Streptococcus pluranimalium meningoencephalitis in a horse

Abstract

A 3-y-old, female Quarter Horse with a history of acute neurologic signs was found dead and was submitted for postmortem examination. Areas of petechial and ecchymotic hemorrhage were present on cross-sections of the cerebrum, cerebellum, and brainstem. Histologic examination of the brain revealed severe, purulent meningoencephalitis and vasculitis with a myriad of intralesional gram-positive cocci. Streptococcus pluranimalium was identified from formalin-fixed, paraffin-embedded tissue obtained from sites with active lesions by PCR and nucleotide sequencing of bacterial 16S ribosomal RNA. S. pluranimalium should be considered as a cause of meningoencephalitis in a horse.

Bacterial meningoencephalitis may be responsible for 9.6% of the cases of neurologic disease in horses. ¹⁰ Neonates are more predisposed to this condition given their lack of a mature immune system and increased permeability of blood-brain barrier compared to adult horses. ¹⁵ In neonatal foals, meningoencephalitis is one of the most common fatal complications of septicemia, occurring in as many as 8–10% of septic foals. ¹⁵ Major clinical signs and clinicopathologic alterations of bacterial meningoencephalitis in foals include obtundation, recumbency, weakness, hyperesthesia of the neck, seizures, hyperfibrinogenemia, hyperlactatemia, and neutrophilic pleocytosis in CSF. ²¹ Pathogenic bacteria such as Escherichia coli, Klebsiella oxytoca, Salmonella spp., Staphylococcus aureus, and Streptococcus equi subsp. equi have been isolated from septic foals with meningoencephalitis.^15,21 On the other hand, bacterial meningoencephalitis is rarely observed in mature horses. ¹²

Common infectious routes associated with meningoencephalitis in adult horses include direct head trauma, hematogenous dissemination, complications of dental disease, and secondary infections of the upper respiratory tract, ear canal, adjacent lymph nodes, orbital tissue, and pituitary gland.^12,18 Various bacterial pathogens have been isolated from infected brain lesions in mature horses, including E. coli, Corynebacterium pseudotuberculosis, Fusobacterium sp., Bacteroides sp., S. equi subsp. equi, S. equi subsp. zooepidemicus, Pasteurella caballi, Actinomyces sp., Actinobacillus equuli, Rhodococcus equi, Proteus sp., and Klebsiella pneumoniae.^15,19 We report here a case of meningoencephalitis in an adult horse caused by Streptococcus pluranimalium, an unusual and emerging zoonotic streptococcal species affecting both humans and animals.

A 3-y-old, racing Quarter Horse mare was presented clinically after flipping over backward and was suspected of head trauma. The clinical history stated that the horse came off the track after training and was observed by caretakers to flip over backward immediately after arriving in its stall as a result of an undetermined cause. No prior history of neurologic deficits or vestibular disease was reported. Upon clinical evaluation, the referring veterinarian reported that the horse was conscious and in lateral recumbency with blood pooling in the right ear canal. Five minutes after intravenous administration of 500 mg flunixin meglumine (Banamine; Merck), 40 mg dexamethasone, hydrocortisone sodium succinate (Solu-Cortef; Pfizer), and mannitol, the horse was able to stand with assistance but showed severe neurologic signs that included incoordination, bilateral horizontal nystagmus, head tilt, and muscle twitching. Drinking and eating capabilities were not affected. The horse improved slightly upon fluid therapy q12h for 2 d with 100 mL of dimethylsulfoxide (DMSO) q12h, 500 mg of flunixin q12h, 20 mg of dexamethasone q12h, and 3 g of gentamicin q24h, but eventually collapsed and died ~44 h after showing clinical signs.

Postmortem examination was performed at the Oklahoma Animal Disease Diagnostic Laboratory (OADDL; Stillwater, OK, USA) per our standard operating procedure. Grossly, the meninges overlying the cerebrum and cerebellum were diffusely expanded by moderate amounts of edema, and meningeal blood vessels were congested. Upon transection of the brain, there were multifocal-to-coalescing areas of petechial and ecchymotic hemorrhage that were most pronounced within the piriform lobe (bilateral), internal capsules, thalamus, and brainstem, and to a lesser extent within the frontal lobe of the cerebrum, cerebellum, and medulla oblongata (Fig. 1). Upon sagittal cross-section evaluation of the skull, no gross evidence of bone fractures (i.e., basisphenoid bone, petrous temporal bones) or contusions were observed within the calvaria and adjacent cranial or cervical structures; radiologic imagining was not performed either antemortem or postmortem. Other gross lesions included severe, diffuse submucosal hemorrhage of the trachea, predominantly affecting the distal half of the trachea and extending into the primary bronchi. There were also large amounts of dark-red, frothy fluid within the tracheal lumen. All lung lobes were discolored dark-red and were markedly wet and heavy, and upon transection, small-to-moderate amounts of dark-red, serosanguineous fluid exuded from the cut surfaces. Collectively, these pulmonary lesions were consistent with exercise-induced pulmonary hemorrhage observed in racehorses following strenuous exercise. ⁶

Figures 1–4.

