CHAPTER 30 Miscellaneous Gram-Positive Bacterial Infections

CORYNEBACTERIUM PSEUDOTUBERCULOSIS

Etiology

Corynebacterium pseudotuberculosis is a gram-positive, pleomorphic, rod-shaped, intracellular, facultative anaerobe with worldwide distribution. It is the etiologic agent of ulcerative lymphangitis, external subcutaneous abscesses, and internal infection in horses. In North America, disease is most prevalent in the southwestern United States, but cases of C. pseudotuberculosis infection have been reported throughout the United States. Infection has been reported in sheep, goats, cattle, buffalo, camelids, equids, and humans.

Corynebacterium pseudotuberculosis grows well at 36° C (96.8° F) on blood agar in 24 to 48 hours, and it forms small, pinpoint-diameter, whitish, opaque colonies surrounded by a weak zone of hemolysis. Because of the high content of lipids in the bacterial cell wall, particularly corynomycolic acid, the colonies spatter in a flame and can be pushed across the agar surface.¹ The high content of lipids may facilitate survival of the organism in macrophages.²

Two species-specific biotypes of C. pseudotuberculosis have been identified based on differences in nitrate reduction,³ and deoxyribonucleic acid (DNA) fingerprinting techniques have revealed multiple strains.⁴^–⁷ Biotypes isolated from small ruminants are nitrate negative, whereas those from horses are nitrate positive. From the result of DNA studies, the terms “biovar equi” for nitrate-positive and “biovar ovis” for nitrate-negative strains were proposed.³ Natural cross-species transmission does not seem to occur between sheep and horses; however, cattle can have infection from either biotype.³

Epidemiology

Three forms of C. pseudotuberculosis infection have been described in horses: ulcerative lymphangitis or limb infection, external abscesses (Fig. 30-1), and internal infection. Ulcerative lymphangitis presents as severe cellulitis, with involvement of lymphatic vessels in one or more limbs and multiple draining ulcerative lesions. In a retrospective study of C. pseudotuberculosis infection in horses from California, horses with ulcerative lymphangitis comprised only 1% of the cases, whereas external abscesses occurred in 91%, and internal abscesses in 8% of the cases.¹¹ Horses of all breeds and gender may develop any of the three forms of disease, although mares may be overrepresented and Thoroughbreds underrepresented in studies by Doherr.¹⁶ In one recent study of internal infection, mares seemed to be overrepresented.²⁰

Fig. 30-1 Typical pectoral abscess caused by Corynebacterium pseudotuberculosis.

The portal of entry of this soil-borne organism is thought to be through abrasions or wounds in the skin or mucous membranes. Many insects have been incriminated as vectors for the transmission of the disease to horses, and recent studies have shown that Haematobia irritans, Musca domestica, and Stomoxys calcitrans can act as vectors for this disease ¹² (Figs. 30-2 and 30-3). The regional location of abscesses suggests that ventral midline dermatitis predisposes to infection. Because of the variable incubation period, ventral midline dermatitis may not be present during maturation of the abscesses.

Fig. 30-2 Houseflies (Musca domestica) feeding on exudate draining from abscess. Flies are vectors for C. pseudotuberculosis infection in horses.

Fig. 30-3 Horn flies (Haematobia irritans) feeding on the ventral midline of horse. Ventral midline dermatitis is a predisposing factor for C. pseudotuberculosis disease in horses, and the bacteria can be found in this species of fly.

