Prognostic Value of Immunoglobulin G (IgG) Patterns by Western Blotting Immunodetection in Treated Dogs Previously Infected with Leishmania infantum

Leishmaniasis is a heterogeneous group of neglected tropical diseases with various clinical syndromes, which is caused by obligate intracellular protozoa of the genus Leishmania and transmitted by the bite of a female phlebotomine sandfly. Humans and several animal species are considered as reservoirs of the disease. Among other animal species, dogs are the most important reservoirs in a domestic environment, maintaining the endemic focus of the parasite. The behavior of the disease progression and the clinical symptoms of the disease in the infected dog is mainly associated with depressed cellular immunity and strong humoral response. This study aimed to assess the role of Western blotting in the analysis of the idiotype expression of the two main immunoglobulins (IgG1 and IgG2) in dogs that are naturally infected with Leishmania infantum (L. infantum) and treated with N-methyl meglumine antimoniate. Interestingly, for the first time, our study identified several L. infantum antigen polypeptides (14, 31, 33, 49, 64, 66, 99, and 169 kDa) that more frequently stimulate an immune reaction in recovered dogs after treatment, whereas in the non-recovered group of dogs, four antigen polypeptides of L. infantum with molecular weights of 31, 49, 66, and 115 kDa with unfavorable prognosis were identified. Clearly, these interesting findings confirm the strong association between the detected immunodominant bands and the successful recovery in treated dogs that can be used for differentiating the treated dogs from the untreated dogs, as well as the markers of a favorable or unfavorable prognosis and, as a consequence, the prediction of the clinical outcome of the disease. Likewise, these data could be helpful in the implementation of novel vaccines from the detected antigens.


Introduction
Leishmaniasis is a group of neglected diseases with a clear endemicity in tropical and subtropical areas, caused by the infection by flagellates' parasites of the genus Leishmania, which occur in all inhabited continents except Australia and Antarctica [1,2]. More than 350 million people in 98 countries around the world are at risk of these diseases, with an annual mortality rate higher than 60,000 people [3]. There are three forms of human leishmaniasis: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis, and fatal visceral leishmaniasis (VL) in the absence of proper treatment [3,4]. The public health and zoonotic importance of leishmaniasis is growing [3,[5][6][7]. In this regard, there is a marked increase in the incidence of human infections (co-infection), especially in immunocompromised cases, as in VL/HIV co-infections cases [5][6][7], and new foci have been reported out of the traditionally endemic areas [6,8]. In accordance with its zoonotic concern, leishmaniasis has two zoonotic forms in Europe: zoonotic CL and zoonotic VL (ZVL) [9]. Importantly, ZVL, caused by L. infantum, is an endemic form of the disease in all countries of the Mediterranean basin, where VL cases in the area account for approximately 5-6% of the global burden [5,[9][10][11]. Among others, Phlebotomus perniciosus (sandfly) acts as the main vector, and dogs are the main reservoirs of infection in this area [6,10,12]. Leishmaniasis in canines is a widespread zoonotic disease that is commonly known as canine leishmaniasis, where the seroprevalence may exceed 40% in certain endemic areas [6,13]. Besides being a common life-threatening zoonotic disease, leishmaniasis in dogs is a very complex pathology that is of particular interest to veterinary practitioners [14].
Hence, a more specific diagnostic tool and an efficient therapy are still needed. Western blotting (WB) analysis is one of the most sensitive and specific techniques that can improve the diagnostic capabilities for canine and human leishmaniasis [23,24]. On the other hand, IgG1 and IgG2 are the two main immunoglobulins that have been related to host Th1/Th2 responses and disease evolution [15,23,[25][26][27][28][29][30][31]. Particularly, IgG1 has been associated with symptomatic infection, whereas IgG2 is more frequently found in asymptomatic dogs [23,26,27,29,30,32]. The present study used WB to analyze the idiotype expression of the total IgG, IgG1, and IgG2 in dogs naturally infected with L. infantum and treated with N-methyl meglumine antimoniate, with the aim of detecting the specific immunoglobulins that can be markers of early infection or early symptomatic phases, as well as prognostic markers. Eventually, the specific antigenic fractions could be used to implement a recombinant antigen used to stimulate an effective antibody response in infected dogs.

