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No. 16 Summer 2003
 

Distemper – Parvo IgM Kit: Practical Aspects 
By Ephraim Keren, VMD
IC News Editor 

Abstract
 A dot - ELISA for the detection of immunoglobulin (IgM) antibodies against canine distemper virus (CDV) and canine parvovirus (CPV) was assessed(1). The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the ImmunoComb® ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot - ELISA technique and the IFA (p<0.001). The dot - ELISA kit was also used to assess the changes in the levels of IgG and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.  

The Editor Comment
The authors suggested that the dogs’ antibody responses to vaccination described in their study should provide a basis for evaluating immune response in naturally infected dogs. As such, the results of the ImmunoComb® in detecting IgM antibodies to CDV and CPV have important practical implications in the clinical situation. Namely, any measurable level of IgM indicates exposure to the specific virus, which was tested. Furthermore, the approximate time of infection may be determined by evaluating both IgM and IgG titers.    


(1) Waner, T., et. al. Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus. Vet Record, 152 (19), 2003, pp. 588-591.    

Sero-Diagnosis of Brucellosis
By Ephraim Keren, VMD

Brucellosis is an important zoonotic disease, which causes abortion in large and small ruminants resulting in economic losses in agriculture and related industries. As such, national programs to control or eradicate brucellosis have been implemented by countries all over the world.  

The disease is transmitted to humans who come in contact with contaminated tissues/discharges or consume unpasteurized dairy products from infected animals. Brucellosis is considered an occupational hazard of slaughterhouse workers and veterinarians. Food sources are associated with a significant number of human cases every year in some countries, notably in Africa and the Middle East, including Israel.  

The two most important pathogenic species of brucella for cattle(1) and small ruminants (sheep and goats) are Brucella abortus and B. melitensis, respectively. The ’Gold Standard’ for confirming the diagnosis of brucellosis is by bacterial culture. However, this method is not convenient for screening herds or flocks and also poses problems for farms that are remotely located from diagnostic laboratories. Thus, serological testing has been used to identify infected animals.  

The serum agglutination test (SAT) has been most widely used for screening purposes and is used as a reference in many countries for international trade. The Complement Fixation (CF) test is regarded to be more sensitive and specific than the SAT and is widely accepted as a confirmatory test. ELISA tests have gained increased acceptance as both screening and confirmatory tests for brucellosis. The sensitivity and specificity of the ELISA is comparable to the CF test and is technically easier to perform.  

National programs to control or eradicate brucellosis involve slaughtering of animals that are identified as infected on the basis of serologic tests. In 1993, the WHO/FAO/OIE organizations published guidelines for a Middle East regional program, which called for a combined approach of vaccination, test and slaughter eradication of infected animals(2). Clearly, the tests must be approved by the international organizations and performed by certified reference laboratories.  

Biogal has developed ImmunoComb® tests kits for serologic diagnosis of brucella infection in cows and sheep, which are offered as an alternative confirmatory assay for this important disease(3).    

References
1. Nielsen, L. Diagnosis of brucellosis by serology. Vet micro, 90, (2002), pp. 447-459.
2. Banai, M. Control of small ruminant brucellosis by use of ’Brucella melitensis’  Rev.1 vaccine: Laboratory aspects and field observations. Vet micro, 90, (2002) pp. 497-519.
3. Cetinkaya, B. and Öngör, H. Evaluation of ImmunoComb® in comparison with other serological tests for ovine brucellosis. Vet Record, Nov. 25, (2000), pp.632-634.         

West Nile Virus Reviewed
By Ephraim Keren, VMD

West Nile Virus (WNV), a flavivirus of the Japanese and St. Louis encephalitis serocomplex has come into the limelight in recent years as a disease agent in horses, domestic birds, and in man.    

Infection is transmitted between birds and from birds to mammals by mosquitoes (Culex spp.). The virus has been associated with human infections for over 60 years throughout Europe, Africa, and Asia, especially in the Mediterranean region and is considered an important cause of equine encephalitis in these regions.  

A rise in the incidence of WNV infection in humans was noted worldwide in the 1990’s. The severity of illness ranged from mild to severe. In 1999, WNV was reported for the first time in the United States in connection with an outbreak that affected 61 human cases of encephalitis (seven fatalities) in the area of New York City. The virus was also isolated from birds and horses in the region. A vaccine for horses was marketed in the USA in 2002.  

From August to November 2000, an outbreak of WNV in Israel affected over 400 people with 29 deaths. During this period, local veterinarians began seeing cases of neurologic illness in horses from various regions in the country. The clinical signs included ataxia, paresis, and death. The virus was also recovered from geese prior to reports of equine disease.  