Temporal and spatial analysis suggests that the disease can be transmitted through horse-to-horse contact or from infected to susceptible horses by insects, other vectors, or contaminated soil; the incubation period is 3 to 4 weeks.¹³ The organism survives for up to 2 months in hay and shavings and more than 8 months in soil samples at environmental temperatures.^14,¹⁵

The incidence of disease fluctuates considerably from year to year, presumably because of herd immunity and environmental factors such as rainfall and temperature. To date, the definitive environmental factors supporting the spread of infection have not been determined.¹⁶ Disease incidence is seasonal, with highest number of cases occurring during the dry months of the year, which is late summer and fall in the southwestern United States, although cases may be seen all year. Horses with internal infection are more frequently seen 1 to 2 months after the peak number of cases with external abscesses.¹⁷

Horses of all ages may be affected, although the low incidence of disease in foals less than 6 months of age suggests that passive transfer of immunoglobulins offers protection in foals born in endemic areas. A case-control study in an endemic area revealed young adults (<5 years) and horses in contact with other horses on summer pasture had increased risk of infection. Horses housed outside or with access to an outside paddock appeared to be at higher risk than stabled horses.¹⁸

Pathogenesis

The pathogenesis of C. pseudotuberculosis infection in horses is poorly understood. The incubation period appears variable; temporal and spatial clustering studies by Doherr et al.¹³ revealed an incubation period of 7 to 28 days. Bacteria are phagocytosed after entry into the host but continue to replicate. Intracellular survival of C. pseudotuberculosis has a key role in the formation of abscesses and is possibly mediated by two virulence factors: bacterial cell wall lipids and phospholipase D (PLD) protein exotoxin. The bacterial cell wall lipids may facilitate survival in macrophages, whereas the PLD exotoxin has profound effects on survival and multiplication within the host. The PLD toxin may directly affect phagocytic cells or inactivate complement and reduce opsonization of the bacteria. The exotoxin also increases vascular permeability, enhancing the spread of infection both locally and through the lymphatics. The subsequent vascular changes permit bacterial spread to additional locations, including regional lymph nodes. Humoral and cell-mediated immune responses ultimately develop, clearing the bacterial infection.¹⁹

Recovery generally is complete within 2 to 4 weeks, although rarely horses develop persistent or recurrent infections lasting for more than 1 year. In one study, 91% horses had complete recovery with no recurrence of infection in subsequent years, implying a long-lasting immunity.¹¹ In 9% of affected horses, however, infections persisted more than 1 year or recurred as external or internal abscesses.¹¹ In sheep and goats, studies have shown that acquired humoral and cellular immune responses develop after infection and that macrophages acquire the ability to kill the organism.¹⁹ Similar studies have not been performed in horses.

Corynebacterium pseudotuberculosis produces various exotoxins, which play a role in virulence; the most studied is PLD. Phospholipases are a heterogenous group of enzymes that share the ability to hydrolyze one or more ester linkages in glycerophospholipids. Although all phospholipases target phospholipids as substrates, each enzyme has the ability to cleave a specific ester bond. Thus, qualifying letters (e.g., A, B, C, D) are used to differentiate among phospholipases and to indicate the specific bond targeted in the phospholipid molecule. PLD is important in the pathogenesis of the disease by its action on cell membranes, causing hydrolysis and degradation of sphingomyelin in endothelial cells and increasing vascular permeability.⁸ The bacterial PLD is similar to the PLD of the brown recluse spider, which explains the presence of pain and edema at the site of infection. Targeted mutagenesis of PLD in C. pseudotuberculosis reduced the ability of this bacterium to establish a primary infection or cause chronic abscess formation in regional lymph nodes. These results indicate that PLD is a virulence determinant of C. pseudotuberculosis, increasing the persistence and spread of the bacteria within the host.⁹ The synergistic activity of C. pseudotuberculosis exotoxins with the exotoxins of Rhodococcus equi in lysing red blood cells in agar forms the basis for the synergistic hemolysis inhibition test.¹⁰

Clinical Findings

External Abscesses

External abscesses may occur anywhere on the body but most frequently develop in the pectoral region and along the ventral midline of the abdomen (see Fig. 30-1). This form of infection is commonly known as “pigeon fever,” because of the large size of the pectoral abscesses with the appearance of a pigeon’s breast, or “dryland distemper,” because of its prevalence in arid geographic regions. Abscesses contain tan, odor-free, purulent exudate and are usually well encapsulated. Additional sites with a predilection for abscess formation include the prepuce, mammary gland, axilla, triceps, limbs, and head. Other less common areas are the thorax, neck, parotid gland, guttural pouches, larynx, flanks, umbilicus, tail, and rectum. Septic joints and osteomyelitis have been reported.¹¹ Horses may have an abscess involving a single site or involving multiple regions of the body. It is common to observe multiple subcutaneous abscesses coursing along a suspected lymphatic.