Ethical Considerations
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Department of Veterinary Sciences (local ethical approval), the University of Turin, Italy, under ethical approval number 2021/2.

Study Population
A total of 70 dogs naturally infected with L. infantum were included in this study. The dogs, which were of different ages, breeds, and genders, were recruited in Liguria, a highly endemic region of northwestern Italy. The dogs were routinely sampled during veterinary clinic visits and diagnosed with Leishmania by veterinary practitioners on the basis of clinical signs, serological tests (IFA), and molecular tests (PCR). The animals that, following ESCCAP (European Scientific Counsel Companion Animal Parasites) guidelines, met the treatment requirements were treated with N-methyl meglumine antimoniate following the standard protocols. All dogs were periodically checked (following the time schedule suggested by ESCCAP guidelines) for a period of 30 months. Successful therapeutic response was achieved upon the complete remission of the clinical signs and negative IFA results for a period of at least 12 months after antimonial therapy. Dogs for whom the complete remission of clinical signs and hematological parameters was not obtained were considered to have an unfavorable prognosis. Whole blood was collected from each animal through the jugular vein puncture in a clean, sterile tube. The samples were then transported to the Laboratory of Parasitology, Department of Veterinary Sciences, the University of Turin, Italy, where they were centrifuged, and the sera isolated and stored at −80 • C until testing.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Serum samples were screened by WB using L. infantum promastigotes as antigens according to the method described elsewhere [34,35] with slight modifications. Electrophoresis was carried out using the Mini PROTEAN III Electrophoresis System (BioRad Laboratories, Hercules, CA, USA) with 14% gels under denaturing and reducing conditions with 1 mg/mL protein extracting/gel. Fractionated proteins were electroblotted at 350 mA for 1 h onto nitrocellulose sheets (BioRad Laboratories, Hercules, CA, USA). Membranes were blocked with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (TBS) (w/v) for 1 h at a temperature of 37 • C and then incubated for 3 h in a Mini PROTEAN II Multiscreen Apparatus (BioRad Laboratories, Hercules, CA, USA) with dogs' sera diluted in a ratio of 1:10 in TBS.
After washing three times with 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) in TBS (v/v), the secondary Rabbit antibody anti-dog IgG-HRP diluted 1:6000 (Sigma-Aldrich, St. Louis, MO, USA) or Goat anti-dog IgG1-HRP diluted 1:3000 (Bethyl Laboratories, Montgomery, TX, USA) or Sheep anti-dog IgG2-HRP diluted in a ratio of 1:7500 (Bethyl Laboratories, Montgomery, TX, USA) were incubated for 1 h. Nitrocellulose membranes were washed twice, and specific anti-Leishmania antibodies were revealed using Amersham ECL WB detection reagents (GE Healthcare, Chalfont, UK). The resulting bands were compared using as marker Prestained Protein Molecular Weight Marker (Fermentas International Inc., ON, Canada) and Biotinylated SDS PAGE Standards broad range (BioRad Laboratories, Hercules, CA, USA). A subset of 18 samples for which serum was available before and after treatment were tested in parallel in WB to exclude possible influence of N-methyl meglumine antimoniate on polypeptide fractions response to crude antigen.

Statistical Analysis
The Yates test was performed on all data using EpiInfo version 6.

