Case ReportMaple Syrup Urine Disease in a Central Indiana Hereford Herd
Mark E. Robarge,1 Jonathan E. Beever,2 Stephen D. Lenz,1
Christopher J. Lynch,3 and William L. Wigle1
1Department of Comparative Pathobiology and Indiana Animal Disease Diagnostic Laboratory, Purdue University,406 S. University Street, West Lafayette, IN 47907, USA2Department of Animal Sciences, University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA3Department of Cellular and Molecular Physiology, Penn State University College of Medicine, 500 University Drive,Hershey, PA 17033, USA
Correspondence should be addressed to William L. Wigle; [email protected]
Maple syrup urine disease (MSUD) and further cases were identified in herd mates of a small Hereford herd in Indiana basedon history, clinical signs, microscopic lesions, and biochemical and genetic testing. This aminoacidopathy has been diagnosed inpolled Shorthorn, polledHereford, andHereford cattle in Australia, Uruguay, Argentina, and Canada and is the result of amutationof the branched-chain alpha-ketoacid dehydrogenase complex. The Indiana index calf case was confirmed by showing the classicaccumulation of ketoacids in liver that results from a defect in the E1-alpha subunit (248 C/T haplotype) in the mitochondrialbranched-chain 𝛼-ketoacid dehydrogenase complex. The presence of the mutation was confirmed in the index case, the dam, andfour related herd mates that represent the first confirmed cases of bovine MSUD mutation in United States cattle.
1. Introduction
Maple syrup urine disease (MSUD) is an inherited, autosomalrecessive, aminoacidopathy resulting from branched-chain𝛼-ketoacid dehydrogenase complex (BCKDH) dysfunction(Skvorak [1]). The BCKDH complex is a mitochondrial mul-tisubunit enzyme composed of three catalytic componentsincluding E1, E2, and E3 [1–3]. MSUD naturally occurs inhumans and polled Shorthorn, polled Hereford, and Here-ford calves resulting in central nervous system dysfunctionapproximately 2–4 days after birth in calves [1, 4]. In polledHerefords, disease is caused by premature termination oftranslation, of the E1-alpha subunit, that is induced by a cyti-dine to thymidine transition at nucleotide 248 (248C-->T)that converts the glutamine codon −6 to a stop codon (Zhanget al. [5]). Since MSUD is an autosomal recessive condition,consanguineous breeding in cattle, as has been shown inhumans, would presumably lead to a greater incidence ofdisease (Skvorak [1]). A deficiency in BCKDH results inan inability to oxidize the branched-chain ketoacids of
the branched-chain amino acids leucine, isoleucine, andvaline (Maxie and Youssef [4]). Loss of this activity resultsin the accumulation of the branched-chain amino acidsalong with their respective ketoacids: ketoisocaproic, keto-𝛽-methylvaleric, and ketoisovaleric acids in cerebrospinal fluid,blood, and tissues. The mechanism by which these metabo-lites cause central nervous system dysfunction is not fullyunderstood (Maxie and Youssef [4]).
MSUD could represent a subset of the hereditary neu-raxial edema disease complex described in Hereford calvesin the United States but these cases were never confirmed tohave the currently known genetic mutations or biochemicalchanges consistent with MSUD and spongy vacuolation wasdescribed in the spinal cord which is not consistent withMSUD (Cordy et al. [6]). A syndrome similar to MSUDwas also reported in Gelbvieh-cross calves in Nebraska butgenetic analysis failed to show a mutation in the E1-𝛼 subunit(O’Toole et al. [7]). To the authors’ knowledge, no case reportof naturally occurring MSUD in cattle has been published inthe United States. In the current report, MSUD in an Indiana
Hindawi Publishing CorporationCase Reports in Veterinary MedicineVolume 2015, Article ID 204037, 4 pageshttp://dx.doi.org/10.1155/2015/204037
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Hereford herd is described to inform veterinarians and own-ers of the presence of this disease mutation in the Herefordcattle population and the possibility of thismutation resultingin clinical disease.
