Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

Introduction dogs were sent by the Municipal Health Department of Barra Mansa to be culled at the Evandro Chagas National Institute of Infectious Diseases (INI-Fiocruz). Culling was performed according to the recommendations of the Brazilian Ministry of Health for the control of VL [2] and the owners provided signed consent for culling. The dogs were not housed for any period of time prior to culling. The sample size included all dogs of the study population, i.e., all dogs that tested seropositive for L. infantum in the town of Barra Mansa and that were sent to INI-Fiocruz during the study period.

Sample collection
Immediately after arrival at INI-Fiocruz, the dogs were restrained mechanically and submitted to clinical evaluation, including inspection of behavior, alertness, posture, gait, skin, and oral and ocular mucosae, as well as palpation of the superficial lymph nodes and organs. The following clinical signs of canine leishmaniosis caused by L. infantum (CanL) were considered: thinness or cachexia; diffuse or localized alopecia; apathy; cutaneous lesions such as ulcers and desquamation; onychogryphosis; enlargement of the superficial lymph nodes, liver or spleen on palpation; keratoconjunctivitis; pale ocular or oral mucosae, and skeletal muscle atrophy [19,20]. The animals were divided into three groups according to the clinical signs of CanL: no clinical signs, few clinical signs (up to three clinical signs), and multiple clinical signs (more than three clinical signs) [21].
After clinical examination, the animals were sedated by intramuscular administration of ketamine hydrochloride (10 mg/kg) plus acepromazine maleate (0.2 mg/kg). Bone marrow was then aspirated from the sternal manubrium. The bone marrow samples were collected into sterile tubes containing EDTA and stored at -20˚C for further analysis by conventional PCR and nested PCR for the detection of Coxiella burnetii DNA. Blood (1 to 3 mL) was collected from the cephalic vein into a sterile vacuum tube without anticoagulant. After coagulation, the blood samples were centrifuged at 1125 x g and the serum obtained was separated and stored at -20˚C until the time of analysis. The serum was used for the detection of antibodies against A. platys/A. phagocytophilum, B. burgdorferi, E. canis/E. ewingii and T. gondii and for the investigation of D. immitis antigens. Next, the dogs were culled with an intravenous overdose of sodium thiopental and potassium chloride in accordance with the guidelines of the Federal Council of Veterinary Medicine of Brazil [22], and immediately necropsied.
During necropsy, the organs were examined macroscopically and samples of skin, spleen, liver, lung, heart valves (tricuspid and mitral), uterus, and mammary gland were collected. These tissue samples were fixed in 10% buffered formalin and embedded in paraffin (FFPE) [23] for immunohistochemistry (IHC) (detection of amastigote forms of Leishmania spp.) and histopathology. In addition, FFPE samples of the spleen, lungs and mammary glands obtained from T. gondii-seropositive dogs in which an inflammatory infiltrate was identified by histopathology were submitted to IHC for the detection of cysts or tachyzoites of this parasite. A second spleen fragment was collected and stored at -20˚C in a sterile RNAse-and DNAse-free tube for subsequent detection of L. infantum DNA by singleplex qPCR. For Leishmania detection by parasitological culture, samples of skin, spleen, popliteal lymph node and bone marrow were collected aseptically and immersed in sterile saline.

