May 2016 Case of the Month

Interpretation

The peritoneal fluid was exudative in nature, with a total protein concentration of 4.0 g/dL (normal: <2.5 g/dL) and a nucleated cell count of 5,900 cells/ul (reported upper limit of 3,000/uL). The nucleated cells were an almost equal mixture of macrophages (48 %, cell depicted by arrow in Figure 1a) and neutrophils (47 %, cell depicted by arrowhead in Figure 1a) admixed with low numbers (5 %) of small lymphocytes. The background was proteinaceous in appearance and there were numerous bacteria noted. The bacteria were a homogeneous population of very small cocci found mostly as individualized cells along with a few pairs and rare short chains. In areas of the smear that had a denser background, a prominent clear zone surrounding the bacteria was evident. The neutrophils were largely nondegenerate. Rare neutrophils and macrophages contained phagocytosed bacteria. The morphologic features of the bacteria was supportive of a Streptococcus strain and the clearing was interpreted as a mucoid capsule. The fluid was interpreted as a septic purulent effusion and the bacteria were presumptively identified as Streptococcus zooepidemicus based on morphologic features and the association of this particular strain of Strep with fibrinous polyserositis in alpacas. The suspicion of a Strep. zooedpidemicus infection on cytological examination was confirmed on aerobic bacterial culture.

Answers to questions:

  1. What is the most likely etiological agent based on morphological features evident in the photomicrographs? The bacteria is a small cocci found in chains. These features are consistent with a Streptococcal spp. The clearing around the bacteria is suggestive of a mucoid capsule, also a feature of many Strep spp. The culture confirmed our suspicions.
  2. What is the likely source of the infection (in terms of route of entry into the host as well as the suspected carrier)? Streptococcus zooepidemicus is a commensal in horses that can be shed to other species, that are often exposed to the organism via the respiratory tract.
  3. Do the cytological features of the neutrophils concordant with the pathological process occurring?There is a left shift of neutrophils in blood and the neutrophils in blood show evidence of toxic change (this occurs as the cells mature faster and are released prematurely from the bone marrow in response to an inflammatory stimulus). In the peritoneal fluid, the nuclei of most of neutrophils have retained a condensed chromatin pattern (i.e. they are nondegenerate). Typically, septic purulent exudates will contain neutrophils that are degenerate (have swollen nuclei from karyolysis) as a result of bacterial toxin-mediated injury at the site of infection and the subsequent loss of the neutrophil’s capacity to maintain osmotic balance. The mucoid capsule of the bacteria helps Streptococcus resist phagocytosis and the majority of the organisms seen in this alpaca’s fluid were indeed extracellular. A few of the neutrophils are minimally swollen, but they are holding up in the battle against infection fairly well. If the effusion were the result of a perforated GI tract, the neutrophils would not look this good! The morphological features of the neutrophils often relate more to the type of bacteria present and the virulence factors the critters possess.  Never rule out a bacterial infection based only on the morphologic features of the neutrophils.

Additional information

Unfortunately, the alpaca’s condition continued to deteriorate. The animal was euthanized and a full necropsy was performed. The main gross findings were a severe fibrinosuppurative epicarditis, pleuritis, and peritonitis. The abdomen contained 2 L of clear, yellow, watery fluid. Fibrin plaques and intra-abdominal adhesions were noted. Histologically, there was evidence of necrosuppurative hepatitis and a moderate, diffuse, fibrinosuppurative peritonitis and pleuritis; both interpreted as a consequence of bacteremia and septicemia. Histologically, there was no evidence of a concurrent respiratory infection, although mucus had been noted on gross examination of the nasal passages. The neurological signs were attributed to septicemia (no other CNS lesions were found).

Discussion

Streptococci are a diverse group of bacteria that include a number of commensal, opportunistic, and primary pathogens. Streptococcus equi subsp zooepidemicus, the offender in the current case, is a commensal in the horse that can cause opportunistic infections in its host, while capable of causing severe disease when transmitted to other species. This bacteria is considered zoonotic and has been transmitted to people from both horses and infected guinea pigs (they are just little horses after all!).1,2,3 In both New and Old World camelids Strep zooepidemicus is an invasive pathogen and often causes very serious disease.4 Polyserositis and septicemia, as seen in this case, is colloquially referred to as “alpaca fever.” Formation of intra-abdominal abscesses has also been documented.5 Systemic Strep zooepidemicus infections bear some clinical similarity to the Strep equi metastatic infections that spread to sites distant from the head and neck in horses known as “bastard strangles.” The factors involved in this phenotypic switch are actively being investigated. The portal of entry from Strep zooepidemicus is often the respiratory tract with subsequent hematogenous dissemination.6 Intratracheal inoculation of the bacteria has been shown to recapitulate severe systemic disease in llamas.7 The capsule Strep zooepidemicus produces is antiphagocytic and produced in a temperature-dependent manner with increased expression of capsular proteins at lower temperatures.8 The bacteria produces fibronectin-binding proteins and is capable of forming biofilms. Loss of these fibronectin-binding proteins reduces the ability of the bacteria to adhere to cells in culture and to cause septicemia in mice.9

Treatment requires antibiotics with high tissue penetration and frequent drainage of affected cavities. In reported cases of managing these infections in people, this has included pericardial drainage. Housing of camelids separate from equids is recommended (as is hand washing if you are an equine enthusiast).

References

  1. Gruszynski K, Young A, Levine SJ, Garvin JP, Brown S, Turner L, Fritzinger A, Gertz RE Jr, Murphy JM, Vogt M, Beall B. Streptococcus equi subsp. zooepidemicus infections associated with guinea pigs. Emerg Infect Dis. 2015 Jan;21(1):156-8.
  2. Held J, Schmitz R, van der Linden M, Nührenberg T, Häcker G, Neumann FJ. Purulent pericarditis and pneumonia caused by Streptococcus equi subsp. zooepidemicus. J Med Microbiol. 2014 Feb;63(Pt 2):313-6.
  3. Pelkonen S, Lindahl SB, Suomala P, Karhukorpi J, Vuorinen S, Koivula I, Väisänen T, Pentikäinen J, Autio T, Tuuminen T. Transmission of Streptococcus equi subspecies zooepidemicus infection from horses to humans. Emerg Infect Dis. 2013 Jul;19(7):1041-8.
  4. Stoughton WB, Gold J. Streptococcus equi subsp zooepidemicus pleuropneumonia and peritonitis in a dromedary camel (Camelus dromedarius) calf in North America. J Am Vet Med Assoc. 2015 Aug 1;247(3):300-3
  5. Rodvold R, Mendez-Angulo JL, Firshman AM, Malone E, Nicholson A. Large intra-abdominal abscess treatment by marsupialization in 2 alpacas. Vet Surg. 2014 Nov;43(8):1004-8
  6. Hewson J, Cebra CK. Peritonitis in a llama caused by Streptococcus equi subsp. zooepidemicus. Can Vet J. 2001 Jun;42(6):465-7.
  7. Cebra CK, Heidel JR, Cebra ML, Tornquist SJ, Smith BB. Pathogenesis of Streptococcus zooepidemicus infection after intratracheal inoculation in llamas. Am J Vet Res. 2000 Dec;61(12):1525-9.
  8. Velineni S, Timoney JF. Capsular hyaluronic acid of equine isolates of Streptococcus zooepidemicus is upregulated at temperatures below 35°C. Equine Vet J. 2015 May;47(3):333-8

Authored by: E. Behling-Kelly

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