2. Case Presentation
The affected male Hereford calf came from a small herd (7cow/calf pairs) in Indiana. This was the owners’ first yearraising cattle and they obtained the herd from the previousowner without any known pedigree information. The cattlewere kept on a small pasture with a small calf barn for shelter.No other calves from this farmwere affected and all calves hadthe same sire. At three days of age, the owner noted that thecalf was “not acting normally.”The following day the calf wasrecumbent and depressed.The calf was given oxytetracyclineand milk replacer with no clinical improvement. On thesubsequentmorning, the animal was presented to the PurdueUniversity Veterinary Teaching Hospital for evaluation.
Upon presentation to the Purdue University VeterinaryTeaching Hospital, the animal was depressed, was laterallyrecumbent, and had bilateral nystagmus. Temperature, pulse,and respiration were within normal limits. Based on the ageof the calf and limited diagnostic work-up, the suspecteddiagnosis at that time was bacterial meningitis. Due to finan-cial restrictions associated with diagnostics and treatment,euthanasia was elected and the calf was submitted to theAnimal Disease Diagnostic Laboratory for necropsy. Neithera chemistry panel nor a complete blood count was performedprior to euthanasia.
Gross necropsy examination was unremarkable. No grossabnormalities were observed in the brain. No obvious urineodor was noted. Specimens of major organs and tissues werecollected, fixed in neutral buffered 10% formalin, routinelyprocessed, embedded in paraffin, sectioned, stained withH&E, luxol fast blue, oligodendrocyte transcription factor(Olig-2), and glial fibrillary acidic protein (GFAP), and exam-ined by light microscopy. Histologic abnormalities on H andE staining were confined to the white matter of the cerebrum,cerebellum, and brain stem and consisted of severe spongyvacuolation of myelin with the long axis of vacuoles parallelto axons (status spongiosus) (see Figure 1). Lesions were notobserved in sections of peripheral nerve. Virchow Robin’sspace in the white matter was also variably expanded byincreased clear space (edema). Astrocytes in the white matterhad an increased amount of eosinophilic cytoplasm (reac-tive). No abnormalities in myelination were observed withluxol fast blue staining compared to routinely used controlbrain sections. The relative number of oligodendrocytes andastrocytes were compared using Olig-2 and GFAP, respec-tively, between the calf with MSUD and an age-matchedcontrol calf. No appreciable difference was observed betweenthe calf with MSUD and the age-matched control calf bytwo observers (William L. Wigle and Mark E. Robarge). Theauthor’s acknowledge that a differencemay have been presentsince edema in the calf with MSUD made observations onnumbers of oligodendrocytes and astrocytes in these areasdifficult to compare to the age-matched control.
Each of the nine amplicons corresponding to the exonsof bovine BCKDHA were successfully amplified using the
Figure 1: Brain from index case: note marked vacuolation of cere-bellar white matter (status spongiosus) with sparing of gray matter.Vacuoles (edema) are empty with the long axis parallel to axons.Bar = 100 𝜇m. Inset: cerebellum at 100x magnification: many astro-cytes in the white matter are reactive with an increased amount ofeosinophilic cytoplasm (arrowheads). Bar = 35𝜇m. Hematoxylinand Eosin stain.
genomicDNA isolated from the original calf, and herdmates,submitted for analysis. Direct sequencing of the PCR prod-ucts revealed that the affected calf was homozygous for thepreviously identified mutation in polled Herefords caused bypremature termination of translation, of the E1-alpha subunit,that is induced by a cytidine to thymidine transition in exon2 (248C-->T) that converts the glutamine codon −6 to a stopcodon (Zhang et al. [5]). In polled Shorthorns, the mutationresults in a substitution of leucine in place of a highlyconserved proline at codon 372 due to a cytidine to thymi-dine transition at nucleotide 1380 (1380C-->T) resulting indysfunction of the E1-alpha subunit (Dennis and Healy [8]).The dam of the affected calf was shown to be heterozygous forthe 248 C/T haplotype, which is consistent with the reportedrecessive inheritance of the disease. Of the eight additionalindividual samples collected from the subject herd, four werefound to be heterozygous for the mutation (corresponding toone carrier cow and three carrier calves) with the remainderbeing homozygous for the normal allele. The sire of theaffected calf had been sold and was unavailable for testing.