Histopathology and immunohistochemistry for detection of Leishmania and T. gondii
For histopathology, the tissues were stained with hematoxylin-eosin (HE) [23]. The inflammatory infiltrate in the tissues was classified as follows: granulomatous, predominance of cells of the monocyte-macrophage system (activated macrophages, epithelioid macrophages, or multinucleate giant cells); pyogranulomatous, predominance of cells of the monocyte-macrophage system amidst a high number of neutrophils; non-granulomatous, predominance of other types of inflammatory cells (lymphocytes, plasma cells, and neutrophils). In addition, inflammatory cells were quantified using a 1-mm 2 optical grid and a manual cell counter under a light microscope. The cells were counted in one field of the HE-stained histological sections at 400× magnification, in the most cellular area of the lesion.
For IHC aimed at detecting amastigote forms of Leishmania spp., the tissues were submitted to the steps of deparaffinization, rehydration, blocking of endogenous peroxidase, antigen retrieval, blockade of nonspecific protein binding, and incubation with polyclonal rabbit anti-Leishmania serum diluted 1:500, according to the protocol of Boechat et al. [24]. The polymerbased HiDef Detection HRP™ Polymer System (Cell Marque, Rocklin, CA, USA) was used for the detection of amastigote forms of Leishmania according to manufacturer recommendations. Histological sections of organs intensely parasitized with amastigote forms of Leishmania were incubated with non-immune homologous serum as negative control and with polyclonal rabbit anti-Leishmania serum as positive control.
For the evaluation of skin parasite load by IHC, Leishmania amastigote forms were quantified as described for the quantification of inflammatory cells. However, the parasites were counted in five fields at 400× magnification in the most parasitized areas. The average number of Leishmania amastigote forms was calculated.
For IHC aimed at detecting T. gondii cysts and tachyzoites, the tissues (lung, mammary glands, and spleen) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked by incubating the histological sections in a solution of 30% hydrogen peroxide and methanol (45 ml of hydrogen peroxide and 55 ml of methanol) for 40 min at room temperature. Antigen retrieval was performed by incubating the histological sections in Declere ™ buffer (Cell Marque, Rocklin, CA, USA) at 110˚C in a Decloaking Chamber™ (Biocare Medical, Pacheco, CA, USA). For the blockade of nonspecific protein binding, the histological sections were incubated with Background Block protein blocking solution (Cell Marque, Rocklin, CA, USA) for 10 min at room temperature. The sections were then incubated with polyclonal anti-T. gondii antibody (rabbit) (Cell Marque, Rocklin, CA, USA) diluted 1:100 for 1 hour at room temperature. The HiDef Detection HRP™ Polymer System (Cell Marque, Rocklin, CA, USA) was used for the detection of T. gondii according to manufacturer recommendations. The enzyme-substrate-chromogen reaction was developed using diaminobenzidine (DAB) plus hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA). The sections were counterstained with modified Mayer's hematoxylin (Thermo Scientific, Fremont, CA, USA) for 2 min. Histological sections of tissues intensely parasitized with parasite forms of T. gondii were incubated with non-immune homologous serum as negative control and with primary polyclonal anti-T. gondii antibody (rabbit) as positive control.

Parasitological culture and identification of Leishmania spp.
The fragments were cultured at 26-28˚C in Novy-MacNeal-Nicolle medium plus Schneider's Drosophila medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and penicillin and streptomycin as antibiotics [25]. The detailed protocol of parasite isolation in culture is registered at https://dx.doi.org/10.17504/protocols.io.22tggen. Parasites isolated in culture were identified as L. infantum by multilocus enzyme electrophoresis [26].
For the quantification of parasite load, a standard curve was constructed with serial dilutions (10 1 to 10 5 parasites) of L. infantum DNA (MHOM/BR/1974/PP75). Positive and negative controls were included in each amplification plate and a threshold of 0.1 was established. The DNA of 1 × 10 5 promastigote forms of L. infantum obtained by parasitological culture was used as positive control and ultrapure water as negative control. Samples in which DNA amplification occurred after the 37th cycle were classified as undetectable. The L. infantum load was expressed as the natural logarithm of the number of parasite genome equivalents (gEq)/ng of DNA.

Conventional PCR for the detection of C. burnetii DNA
Forty-five bone marrow samples were submitted to conventional PCR as previously described [27]. The QBT-1 (5'TATGTATCCACCGTAGCCAGT C-3') and QBT-2 (5'-CCCAACAACA CCTCCTTATTC-3') primers were used for the amplification of a 687-bp fragment. The reaction mixture contained 0.2 μM of each primer (Invitrogen, Life Technologies, São Paulo, Brazil), 200 μM of dNTP (20 mM of each deoxynucleotide triphosphate), 1.5 mM MgCl 2 , 0.5 U Platinum 1 Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 4 μL DNA, and nucleasefree water (Promega, Madison, WI, USA) in a final volume of 25 μL. The cycling conditions were described previously [28]. To confirm the amplification, the generated products were separated on an agarose gel stained with 10 μL of Gel RED solution (10,000X) per 100 μL of agarose gel, visualized, and recorded with a photodocumentation system. The target products were then purified using the BigDye Terminator 1 X-Purification kit (Applied Biosystems, Foster City, CA, USA). The products obtained were sequenced using the BigDye 1 Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and the ABI PRISM 1 3100 software (Applied Biosystems, Foster City, CA, USA). Partial sequences were compared with the htpAB gene from BLAST 1 .

