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Brief Communication |
Correspondence: 1Corresponding Author: John S Munday, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand
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Key Words: Bovine • encephalitis • fungi • Mortierella wolfii • zygomycosis
The zygomycete class of fungi includes the genera Absidia, Rhizomucor, Rhizopus, Mucor, Mortierella, Basidiobolus, and Conidiobolus.9,12 In cattle, zygomycosis most commonly develops within the lungs or gastrointestinal tract as a result of impairment of the body's defenses (e.g., because of ruminal acidosis).9,18 Zygomycosis is also a significant cause of mycotic abortion in cattle.12 This is a report of a cow that developed clinical signs of neurologic disease. Necropsy revealed marked meningoencephalitis with myriad intralesional zygomycete fungi. Polymerase chain reaction (PCR) and subsequent gene sequencing identified the fungi as Mortierella wolfii. To the authors' knowledge, this is the fourth report of fungal encephalitis in an adult cow3,6,13 and the first report in which an etiologic diagnosis was made. Additionally, this is the first time that PCR has been used to investigate fungal disease in cattle.
A 7-year-old Jersey dairy cow presented with clinical signs of lethargy, hypermetria, ataxia, and blindness. The cow was from a herd of 400 that was being fed fresh pasture supplemented with small amounts of grass silage. The temperature, heart rate, and respiratory rate of the cow were within normal limits. Auscultation of the lungs did not reveal significant findings; however, rumen movements were not audible. Although breeding records and previous pregnancy testing indicated that the cow should have been 5 months pregnant, no fetus was detectible on rectal examination. Blood chemistry was within normal limits, whereas hematology revealed increased band neutrophils (1.4 x 109/liter, reference range 0.0–0.1 x 109/liter) and fibrinogen (14.8 g/liter, reference range 2.0–7.0 g/liter). Mild toxic changes were observed within neutrophils. A presumptive diagnosis of bacterial meningitis was made, and the cow was treated with 15 mg/kg IM procaine penicillin.a An additional dose of procaine penicillin was given 24 hours later; however, the cow died 36 hours after initial presentation. During the next month, 2 abortions were observed within the herd. M. wolfii was cultured from 1 calf, and no cause of abortion could be identified in the other.
Necropsy examination revealed that the cow had good body condition. Examination of the meninges revealed diffuse thickening and opacity. Meningeal blood vessels were prominent, and numerous petechial hemorrhages were visible. Cut sections of the cerebrum revealed multiple, variably sized, poorly defined foci of reddening and hemorrhage (Fig. 1). The largest of these foci measured 2 cm in diameter. The lungs contained foci of consolidation and hemorrhage. The affected areas comprised approximately 30% of both lungs. An examination of the endometrium revealed diffuse thickening with a roughened texture. A fetus was not present. Representative samples were taken from multiple organs, fixed in formalin for 24 hours, and routinely processed for histologic examination.
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The DNA was extracted from paraffin-embedded tissue as previously described.8 The D1, D2, and D3 region of the 25–28 S (large subunit ribosomal RNA) was amplified with primers LROR and LR16.2 Amplification was performed in a 0.2-ml PCR tube with a total reaction volume of 50 µl by using a GeneAmp PCR system 9700 thermocycler.b Each reaction tube contained: 2.5 U Taq DNA polymerase; 250 µM (each) dATP, dCTP, dGTP, and dTTP; 20 mM Tris-HCl; 50 mM KCl; 2.0 mM MgCl2; primers, and sample DNA. A PCR round was conducted with an initial 2-minute denaturation step at 94°C coupled to repeating cycle of 45 seconds at 94°C, 45 seconds at 50°C, and 45 seconds at 72°C for 40 cycles, followed by a 2-minute final extension step at 72°C.
The oligonucleotide primer LROR (5 µM) was used for sequencing. PCR products used as templates for sequencing were purified using the High Pure PCR Purification Kit.c Sequencing reactions were performed by using an ABI Prism BigDye terminator cycle sequencing kitb in a GeneAmp PCR System 9700 thermal cycler and were electorphoresed by using an ABI Prism 310 genetic analyzer.b The sequence data were edited using DNASTAR software.d A blast search was performed using the National Center for Biotechnology Information database. This revealed 100% homology with 3 strains of M. wolfii. The PCR analysis enabled an etiologic diagnosis of M. wolfii meningoencephalitis in this case.