Results
Out of the 70 examined dogs, 25 (35.7%) responded positively to the treatment, reaching complete remission of the clinical signs and the normalization of hematological parameters. For the remaining 45 animals (64.3%), the standard treatment with N-methyl meglumine antimoniate was never allowed to reach complete disease remission. Total IgG, IgG1, and IgG2 in all 70 analyzed sera recognized a total of 27 polypeptide fractions of the L. infantum crude antigen, with molecular weights ranging from 10 to 286 kDa. As shown in Table 1, a significant correlation emerged between eight L. infantum antigens (14,31,33,49,64,66,99, and 169 KDa) and the total IgG in dogs with favorable prognoses. On the other hand, no significant IgG1 or IgG2 patterns could be identified. On the basis of this first set of data, the Yates corrected chi-square test has been repeated considering all eight immunodominant bands together. The results are reported in Table 2. Moreover, the different combinations between the immunodominant bands have been analyzed. We chose four antigens fractions, 31, 33, 49, and 66 kDa, as the most frequently recurrent among the examined dogs. (Table 3).    Although the non-recovered group of dogs had a statistically significant difference in the immune reaction, four antigen polypeptides of L. infantum were identified with molecular weights of 31, 49, 66, and 115 kDa with relative risk values of 3.6, 3.6, 2.6, and 2.5 and odds ratios of 22.67, 6.91, 7.79, and 10.50, respectively (p < 0.001). Out of these four antigen strains, and after 30 months' duration of following up all the dogs, the significant difference persisted only for the 31 and 115 kDa bands with relative risk values of 1.8 and 2.65 (p < 0.001), respectively. Moreover, no significant IgG1 or IgG2 patterns could be identified in this group.

Discussion
The development of novel prognostic patterns for infectious diseases is an important step toward the control of this category of diseases [36][37][38]. The present study reports for the first time eight L. infantum antigen polypeptides (14,31,33,49,64,66,99, and 169 KDa) of valuable prognostic value in dogs previously infected by the parasite and treated with N-methyl meglumine antimoniate. Interestingly, the statistical data elaboration confirmed a strong association between these immunodominant bands and successful recovery in treated dogs. Among the detected antigens, fragments of 31, 49, and 66 kDa were found most frequently. More specifically, a recombinant antigen made by 31-33 kDa would be technically easier to make and still highly associated with a favorable clinical outcome.
Despite the existence of several studies on leishmaniasis, no effective vaccine is currently available for humans or dogs; thus, the control of the disease mainly relies on chemotherapeutic trials that are challenged by toxicity and/or the development of drug resistance [19,39,40]. There is a genuine need for a specific diagnostic test to distinguish between the relapsed dogs and the recovered dogs [38,41]. In fact, an understanding of the immunological mechanisms and the host immune response is very important for studying the design of novel drugs, drug targets, and vaccine preparation [17,31,42,43]. The immunological response to the Leishmania infection seems complex, and differs with the species that caused the infection, the virulence factors of the species, and the differences in the mechanisms of susceptibility and resistance to the infection [17,31,44,45]. Interestingly, the protective immune response to Leishmania infection is primarily cell-mediated, which means that it mainly involves the induction of protective immune responses and the precise migration and localization of the effector cells [16,24,46,47]. The humoral immune response has no central role in parasite clearance [31,48]; however, it has a great role in serological diagnosis, since it is expressed by high titers of IgG and IgM levels, which are highly variable before and after treatment [49][50][51][52][53][54]. Despite the existence of several studies on the relationship between cytokines, T-cell subsets, and immunoglobulin classes and subclasses, and the persistence and multiplication of Leishmania in experimental models and in humans [16,27,28,31,47,55], the exact mechanisms of the control immune response during and after the chemotherapy of the infected animals remain unclear, especially in dogs. Indeed, the mammalian host protection against VL is dependent on the development of Th1-type immunity, which triggers the enhanced leishmanicidal activity by the infected macrophages, whereas a Th2 response is associated with the susceptibility to infection and a strong ineffective humoral response [31,[56][57][58].
As previously mentioned, both IgG1 and IgG2 are the two main immunoglobulins that have been related to host Th1/Th2 responses and disease evolution [15,23,[25][26][27][28][29]. Concerning the potential prognostic value of the IgG isotypes, several studies have demonstrated that IgG1 has been associated with symptomatic infection, whereas IgG2 is more frequently found in asymptomatic dogs infected with Leishmania [23,26,27,29,30,32,54,59]. In this regard, it was reported that immune reactions in the early phases of infection (IgG2) recognize mainly low-weight polypeptides, such as 14, 16, and 18 kDa antigen strains, while the immune reactions against 24 kDa polypeptides characterize the early symptomatic stages of the disease [16,23]. In symptomatic dogs, IgG2 reacts against seven main antigen strains: 26, 29, 34-35, 42, 45, 50-57, 67 kDa; however, the 67 kDa fragment is also the main target for IgG1 [27,60]. The levels of both immunoglobulin and IgG2 are drastically decreased after treatment, especially IgG1 and IgG2 anti-67 kDa [60]. Given the above information, the use of an accurate serological technique to check the alteration in IgG and IgG2 levels during the course of the infection and/or after treatment might be of promising prognostic value [16,30,59]. Several published works have revealed the major role played by many serological tests in the diagnosis of the disease [4,61,62]. Among the other serological assays, WB has been identified as a highly sensitive and specific serological test for the detection of specific anti-Leishmania antibodies in many animals, especially in dogs [34,35,[63][64][65][66]. However, some previous studies on symptomatic dogs have distinguished between the immunodominant and the non-immunodominant protein bands through the WB analysis of antibody response during experimental canine L. infantum infection [60,67]. In the same study of 12 antigenic bands examined, including the immunodominant bands (12,14,24,29,48, and 68 kDa), it revealed significantly increased intensities and higher frequencies of recognition compared to the non-immunodominant bands [67]. Reactivity with the 14, 48, and 68 kDa bands signified early infection, whereas increased reactivity with the 14, 24, and 29 kDa bands was associated with post-treatment parasite persistence and potential unfavorable prognosis, which was consistent with our results [67].
As depicted in Tables 1-3, several antigenic molecules have been detected using WB, which is consistent with the findings of some studies on sera from dogs challenged with wild-type L. infantum with prior vaccination with L. infantum H-line, which concluded on the valuable diagnostic role of various antigenic molecules, i.e., 21, 85, and 110 kDa of L. infantum in dogs infected with VL [63,66,[68][69][70][71]. Another study concluded on the high sensitivity (99.1%) of WB in the detection of several bands between 15 and 118 kDa in two groups of CL patients, especially the 63 kDa band, which was found to be more sensitive [66,68,69]. Based on these findings, we also suggest that the control of infection after successful chemotherapy in dogs involves a particular and specific antibody response and clear statistical correlation with the detected bands and total IgG level (not IgG1 or IgG2), which is consistent with some previous studies that concluded that a measurable antibody response was detected in most cases of CL and VL [24,28,66,72].

