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No. 22 Summer 2006
 
Canine Neosporosis: A Diagnostic Challenge                                   
by Ephraim Keren, VMD, IC Newsletter Editor   

Natural infection with Neospora caninum , is known to occur in cows, sheep, goats, horses, cats and dogs. A previous article (in ImmunoComb ® News # 21 ) described neosporosis as a major cause of abortion in cattle. Canine neosporosis is reviewed in this sequel article. N. caninum is a protozoan parasite related to Toxoplasma gondii . Its life cycle is not fully understood, but the dog has been described as the definitive host.

Transmission    
Vertical transmission is probably the most important route of transmission in dogs, as canine clinical cases have been most commonly reported in puppies and young dogs. Infected dogs can perpetuate N. caninum in the environment and horizontal transmission to other animals is thought to occur via ingestion of contaminated feedstuffs. Neosporosis can be transmitted by inapparently infected animals.

Clinical Signs  
Neosporosis is associated with fatal disease in dogs of all ages. Pups that have been infected in utero typically develop an ascending paralysis. Other signs include difficulty in swallowing, head tremors, pneumonia, and/or heart failure, often with a fatal outcome. Several litter mates may be affected.

A variety of neurological disorders have been described in mature dogs. The first signs of infection may be mild ataxia and knuckling over on the hind feet. In time, the joints can become fixed in extension with loss of pain sensation and hindquarter muscle atrophy. Other neurologic signs include nystagmus and encephalomyelitis. Several cases of cutaneous neosporosis in dogs (pyogranulomatous and nodular dermatitis) have been reported in the literature.

Diagnosis  
Serologic testing (IFA, ELISA) provides strong evidence of prior exposure to N. caninum and may be the only reliable way to identify subclinically infected animals. Antibodies do not cross react with T. gondii .  Definitive diagnosis of clinical neosporosis is based on histological diagnosis, which may be confirmed by immunohistochemistry and PCR techniques.  The parasite encysts in the brain and other organs. The failure to detect N. caninum organisms may result in false negative histologic findings. Tachyzoites and tissue cysts containing bradyzoites are the only known stages of N. caninum . Tachyzoites have been found in many cell types, whereas tissue cysts have only been found in neural tissues.

Treatment  
Clindamycin hydrochloride (25 mg twice daily) is the antibiotic of choice for both toxoplasmosis and neosporosis. It has been found to work successfully in some (dermatologic) cases of neosporosis. However, if ascending paralysis has developed before treatment is started, the prognosis for recovery is poor. 

Prevention and Control  
Neosporosis is a diagnostic challenge for the veterinarian, particularly when inapparently infected dogs are involved. This poses a particular problem for breeding programs since transplacental infection is known to occur from subclinically infected animals. The problem is often recognized too late, after pups develop clinical disease or die. Serologic testing for N. caninum is therefore important in identifying breeding bitches and is recommended as a preventive health measure in breeding programs.

References
1. Dubey J. P. (2003). Review of Neospora caninum and neosporosis in animals. The Korean Journal of Parasitology , 41(1) , 1-16.
2. Fritz, D. et al. (1997). Neospora caninum nodular dermatitis. Canine Practice , 22(4) .
3.  Steinman, A., Shpigel, N.Y., Mazar, S. et al. (2006). Low seroprevalence of antibodies to Neospora caninum in wild canids in Israel. Veterinary Parasitology , (in press).  
  
Serologic Diagnosis of Canine Anaplasmosis

We thank the Veterinary Forum ( www.ForumVet.com ) for permission to publish the following abstract of an original article, entitled “Anaplasmosis: Tick-Transmitted Infectious Disease”, by Rebeka Gunn, and A. Rick Alleman, which appears in the January 2006 issue, pages 35-40.    

Case Presentation
An 8-year old intact female German shepherd presented to a local veterinarian with a history of anorexia, lethargy for several days, and progressive exercise intolerance. The dog had recently been in heat and upon physical examination it was panting, had a slight fever 103.4º F (39.6º C), and exhibited moderate abdominal discomfort and pale mucous membranes.

Diagnostic Work Up
A complete blood count (CBC) revealed thrombocytopenia (83 x 103 / μl) and mild leucopenia (5.61 x 103 / μl). The veterinarian initiated antibiotic therapy (cephalexin 30 mg/kg PO q12hr) and referred the case to Florida Veterinary Medical Teaching Hospital (VMTH) for further diagnostic evaluation. Early pyometera was a consideration, despite the absence of any vaginal discharge or distinct radiographic evidence to support this diagnosis.