Fungal encephalitis is rare in cattle, with most cases reported in aborted14 or neonatal5,19 calves. To the authors' knowledge, fungal encephalitis has only been previously reported in 4 adult cows. All 4 previously reported cows had a necrotizing vasculitis with intralesional zygomycete fungi.3,6,13 Three of the cows developed encephalitis soon after calving.3,6 Two cows were found to have predisposing factors; 1 had traumatic reticulopericarditis,3 the other had anemia caused by theileriasis and anaplasmosis.13 Fungi were detected in the lung, as in the presently reported case, in 1 of the 4 previously described cases.6 No etiologic diagnosis was made in the 4 previously reported cases of fungal encephalitis in adult cattle.
As is likely in the currently reported case, all previously reported cases of bovine fungal encephalitis developed as a result of hematogenous spread of fungi to the brain.3,5,13,19 In contrast, 69% of human central nervous system zygomycoses occur as a result of direct spread of fungi from the nose or nasal sinus.15 Ketoacidosis promotes fungal growth while inhibiting neutrophil function, and 80% of patients with rhinocerebral zygomycosis have underlying diabetes mellitus.15,16 In humans, only 15% of central nervous system zygomycoses develop because of hematogenous fungal dissemination.15,17 The initial fungal infection usually involves the lungs,15,17 and hematogenous cerebral mycosis is strongly associated with underlying malignancies, bone marrow transplant, and treatment with deferoxamine.15 Deferoxamine is an iron-chelating agent.17 It reduces iron availability within the body, but promotes fungal iron uptake.17 Of the predisposing factors in humans, only ketoacidosis is likely in an adult dairy cow. In this case, serum biochemistry did not indicate ketoacidosis at the time of presentation, but it could have occurred earlier in the course of the disease. M. wolfii has not been reported to cause disease in humans.
The mycotic endometritis and the absence of the expected fetus in this cow suggest the cow aborted before developing encephalitis. Fungal infection causes around 6% of bovine abortions in North America.10–12 Aspergillus species are responsible for 71% of these.12 Although zygomycetes cause 21% of bovine mycotic abortions in North America, M. wolfii has only been identified in 0.5% cases.12 In contrast, M. wolfii is the most common cause of bovine mycotic abortion in some parts of New Zealand.1 This saprophytic fungus has a worldwide distribution and is widespread in the environment.7 Therefore, it is likely that environmental (a warm wet climate) and management (frequent use of grass silage) factors result in the high incidence of M. wolfii in mycotic abortions in New Zealand.1
Unlike other fungal causes of abortion, 20% of cows that abort because of M. wolfii will subsequently develop fatal mycotic pneumonia.4 Although the pathogenesis of M. wolfii abortion and pneumonia is not fully resolved, it is most likely that fungi establish a primary focus of infection in the lungs.4 From here, the fungi travel through the blood to the uterus, resulting in endometritis and abortion.4 After abortion, in 20% of cows, large numbers of fungi then re-enter the circulation, resulting in embolic pneumonia.4 Why only a proportion of cattle develop embolic pneumonia is unknown. It is possible that cows that develop embolic pneumonia have ketoacidosis or are immunosuppressed. However, it is also possible that the number of fungi entering circulation from the uterus is dependent on the degree of damage to uterine blood vessels.
The currently reported cow was unusual because of the extensive involvement of the brain and the predominance of neurologic clinical signs on presentation. To the authors' knowledge, this is the first report of neurologic disease that is attributable to M. wolfii in an adult cow. Although the currently reported case did have embolic pneumonia, the brain was the most severely affected organ. It is hypothesized that the cow developed a primary pulmonary M. wolfii infection. Fungi then travelled through the blood to the uterus. After abortion, a shower of fungi was released into the circulation, resulting in embolic meningoencephalitis.
Fungal culture was not possible in this case. However, definitive diagnosis was possible using PCR on formalin-fixed, paraffin-embedded sections. To the authors' knowledge, this is the first time PCR has been used as a diagnostic test to identify M. wolfii. Although rare, fungal encephalitis should be considered as a possible cause of neurologic disease in adult cattle, especially if there is a history of abortions or feeding silage within the herd. Polymerase chain reaction is a valuable tool that enables an etiologic diagnosis of fungal disease in cattle, even when only formalin-fixed tissue is available.
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a. Phoenix Pharmacillin, Phoenix Pharmaceuticals, Auckland, New Zealand.
b. PE Applied Biosystems Corp., Foster City, CA.
c. Roche Diagnostics GmbH, Mannheim, Germany.
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