Canine heartworm disease (HWD) is a potentially life-threatening mosquito-borne disease caused by the nematode Dirofilaria immitis, affecting both wild and domestic dogs with worldwide distribution and global significance (1, 2). Heartworms reside in the pulmonary arteries of the canine definitive host, causing proliferative endarteritis which can progress to chronic vascular remodeling, pulmonary hypertension, and right-sided congestive heart failure (1, 3). The pathophysiology of the HWD damage is based on the host’s response to the parasite [i.e., adults and microfilariae (mfs)] and the bacterial endosymbiont Wolbachia pipientis, which resides in all biological stages of D. immitis, triggering the release of proinflammatory and chemotactic cytokines and leading to cellular infiltration and amplification of the inflammatory response (4, 5). The host’s initial immune interaction against the parasite falls under the umbrella of the innate immune response which includes the development of an acute phase response (APR) (6, 7), being a nonspecific and complex reaction that happens shortly after any tissue injury (e.g., trauma, infection, or inflammation) trying to restore the homeostasis and eliminate the underlying cause of the disturbance (8, 9). In this context, an APR characterized by a variation in the acute phase proteins (APP) concentrations such as an increase in C-reactive protein (CRP) not accompanied by increases in haptoglobin as positive APP, and a decrease in albumin and paraoxonase-1 (PON-1) as negative APP have been described in dogs infected by D. immitis (6, 7, 10). Moreover, a CRP increase according to the degrees of the disease and a correlation between positive APP such as CRP and the severity of pulmonary arteriola damage and pulmonary hypertension have been also reported (11–13), indicating inflammatory processes could contribute to the progression of the disease. Therefore, though more research is needed, inflammation biomarkers such as APP seem promising in assessing the status of the heartworm-infected animals, diagnosing pathological aspects of the HWD and/or helping in the establishment of an accurate prognosis (7).

In this scenario, the ESR measurement is a hematology test commonly performed in human medicine to measure the speed at which red blood cells (RBC) settle into a tube of anticoagulated blood in a specific unit of time, most commonly an hour, to highlight the occurrence and extent of inflammation (14, 15). This determination has recently returned to the spotlight in veterinary medicine, being identified as a useful and timely inflammatory biomarker in cases of canine rheumatoid arthritis, osteoarthritis, babesiosis, ehrlichiosis, and feline chronic kidney disease (16–20).

In addition, ESR proved to be a sensitive biomarker for the diagnosis of active forms of canine leishmaniosis by Leishmania infantum (CanL) (21), a sand fly-borne disease that recognizes an APR as part of innate immunity. Similarly to HWD, the inflammatory response that characterizes CanL in active form consists of increases in positive APP, such as CRP, and decreases in negative APP such as albumin, paraoxonase 1, or apolipoprotein 1. Moreover, the endemicity of CanL frequently overlaps with that of HWD (22), leading to the high possibility of co-infections in endemic areas (23).

Hence, this study aims: (i) to evaluate the ESR in dogs naturally infected by D. immitis; (ii) to compare the ESR level in heartworm-infected dogs with a population of heartworm-infected and L. infantum seropositive dogs as well as with a group of clinically healthy dogs; (iii) to assess the existence of a correlation between ESR and other laboratory parameters (e.g., APP) in heartworm naturally infected dogs.

A total of 98 shelter dogs were screened for enrollment. Twenty-nine dogs were excluded being heartworm-negative and not clinically healthy (n = 27), or positive to anti-Anaplasma antibodies (n = 2). Sixty-nine [n = 36 males and n = 33 females; 7.0 (1.3–9.0) years] out of 98 dogs met the inclusion criteria and were consequently enrolled in the study, including 22 animals in the control group, 18 in the Dirofilaria group, and 29 in the Dirofilaria/Leishmania group. Of the last group, 21 out of 29 dogs were affected by a CanL non-active form (i.e., Dirofilaria/Leishmania non-active group) whereas the remaining 8 were diagnosed with an active form of CanL (i.e., Dirofilaria/Leishmania active group).

Erythrocyte sedimentation rate level and the proportion of animals with ESR values higher than 8 mm/h in Dirofilaria, Dirofilaria/Leishmania, and control groups were statistically compared and reported in Table 1. The same comparison including the subclasses of the Dirofilaria/Leishmania group is detailed in Table 2 and Figure 1.

Dirofilaria and Dirofilaria/Leishmania groups presented a significantly higher ESR level (p = 0.0002 and p < 0.0001, respectively) compared to healthy dogs (Table 1). Dirofilaria/Leishmania group did not show a significant difference in ESR median value than the Dirofilaria group (Table 1). However, when the CanL subclasses were considered, heartworm-infected dogs affected by a CanL active form (i.e., Dirofilaria/Leishmania active group) had a significantly higher ESR level compared to all the other groups examined (Table 2). Differently, heartworm-infected dogs affected by a CanL non-active form (i.e., Dirofilaria/Leishmania non-active group) revealed an ESR similar to the Dirofilaria group, but significantly higher and lower compared to the control and the Dirofilaria/Leishmania active group, respectively (Table 2).