Streptococcus pluranimalium meningoencephalitis in a Quarter Horse mare. Figure 1. Multifocal-to-coalescing areas of petechiation and ecchymotic hemorrhage are evident in the transections of thalamus, piriform lobes, and brainstem. Figure 2A. The leptomeninges over the cerebrum are expanded by densely cellular, inflammatory infiltrates. Figure 2B. The infiltrates are composed of intact and degenerate neutrophils intermingled with cellular and karyorrhectic debris and fibrin, extending into the cerebral parenchyma. H&E. Figure 3. Necrosuppurative vasculitis in an area of hemorrhage in the cerebrum. H&E. Figure 4. Large numbers of gram-positive cocci are present within foci of inflammation and necrosis in the brain. Gram stain.

Microscopically, lesions within the cerebrum and cerebellum were characterized by necrosuppurative meningoencephalitis and vasculitis with myriad intralesional cocci. Markedly expanding the leptomeninges and Virchow–Robin spaces, with variable extension to the adjacent neuroparenchyma of both gray and white matter, were multifocal-to-coalescing areas of intense perivascular cuffing, hemorrhage, and necrosis (Fig. 2A, 2B). The perivascular cuffs were composed of intact and degenerate neutrophils, with lesser numbers of macrophages intermingled with eosinophilic cellular and karyorrhectic debris and fibrin (Fig. 2B). Necrosuppurative vasculitis was present in areas of hemorrhage (Fig. 3). Occasionally, the endothelial cells of affected blood vessels were hypertrophic. Within adjacent areas, the neuropil was rarefied, and neurons were degenerate or necrotic. Axonal spheroids were also present within the white matter. Myriad gram-positive cocci were present within the inflammatory and necrotic foci, mainly around the blood vessels (Fig. 4). Severe pulmonary edema and mild lymphocytic portal hepatitis were also identified microscopically.

Total DNA was extracted from 35-µm thick scrolls of formalin-fixed, paraffin-embedded brain tissue from sites with active lesions (DNeasy tissue and blood extraction kit; Qiagen). The tissue scrolls were first washed by vortexing in 1 mL of xylene followed by 1 mL of ethanol. After each wash step, the scrolls were centrifuged at 10,000 × g for 5 min and the supernatant discarded. The wash steps were then repeated a second time, and after a second ethanol wash, the scrolls are allowed to dry for 10 min before proceeding with DNA extraction following the manufacturer’s instructions. PCR was performed on the extracted DNA using universal primers P515FPL (5′-GCGGATCCTCTAGACTGCAGTGCCAGCAGCCGCGGTAA-3′) and P13B (5′-CGGGATCCCAGGCCCGGGAACGTATTCAC-3′) ¹⁶ and a commercial kit (HotStar; Qiagen). Each 50-µL reaction contained 5 µL of 10× PCR buffer, 4 µL of 25 mM magnesium chloride, 1 µL of 10 µM forward and reverse primer, 0.5 µL of Taq DNA polymerase (5 U/µL), 0.5 µL of 10 mM dNTP mix, 5 µL of DNA template, and 33 µL of nuclease-free distilled water. The thermocycling profile was as follows: 15 min at 95°C for initial denaturation followed by 40 cycles of 94°C (30 s) and 72°C (110 s), and a final elongation step at 72°C for 10 min. PCR products were electrophoresed on 2% agarose gel to visualize the amplicon. The amplicon was purified (Wizard SV; Promega), and nucleic acid sequencing was performed (Eurofins Genomics, Louisville, KY, USA). The nucleotide sequences (799 bases) were then analyzed (BLAST, https://blast.ncbi.nlm.nih.gov) and had 100% identity to S. pluranimalium with 100% query coverage (GenBank accessions KT943470.1, KR819496.1, EU391530.1, EU391526.1).

