History
A 1-month-old 57.3-kg (126.1-lb) sexually intact male Durham Shorthorn was evaluated at the large animal clinic of the University of Illinois Veterinary Teaching Hospital because of dull mentation, weakness, tachypnea, and hypothermia.
Clinical and Clinicopathologic Findings
On admission (day 0), the calf was mentally dull but responsive to auditory stimuli and able to walk. Rectal temperature was 38.3°C (100.9°F). Initial major serum biochemical changes included hypoglycemia (33 mg/dL; reference interval, 48 to 73 mg/dL), hyperlactatemia (4.9 mmol/L; reference interval, 0.6 to 2.2 mmol/L), and hypoproteinemia (4.5 g/dL; reference interval, 5.9 to 8.3 g/dL). Hematologic changes included mild leukopenia with left shift (WBC count, 3.69 × 103/μL [reference interval, 4 × 103 WBCs/μL to 12 × 103 WBCs/μL], neutrophil count, 2.36 × 103/μL [reference interval, 2.5 × 103 neutrophils/μL to 5.5 × 103 neutrophils/μL], and band cell count, 0.59 × 103/μL [reference interval, 0 × 103 band cells/μL to 0.1 × 103 band cells/μL]). It should be noted that adult bovine reference intervals were used for biochemical and hematologic data because age-specific reference intervals had not been adequately determined in the laboratory in which the assessments were made. Intravenous administration of crystalloid fluidsa with dextrose, ampicillin, and flunixin meglumine was initiated, but the calf's condition deteriorated to obtunded mentation, ataxia, circling, and head pressing with a decreased suckle reflex by day 2 of hospitalization. The calf was dyspneic with an increased expiratory effort and respiratory rate of 24 to 100 breaths/min.
On day 2, a CBC revealed marked neutrophilic leukocytosis with left shift that extended to myelocytes (WBC count, 30.9 × 103/μL; neutrophil count, 16.69 × 103/μL; band count, 8.96 × 103/μL; metamyelocyte count, 0.9 × 103/μL [reference interval, 0 × 103 metamyelocytes/μL]; myelocyte count, 0.31 × 103/μL [reference interval, 0 × 103 myelocytes/μL]) and monocytosis (monocyte count, 1.55 × 103/μL [reference interval, 0.3 × 103 monocytes/μL to 0.9 × 103 monocytes/μL). Serum biochemical analysis revealed that the calf was hypoproteinemic (total protein concentration, 4.9 g/dL), hypernatremic (sodium concentration, 157 mmol/L; reference interval, 132 to 142 mmol/L), and hyperchloremic (chloride concentration, 116 mmol/L; reference interval, 95 to 106 mmol/L).
A CSF sample was collected from the cerebromedullary cistern at the atlanto-occipital site. Grossly, the fluid was colorless and opaque (unable to read print through the sample [Figure 1]).
Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→
Analysis of CSF sample
The CSF total protein concentration was 512 mg/dL. A hemacytometer count was performed; the nucleated cell count was 13,882 cells/mm3 and the RBC count was 88 cells/mm3. A cytocentrifuged sample of the fluid was stained with Wright-Giemsa stain and examined microscopically. The prepared cytocentrifuged sample was cellular with minimal peripheral blood contamination. A 200-cell differential count revealed 89.5% nondegenerate to moderately degenerate neutrophils, 8% large mononuclear cells, and 2.5% small lymphocytes with low numbers of ruptured cells and moderately proteinaceous basophilic background (Figure 2). Very rare rod-shaped intracellular bacteria were detected within neutrophils.
Additional Findings
Treatment of the calf with ampicillin and flunixin was continued along with systemic support (IV fluid administration and nasal oxygen supplementation). Initially, the calf's clinical signs appeared to stabilize. Five days following the initial CSF analysis (day 7 of hospitalization), a follow-up CSF sample was collected. The CSF sample had a high total protein concentration (559 mg/dL) and persistent but decreased pleocytosis (2,488 cells/mm3) with neutrophil predominance (52% nondegenerate neutrophils), an increase in large mononuclear cells (47% large mononuclear cells), and rare lymphocytes (1% small lymphocytes). Increased numbers of rod-shaped bacteria were noted.