Clinical signs most frequently associated with external abscesses are edema, fever, and nonhealing wounds. Other clinical signs include lameness, ventral dermatitis, weight loss, depression, anorexia, and mammary gland or preputial swelling. Generally, horses with external abscesses do not develop signs of systemic illness, although one-quarter will develop fever.¹¹ If signs of systemic illness are present, further diagnostic testing to rule out internal infection is warranted. In cases of external abscessation, a large area of edema is often observed in the area of abscess formation. As the abscess matures, this area becomes hard and painful. Some abscesses become quite large, particularly in the pectoral region. Abscesses typically have a thick capsule and can cause severe lameness if located in the axillary, triceps, or inguinal region.¹¹ Maturation can be slow and drainage difficult to establish if the abscess lies deep to muscle.

After drainage is established, either by spontaneous rupture or lancing, most horses recover within 10 to 14 days without complications. The abscesses may contain from 5 to 400 mL of thick, tan, purulent exudate. Ultrasonography may aid in determining the best location for drainage of external abscesses (Fig. 30-4). The case fatality for horses with external abscesses is very low (0.8%).¹¹

Fig. 30-4 Ultrasound image of pectoral abscess demonstrating exudate with mixed echogenicity surrounded by thin hyperechoic capsule (arrows). The abscess is approximately 3 to 4 cm in diameter.

Clinical pathologic abnormalities that may be observed include anemia of chronic disease, leukocytosis with neutrophilia, hyperfibrinogenemia, and hyperproteinemia. These hematologic parameters can occur with either internal or external abscesses but are more consistently observed with internal abscesses.

Internal Infection

Approximately 8% of affected horses develop internal infection, which is associated with a case-fatality rate of 30% to 40%.¹¹ In a retrospective study, infection was localized to a specific organ(s) in 90% (27/30) of horses. Involvement of multiple internal organs was identified in 37% (10/27) of horses. The organs most often involved were liver and lungs, with kidney and spleen being affected less often. Abdominal ultrasonography was a useful diagnostic tool to identify affected abdominal organs specifically^17,²⁰ (Figs. 30-5 to 30-8).

Fig. 30-5 Abnormal right liver lobe showing multiple hypoechoic abscesses producing mottled appearance to liver. The liver lobes were enlarged with rounded margins. Ultrasound image was obtained from the right 13th intercostal space with a 3.5-MHz sector transducer at a scanning depth of 14 cm.

Fig. 30-6 Solitary right renal abscess (arrows) in mare with history of weight loss. Aspiration of this abscess yielded a large quantity of purulent material. The size of this abscess did not change with antimicrobial therapy. Ultrasound image was obtained from the right 16th intercostal space with a 3.5-MHz curvilinear transducer at a scanning depth of 25 cm.

Fig. 30-7 Abnormal right kidney with two small hypoechoic abscesses, one involving the cortex and one involving the renal medulla (arrows). Urine was culture-positive for C. pseudotuberculosis. This mare was pregnant at the time of the examination and aborted her fetus within 48 hours. The renal abscesses resolved with antimicrobial therapy. Ultrasound image was obtained from the right 17th intercostal space with a 3.5-MHz curvilinear transducer at a scanning depth of 20 cm.

Fig. 30-8 Abnormal spleen with area of increased echogenicity surrounding small hypoechoic area, consistent with splenic abscessation (arrows). Clinical signs resolved after a long course of antimicrobial therapy. Ultrasound image was obtained from the left 11th intercostal space with a 3.5-MHz curvilinear transducer at a scanning depth of 17 cm.

