Antigen-blocking phenomenon in dogs affected by Dirofilaria immitis: frequency, reversion, associated immune response and co-positivity with other vector-borne pathogens

Detection of Dirofilaria immitis antigens is a cornerstone for diagnosis of heartworm disease (HWD). However, no antigen detected (NAD) results may occur secondary to various biological phenomena, including antigen-blocking (AB). Serum heat treatment has been proposed as a strategy to dissociate circulating immune complex (CIC) and reverse AB. This study aimed to: (i) determine the frequency of NAD results; (ii) evaluate the efficacy of heat treatment in reversing NAD results; (iii) investigate potential associations between AB, immune response and seropositivity to other vector-borne pathogens (VBPs). Blood and serum samples were collected from 101 D. immitis-infected dogs from Sardinia (n = 73) and Sicily (n = 28), as well as from 7 healthy dogs serving as controls. Dirofilaria immitis microfilariae and antigens were detected using Knott's test and IDEXX SNAP Leish 4Dx®, respectively. Seropositivity with Anaplasma spp., Ehrlichia spp. and Leishmania infantum, was evaluated simultaneously. Dirofilaria immitis antigen-negative serum samples underwent heat treatment. Serum concentrations of interferon gamma (IFN-γ), interleukin 12 (IL-12), and interleukin 4 (IL-4) were quantified via enzyme-linked immunosorbent assays (ELISA) across three groups: antigen-blocked dogs (AB, n = 8), SNAP-positive/Knott-positive dogs (DC, n = 20), and healthy non-infected dogs (C, n = 7). Initial antigen negativity was observed in 11 out of 101 samples (10.9%), with heat treatment successfully reversing NAD in 8 of the 11 cases (72.7%) yielding an overall confirmed AB frequency of 7.9% (8/101). Interleukin analysis revealed significantly higher IL-4 levels in the AB group [median 53.71 pg/mL (Q1–Q3: 35.94–71.00); P < 0.001] and significantly higher IL-12 levels in the DC group (49.85 ± 14.56 pg/mL; P = 0.001). Overall VBPs seropositivity was significantly higher in the AB group (4/8, 50.0%) than in the DC group (2/20, 10.0%; P = 0.0384). Specifically, L. infantum seropositivity was detected in 37.5% (3/8) of AB dogs compared to 5.0% (1/20) of DC dogs. Antigen blocking emerged as a relevant cause of NAD results in canine HWD, occurring in approximately 8% of infected dogs in this study. Heat treatment successfully restored antigen detection in most NAD samples. The association between antigen blocking and increased IL-4 concentrations, potentially amplified by co-positivity with other VBPs, suggests that a Th2-biased immune response may contribute to CIC formation and subsequent antigen masking.