi:10.1053/j.jepm.2010.07.007
Journal of Exotic Pet Medicine, Vol 19, No 3 ( July), 2010: pp 226 –230
227
Clinical Testing for E. cuniculi in Symptomatic Rabbits
Figure 1. Disorientation and rolling behavior due to central nervous system manifestations of E. cuniculi infection.
rologic signs comprise the greatest percentage of patient presentations relative to a presumed encephalitozoonosis diagnosis. Vestibular disease and seizures are the most obvious clinical signs associated with a rabbit infected with E. cuniculi, although intermittent aggressive behavior, ataxia, seizures, and swaying may also be present.1 Posterior paresis and severe muscle wasting have been associated with late-stage disease presentations. Vestibular signs range from a mild head tilt to a severe inability for the patient to “right” itself, creating a constant rolling presentation.1,4,5 The neurological manifestations are presumed to occur as brain cells rupture and E. cuniculi organisms are released into surrounding tissues. Common terms used to describe the vestibular disease include wryneck, torticollis, head tilt, or labyrinthitis (Fig 1).1,5 Renal manifestations are equally quite varied.1-5 Subtle clinical signs such as urine scald in the perineal region and along the medial aspect of the
Figure 3. Cataract formation in an adult rabbit.
rear legs and “bladder sludge” have been noted in cases testing positive for E. cuniculi. Dysuria is thought to be secondary to the neurologic manifestations of the disease or from muscle wasting associated with the patient’s general illness. Mild azotemia and elevations in serum/plasma creatinine are occasionally noted, but these abnormal findings are not reliable indicators for E. cuniculi infections (Fig 2). Intraocular disease, such as cataract formation, hypopyon, and/or uveitis, is commonly diagnosed in pet rabbits.1-5 A large percentage of rabbit cases with intraocular disease have tested positive for E. cuniculi using serologic, histopathologic, or polymerase chain reaction (PCR) testing.1-5 Separate studies by Kunzel and Csokai revealed that a high percentage of cases with phacoclastic uveitis secondary to lens rupture tested positive for E. cuniculi on PCR analysis of lens material (Fig 3).7,9 Nonspecific disease presentations of general malaise, weight loss of unknown origin, anorexia, sore hocks, and diarrhea are common complaints of owners with sick rabbits. Diagnostic testing for E. cuniculi in these cases may identify this organism as a factor in these nonspecific disease processes, but should not preclude a complete medical workup.
Diagnostic Features
Figure 2. Urine scald caused by renal disease and dysuria from E. cuniculi infection.
Encephalitozoon cuniculi causes a wide range of disease manifestations in pet rabbits. Subclinical carriers are a primary source of disease exposure in multiple rabbit households, pet stores, and breeder colonies.1 A susceptible rabbit can become infected by ingesting food contaminated with urine. There has been verification
228 that young rabbits can be infected by a diseased parent during their first few days of life.1 As mentioned earlier, vertical transmission in utero may also occur. Natural infection in young rabbits results in seroconversion at 8 to 10 weeks of age.1 Under normal circumstances, natural infection with E. cuniculi produces an immunoglobulin M seroconversion. High antibody levels can be detected for up to 6 to 9 weeks postinfection, whereas spores can be detected in an infected rabbit’s urine for up to 3 months. The presence of increased antibody levels precedes the demonstration of intracellular organisms in renal tissue by 2 weeks, histologic changes in renal tissue by 4 weeks, and histologic changes in brain tissue by 8 weeks.1 Similar to other chronic infectious diseases (e.g., feline infectious peritonitis) and obligate intracellular pathogens (e.g., avian chlamydiosis), a positive antibody titer to E. cuniculi only confirms exposure. It has been shown that a considerable percentage of rabbits have high titers even in the absence of clinical signs.7 Numerous attempts to use current DNA-probe diagnostic testing have been performed to allow quicker, more accurate confirmation of the disease process. PCR analysis of rabbit encephalitozoonosis cases have been studied7-10; however, both urine and cerebrospinal fluid samples tested with PCR analysis were found to be unreliable for detection of E. cuniculi in either subclinical patients or those showing overt disease signs.9 PCR testing of phacoemulsified lens material from rabbits with phacoclastic uveitis did prove to be a sensitive sample source. Scientific investigations have shown that even postmortem identification of E. cuniculi in infected rabbits may be challenging.10 In a study by Csokai and coworkers,10 the authors could identify very few spores in confirmed encephalitozoonosis cases with severe cerebral histologic lesions using Ziehl-Neelsen and acid-fast trichrome stains. In renal tissue, organisms may often be difficult to identify because of the severe fibrotic or granulomatous reaction caused by the infected cells. E. cuniculi infection should be considered in the differential for rabbit cases in which granulomatous inflammatory lesions are present in renal or brain tissue samples.1,3,7
Serology Using Enzyme-linked Immunosorbent Assay (ELISA) and Protein Electrophoresis Using laboratory submissions to the University of Miami, Division of Comparative Pathology, Miller
Rich
