• AT THE WSAVA CONGRESS •
Antibody Testing Confirms Immunization in Puppies
Ephraim Keren, VMD
Canine parvovirus (CPV-2) and canine distemper (CDV) virus are two important pathogens causing potentially fatal diseases of dogs, which can be prevented by vaccination. However, vaccination with effective vaccines does not always guarantee successful immunization. Interference by maternal antibodies is regarded as a major cause of vaccination failure in young dogs. These passive antibodies are transferred to the pups from the dam through the colostrum. After nursing, the pups’ antibody titers average about 75% of that of the dam. The absorption of IgG colostral antibodies takes place in the first 48 hours after birth.
These maternally derived antibodies decline exponentially and have an average half life of 8-12 days. Therefore, the greater the initial concentration of IgG in the colostrum, the longer the period of "protection" against both pathogenic and vaccine virus.
The "fate" of the vaccination in the pup is dependent on the amount of maternal IgG present at the time of vaccination. If the maternal IgG level is low, the vaccine virus will be able to stimulate the immune system to produce first IgM and then IgG humoral antibodies. When the maternal antibody concentrations are high, they will neutralize the vaccination, and no immune response will occur in the pup.
One of the immunization strategies used to overcome the problem of "vaccination failure" due to maternal antibodies is the administration of multiple vaccinations starting from about 6 weeks until 4 months of age. By vaccinating frequently, at some point the maternal antibody titer of the puppy has dropped to low enough to allow successful immunization. This approach is costly to the client in terms of the number of vaccinations and leaves the veterinarian in doubt as to the success of immunization until about 4 months of age, when most pups have lost their passive immunity.
An alternative approach to multiple vaccinations would be to determine the level of humoral immunity in the individual pups as part of the vaccination protocol. The development of a standardized in-clinic rapid dot-ELISA procedure for CPV IgM and IgG antibodies could allow the veterinarian to assess the actual immunization status of individual puppies after vaccination.
The dot-ELISA (ImmunoComb®) and other ELISA techniques are being used with increased frequency in veterinary medicine for the assay of antibodies to a growing number of pathogens.
Abstracted from lecture by: Waner, T. "Response of puppies to vaccination for Canine Distemper Virus and Canine Parvovirus". 27th WSAVA Congress, October 2002, Granada, Spain
DISEASE BACKGROUND - Feline Corona Virus (FCoV)
FCoV is commonly found in places where lots of cats are gathered, like catteries. The virus infects the digestive system and usually causes no harm. The presence of antibodies indicate that the cat has been exposed, however, clinical disease does not result until the virus mutates and changes its tropism from gut epithelial cells to macrophages. The disease, Feline Infectious Peritonitis (FIP), is fatal.
• AT THE 2ND INTERNATIONAL FCOV/FIP SYMPOSIUM •
Researchers Meet to Discuss FCoV /FIP Shlomit Mazar, Biogal’s R & D Manager This past August, I attended the 2nd International FCoV/FIP symposium in Glasgow, Scotland. The participants were mostly investigators from various research institutions. Clinical veterinarians and representatives from commercial companies and feline organizations attended as well. The purpose of the conference was to present the accumulated knowledge about Feline Corona Virus and Feline Infectious Peritonitis. The symposium dealt with 3 main issues: diagnostics and treatment, epidemiology and vaccination prospects. While a large amount of work was presented, researchers were unable to propose very many recommendations for dealing with this challenging disease.
One recommendation was agreed upon with regard to the diagnosis of FIP. Before euthanizing a clinical case (even in a cat with PCR and high antibody titers), a biopsy for histopathologic confirmation should be performed.
Living with Feline Coronavirus
Lecture Abstract by: Perry, S. The 2nd International FCoV/FIP Symposium, August 2002, Glasgow, UK.My first reaction, back in 1995, to hearing that my cats were infected with feline coronavirus was sheer horror, closely followed by panic. There were 9 of them, and 4 of those tested positive. My veterinarians advice was uncompromising and stark – all four that had the virus must be euthanased immediately, to stop them spreading it to the rest of mine and to others in the neighborhood. On top of that, the others were probably already infected, so I had either to put them down at the same time or to re-test in six months, but he thought that was only delaying the inevitable. There was nothing to be done about the disease - those cats who had it were certain to die and in a particularly unpleasant way.
Fortunately for all of us, I was too upset to make a decision for several days, and during that time the vet’s young locum contacted Dr. Diane Addie at Glasgow, who asked if I would be willing to take part in her research. Her view was that the antibody positive cats were in fact unlikely either to develop the disease or to infect the others if I took certain precautions, and that to euthanase them was unnecessary. I jumped at the chance.