The percentage of animals with an ESR level >8 mm/h was significantly higher in Dirofilaria and Dirofilaria/Leishmania groups including the CanL subclasses than in healthy dogs (Table 1), while no other differences among groups have been found (Table 1).

Correlations between ESR and laboratory parameters including inflammatory markers are shown in Table 3. A significant from moderate to strong positive correlation between ESR and CRP has been found in all groups except for the Dirofilaria/Leishmania non-active group. Furthermore, in Dirofilaria/Leishmania group a moderate positive correlation between ESR and globulins (p = 0.008) and beta globulins percentage (p = 0.04) as well as a moderate negative correlation between ESR and HCT (p = 0.03), albumin (p = 0.04), albumin percentage (p = 0.01), and albumin/globulins ratio (p = 0.01) were detected (Table 3). In Dirofilaria/Leishmania active group a strong positive correlation between ESR and gamma globulins percentage (p = 0.02) as well as a strong negative correlation between ESR and albumin (p = 0.01), albumin percentage (p = 0.01), albumin/globulins ratio (p = 0.04) were found (Table 3). In Dirofilaria/Leishmania non-active group, no correlations between ESR and the laboratory parameters considered were present (Table 3).

Values of the main inflammatory markers evaluated (i.e., CRP, HPT, ferritin, albumin, and ESR) in Dirofilaria group, and Dirofilaria/Leishmania group including the two subclasses were statistically compared and reported in Table 4. Among positive APP, CRP level was significantly increased in the Dirofilaria/Leishmania active group where also the number of the dogs with an increased value was statistically higher compared to the other two groups (Table 4). In the Dirofilaria/Leishmania active group, the number of animals with increased ferritin was also significantly higher than in the Dirofilaria/Leishmania non-active group (Table 4). No statistically significant difference was found between the groups for haptoglobin (Table 4). Albumin was significantly decreased in the Dirofilaria/Leishmania active group in which 87.5% of the animals had a significantly reduced value (Table 4).

From a clinical point of view, dogs presented a good state of health with BCS and MCS mean values of 2.7 and 1.3, respectively. Moreover, enrolled dogs were considered asymptomatic for what concerns HWD, while animals in the Dirofilaria/Leishmania group showed a mean CanL clinical score of 1.0 (1.0 in the Dirofilaria/Leishmania non-active group and 1.2 in the Dirofilaria/Leishmania active group).

In the present study, the ESR level has been evaluated in dogs naturally infected by D. immitis and compared to the values obtained from a group of healthy animals and a population of heartworm-infected and L. infantum seropositive dogs subclassified according to the CanL clinical form. Furthermore, the potential correlations between ESR and other inflammatory markers usually implied in clinical practice were also studied.

In the Dirofilaria group, the ESR median value (i.e., 12.5 mm/h ca.) was significantly higher compared to the healthy group (i.e., 7.5 mm/h ca.), confirming the existence of an APR in naturally heartworm-infected dogs which is known to be characterized by an increase in APP circulating in the blood, such as CRP. Indeed, in this group of heartworm-infected dogs, a positive correlation between ESR and CRP was found. However, while the CRP was increased in 38.9% of the dogs in the Dirofilaria group, the ESR level was higher in almost 89% of the cases.

When compared to the Dirofilaria/Leishmania group (ESR = 13 mm/h ca.), heartworm-infected dogs presented a slightly lower and not statistically different ESR value. However, this comparison yields markedly different results when considering the subgroups based on the clinical form of CanL within the Dirofilaria/Leishmania group. Indeed, while dogs in the Dirofilaria group exhibited an ESR value that was not statistically dissimilar from that of the heartworm-infected dogs affected by a CanL non-active form (i.e., 11 mm/h ca.), both these groups of animals showed significantly lower ESR levels compared to heartworm-infected dogs affected by a CanL active form (i.e., 46 mm/h ca.). Likely, these results mirror the clinical condition of the animals enrolled in this study. Considering that heartworm-infected dogs were asymptomatic for what concerns HWD, it has already been reported that the increase in APP such as CRP is more notable according to the severity of the disease, though the inflammatory response is significant from the early stages of the infection probably due to the endosymbiont Wolbachia spp. and the vascular damage (7, 11, 13). Similarly, dogs included in the Dirofilaria/Leishmania non-active group were asymptomatic for HWD and showed a low CanL clinical score.