WNV was previously identified in migratory birds in Israel and in a flock of domestic geese in 1997. Subsequently, it caused clinical disease and mortality in domestic geese flocks resulting in significant economic losses for the raisers. In 2001, a program in which a homologous inactivated vaccine was administered to goslings was effective. Several outbreaks of WNV occurred in unvaccinated flocks, while vaccinated flocks that showed positive antibody titers remained healthy.  

Serology for WNV has been suggested as a diagnostic procedure in ill horses that show central nervous symptoms. Paired blood samples (collected 10 to 14 days apart) are tested for antibodies to WNV.  If the horse dies, post mortem examination of the brain for rabies is recommended, as well.    

References  
1. The Israel Veterinary Services, Ministry of Agriculture Bimonthly Report, September - October 2002.    
2. Perl, S. et. al. West Nile Virus Encephalitis in Horses in Israel. Israel Jl of Vet Med, (2002), Vol. 57(2), pp. 65-68     
3. Preston, A. West Nile Fever Update. Israel Jl of Vet Med, (2002), Vol. 57(4), pp. 169-70.    

From the Editor: Applications of Serology
By Ephraim Keren, VMD                

Serology is the determination of antibody levels in blood or serum to specific disease agents (notably viruses) has long been recognized as a diagnostic method with a wide range of applications for infectious diseases. A number of applications using serology are addressed in this issue of the ImmunoComb® News:

1.  Diagnosis of Clinical Illness – The major clinical application of serology is in helping diagnose clinical disease, as described in the review on West Nile Virus (WNV) and brucellosis. Biogal’s development of ImmunoComb® kits for these 2 diseases is being considered; we invite further comments from our readers.

2.  Vaccine Efficacy – The controversy continues over the value of serology for determining whether an animal is immune or should be vaccinated against a particular disease agent. Challenge studies using virulent virus are not appropriate in the clinical setting to assess whether a vaccination program ‘has worked’. However, this type of ‘field trial’ was observed following a vaccination program in geese for WNV. Vaccinated sero-positive goslings resisted disease while non-vaccinated birds became sick.

3.  Immunity Status – Antibody titers may be used to evaluate if an animal has a protective level of immunity to a particular disease. While serology cannot necessarily determine how this immunity was achieved (maternal antibodies, vaccination, or natural infection), it can provide important information for veterinarians about animal’s immunity status.  

In summary, veterinarians should be familiar with serology, which continues to be a valuable tool for diagnosis, prevention and control of infectious diseases.    

Vet Forum: Interpretation of Results

We recently bought some ImmunoComb® kits and are pleased with the results. However, we need some information regarding interpretation.
  
1. Can the ImmunoComb® test distinguish between vaccine and wild type strains of canine Distemper and Parvovirus, or does the kit detect antibodies to all strains of these viruses?
2. What is considered a ‘protective’ antibody titer?
3. Does a high IgG antibody titer reflect current disease or do we need to perform a follow up test?     

Thank you
Sincerely, Dr Z. Asgarali
School of Vet Medicine
University of the West Indies, Trinidad  

Dear Dr. Asgarali,  

1.  The ImmunoComb® kits for Canine Distemper Virus (CDV) and Canine ParvoVirus (CPV) test for antibodies that are produced in  response to vaccination and/or natural infection. As with most other serologic tests, the IC cannot determine the origin of viral exposure. The CDV kit uses the Rockborn strain antigen and the CPV kit uses the CPV-2 antigen.
  
2.  We consider an ImmunoComb® result of S3 to be the positive “cutoff” (for IgG antibodies) for both CDV and CPV. Based on tests conducted by the Cornell University Diagnostic Labs an S3 is approximately equivalent to VN 1:16 (CDV) and HI 1:80 (CPV). Virus challenge studies reported in the literature suggest that these antibody titers are “protective”. The decisions and recommendations regarding revaccination with the help of serologic information is the veterinarian’s responsibility.  
    
3.  A high IgG titer may reflect current or past disease. Two tests (acute and convalescent samples) are usually recommended in clinical cases. IgM evaluation is suggested to evaluate current/recent exposure to the antigen. IgM levels may remain
elevated(1) for up to 3 months. IgG is much longer lived  (years), but its production starts after the initial IgM response.  

I hope that these comments have addressed your questions. If you require any further clarification, please do not hesitate to let me know.  

Sincerely,  
Ephraim Keren, VMD  


(1) Appel M. and Summers B. Canine Distemper: Current Status. In Recent Advances in Canine Infectious Diseases, Carmichael L. (Ed). www.ivis.org/advances/Infect_Dis_Carmichael/appel/chapter_frm.asp