Nested PCR for the detection of C. burnetii DNA
To increase the sensitivity and specificity of PCR for the detection of C. burnetii DNA, all samples were submitted to a second reaction ("nested") using the QBT N3+ (5'-AAG CGT GTG GAG GAG CGA ACC-3') and QBT N4-(5'-CTC GTA ATC ACC AAT CGC TTC GTC-3') primer pair [29]. The positive controls used in PCR were extracted from cultures of cells infected with C. burnetii. A volume of the DNA solution of 4 μL was used in the conventional PCR assay and of 2 μL in the nested PCR assay.
For amplification, the reaction contained 1X PCR buffer 10 X, 0. Veriti 96) using the previously described cycling conditions [29]. To confirm the amplification, the generated products were separated on a 1% agarose gel stained with 10 μL of RED Gel solution (10,000X) per 100 μL of agarose gel. The target products were purified, sequenced, and compared as described in the previous item for conventional PCR. Serological detection of antibodies to Anaplasma spp., B. burgdorferi and Ehrlichia spp., and of D. immitis antigens The ELISA 4 Dx 1 Plus test (IDEXX 1 , Westbrook, ME, USA) was used for the serological detection of antibodies to A. platys/A. phagocytophilum, B. burgdorferi and E. canis/E. ewingii, and of D. immitis antigens following the recommendations of the manufacturer. According to the manufacturer, this test has the following sensitivities and specificities, respectively: 90.3% and 94.3% for Anaplasma spp., 94.1% and 96.2% for B. burgdorferi, 99.0% and 99.3% for D. immitis, and 97.1% and 95.3% for Ehrlichia spp.

Detection of anti-T. gondii antibodies in serum
The indirect immunofluorescence antibody test (IFAT) was used as described previously [30]. Anti-dog IgG (whole molecule) FITC antibody produced in rabbits was used as conjugate (Sigma-Aldrich Co., MO, USA). The sera were first diluted 1:16 and then at a ratio of four until 1:256. Samples with a titer of 1:16 or higher were considered positive. Previously known positive and negative controls were included in each analysis. IFAT is considered to have good specificity and sensitivity and is recommended for the immunodiagnosis of T. gondii infection in dogs, where it can be used as the only test [31].

Statistical analysis
Data were analyzed using the free R software, version 3.5.1 [32]. Clinical signs, positivity in the diagnostic tests, and histological changes are described as simple frequencies. For the description of continuous variables (number of inflammatory cells and parasite load), the median (50th percentile) was calculated and the minimum and maximum values are reported. For inflammatory cells, the 75th percentile was also calculated.
The normality of the quantitative variables (parasite load and inflammatory cells) was rejected by the Shapiro-Wilk test at a significance level of 5%, which indicated the use of nonparametric tests for the analysis of these variables. The Mann-Whitney test and Fisher's exact test were used for comparison of the quantitative and qualitative variables, respectively, between the monoinfected and co-seropositive groups. Boxplots were constructed for the graphical presentation of the comparison of parasite load. The median parasite load in the spleen was expressed as the natural logarithm of the parasite genome equivalents/nanogram of DNA (gEq/ng). A p-value < 0.05 indicates statistical significance.

Ethics statement
This study was carried out in strict accordance with the recommendations of the Brazilian Ministry of Health and the Federal Council on Veterinary Medicine, with permission of the owners. The study protocol was approved by the Ethics Committee on Animal Use of the Oswaldo Cruz Foundation (CEUA/Fiocruz; Permit Numbers: LW-54/13 and LW-24/17). The dogs were sedated by intramuscular administration of ketamine hydrochloride and acepromazine maleate and culled with an intravenous overdose of sodium thiopental and potassium chloride. All efforts were made to minimize suffering.

