Systemic disseminated Neospora caninum infection with cutaneous lesions as the initial clinical presentation in a dog

Abstract

This report describes a disseminated Neospora caninum infection with cutaneous involvement as the primary presenting clinical sign, in an apparently immunocompetent 7-year-old, spayed female boxer dog. The dog had an 8-day history of progressive lethargy associated with the appearance of multiple cutaneous and ulcerated masses, followed by an acute deterioration of her clinical status. Blood analysis revealed thrombocytopenia, increased liver enzyme activity, and partial thromboplastin time. Disseminated intravascular coagulation was suspected. Tachyzoites were identified on cutaneous cytology and species was determined by polymerase chain reaction (PCR) assays on blood and cerebrospinal fluid. The post-mortem evaluation revealed involvement of the neurological system, liver, lung, and skin.

Neospora caninum is an obligate intracellular tissue cyst-forming coccidian parasite of the phylum of Apicomplexa (1) and is morphologically similar to Toxoplasma gondii. This parasite has a global distribution and, although principally recognized as a neuromuscular disease in dogs, neosporosis can induce a variety of clinical signs and lesions including myocarditis, polymyositis, pancreatitis, dermatitis, and interstitial pneumonia (1–4). In the adult dog, clinical manifestation has mainly been described in immunocompromised dogs (3,5–8).

Case description

A 7-year-old, spayed female, boxer dog was presented to the emergency service with an 8-day history of progressive lethargy and the appearance of multiple cutaneous and subcutaneous masses. These masses, noted over the entire body, were of a medium to large size (2 to 10 cm in diameter), most were ulcerated, with a significant number oozing a serosanguinous to yellow-brown fluid. The skin lesions were not associated with alopecia or erythema and were not pruritic. The depth of the ulceration appeared to be variable. Seven days before presentation, corticosteroid therapy, (unknown dose and medication) had been implemented by the referring veterinarian. A complete blood (cell) count (CBC) and serum biochemistry were performed at the referring clinic and revealed moderately elevated activity of liver enzymes (Table 1). Melena, hematuria, and an acute decline in her overall clinical status had developed 48 h before referral. The dog was significantly clinically more depressed and could no longer ambulate at this time. The dog lived in a suburban area and contact with coyotes may have been a possibility according to the owners. Four years previously, the dog had been treated for bacterial folliculitis and Malassezia dermatitis, secondary to a suspected environmental or food allergy. A definitive diagnosis for the dermatological disease was not obtained; however, it did resolve with a combination of cephalexin and ketoconazole therapy. At the time of presentation, the dog had no evidence of a chronic medical condition nor was she receiving any medical treatments.

Table 1.

Comparison of selected biochemical parameters from the referring veterinary clinic to those performed at the referral hospital.

Parameter	6 days prior to referral	Day 3 of hospitalisation	Reference interval
Albumin (g/L)	33.0	12.0	26.7–35.4
Calcium (mmol/L)	2.36	1.93	2.33–2.69
ALT (U/L)	574	3580	21–80
ALP (U/L)	290	2067	10–113
GGT (U/L)	62	28	1–8
Bilirubin (μmol/L)	17.0	26.2	1.8–8.8
Urea (mmol/L)	8.70	24.33	3.26–9.44
Phosphorus (mmol/L)	1.37	2.08	0.79–1.51

ALT — alanine aminotransferase; ALP — alkaline phosphatase; GGT — gammaglutamyl transpeptidase.

On physical examination, the patient was severely mentally depressed, recumbent, and non-ambulatory. A mild tachycardia (120 beats/min) with a strong and synchronous femoral pulse, eupnea (28 breaths/min) and normothermia (37.8°C) were noted. Capillary refill time was 3 s and dehydration was estimated to be 8%. Several areas of petechiation were noted in the oral cavity along with generalized facial edema. Melena was observed upon rectal examination. Numerous ulcerative, necrotic subcutaneous nodules of various sizes were distributed over the entire body, with a higher incidence in the facial area.

Neurologic evaluation revealed a non-ambulatory animal with generalized weakness, a decrease in mental awareness but responsiveness to external stimuli. No significant findings were noted on a cranial nerve examination. Proprioceptive deficits were noted in all 4 limbs with a significant decrease in the muscular tone of all limbs. The remainder of the spinal cord reflexes were within normal limits, with the exception of bilaterally decreased patellar reflexes and there was no evidence of neck or back pain. Given the clinical presentation and the neurological examination, a preliminary diagnosis of polyradiculoneuritis was made.