Sections of frozen liver from the calf affected withMSUDand three “healthy” age-matched but not breed-matchedcontrol calf frozen livers were evaluated for branched-chainketoacid levels (Olson et al. [9]) of ketoisovaleric (KIV),ketoisocaproic (KIC), and keto-𝛽-methylvaleric (KMV) acidswhich are the ketoacids of valine, leucine, and isoleucine,respectively, and showed marked elevations consistent withMSUD. “Healthy” control calves of a comparable age butdifferent breed were diagnosed with pneumonia, ventricularseptal defect, and coccidiosis, respectively, which presumablyshouldminimally affect the branched-chain ketoacid levels inthe liver. Results are summarized in Table 1.
3. Discussion
The history, histologic findings, homozygous mutation inthe E1-𝛼 subunit, and increased branched-chain ketoaciddetection in liver of submitted Hereford calf are consistentwith MSUD. Multiple animals (one cow and three calves)
Ketoacid concentrations in the liver of the Hereford calf with maple syrupurine disease versus the average of three “healthy” age-matched but notbreed-matched control calves are summarized. KIV: ketoisovaleric, KIC:ketoisocaproic, and KMV: keto-𝛽-methylvaleric acids, respectively.
in the herd were heterozygous for the mutation in the E1-𝛼subunit as well.
Status spongiosus, the histologic hallmark of MSUD, isa term that describes neural tissue that has a microvacuolar,sieve-like change by light microscopy [2, 10, 11]. This appear-ance may be from swelling of astrocyte/oligodendrocytecytoplasm, processes in neuropil, or myelin sheaths [4, 11, 12].Although the presence of this lesion inmyelin is distinctive onlight microscopic evaluation, electron microscopy is neededto definitively define the change (Maxie and Youssef [4]). InMSUD,myelin vacuolation is due to splitting ofmyelin lamel-lae at the intraperiod line which produces vacuoles withinthe myelin sheath mostly involving the outer myelin lamellae[13]. Status spongiosus has many causes in animals includingidiopathic, toxic, metabolic, and infectious conditions [4, 5,7, 11, 14, 15].
Many different categories of disease, as mentioned above,in calves can cause status spongiosis of white matter andwere considered in this index case before ancillary testingconfirmed this calf to have MSUD. One such cause is idio-pathic spongiform myelinopathy that has been documentedin horned Hereford calves in New Zealand and in polledHereford calves in Britain (Maxie andYoussef [4]). Idiopathicmyelinopathies were ruled out after positive ancillary testingconfirmed MSUD. Hepatic and to a lesser extent renalencephalopathy are possible etiologies; however, the kidneyand liver were normal in this case, andAlzheimer type II cells,reactive astrocytes in the gray matter with a clear nucleus,and increased amount of cytoplasm found singly or ingroups were not observed [4, 11]. Hexachlorophene, a poly-chlorinated phenolic compound used as a topical antisep-tic, and halogenated salicylanilide, an anthelmintic, cause sta-tus spongiosus of whitematter in both the central and periph-eral nervous system (Maxie and Youssef [4]). Toxic com-pounds like these can be ruled out based on lack of exposureto the compound and no lesions seen in peripheral nervoustissue. Ingestion of corn towards the end of growing seasoninfected with Stenocarpella maydis can cause status spongio-sus as a result of mycotoxicosis but was ruled out since thisfungus is found in southern Africa and Argentina (Maxieand Youssef [4]). Many toxic plants should be consideredincluding Stypandra sp.,Hemerocallis sp., Tylecodon wallichii,Ornithogalum toxicarum, andHelichrysum sp.; however, theseplants grow in various countries around theworld and are notreported in Indiana (Maxie and Youssef [4]).
To the authors’ knowledge, this represents the first doc-umented naturally occurring case of MSUD in cattle in theUnited States. This finding indicates the presence of thegenetic mutation for MSUD within the US cattle populationand suggests the possible need for genetic screening toeliminate the trait.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
Acknowledgment
The authors would like to thank Dr. Margaret Miller for helpin histological interpretation and IHC selection.
References
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