Results
All 66 L. infantum-seropositive dogs investigated in this study had infection with L. infantum confirmed by qPCR and/or culture, as described below. Among these 66 dogs, 50 (76%) had antibodies against at least one pathogen other than L. infantum (co-seropositive group). The remaining 16 dogs (24%) did not have antibodies against certain pathogens other than L. infantum and were thus considered monoinfected with L. infantum (monoinfected group). The following frequencies of co-seropositivity for certain pathogens were observed in the 66 dogs investigated: T. gondii (59%), Ehrlichia spp. (41%), and Anaplasma spp. (18%). None of the animals was positive for B. burgdorferi, C. burnetii, or D. immitis. Among the 50 co-seropositive dogs, 19 (38%) were co-seropositive only for T. gondii, 12 (24%) for T. gondii and Ehrlichia spp., 7 (14%) only for Ehrlichia spp., 5 (10%) for Ehrlichia spp., Anaplasma spp. and T. gondii, 3 (6%) for Ehrlichia spp. and Anaplasma spp., 3 (6%) for Anaplasma spp. and T. gondii, and 1 (2%) only for Anaplasma spp. Statistical analysis of the association of breed with the monoinfected and co-seropositive groups was not possible because of the small number of breed dogs.
Clinical signs compatible with CanL were present in 60 (91%) of the 66 dogs evaluated and absent in six (9%). Clinical signs were observed in 15 (94%) of the 16 dogs monoinfected with L. infantum and in 45 (90%) of the 50 co-seropositive dogs. When the frequency of clinical signs of CanL was compared between monoinfected and co-seropositive dogs, no significant difference was observed (p = 0.407). Table 1 shows the frequency of each clinical sign observed in monoinfected and co-seropositive dogs and no significant difference was observed between the two groups.
Histopathology revealed changes in at least one of the organs studied in 15 (93.8%) of the 16 monoinfected dogs. All 50 (100%) co-seropositive dogs exhibited some type of histological alteration in the examined tissues. Table 2 shows the frequencies of the main histological changes observed in monoinfected and co-seropositive dogs.
The number of inflammatory cells observed in each organ of monoinfected and co-seropositive animals is shown in Table 3. The number of inflammatory cells/mm 2 observed in each organ according to the co-seropositivity for certain pathogens is given in the S1 Table. Immunohistochemistry revealed the presence of amastigote forms of Leishmania spp. in all types of organs studied. In addition, amastigote forms were detected in at least one of the organs in 12 (75%) monoinfected dogs and in 39 (78%) co-seropositive dogs. There was no difference in the positivity for amastigote forms of Leishmania spp. between monoinfected and co-seropositive dogs in any of the organs studied ( Table 4).
The histological changes and amastigote forms detected by IHC in monoinfected and coseropositive dogs are shown in Fig 2A-2H.
Among the 39 T. gondii-seropositive dogs, an inflammatory infiltrate was observed in 33 spleens, 26 lungs, and 14 mammary glands. These samples were processed for IHC to detect T. gondii cysts or tachyzoites. One spleen sample and two lung samples were lost during processing. None of the samples examined by IHC were positive for this parasite.