School of Medicine, along with input from several private practitioners, a testing protocol for antemortem diagnosis of E. cuniculi using commercially available ELISA plates and standard agar gel electrophoresis was established.4 An adaptation to the existing qualitative “positive or negative” reporting format was used by determining a titer through serial dilutions. Titers were reported as the last sample dilution that resulted in a positive reaction on the ELISA plate for that patient. As has been the process for case follow-up while the patient is being treated and after therapy has ceased for a variety of other infectious diseases, titers hopefully allow the veterinary practitioner to observe a drop in antibody titers in relation to a positive treatment response. The basis of titer analysis is that a high initial titer correlates to the severity of the infectious disease state of the patient on presentation, and a drop in titer during and after treatment is indicative of an improved health status owing to a diminished immune response because of a reduced population of organisms present in the patient’s body.4 The use of serum/plasma protein electrophoresis to monitor disease states has been in place for more than 2 decades. As far back as 1977, it has been possible to separate cat sera into 10 fractions.11 Today, more than 200 plasma proteins have been described and quantified in man and animals. Proteins, being charged particles, move across a gradient in an electrically charged field. The following are the general bands produced in most animals on an electrophoretic screening: albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin. Some avian species produce a prealbumin fraction. For rabbits, the plasma proteins of interest include albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin.4 The albumin to globulin (A/G) ratio is also a useful parameter to measure in suspected E. cuniculi cases. In a recent article it was noted that E. cuniculi suspect cases had a lower A/G ratio than clinically normal rabbits and higher gamma globulin levels than clinically normal rabbits.4 These findings were not unexpected because albumin is a “negative acute phase protein,” meaning albumin is expected to decrease in the acute phase of certain inflammatory processes. In addition, even though E. cuniculi infections may appear clinically acute to the owner, they are classically a chronic inflammatory disease process; hence a rise in gamma globulins is expected in active cases. Using the combination of E. cuniculi antibody titer testing and protein electrophoresis,
229
Clinical Testing for E. cuniculi in Symptomatic Rabbits
a veterinary clinician may be able to determine the severity of the case at presentation and provide some prognostic evaluation for the patient and owner. Sequential serum/plasma analysis during or at the end of a treatment protocol may allow the practitioner the ability to determine if antibody levels and gamma globulin levels have dropped to a point where treatment can be discontinued and the clinical aspect of the case considered to be resolved. In a recent article by Dr. Carolyn Cray submitted to Exotic DVM as a Special Report, the following tables were included to further enhance the benefit of using specific antibody titers with protein electrophoresis (Tables 1 and 2).4,12 The conclusions drawn from these data support that an antemortem diagnosis of an active E. cuniculi infection could be made when the patient has a high E. cuniculi titer, a decreased A/G ratio, and a normal beta globulin level.4,12
Conclusion Continued research on antemortem diagnostic profiles, treatment protocols, and histological analysis of both treated and untreated E. cuniculi– infected animals will be critical to further our understanding of the total clinical picture of the affects of this organism on the overall health of rabbits. There is much more information that needs to be determined concerning posttreatment infectivity of rabbits that test positive for E. cuniculi. Necropsy analysis of renal and brain tissue from rabbits that either died exhibiting clinical signs consistent with encephalitozoonosis or old age, as well as those euthanized because of a severe disease state, will be essential to develop a true understanding of the epidemiological results of
Table 2. Protein electrophoresis changes by fraction in ECUN suspect and non-ECUN clinically abnormal clinical groups
Clinical Group
Titer
Clinically normal Non-ECUN clinically abnormal ECUN suspect Neurologic (n ⫽ 54) Ocular (n ⫽ 19) Renal (n ⫽ 12)
794.0 ⫾ 171.9 742.3 ⫾ 245.0 1324.0 ⫾ 138.0 1679.0 ⫾ 218.5 344.5 ⫾ 382.7 628.6 ⫾ 560.2
ECUN, Encephalitozoon cuniculi; ELISA, enzyme-linked immunosorbent assay.
ECUN Suspect
Non-ECUN Clinically Abnormal
Total protein A/G ratio Albumin Alpha-1 Alpha-2 Beta Gamma
— Decreased Decreased
— Decreased Decreased
Decreased — Increased
Decreased Increased Increased
ECUN, Encephalitozoon cuniculi; EPH, protein electrophoresis; A/G, albumin to globulin.
our current and future testing and treatment regimens.
Acknowledgments I wish to thank Drs. Carolyn Cray and Susan Kelleher for their support and the use of their library materials in accumulating the data needed for this article.
References 1. 2. 3. 4.
Table 1. Mean ⴞ SEM antibody titer results of ELISA by clinical group
EPH Value
5.
6. 7. 8.
Harcourt-Brown FM: Textbook of Rabbit Medicine. Oxford, UK, Butterworth-Heinemann, 2002 Harcourt-Brown FM: Encephalitozoon cuniculi infection in rabbits. J Exotic Pet Med 13:86-93, 2004 Harcourt-Brown FM, Holloway HK: Encephalitozoon cuniculi in pet rabbits. Vet Rec 152:427-431, 2003 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 Deeb BJ, Carpenter JW: Neurologic and musculoskeletal diseases, in Quesenberry KE, Carpenter JW (eds): Ferrets, Rabbits and Rodents: Clinical Medicine and Surgery (ed 2). St. Louis, MO, Elsevier/ Saunders, pp 203-211, 2004 Keeble EJ, Shaw DJ: Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in the United Kingdom. Vet Rec 158:539-544, 2006 Csokai J, Joachim A, Gruber A, et al: Diagnostic markers for encephalitozoonosis in pet rabbits. Vet Parasitol 163:18-26, 2009 Dipineto L, Rinaldi L, Santaniello A, et al: Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy. Zoon Public Health 55:173-175, 2008
230 9. 10.
Rich Kunzel F, Gruber A, Tichy A, et al: Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits. Vet Parasitol 151:115-124, 2008 Csokai J, Gruber A, Kunzel F, et al: Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation. Parasitol Res 104:629-635, 2009
11.
12.
Kaneko JJ: Serum proteins and the dysproteinemias, in Kaneko JJ, Harvey JW, Bruss ML (eds): Clinical Biochemistry of Domestic Animals (ed 5). San Diego, CA, Academic Press, pp 117-138, 1997 Cray C: New testing option for the diagnosis of Encephalitozoon cuniculi in rabbits. Exotic DVM 11:27-28, 2009