Treatment and Additional Diagnostics
The following day at VMTH, the dog’s condition was worse; fever of 104.3º F (40.2º C) and thrombocytopenia of 57 x 103 / μl. The dog was hospitalized for 4 days for supportive fluid therapy and initiation of a 10-day course of doxycyline (5 mg / kg PO q12hr). During this time the dog’s physical condition improved and the thrombocytopenia began to resolve. The diagnosis was still open upon discharge from VMTH; results of serology tests for Ehrlichia canis , Rickettsia rickettsii, Anaplasma phagocytopilium, and Anaplasma platys were pending.

Relapse and Further Evaluation
Seven days later, a repeat CBC by the referring veterinarian showed a normal thrombocyte (platelet) count of 274 x 103 /μl. The dog remained free of clinical signs up at a follow up exam one month following discharge from VMTH. Then, 2 days later the dog returned to the emergency service with the same clinical signs as those on the initial presentation, including severe thrombocytopenia. Blood smears were prepared from venous and capillary blood samples.  Upon microscopic examination, particularly from the capillary blood samples, many platelets were noted to contain small, round, distinct, basophilic structures identified as morulae. Large and giant platelets, some of which contained inclusions, were also observed. Based on clinical signs, cyclic thrombocytopenia, and intrathrombocytic organisms identified in the blood smears, a diagnosis of A. platys was made.

Outcome
The patient was hospitalized for further care, and full month of doxycycline therapy was prescribed in accord with the working diagnosis of anaplasmosis. The dog responded to initial treatment and was discharged 4 days later.  A blood sample was resubmitted for serologic evaluation. This time, the result was positive for A. platys  at an antibody titer of ≥ 1:40 by the Immunoflourescent Antibody test (IFA) – a finding that correlates with active infection or previous exposure. Based on the change in titer between acute and convalescent sera and observation of intraplatelet organisms, active infection was confirmed. Follow up visits during the next 2 months revealed normal platelet counts and indicated that the dog had responded well to therapy. The cyclic thrombocytopenia did not recur following completion of the final course of antibiotics. The dog remains clinically healthy today, except for pannus in both eyes that was resolving under separate treatment.

Discussion
In 1978, Ehrlichia platys was identified as the causative agent of canine infectious cyclic thrombocytopenia. The organism was named Ehrlichia platys because of its morphologic similarity to other ehrlichial organisms. However, molecular analysis later revealed that some species of Ehrlichia were more closely related to Anaplasma marginale , so the organism was renamed Anaplasma platys . Both Ehrlichia and Anaplasma species belong to a family of small gram-negative, pleomorphic, obligate intracellular bacteria. A. platys organisms can be identified by microscopic examination of blood smears and appear as dark blue staining inclusions inside platelets. However, this method is tedious, time consuming, and often unrewarding because inclusions tend to occur transiently and in low numbers. Furthermore, inclusions must be differentiated from normal platelet granules and (Romanovsky-type) stain precipitate. PCR amplification is useful in confirming active infections, but is often not available to many veterinarians. Thus, detection of serum antibody by IFA testing remains the most practical and frequently used method of diagnosis in a clinical setting.

As was the case in this animal, serologic test results for serum collected early in the disease process may yield a negative titer. Therefore, paired serum samples of acute and convalescent phase (taken 3 or more weeks later) sera are preferred to obtain a conclusive diagnosis. A four-fold increase in antibody titer in the convalescent sample is generally held as confirmatory for infection, whereas a single sample with a high titer (≥ 1:64) may indicate either infection or exposure. 

Editor’s Note
The above case illustrates how a “false negative” may result when a serologic test is performed in an animal during the acute stage of infection. Detectable levels of antibodies develop later and thus underlines the importance of evaluating paired serum samples in serologic diagnoses. We would like to remind our readers that positive antibody titers to E. canis indicates previous exposure and/or current infection. This finding may be of diagnostic significance even if the animal is not currently showing clinical signs.  

From the Editor: Serology is not always black or white 
by Ephraim Keren, VMD  

Veterinarians today have a greater and more sophisticated choice of diagnostic tools to use in their every day practice than when I graduated from veterinary school over twenty five years ago and certainly compared to my father who received his VMD in 1947. Despite these advances in modern technology, our approach to making a diagnosis has remained basically the same.  