Discussion
A high frequency of co-seropositivity for certain pathogens in L. infantum-seropositive dogs with infection with this parasite confirmed by PCR and/or culture was found, especially coseropositivity for T. gondii. Considering the breed, the results of this study can only be extrapolated to other similar populations composed mainly of mongrel dogs (85%). A high frequency of anti-T. gondii (62.9%) antibodies was also detected in dogs from a CanL-endemic area in the state of São Paulo, Brazil [33]. However, in studies conducted in areas not endemic for CanL in Brazil, the authors found frequencies of anti-Leishmania and anti-T. gondii antibodies of 11.6% in dogs from Londrina, state of Paraná [34], and of 10.2% in dogs from the central region of the state of Rio Grande do Sul [35]. These percentages are lower than those observed in the present study. Paulan et al. [33] found a significant association between reactivity to anti-L. infantum and anti-T. gondii antibodies in dogs. The authors suggested that the immunosuppression caused by Leishmania may increase the susceptibility of these animals with CanL to this coccidium. Additionally, Zulpo et al. [34] observed a higher frequency of anti-Leishmania antibodies in dogs seropositive for T. gondii and Neospora. However, in the present study, it was not possible to assess whether dogs with L. infantum are at increased risk of infection with T. gondii or vice-versa because of the lack of a control group of dogs not infected with L. infantum. The presence of L. infantum may have not increased the susceptibility to T. gondii infection in this study, as observed in cats [36,37], since a high frequency of anti-T. gondii antibodies has been reported in pigs (65.8%) and chickens (47.8%) in the same area [38]. These results suggest high environmental contamination with T. gondii in Barra Mansa, which may explain its high frequency also in dogs.
In the present study, the frequency of L. infantum and Ehrlichia spp. co-seropositivity was high (41%) and higher than those reported in studies carried out on dogs from Cyprus (36.2%) PLOS ONE [39], Côte d'Ivoire (4.9%) [12], and Portugal (2.2%) [40]. This frequency was also higher than those found in dogs from Campo Grande, Mato Grosso do Sul (31.7%) [41], and Cuiabá, Mato Grosso (2.5%) [42], Brazil. On the other hand, higher frequencies of L. infantum and Ehrlichia spp. co-seropositivity or co-infection than that observed here were reported in Ilha Solteira, São Paulo, Brazil (74.3%) [33], and Catalonia, Spain (55.7%) [7]. Furthermore, the frequency of L. infantum and Anaplasma spp. co-seropositivity in the present study was higher than that reported in dogs from Cyprus (10.6%) [39] and Portugal (1.1%) [40], but lower than the 52.5%  [10]. Taken together, the results of these surveys and the present findings demonstrate that co-infection with or co-seropositivity for Ehrlichia spp. and Anaplasma spp. is common among dogs infected with L. infantum in different parts of the world. The frequency of co-infection with or co-seropositivity for Ehrlichia spp. and Anaplasma spp. can be quite high in regions with favorable environmental conditions for ticks, which are Table 3   the vectors of these pathogens [43]. According to Figueredo et al. [43], the presence of ticks and the occurrence of pathogens transmitted by these vectors tend to be more common among dogs in regions where the socioeconomic status of the population is low and dogs are not in close contact with their owners (kept in the backyard or semi-restricted). In the city of Barra Mansa, seropositivity of dogs for the proteobacteria Rickettsia rickettsii and/or R. parkeri, which are also transmitted by ticks, has been demonstrated, with a frequency of 19.2% [44]. In addition, anti-R. rickettsii antibodies and anti-L. infantum antibodies were simultaneously detected in 7.7% of the examined dogs [45]. These studies and the present results suggest that dogs from Barra Mansa are frequently exposed to ticks and to the agents transmitted by these arthropods. The investigation of these co-infections or co-seropositivities is important since dogs with CanL can exhibit clinical signs similar to those caused by tick-borne diseases, a fact that makes the diagnosis of L. infantum difficult [39,46]. Consequently, the dog remains in the environment for a longer period and thus serves as a source of L. infantum infection for sandflies.