S. pluranimalium was first identified in several species of domestic animals in 1999 and designated as a new species of the Streptococcus genus. ¹ The term “pluranimalium” is a combination of “plura” (many) and “animalium” (from animals), which indicates that this Streptococcus sp. has the potential to infect many different animals. ¹ The strains were recovered from the milk and genital tracts of cows with subclinical mastitis, the tonsils of calves, goats, and cats, and the crops and lungs of canaries. ¹ S. pluranimalium has been further isolated from abortion materials of cattle and septic calves, indicating the pathogenic potential of this organism. ³ Identification of S. pluranimalium in the brain and CSF from a neonatal calf with septicemia and severe meningoventriculitis suggested this bacterium can induce CNS lesions in domestic animals. ¹⁷ S. pluranimalium has also been regarded as a contributory agent to valvular endocarditis and sepsis in broiler chickens, mastitis in goats and cows, a respiratory syndrome in dogs, and may be associated with abortion in goats, infertility in alpacas, conjunctivitis in pheasants, and mastitis in Mediterranean buffaloes (Bubalus bubalis).^4,5,8,20 This organism has also been isolated from nasal swabs of healthy horses. ⁷

S. pluranimalium has also been detected from the blood of a septicemic human patient with neutropenic fever. ¹⁴ Furthermore, S. pluranimalium was implicated as a cause of subdural empyema in an immunocompetent human patient, and a possible complication of asymptomatic frontal sinusitis. ¹¹ S. pluranimalium was first reported in humans as a causative agent of brain abscesses in a child with congenital cyanotic heart disease who had a history of contact with chickens 4 d prior to admission. ¹¹ Several case reports have also described the contributory potential of this bacteria to human brain abscesses, ² and S. pluranimalium has been isolated from human patients with periodontitis and endocarditis.^2,11

Bacteria of the Streptococcus genus comprise more than 100 species of gram-positive, non-motile, chain-arranged, facultatively anaerobic or obligately anaerobic cocci, of which the majority inhabit human and animal mucous membranes as commensal microflora, from which they may become opportunistic pathogens. ⁹ Various Streptococcus spp. have different levels of host predilection, virulence potential, and biochemical characteristics. ⁹ Based on 16S rRNA gene sequencing and phylogenetic analysis, the strains of S. pluranimalium isolated from cattle, broiler chickens, and humans are genetically related to S. hyovaginalis, S. thoraltensis, and S. halotolerans.^1,5,11,13

Although the first case of S. pluranimalium infection was documented >20 y ago, the pathogenesis of this bacterium remains unclear. Whole-genome analysis of S. pluranimalium isolated from cattle (strain TH1417) showed at least 5 potential virulence factors, ¹³ including fibronectin-binding protein and LPXTG-anchored protein, which are responsible for bacterial adhesion; hemolysin III homolog, which may contribute to the formation of transmembrane pores on host cells; cell wall–anchored protein sortase, an enzyme associated with the interaction between bacteria and the microenvironment; and immunoglobulin A1 proteinase, which protects against host immune attack. This strain also contains some antibiotic-resistant genes such as macrolide efflux (MEF) gene, methionine sulfoxide reductase (MSR) gene, and lincosamide nucleotidyltransferase (LNU) gene that confers the potential for resistance to macrolides and lincomycin. ¹³ Mice experimentally infected with S. pluranimalium via an intraperitoneal route had significant lesions in brain and lung but not in other organs (e.g., liver, kidney, spleen, or heart), indicating potential tissue tropism of this bacterium. ⁸

In our case, we did not definitively determine the route of infection by S. pluranimalium, and the correlation between infection and clinical history of suspected head trauma remains undetermined. The clinical history suggests that coup contrecoup injury may account for meningeal hemorrhage in the absence of calvarial fractures, and the documented blood pooling in the horse’s right ear after falling may suggest that such peracute head trauma occurred. Potential damage to structures within the internal ear canal or other cranial structures may have allowed opportunistic introduction of commensal S. pluranimalium into the CNS, as has been described for other pathogens. ¹⁹ However, were this sequence true, neurologic signs secondary to opportunistic S. pluranimalium infection would have been delayed and not observed immediately after falling, as occurred in our case. Alternatively, the distribution of vasculitis and meningoencephalitis suggests that hematogenous infection with S. pluranimalium was the cause of neurologic clinical signs and ultimately the demise of this animal, particularly given that no overt evidence of cranial fracture was observed at postmortem examination. Regardless of pathogenesis, S. pluranimalium has been isolated from nasal swabs of heathy horses and hence is available to act as an opportunistic pathogen. Infection by S. pluranimalium should be added to the list of causes of equine meningoencephalitis.