At the time of admission, the following diagnostic testing was performed: CBC (Advia 120; Siemens Healthcare, Tarrytown, New York, USA), serum biochemistry (Beckman DxC 600; Beckman Coulter, Fullerton, California, USA), coagulation times (PT/PTT) (Synbiotics SCA 2000 Coagulation analyzer; Idexx, Lachine, Quebec) and a Global FAST3 (focused assessment with sonography in trauma, triage, and tracking) (iU22 Ultrasound and C8-5 probe; Philips Healthcare, Markham, Ontario) examination. The CBC revealed an inflammatory leukogram [leucocytes: 31.5 × 10⁹ cells/L; reference interval (RI): 5.10 to 14.20 × 10⁹ cells/L] characterized by a mature neutrophilia (27.12 × 10⁹ cells/L; RI: 2.70 to 9.80 × 10⁹ cells/L) and a moderate left shift (bands: 3.15 × 10⁹ cells/L; RI: 0.0 to 0.30 × 10⁹ cells/L). Disseminated intravascular coagulation was suspected considering the moderate thrombocytopenia (80.00 × 10⁹ cells/L; RI: 153.00 to 400.00 × 10⁹ cells/L), increased partial thromboplastin time (PTT prolonged at 143 s; RI: 58 to 97 s) and the clinical presentation (e.g., melena and petechiae). Serum biochemistry abnormalities (Table 1) included a significant increase in the activity of liver enzymes relative to the results obtained at the referring veterinary clinic and were consistent with worsening of the hepatic dysfunction. A urinalysis revealed a cloudy, brownish and moderately concentrated urine (urine specific gravity was 1.048). The observed pigmenturia may have been due to hemoglobinuria or myoglobinuria; however, no further testing was performed to investigate this abnormality. No significant abnormalities were noted on a Global FAST3 cage-side ultrasound examination.

Fine-needle aspirates of the cutaneous lesions were submitted for cytological evaluation. Most smears contained slightly to moderately degenerative neutrophils and some macrophages. Numerous spindle to crescent-shaped structures (approximately 5 to 7 μm × 2 μm in size) with light blue cytoplasm and a small purple to pink nucleus were observed. Several of these structures had been phagocytized by neutrophils and macrophages (Figure 1). Morphologically these structures were compatibles with tachyzoites, and the differential diagnosis consisted of Neospora sp. and/or Toxoplasma gondii. As cytological differentiation of these protozoal species is not possible, polymerase chain reaction (PCR) analyses for both organisms were performed on whole blood, cerebrospinal fluid (CSF) and skin, the latter 2 were on post-mortem samples. Polymerase chain reaction (Real-Time PCR for N. caninum; Laboratoire de diagnostic virologique vétérinaire et moléculaire, University of Montreal) revealed the presence of N. caninum DNA in blood and CSF suggestive of the presence of parasitemia and central nervous system (CNS) infection with this parasite. The PCR assay for T. gondii (9) was negative on these 2 matrices. The CNS infection with N. caninum was later confirmed on post-mortem examination by histopathology and immunohistochemical staining. Furthermore, PCR analysis on formalin-fixed (10% neutral buffered formalin) paraffin-embedded skin tissue also revealed the presence of N. caninum DNA and was negative for T. gondii DNA (9). DNA was extracted from formalin-fixed, paraffin-embedded tissue by dissolution of the paraffin in toluene followed by proteinase K digestion followed by ethanol precipitation without phenol-chloroform extraction (10–12). The presence of N. caninum DNA in the blood, skin and CSF fluid confirmed a diagnosis of systemic disseminated N. caninum infection with cutaneous involvement.

Figure 1.

Photomicrographs of fine-needle aspirates of the cutaneous lesions. The smears contained mainly, slight to moderately degenerated neutrophils and macrophages. Numerous spindle- to crescent-shaped structures (approx. 5–7 μm × 2 μm) with light blue cytoplasm and a small purple to pink nucleus were present. Several of these structures had been phagocytized by neutrophils (A) and macrophages (B). Morphologically, these structures were compatible with tachyzoites of either Neospora sp. or Toxoplasma gondii. Wright-Giemsa, ×100 objective, scale bar = 20 μm.