. Number of inflammatory cells/mm 2 observed in tissues of
In the present study, the absence of co-seropositivity for B. burgdorferi, C. burnetii or D. immitis in L. infantum-seropositive and infected dogs suggests that the circulation of these pathogens is low or does not occur in the area studied. Additional tests such as PCR for the detection of D. immitis and B. burgdorferi may be useful to identify possible infected and nonseropositive dogs. The absence of C. burnetii-positive dogs may be explained by the lack of proximity of the dogs studied with infected sheep and goats, which act as important sources of co-seropositive for T. gondii. Note the large numbers of lymphocytes and plasma cells and few neutrophils. HE. (H) Multifocal granulomatous endocarditis and myocarditis consisting mainly of macrophages in a dog co-seropositive for T. gondii. Note the large numbers of lymphocytes and few plasma cells. HE.
https://doi.org/10.1371/journal.pone.0247560.g002 contamination with this bacterium [28]. However, co-infection with D. immitis and L. infantum has been reported in dogs in Brazil [47,48]. In addition, studies on L. infantum-seropositive dogs conducted in Europe demonstrated co-infection with or co-seropositivity for D. immitis and B. burgdorferi, but the prevalence was low [10,14,39,40,[49][50][51][52]. In these surveys, the reported prevalence of D. immitis and L. infantum co-infection or co-seropositivity in dogs was 1.2% to 8.3% in Portugal [40,49], 4.3% in Cyprus [39], and 0.16% to 0.6% in Greece [11,52]. Additionally, clinical cases of this co-infection in three dogs were reported in Italy [50]. Twenty-nine out of 98 (29.6%) dogs were co-infected with L. infantum and filariae, including D. immitis, in Spain [14]. Co-seropositivity of dogs for L. infantum and B. burgdorferi was observed in 14 of 2,620 dogs (0.5%) in Greece [11] and in 1 of 1,185 dogs (0.08%) in Portugal [40]. However, this co-seropositivity or co-infection was not found in Cyprus [39] or Côte d'Ivoire [12], in agreement with the present study. Although C. burnetii, D. immitis and B. burgdorferi were not found in the area studied, the detection of C. burnetii in two dogs [28] and a prevalence of D. immitis of 16.3 to 62.2% [53] and of B. burgdorferi of 1.4 to 41.9% [54] have been reported in other cities in the state of Rio de Janeiro. Further studies on the occurrence of co-infection with these three pathogens in L. infantum-seropositive dogs are necessary not only because they are zoonoses, but also because co-infections with filariae have been shown to increase the severity of clinical signs of CanL [14].
In the study of Toepp et al. [10], the risk of L. infantum seropositivity was 1.68 times higher among dogs seropositive for agents transmitted by ticks, such as Ehrlichia spp. and Anaplasma spp., than among dogs that were not co-infected with these agents. In addition, in a study carried out in the Campania region in Italy, the presence of antibodies against Neospora caninum was the main risk factor for L. infantum seropositivity, while the presence of antibodies against L. infantum was the main risk factor for N. caninum seropositivity [4]. Investigating the occurrence of co-infections in dogs living in VL-endemic areas is important since Leishmania infection can trigger an ineffective immune response that renders dogs more susceptible to other infections, while co-infections can increase the dog's susceptibility to Leishmania [4,7,10]. These hypotheses are reinforced by the high frequency of co-seropositivities among dogs seropositive for and/or infected with L. infantum found in this study and in other studies [4,7,10].
Toepp et al. [10] also observed a significant association between exposure to agents transmitted by ticks, such as Ehrlichia spp. and Anaplasma spp., and the progression of clinical signs of CanL and a higher mortality rate in these dogs. These authors believe that co-infections alter the immunity of dogs, allowing L. infantum infection to thrive within the phagocytes leading to clinical disease. In another study [7], the frequency of anti-A. phagocytophilum antibodies was significantly higher in dogs with CanL than in healthy dogs; in addition, these antibodies were more frequent in dogs with more severe clinicopathological changes and with more severe CanL. A study using histopathology found that the percentage of dogs with L. infantum amastigote forms on the skin was almost double (36%) in the group of dogs coinfected with L. infantum and E. canis when compared to the group of L. infantum-monoinfected dogs (19%) [55]. The authors attributed this result to the fact that E. canis caused a reduction in the class II histocompatibility complex, leading to depression of the immune system and favoring the multiplication of L. infantum. An experimental study on mice showed that Ehrlichia spp. is able to reduce autophagy in infected macrophages, allowing the growth and replication of bacteria and, consequently, of other intracellular agents such as Leishmania [56]. However, in the present study, no significant association was found with clinical signs, frequency of positivity for amastigote forms of Leishmania spp. in different tissues or L. infantum load in co-seropositive dogs when compared to monoinfected dogs. This divergence to the literature may be due to the small number of monoinfected animals in the present study, which may have influenced the statistical analysis. Another explanation is the fact that we did not perform a longitudinal analysis, unlike Toepp et al. [10] who evaluated dogs for the progression of clinical signs and death over a 10-month period. Evaluation of the dogs for the same or a longer period of time than that used by Toepp et al. [10] might have resulted in the observation of worsening of the clinical status and a higher L. infantum load in co-seropositive dogs.