Heartworm-infected dogs affected by a CanL active form showed the highest ESR median value (i.e., 46 mm/h ca.) among the groups considered in the present study. This finding is not surprising given that recent evidence demonstrated that dogs with a CanL active form had a higher ESR level compared to L. infantum exposed and healthy dogs (21). Additionally, the majority of these dogs in active form (92%) presented an increased ESR (21), as also herein reported in the Dirofilaria/Leishmania active group (100%). While direct comparison of ESR values between these two studies is precluded by the distinct descriptive statistics employed for data presentation (mean vs. median), it is noteworthy in the current investigation that the median ESR value is notably elevated. Then, based on the clinical condition and the type of laboratory alterations detected in the Dirofilaria/Leishmania active group, it is reasonable to suppose that the multisystemic effect of L. infantum in its active form may have had the greatest impact on the APR.

In the Dirofilaria/Leishmania group, the correlation between the ESR level and the other parameters, including markers of inflammation, differs greatly when considering the group as a whole or the subgroups based on the clinical form of CanL. In the Dirofilaria/Leishmania non-active group, ESR does not appear to correlate with any of the parameters. This is similar to the Dirofilaria group where the only correlation identified was with CRP. This finding may indicate the involvement of additional factors contributing to the observed increase in ESR within this group.

In the Dirofilaria/Leishmania active group, a strong and positive correlation between ESR and CRP, which is the main positive APP in dogs, as well as gamma globulins has been found. Increased concentrations of these serum proteins are known to occur in both CanL and HWD, being able to influence the ESR hastening agglutinations of erythrocyte as known from human medicine (38, 39). Additionally, a strong and negative correlation was discovered in the Dirofilaria/Leishmania active group between ESR and albumin, a negative APP whose reduction is expected during both CanL and HWD (7, 31, 40, 41). Albumin is thought to break up rouleaux and slow down red cell aggregation, resulting in a lower ESR and potentially explaining the correlation between clinical hypoalbuminemia and elevated ESR (42). However, this result varies from that obtained in heartworm-negative dogs affected by an active form of CanL, where no such correlation was found (21).

In the context of positive APP, a mention should be made of haptoglobin and ferritin. Although no statistically significant differences were found, it is noteworthy that in all groups herein considered, the number of animals with reduced haptoglobin was greater than those with an increased value (Table 4), despite being a positive APP. This paradoxical result is consistent with previous reports in heartworm-infected dogs (6, 10, 41), but further investigation is needed.

Ferritin has only been evaluated once in dogs with HWD, showing a moderate increase (41). Conversely, in dogs with leishmaniosis, an increase in ferritin is known (43, 44). In the canine population herein considered, no statistically significant differences were found, except for the number of animals with the increased parameter, which was significantly higher in the Dirofilaria/Leishmania active group. Considering that the precise mechanism underlying increases in serum ferritin is not well understood (45, 46) and the limited data available for specific diseases such as HWD, the role of ferritin as APP in heartworm-infected dogs as well as in L. infantum co-infected animals deserves additional research.

This study has a few limits, such as the small number of dogs included in the Dirofilaria/Leishmania active group. However, asymptomatic infection is the most common outcome of exposure to Leishmania parasites (47–49). Furthermore, the population enrolled in this study did not present overt clinical forms of HWD. Therefore, it is reasonable to suppose that the increase in ESR levels may be higher in heartworm-infected dogs with more severe clinical forms (e.g., pulmonary hypertension and caval syndrome), as has been demonstrated for CRP which increases according to the disease severity (11–13). Finally, the measurement of ESR in dogs has currently some limitations. Correction factors based on hematocrit values are lacking, and there is no information on the potential influence of key factors such as age, gender, and reproductive status of dogs on the ESR level. Additional research is required to elucidate these aspects which are known to affect the ESR, as reported in human medicine (50).

In conclusion, this study presents the first evaluation of ESR measured by a point-of-care device in dogs naturally infected by D. immitis and in a population of heartworm-infected and L. infantum seropositive animals. The ESR was confirmed to be an inflammation marker as well as a helpful disease index, being notably increased in heartworm-infected dogs affected by an active form of CanL, as previously reported (21). Further studies will be needed to assess the use of ESR in clinical settings, such as its potential as a follow-up tool for post-treatment monitoring.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal studies were approved by the Ethics Committee of the Department of Veterinary Medicine of the University of Bari (Prot. Uniba 24-2022). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

MAC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Visualization, Writing – original draft. OG: Investigation, Data curation, Writing – review & editing. AU: Investigation, Writing – review & editing. FG: Writing – review & editing. VT: Investigation, Writing – review & editing. RD: Formal analysis, Writing – review & editing. MS: Writing – review & editing. AZ: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.