Despite a change of antimicrobial treatment to florfenicol, the calf's condition continued to worsen and it was euthanized by IV administration of pentobarbital on day 8; a necropsy was performed. Grossly, the leptomeninges of the brainstem were markedly thickened and cloudy, with flakes of yellow opaque friable material present (Figure 3). Histologically, the leptomeninges of the cerebrum, cerebellum, and brainstem were thickened with marked fibrinosuppurative inflammation (Figure 4). The thalamus and brainstem had rarified and vacuolated foci with large accumulations of neutrophils, foamy macrophages (gitter cells), and high numbers of glial cells (gliosis). Swollen axons (spheroids) were also noted, and many blood vessels were partially to completely occluded by large adherent aggregates of fibrillar material (fibrin thrombi). The perivascular, or Virchow-Robin, spaces were distended by fibrin, edema, and neutrophils. Bacterial culture of samples of brain tissue failed to recover any organisms. Brain tissue underwent PCR assay analysis to detect Listeria DNA and reverse-transcription PCR assay analysis to detect bovine viral diarrhea virus RNA; results of these tests were negative.
Morphologic Diagnosis and Case Summary
Morphologic diagnosis: marked, acute to subacute, diffuse, fibrinosuppurative meningoencephalitis with thrombosis and multifocal encephalomalacia of the brain.
Case summary: thrombotic meningoencephalitis (TME) in a calf, the suspected etiologic agent of which is Histophilus somni.1
Comments
Regardless of species, CSF samples should be clear, and it should be very easy to read newsprint through the sample. Increased turbidity, including the frank opacity in the case described in the present report, often indicates an increase in cellularity, which is termed pleocytosis. Although appropriately sourced reference intervals for CSF variables have not been determined in our laboratory or reported for healthy calves, expected findings in adult cow CSF samples reportedly include low cellularity (< 10 cells/mm3) with lymphocyte predominance, lower numbers of large monocytoid cells, and only rare neutrophils.2 Total protein concentration has been reported as < 67 mg/dL; however, < 30 mg/dL is commonly used as a cutoff for normal cerebromedullary samples in our laboratory.2,3 In most clinical pathology laboratories, specialized centrifuges (cytocentrifuges) are used to prepare cytologic preparations. A satisfactory, though somewhat cumbersome, method for preparing cytologic samples from low-cellularity fluids has been reported and may be used in practice settings.4
In studies3,5 of bovine CSF samples, including investigation of findings from 102 cattle,3 neutrophil predominance was noted commonly in cases involving bacterial infection (eg, cases of TME, neonatal meningitis, older-calf meningitis, otitis-related meningitis, and CNS abscess). Similar to the findings for the calf of the present report, 2 other cases with neutrophilic pleocytosis that were monitored by repeated CSF analysis revealed progression to monocytic pleocytosis.3 This pattern of change is indicative of maturation of an acute neutrophilic inflammatory process to a more chronic histiocytic process. Interestingly, among the 3 bovids with TME in that study,3 the highest reported CSF total nucleated cell count was 462 cells/mm3, which was much lower than the CSF total nucleated cell count in the calf of the present report; the highest CSF total nucleated cell counts were found in cases of neonatal or older- calf meningitis and abscess formation.
Neutrophilic pleocytosis is not commonly associated with listeriosis or noninfectious causes, such as salt toxicosis, neoplasia, and trauma.3 Listeriosis in cattle typically results in histiocytic pleocytosis, but mixed or lymphocytic pleocytosis has been reported.3 Of 22 bovine CSF samples from confirmed listeriosis cases, none had a neutrophilic pleocytosis; however, in humans with listerial meningitis, neutrophilic pleocytosis is an expected finding.3,5 In concordance with the neutrophilic leukocytosis identified in the initial CSF sample from the calf of the present report, the result of the Listeria PCR assay was negative.