Conclusions
Given the above data, these L. infantum antigenic fragments weighting can be considered as favorable prognostic indicators and might play an important role in the modulation of the host's immune response. These findings also suggest that WB analysis of antibodies against those detected antigens represents a specific prognostic test for distinguishing between dogs that recovered from the infection and those that did not recover. In addition to its diagnostic-prognostic value, a valuable future application of our data is a recombinant antigen made out of the eight indicated fragments, which could be very promising in the implementation of novel canine vaccines that can be used later in humans for combating this neglected disease. Further research is suggested to further explore the topic on a large-scale level in other reservoirs and various parasite species.
Author Contributions: E.K.E., S.Z., A.T. and A.T. E.F. conceived the study, designed it, and contributed their scientific advice and supervision besides revising the manuscript. E.K.E., S.Z., M.P. and A.T. contributed to the methodology, formal analysis, and data curation. E.K.E. and S.Z. drafted the manuscript. M.S.L. and K.F.A. contributed their scientific advice, data analysis, prepared the manuscript for publication, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: Not applicable.
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the institutional review board of the Department of Veterinary Sciences (local ethical approval), the University of Turin, Italy, under ethical approval number 2021/2.

Informed Consent Statement:
The data that support the findings of this study is contained within the article.
Data Availability Statement: Blood samples from dogs were collected by licenced veterinarians within routine clinical diagnostic activities and after informed consent of the animals' owner.