A diagnosis of internal infection is based on clinical signs, clinicopathologic data, serology, diagnostic imaging, and bacterial culture. The most common clinical signs are concurrent external abscesses, decreased appetite, fever, lethargy, weight loss, and signs of respiratory disease or abdominal pain. Other signs observed in horses with internal abscesses include ventral edema, ventral dermatitis, ataxia, hematuria (caused by renal abscesses), and infrequently, abortion. The median age in two studies was 7 and 8 years, with a range of 1 to 23 years.^11,²⁰

Anemia of chronic disease, leukocytosis with neutrophilia, and elevated fibrinogen are common features of infection, particularly in horses with internal abscesses ¹¹ (Table 30-1). Leukocytosis with neutrophilia was seen in 36% and 76% of horses with external and internal abscesses, respectively. Hyperproteinemia, caused by increased serum globulin concentrations, was observed in 38% and 59% of horses with external and internal abscesses, respectively.¹¹

Table 30-1 Values of Key Clinicopathologic Data in 30 Horses with Internal Corynebacterium pseudotuberculosis Infection

CLINICOPATHOLOGIC DATA	MEAN ± SD	RANGE
Hematocrit (%)	33.9 ± 7.2	23.4–50.0
Total white blood cell (WBC) count (cells/μL)	19,297 ± 9485	6400–53,420
Neutrophil count (cells/μL)	16,080 ± 9109	2944–49,627
Platelet count (cells/μL)	228,967 ± 86,280	15,000-449,000
Fibrinogen (mg/dL)	670.0 ± 306.4	100–1600
Total protein concentration (g/dL)	8.7 ± 1.5	6.3–12.1
Globulin concentration (g/dL)	6.7 ± 1.8	3.5–10.5
Synergistic hemolysis inhibition (SHI) titer	2611	64–20,480

Data from Pratt SM, Spier SJ, Vaughan B, et al: J Am Vet Med Assoc 227:441-448, 2005.

Peritoneal fluid is frequently abnormal in horses with abdominal abscesses. However, peritoneal fluid analysis may be normal if abscesses are located retroperitoneal in the kidneys without involvement of other abdominal structures. C. pseudotuberculosis was isolated from 32% of samples of peritoneal fluid from affected horses.¹¹ Failure to isolate the organism from peritoneal fluid does not rule out the disease. The organisms could be located retroperitoneal, sequestered within a thick capsule, or suppressed by local factors or nucleated cells.

Ulcerative Lymphangitis

Ulcerative lymphangitis is the least common form of C. pseudotuberculosis seen in horses in North America, although this form of disease has been reported worldwide. Limb swelling, cellulitis, and draining tracts following lymphatic vessels are seen. Horses often develop severe lameness, fever, lethargy, and anorexia. Aggressive medical therapy is necessary, or the disease may become chronic, resulting in limb edema, lameness, weakness, and weight loss.¹

Diagnosis

Culture

The typical clinical presentation of single or multiple, maturing pectoral abscesses, with or without ventral midline abscesses, is highly suspicious of C. pseudotuberculosis infection. Culture of the characteristic tan or blood-tinged, odorless exudate is diagnostic. Bacteriologic culture of aspirates or draining abscesses readily yields growth of moderate to large numbers of organisms on blood agar in 24 to 48 hours. The colonies appear small, white, and opaque. Gram stain reveals gram-positive pleomorphic rods. Equine isolates reduce nitrate (unlike strains from small ruminants). In horses with internal infection, samples obtained by ultrasound-guided aspiration from affected organs can also yield a positive culture.¹⁷^–²⁰