One good thing that came out of this research is that my veterinarian, who was initially very scathing and said I was only delaying the inevitable deaths of my household, has become an enthusiastic convert. He now tries to persuade any owner whose cat tests positive without apparently showing symptoms of FIP not to opt for euthanasia, but to keep the cat away from others and to re-test in six months or a year. The best thing, though, is that nearly all my cats are still alive and thriving happily. They seem untroubled by being restricted to their own house and garden, and at least I no longer have to worry that one will be killed or injured on the roads. In the seven years since those first tests, only one of my cats has gone on to develop FIP, which was as horrible as I had been led to believe, and she was put to sleep. Five of the original group are alive and well, with only two still testing positive for feline coronavirus. Though four have died over the years, all but Pippa have done so of other ailments associated with old age. I mourned and still miss each of them, but their deaths seem somehow in keeping with the natural order of life, rather than the ’humane solution’ that I was told back in 1995.
Evaluation of the Feline Coronavirus Antibody ImmunoComb®
Lecture Abstract by: Addie, D., McLachlan, S. A. & Oswald, I. The 2nd International FCoV/FIP Symposium, August 2002, Glasgow, UK. Aims: To evaluate a commercially available feline coronavirus (FCoV) antibody ImmunoComb
® (Biogal Galed Laboratories) as an in-house test for veterinary surgeons.
METHODSamples: 110 serum, plasma and effusion samples submitted to a diagnostic laboratory for FCoV immunofluorescent antibody (IFA) testing. The ImmunoComb
® tests were used according to the manufacturer’s instructions using a 5µl sample. Results were read blind by two individuals and compared to the IFA titer. The correlation between the kit results and the IFA titer was calculated to evaluate the sensitivity of the kit.
Results: The ImmunoComb
® results correlated well with the IFA titers (the correlation between reader 1 and IFA titer was 0.84, and the correlation between reader 2 and IFA titer was 0.85. The correlation between each reader was 0.95).Two samples with an IFA titer of zero and 3 samples with an IFA titer of 10 gave positive ImmunoComb
® results, indicating that false positive results might occur by Immunocomb.
Conclusions: The ImmunoComb
® is a useful in-house kit for screening for FCoV antibodies. A few false positive results did occur, so a positive result in a healthy cat would have to be confirmed using another technology. The ImmunoComb
® is a useful diagnostic aid in FIP (within the usual limitations of serology in FIP diagnosis). The correlation of ImmunoComb
®-negative results with IFA seronegativity was 100%, so it can be used with confidence for screening cats prior to entry into a FCoV free cattery or stud.
From the Editor: Help in Understanding ImmunoComb® Results
Ephraim Keren, VMD
Thank you, to our customers and agents for your questions, some of which are reviewed below for the interest of our readers. Cut-Off Titer
Some veterinarians are apparently uncomfortable with calling low positive (S1 or S2) results "suspicious" or "not significant". The key to interpreting results of the Immunocomb or any other serologic test is understanding the meaning of a "cut-off" titer.
Normal animals have many immunoglobulins (antibodies), some of which cross react with different antigens and are not specific to the antigen being tested. As such, low levels of antibodies are not considered to be ’significant’ enough in order to call the result positive. The cut-off value is the minimal level of antibodies that is considered to be a ’significant’. Therefore, a result that is more than zero is not always considered to be ’positive’ unless it is greater than the ’cut off ’ titer. (This is also the case with antigen tests when detection of minute amounts of antigen is not always considered positive).
ImmunoComb® Results Read by Eye Some customers complained that it was difficult to read low positive results. One of the strong selling points of the ImmunoComb® test is that results can be read by eye. After years of experience using the Immunocomb in my own clinic, I employ the following rule of thumb when reading results:
S0 - No or trace color.
S1 - A light shade of grey (more discernable than ’trace’).
S2 - A shade of grey which is lighter than S 3, but darker than S 1.
S3 - A shade of grey comparable with the positive reference spot.
I use the CombScale to grade color results from tests performed at different times to evaluate if the score has changed (i.e. seroconversion).
If the customer would prefer more exact numerical results, we can offer the ’CombScan’ which will read the color result with a scanner and provide numerical values which are comparable with the standard laboratory ELISA’s.
Fluorescent Antibody Test for Canine Distemper This test has been used for identifying Distemper viral antigen, which may be present in surface epithelium (conjunctiva or other mucous membranes). ’False’ positives have been attributed to non-specific artifacts and care needs to be taken to identify viral antigen in intact cells. And of course, the FA test requires trained laboratory personnel and special equipment to perform.
The FA test is often positive in acute (fatal) cases of Distemper, however, viral antigen has often disappeared from peripheral cells in subacute cases. The antigen may be masked by viral neutralizing antibody, which is typically present in dogs that survive the acute illness
(1). These cases could produce ’false’ negative results for the FA test and would more likely be detected by an antibody test.
(1) Appel, Max (Cornell Univ.) ’Canine Distemper’. In Kirk’s Current Veterinary Therapy VI,
Small Animal Practice, pp. 1308-13. W. B. Saunders Co. (1977).