In humans and cattle, bacteria have been detected in only 40% to 45% of CSF samples from individuals with neonatal meningitis and in 66% of CSF samples from individuals with TME.3,6 The calf of the present report received ampicillin for approximately 36 hours before collection of the initial CSF sample, which may have decreased the number of bacteria; however, the bacterial population was more apparent on examination of the second CSF sample. A lack of visible bacteria or degenerate neutrophils in a CSF sample should not exclude bacterial infection from the differential diagnosis list for patients with neutrophilic pleocytosis but may indicate the need for further diagnostic procedures (including bacterial culture of a CSF sample) in cases with a high index of suspicion.
The pathogenesis of TME is multifactorial. Initially, H somni (basonym Haemophilus somnus) is inhaled on water droplets or fomites and initiates a commensal infection in the upper airway tract.7 Subsequent to stress, colonization of the lungs allows the bacteria to access the vascular system, where the organisms circulate until perturbed endothelium is encountered. This commonly happens in the area of turbulent blood flow in the cerebral cortex at the junction of white and gray matter. Histophilus somni can adhere to disrupted bovine endothelium and produce virulence determinants (including lipooligosaccharide, tissue factor [coagulation factor III], E-selectin, and intercellular adhesion molecule 1 [ICAM-1]) that activate platelets and induce thrombi formation.1,7 The major lesions associated with TME relate to thrombosis of cerebral vasculature and resultant ischemia. The hypoproteinemia in the calf of the present report may have been indicative of failure of passive transfer, a condition which would have predisposed the calf to infectious diseases and contributed to the failure of treatment.
Bacterial culture of samples of brain tissue from the calf of the present report yielded no growth. Negative results of bacterial culture can be expected when animals have been treated with antimicrobials. Moreover, H somni can be challenging to grow in culture. A recent evaluation of a quantitative real-time PCR (qPCR) assay for detection of H somni in bovine pneumonic lung samples from 150 bovids and comparison of the assay results with those of standard tissue culture techniques revealed that only 6 samples were culture positive (4 of which were also qPCR assay positive), whereas 31 samples were qPCR assay positive and culture negative.8 The investigators noted that the animals that were qPCR assay positive and culture negative for H somni most commonly had received antimicrobials prior to necropsy or were infected with a second bacterial agent that may have overgrown the H somni in culture.8 Given the unusually high pleocytosis in the case described in the present report, a dual infection with both H somni and another agent causing neonatal meningitis could be posited. Most commonly, Escherichia coli is isolated in cases of septic meningitis.3,6,7 However, the result of bacterial culture of the calf's brain tissue was negative and overt bacterial meningitis was not evident histologically; therefore, infection with a second agent can only be suspected. Nevertheless, CSF analysis of calves with CNS signs can be helpful in characterization of inflammation as well as the identification of infectious organisms, which may be invaluable in subsequent diagnostic and therapeutic decisions.
Footnotes
Plasmalyte, Baxter, Deerfield, Ill.
References
1. Zachary JF. Mechanisms of microbial infections In: Zachary JF, McGavin MD, eds. Pathologic basis of veterinary disease 5th ed. St Louis: Elsevier Mosby, 2012;147–241.
2. Welles EG, Tyler JW, Sorjonen DC, et al. Composition and analysis of cerebrospinal fluid in clinically normal adult cattle? Am J Vet Res 1992; 53:2050–2057.
3. Stokol T, Divers TJ, Arrigan JW, et al. Cerebrospinal fluid findings in cattle with central nervous system disorders: a retrospective study of 102 cases (1990–2008)? Vet Clin Pathol 2009; 38:103–112.
4. De Lorenzi D, Mandara MT. The central nervous system In: Raskin RE, Meyer DJ, eds. Canine and feline cytology: a color atlas and interpretation 2nd ed. St Louis: Saunders Elsevier, 2010;325–365.
5. Scott PR, Penny CD. A field study of meningoencephalitis in calves with particular reference to analysis of cerebrospinal fluid Vet Rec 1993; 133:119–121.
6. Drevets DA, Bronze MS. Listeria monocytogenes epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 2008; 53:151–165.
7. Green SL, Smith LL. Meningitis in neonatal calves: 32 cases (1983–1990) J Am Vet Med Assoc 1992; 201:125–128.
8. Bell CJ, Blackburn P, Elliott M, et al. Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease? J Vet Diagn Invest 2014; 26:631–634.