Based upon the presence of N. caninum DNA in the blood and the clinical appearance and cytological analysis of the skin lesions a clinical ante-mortem diagnosis of dermal neosporosis was highly suspected. Therapy with clindamycin (Clindamycin; Sandoz Canada, Boucherville, Quebec), 10 mg/kg body weight (BW), IV, q12h, and trimethoprim-sulfamethoxazole (Trimidox; Vétoquinol, Lavaltrie, Quebec), 15 mg/kg BW, IV, q8h was initiated. The antibiotic coverage was extended with ampicillin (Ampicillin, Fresenius Kabi), 22 mg/kg BW, IV, q8h due to the presence of the necrotic skin lesions and the melena. Pantoprazole (Pantoprazole; Sandoz Canada), 1 mg/kg BW, IV, q12h, S-adenosylmethionin (Zentonil advanced; Vétoquinol), 20 mg/kg BW, PO, q12h, fluid therapy (Plasmalyte; Baxter, Mississauga, Ontario) and vitamin K1 (Phytonadione; Vétoquinol), 1 mg/kg BW, SQ, q8h were also administered as supportive care considering the hepatic injury and suspected gastrointestinal hemorrhage. Given the suspicion of disseminated intravascular coagulation, a total of 30 mL/kg BW of fresh frozen plasma was administered during the first 24 h of hospitalization with resulting normalization of the clotting times.

Unfortunately, no clinical improvement was observed following the implementation of these therapies and by 72 h of hospitalization severe dyspnea had developed. A thoracic radiograph revealed a megaoesophagus, a nodular bronchointerstitial lung pattern, and mild pulmonary edema consistent with pulmonary involvement and acute lung injury. Due to the lack of clinical improvement, the worsening of the respiratory clinical signs, multi-organ involvement, and a presumptive diagnosis of sepsis secondary to N. caninum infection, euthanasia was elected. A sample of CSF was obtained from the cerebellomedullary cistern immediately following euthanasia. Analysis of the CSF fluid revealed a marked mononuclear pleocytosis (122 cells/μL; RI: < 4 cells/μL) and a positive PCR test for N. caninum DNA. Tachyzoites were not observed on cytological evaluation.

A post-mortem examination was performed on the dog. Microscopic evaluation of the brain and spinal cord revealed diffuse, moderate to severe inflammation characterized by perivascular infiltration of lymphocytes, plasma cells, and macrophages. In addition, multifocal foci of necrosis were present in the liver, cardiac and skeletal muscles and there was mild mononuclear inflammation. Severe panniculitis and pyogranulomatous dermatitis were also noted. Several ovoid to round organisms compatible with zoites (tachyzoites and/or bradyzoites) were noted in the brain, liver, lungs, and skin lesions. Immunohistochemistry (Polyclonal goat antibody; 1:5000, VMRD, Pullman, Washington, USA) of the cerebellum demonstrated positive immunoreactivity for N. caninum (Figure 2). All of these findings were consistent with a septic inflammatory process due to severe disseminated N. caninum infection.

Figure 2.

Photomicrograph showing immunohistochemical reaction of the cerebellum. Intense immunoreactivity of several ovoid to round Neospora/Toxoplasma-like organisms (tachyzoites). Immunohistochemistry with anti-Neospora caninum antibody and hematoxylin counterstain, ×100 objective, scale bar = 20 μm.

Discussion

Although N. caninum has a wide geographical distribution and exposure to the organism is not uncommon, development of clinical N. caninum infection in dogs is rare (1,4,13,14). Following infection, the severity and progression of the clinical signs tend to be dependent on the age and immunocompetence of the host. Three infectious life stages are reported: sporozoites within sporulated oocysts, rapidly dividing tachyzoites, and slowly proliferating bradyzoites within tissue cysts. Tachyzoites may be found in all hosts tissues, in particular during the acute phases of the infection. In the immunocompetent dog, the replication of tachyzoites is estimated to take 20 divisions before differentiation into bradyzoites (15), the quiescent life stage of the parasite which produces tissue cysts. In the acute stages of the infection, a T-helper 1 (Th-1)-type response is induced, which appears to be essential for limiting parasite replication. It is for this reason that a chronic asymptomatic infection is usually not detected. However, in an immunocompromised patient, reactivation of the bradyzoites and conversion to tachyzoites may occur, which may result in either vertical transmission of the parasite or clinical disease (3,15,16).

Neosporosis due to transplacental transmission of the organism most commonly occurs in young dogs and clinical signs are apparent between 4 wk to 6 mo of age. Recrudescence of clinical signs may occur in dogs as they age; however, clinical manifestation tends to be subtler or even subclinical. Horizontal acquisition of the organism is also possible and tends to occur through consumption of raw meat and infected intermediate hosts containing tissue cysts, or from sporulated oocyst-contaminated food or water (3,14,16). A domestic cycle between dogs and cattle seems to maintain the N. caninum life cycle. Unfortunately, in this clinical case reported we were unable to determine the method of transmission. The clients reported that the dog did not have contact with farm animals and was not fed a raw meat diet. She was fed a commercial dog food (unknown brand). However, the owners did report that contact with coyotes was possible.