Serology

Without a positive bacterial culture, the practitioner must rely on hematology, clinical chemistry, and serologic testing to support a diagnosis. Hematologic changes are nonspecific and indicative of a chronic inflammatory response. Serologic testing using the synergistic hemolysis inhibition (SHI) test can be a useful aid in the diagnosis of internal abscesses.^10,¹¹ The SHI test measures immunoglobulin G (IgG) to the exotoxin of C. pseudotuberculosis and is available through the California Animal Health and Food Safety Laboratory System in Davis, California. Serology is generally not helpful for diagnosis of external abscesses and may be negative early in the course of disease and even at the time of abscess drainage. Positive SHI titers must be interpreted carefully, after appropriate consideration of clinical signs, to distinguish active infection from exposure or convalescence. Both published and unpublished data from the University of California suggest that a reciprocal titer of 256 or greater is indicative of active infection. Horses with internal abscesses generally have SHI titers of 512 or higher. In one study of internal infection, SHI titers ranged from 512 to 20,480.²⁰ Titers of 16 or lower are considered negative, whereas titers between 16 and 128 are considered suspicious or indicative of exposure.²¹ These are only general guidelines, however, because there is considerable overlap in results from horses with active disease, exposure, and recovery from infection. Experimental inoculation of horses with a bacterin-toxoid developed from C. pseudotuberculosis produced increased SHI titers equivalent to those seen with active infection; the protection offered from this bacterin has not been proved to date.

Ultrasonography

Ultrasonography is extremely useful for diagnosis of internal infections in horses, not only for identifying affected organs, but also for defining the nature and extent of involvement of abdominal viscera. Abdominal ultrasound permits documentation of involvement of specific organs in the absence of clinicopathologic evidence of disease involving these organs. Abdominal ultrasound may be used for collection of transcutaneous liver and kidney biopsies and aspirates of abscesses for a definitive diagnosis (see Fig. 30-8). Thoracic ultrasound should be performed in affected horses to determine the severity of pulmonary disease (Fig. 30-9). Serial examinations can be used to evaluate response to treatment. Examinations should be performed using low-frequency (2.5-3.5 MHz) and medium-frequency (5 MHz) ultrasound transducers. Complete examination of the abdomen consists of both right and left paralumbar fossa regions, all intercostal spaces from the ventral lung margins to the costochondral junctions, and the ventral abdomen from the sternum to the inguinal region. Presence of peritoneal fluid and abnormalities of the liver, spleen, kidneys, stomach, duodenum, small intestine, cecum, and large colon can be observed.

Fig. 30-9 Cranioventral consolidation with moderate pleural effusion in mare with pneumonia secondary to C. pseudotuberculosis. The small hyperechoic areas within the consolidated lung represent air trapped within the small airways. The image was obtained from the right sixth intercostal space with a 3.5-MHz curvilinear transducer at a scanning depth of 16 cm.

Hepatic abnormalities associated with C. pseudotuberculosis infection in horses include hepatomegaly, multiple small hypoechoic areas resulting in a “moth-eaten” appearance, and discrete, circular, anechoic to hypoechoic areas (see Fig. 30-5). Renal abscessation may appear as a single large (10-15 cm in diameter) area or multiple anechoic to hypoechoic areas involving either the cortex or the medulla¹⁷ (see Figs. 30-6 and 30-7). Splenic abnormalities may also be observed and include small, irregularly shaped, hypoechoic areas without obvious encapsulation (see Fig. 30-8). In horses with pulmonary involvement, examination of the thorax may reveal presence of pleural defects (“comet tails”), consolidation, pleural effusion, or pericardial effusion (see Fig. 30-9).²⁰

Early diagnosis of internal infection caused by C. pseudotuberculosis is important for a successful outcome but is often difficult because of the insidious onset and nonspecific nature of clinical signs, which may include anorexia, fever, lethargy, and weight loss. In endemic areas, horses that have had an external abscess in the previous 6 months and then develop signs of systemic illness should be suspected of having internal C. pseudotuberculosis infection, as should horses with compatible signs residing on a property where other horses have had external abscesses. For horses with this history, ultrasonography and serologic testing are recommended as an aid for diagnosis of internal infections.