After receiving a thorough history and conducting a complete physical examination, the clinician may elect to perform further diagnostic tests in pursuit of a diagnosis. As such, he or she must then know how to interpret the results of these diagnostic tests. Some tests may provide absolute “Yes” or “No” answers. Others do not. Hematology and serology for example, provide values (i.e. quantitative or semi-quantitative results) that must be interpreted as normal or abnormal. Such findings may sometimes be more helpful to the veterinarian than a simple “Yes” or “No”.  

This issue comes up frequently when I am called upon to assist veterinarians in interpreting results of the ImmunoComb ® (IC) test. The IC’s grey color result indicates that the animal has detectable levels of antibodies, i.e. a positive result. However, the interpretation of this finding is not necessarily black and white.   

The case reports featured in this issue illustrate this point. In one case, a diagnosis of Canine Distemper was supported by a sero-negative finding. This result indicated that a previously administered vaccination did not induce a protective level of antibodies in the dog. As such, the animal was not immunized and subsequently became sick.  

In a different case, a sero-negative finding in the acute stage of illness did not initially support the diagnoses of canine Anaplasmosis. However, subsequent testing of paired serum samples did show that the dog mounted an antibody response to the agent. That finding together with microscopic evidence confirmed the diagnosis. These examples underscore how important it is for the veterinarian to be familiar with the limitations of a diagnostic test for a particular disease and know how to interpret the test results.    

I will conclude my column with the following anecdote. Several years ago, a relative of mine, MK, asked me to review some laboratory tests that she recently underwent. Based on the results of serologic tests, her physician arrived at a diagnosis of Lyme Disease, an infectious disease transmitted to animals and people by ticks. MK was relieved about finding an answer regarding the illness that she had been suffering from for some time, but she consulted with me, as she was about to begin a long course of antibiotics. I disagreed with the physician’s interpretation of the serology. At my suggestion, MK sought a second opinion from another physician who concluded that the serology findings did not support the diagnosis of Lyme Disease. Another diagnosis made and MK was put on an entirely different course of treatment, which proved successful.   
   
Vet Forum  
Ephraim Keren-Kornblatt, VMD

I thank Dr. Ram Furer of Ashkelon, Israel for permission to publish the following case report.   

Case History
A sick 4-month-old male mixed breed pup presented to Dr. Furer’s Animal Medical Center. Eleven days earlier, the pup was vaccinated with a multivalent vaccine (DHL-P*) and kept outdoors.  The owners reported that the pup started coughing four days ago. Yesterday, the dog refused to eat and started barking at its food. Today, it had excessive salivation, diarrhea and vomited several times.    

Upon physical examination, the pup exhibited conjunctivitis, muscular tremors of the mouth, and a fever 40.6º C. A complete blood count showed a leucocytosis (35.1 x 109 / µl) with a left shift.   *DHL-P = Distemper-Hepatitis-Lepto-Influenza and Parvovirus  

Question
Dr. Furer suspected the pup was ill with canine distemper virus (CDV). He wondered whether serology would be helpful in confirming the diagnosis since a CDV vaccination was recently given.   

Answer
I advised Dr. Furer to perform a serologic test to determine an antibody titer to CDV and to help him in arriving at a diagnosis. Finding a significant antibody titer (i.e., ImmunoComb ® Scale Score > S3) would indicate that the pup mounted an appropriate immune response to either the vaccination or field exposure to CDV and would be a good prognostic sign. A sero-negative result would be expected in a non-immunized pup during the acute stage of infection. (Significant levels of maternal antibodies are usually no longer present by 4 months of age.)     

An ImmunoComb ® antibody test for serum IgG to CDV (and canine parvovirus) was performed. A suspicious-low positive result (S1) was determined for both antigens.  This result indicates that the vaccination administered 11 days previously did not confer immunity and supports Dr. Furer’s clinical diagnosis of CDV. Most pups that fail to mount a good immune response to CDV exposure develop clinical signs and die 2 to 4 weeks following infection.  

In light of the poor clinical condition and low chances for recovery, the owners elected to euthanize the pup. An Immunoflourescent Antibody test was performed on the pup’s brain tissue to rule out rabies as a cause for the pups’ neurologic symptoms. The result was negative.