Another hypothesis for the lack of aggravation of CanL due to co-seropositivity for certain pathogens in the present study is that the seropositive dogs may not be co-infected or infection with these pathogens is not active, but they were previously exposed to the pathogens and exhibited detectable memory antibodies. Anti-E. canis antibodies can persist in the circulation for a long time even after elimination of the agent [57]. In contrast, very recent infections may have also contributed to false-negative results of the serological tests since most of them detect IgG antibodies that are produced later [57]. As a result, dogs classified as monoinfected with L. infantum may be infected with the other pathogens investigated but did not have enough time to produce late-phase antibodies against these pathogens.
The possibility of a serological cross-reaction of other pathogens with L. infantum must also be taken into account. Other Leishmania species [58], E. canis, Babesia canis, N. caninum, T. gondii [59], Leptospira interrogans [58], Trypanosoma cruzi [59], and T. caninum [60] can cross-react, thus causing false-positive results in serological L. infantum tests. However, previous studies reported that cross-reactivity with L. infantum in serological tests for B. canis and E. canis is rare [61,62] and the ELISA 4 Dx 1 Plus test has high sensitivity and specificity. Additionally, in the present study, about 90% of the dogs studied tested positive for Leishmania by culture and/or IHC, and all of them tested positive for L. infantum by qPCR. Therefore, cross-reactivity with antibodies of other agents is unlikely.
The possibility that the L. infantum-monoinfected dogs were actually co-infected with other pathogens not investigated here, such as Babesia spp. and helminths, cannot be ruled out. This fact may have contributed to the similarities in clinical signs, L. infantum positivity and parasite load between monoinfected and co-seropositive dogs. However, the present results do not rule out the possibility that co-infection with L. infantum and other pathogens may exert immunomodulatory activity, preventing a synergistic effect on the aggravation of diseases caused by these agents. Therefore, further studies are needed to better understand the role of co-infections in modulating the immune system of dogs.
Although the number of clinical signs or parasite load was not higher in co-seropositive dogs, a larger number of inflammatory cells was observed in the spleen, lung and mammary gland of these dogs. Studies have associated the inflammatory reaction observed in these organs with L. infantum infection in dogs [24,63]. However, this inflammatory reaction is not a specific histological alteration and it was not possible to correlate its presence with L. infantum parasitism in all cases. Nevertheless, the participation of L. infantum in these histological changes cannot be ruled based on the lack of detection of amastigote forms of this parasite since these forms are heterogeneously distributed and may not be visualized depending on the histological section examined, even if they are present in the tissue. In addition, even in the absence of amastigote forms, the inflammatory reaction can be triggered by peripheral stimuli such as parasite antigens, DNA or inflammatory mediators, as well as by the deposition of immune complexes in tissues [64,65]. In a previous study [6], the intensity of the inflammatory infiltrate was higher in the central nervous system of dogs co-infected with T. gondii and E. canis when compared to dogs infected only with L. infantum. Similarly, co-infections may have contributed to aggravate the intensity of the inflammatory infiltrate in the spleen, lung and mammary gland of the co-seropositive dogs studied here. However, the inflammatory infiltrate in these organs was not associated with the detection of T. gondii by IHC. This result suggests that T. gondii infection was latent in these animals, which is commonly seen in dogs [66] and was not reactivated by co-infection with L. infantum. On the other hand, co-infection with Ehrlichia spp. and Anaplasma spp. may have contributed to a more intense inflammatory infiltrate in the spleen and lung of the co-seropositive dogs in this study. This can be explained by the fact that a perivascular inflammatory infiltrate consisting of macrophages and lymphocytes is the main histological alteration observed in the lungs and spleen of dogs infected with E. canis and Anaplasma spp. [67,68]. These dogs may have been in an early stage of the histological changes associated with L. infantum co-infections and the time was not sufficient to develop apparent clinical signs related to the affected organ(s). This hypothesis could explain why co-seropositivity for certain pathogens did not exacerbate the intensity of clinical signs in the dogs studied.

Conclusions
The present results suggest that co-seropositivities for Anaplasma spp., Ehrlichia spp. and T. gondii are common among dogs infected with L. infantum in the region studied. However, these co-seropositivities did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.