Canine leishmaniasis is a widespread and severe zoonotic disease caused by L. infantum. The inefficacy of drugs used to treat this disease by completely eliminating the parasite stresses the need for follow-up and predictive markers that enable treatment to be tailored to the patient. In this study, we analyze the usefulness of two recombinant antigen-based ELISAs in monitoring and predicting the clinical success of the most frequently used therapy against leishmaniasis, the combination of meglumine antimonite and allopurinol, in dogs with canine leishmaniasis.
At diagnosis, antibodies against rKMPII showed the highest seroprevalence among the L. infantum
antigens studied, thus supporting previous results that indicated that this antigen acts as a potent B-cell immunogen in dogs.23,24
Seropositivity against rTRYP was also significant because it was detected in half of the dogs studied, as found elsewhere.23
When the two recombinant antigen-based ELISAs were evaluated in parallel, 80% of dogs with leishmaniasis showed seroreactvity against rKMPII or rTRYP. In addition, concentrations of antibodies against rKMPII and rTRYP correlated significantly with canine leishmaniasis–related biochemical parameters (hypergammaglobulinemia and hypoalbuminemia) and with the number of clinical signs recorded in dogs. These results suggest that detection of antibodies against KMPII and TRYP antigens could be components of a standardized tool for the diagnosis of canine leishmaniasis.
When the entire group of dogs was considered, the results show that treating dogs with leishmaniasis leads to a clinical improvement and a parallel significant decrease in concentrations of specific antibodies against rKMPII, rTRYP, and CTLA one year after the beginning of treatment. However, concentrations of antibodies against rKMPII and rTRYP decreased significantly as early as one month after treatment was started, at the same time that the first reduction in the number of clinical signs, whereas the decrease in concentrations of antibodies against CTLA occurred at six months. In addition, the proportion of dogs that seroreverted one year after the beginning of treatment to recombinant proteins was significantly higher than those that seroreverted for CTLA. Thus, our results suggest that the dynamics of antibodies against recombinant proteins may be more useful than the dynamics of antibodies against CTLA for assessing clinical improvement after treatment, as described in human patients treated for visceral leishmaniasis. In accordance with our results, antibodies against the recombinant proteins rKMPII25
cTXNPx, a protein similar to TRYP,26
decreased significantly after treatment of patients with visceral leishmaniasis, conversely to serologic results with crude Leishmania
A significant reduction in concentrations of antibodies against CTLA has been extensively reported in dogs with leishmaniasis after treatment.8,15,21,22
This reduction is thought to be related to attenuated antigenic stimulation resulting from the decrease in parasite load. Our results might indicate that antigens such as rKMPII or rTRYP could be more sensitive than CTLA in detecting early reduction of Leishmania
load in treated dogs, although the presence of seropositive dogs for these recombinant proteins at the end of the study could indicate persistence of subclinical infection.14,15,21
Unfortunately, parasitologic data for these dogs during follow-up or at the end of the study were not available.
Because of the lack of parasitologic data, dogs were split in two groups according to only their clinical status one year after the beginning of therapy. We evaluated the value of rKMPII, rTRYP, and CTLA as predictive markers for long-term disease-free survival in dogs with canine leishmaniasis. One month after treatment was started, concentrations of antibodies against rKMPII had decreased significantly in disease-free survivor dogs. Conversely, in those remaining clinically ill, concentrations of antibodies against rKMPII did not decrease significantly until six months after the beginning of therapy. Thus, the dynamics of antibodies against rKMPII might be an early predictive marker for long-term disease-free survival after therapy for canine leishmaniasis.
Different profiles of antibodies against rKMPII between the two groups of dogs could be caused by different parasite loads related to different immune responses, but further studies are needed to confirm this possibility. Quantification of parasite burden in bone marrow samples by real-time polymerase chain reaction and further immune characterization could be helpful for answering this question. Information from other studies that used bone marrow aspiration showed that there was no sterile cure in most treated dogs, despite clinical improvement.14,15,21,27
Thus, the possibility of relapse as an epidemiologic risk28,29
still exists in disease-free survivor dogs, although to a lesser extent. For this reason, it has been recommended to use preventative measures against parasite transmission in animals and to monitor them on a regular basis to assess future clinical relapse.30
Concentrations of antibodies against CTLA did not decrease significantly until six months after the beginning of therapy, and this decrease occurred in disease-free survivors and dogs that remained clinically ill. Regarding concentrations of antibodies against rTRYP, although the decrease occurred one month after treatment was started, this decrease was observed in both groups of dogs. Consequently, ELISAs for CTLA8,22
and rTRYP could not predict long-term clinical success of therapy.
Some parameters have been suggested as predictive markers of clinical outcome in canine leishmaniasis,8,31,32
although, to date, none has been used in veterinary clinical practice. Dogs showing high levels of IgG against Leishmania
at diagnosis showed a poor prognosis after chemotherapy.8
In addition, several studies have reported different dynamics of antibodies against Leishmania
-specific antigens between responsive and nonresponsive dogs. In accordance with our results, Western blot showed a reduction in the intensity of low molecular mass (12–30 kD) bands in serum samples of dogs that showed clinical improvement after treatment against Leishmania
. In contrast, persistence of 14-kD, 24-kD, and 29-kD bands has been related to persistence of the parasite and a potential unfavorable prognosis.32
Interestingly, two studies have described specific seroreactivity against a band of 26 kD, approximately the molecular mass weight of rTRYP, in untreated dogs in the acute phase of disease and in unsuccessfully treated dogs,31,33
thus suggesting that such seroreactivity can be a marker for active canine leishmaniasis and a potential prognostic marker.
The results reported in the present study suggest that ELISAs based on the insect-derived antigens rKMPII and rTRYP from L. infantum may be useful for assessing clinical improvement after treatment of canine leishmaniasis. Also, dynamics of antibodies against rKMPII could be useful for predicting long-term disease-free survival after one year of the beginning of therapy against this parasitic disease in dogs.