Scientific information concerning neosporosis in dogs is scant and only several case reports describe systemic or cutaneous N. caninum infections in this species. The common thread in these case reports was that the dogs had an underlying immunosuppressive disease or received treatment with immunosuppressive drugs for immune-mediated thrombocytopenia, immune-mediated hemolytic anemia, pemphigus foliaceus, or hyperadrenocorticism (3,5–8,17). Immunosuppressive therapy may lead to parasitic reactivation accompanied by rapid multiplication of tachyzoites in several organs resulting in systemic infection and multi-organ dysfunction. Moreover, it has already been postulated that down-regulation of the cellular immune response induced by immunomodulatory therapy facilitates the survival of the parasite in the host and favors the development of disseminated lesions (17). In the present report, there was nothing in the history, clinical evidence, or necropsy findings that suggested an underlying disease that would result in an immunosuppressive state. A short course of corticosteroid therapy had been administered by the referring veterinarian, but the cutaneous lesions and clinicopathologic signs of hepatic impairment were present before administration of corticosteroid drugs.

Neosporosis most frequently manifests as neuromuscular disease in young dogs: a polymyositis-polyradicuoneuritis is the typical clinical presentation (2,18,19). Affected dogs are presented with exercise intolerance, ataxia, urinary incontinence, muscle pain, and muscle atrophy. Occasionally, the clinical signs may progress to lethargy, tetraplegia, dysphagia, dyspnea, regurgitation, or vomiting. In contrast to the clinical presentation of juvenile dogs, adults typically develop meningoencephalitis. The cerebellum appears to be a favored site of infection in the adult dog, as was present in the current case; this accounts for the common clinical presentation in the adult dog consisting of limb paresis or paralysis, ataxia, head tilt, ocular abnormalities (including miosis, diminished pupillary light reflexes or anisocoria), and seizures (3,4,14). Although principally recognized as a neuromuscular disease, disseminated infections involving the myocardium, liver, pancreas, lungs, or eyes may also occur, particularly in adult dogs (3,14). Dermal neosporosis has also been described as a specific clinical entity and is becoming increasingly recognized as a unique presentation of N. caninum infection, especially in immunocompromised patients (5–8,14,18).

In this clinical report the dog had a diffuse, severe meningoencephalitis with a mild meningomyelitis and intralesional zoites. There was evidence of multifocal necrotizing hepatitis, pneumonia, and deep pyogranulomatous dermatitis associated with a severe panniculitis. Organisms were noted in each of these affected areas. Furthermore, there was evidence of myocarditis, nephritis, myositis, and muscular necrosis. However, organisms were not identified at these sites. Of further interest was the presence of a megaesophagus in this dog. Previous clinical reports have also mentioned the association of N. caninum infection and megaesophagus (2,4). Therefore, neosporosis should be considered in the etiological differential diagnosis of megaesophagus in the dog.

Diagnosis of neosporosis most often relies on serology or detection of protozoal organism in body fluids. Detection of the organism may be accomplished by microscopic evaluation of fine-needle aspirates, tissue biopsies, or via PCR assays. In this clinical report the ante-mortem diagnosis was based on cytological detection of the organism in the cutaneous lesions and confirmed, ante-mortem with PCR assays on whole blood. Further confirmation of the systemic infection was accomplished on post-mortem examination by PCR assays (positive PCR assays on the CSF and skin), microscopic detection of organisms in various organs, and immunohistochemistry in the cerebellum (20).

Long-term antibiotic therapy with clindamycin, for a minimum of 8 wk and continued as long as clinical improvement is noted, is the current recommended therapy for N. caninum infection in the dog. Trimethoprim-sulfadiazine-pyrimethamine, combinations of clindamycin and trimethoprim-sulfadiazine, or combinations of clindamycin and pyrimethamine have also been described as alternative treatment options. There may be complete or partial response to the antibiotic therapy as bradyzoites cysts may persist despite appropriate treatment (2,20). Response to therapy appears to be most effective for cutaneous neosporosis rather than for the other clinical syndromes. In the present case, the dog was diagnosed with fulminant systemic neosporosis with a cutaneous presentation. Unfortunately, despite the initiation of appropriate treatment and intensive care, monitoring, and support, the patient developed sepsis, disseminated intravascular coagulation, and multiple organ failure resulting in euthanasia. There is relatively scant data in the veterinary literature concerning disseminated N. caninum infection in the dog and its expected overall prognosis. However, in this clinical case the prognosis was grave due to sepsis and multiple organ failure.