0b2cbeffa80d80718139a1952193e56a pathogens-14-00576.pdf 2f26d06c8fbd229a464bfcf74ae869fa6cd787d6 pathogens-14-00576.pdf 15355534358a709a1ef4b340d80e1a5c2f0a879af2b721cd1e65a9573dddacff pathogens-14-00576.pdf Title: Humoral and T-Cell-Mediated Immunity Against Phlebotomus perniciosus Salivary Proteins in Dogs from a Leishmaniosis-Endemic Area Subject: Compounds in sand fly saliva elicit specific immune responses that may play a role in the establishment of canine Leishmania infection. Although canine antibodies to anti-sand fly saliva antigens have been extensively studied, little is known about cellular immune responses against Phlebotomus perniciosus salivary proteins. This study aimed to explore humoral and T-cell-mediated immunity against P. perniciosus salivary proteins in dogs (n = 85) from Mallorca (Spain), a leishmaniosis-endemic area, and find correlations with demographic (age, sex, and breed) and parasite-specific immunological parameters. Anti-sand fly saliva IgG was examined using a P. perniciosus whole salivary gland homogenate (SGH) ELISA and recombinant salivary protein rSP03B ELISA. Interferon gamma (IFN-) release whole blood assays with L. infantum soluble antigen (LSA), SGH, and rSP03B were also performed. Positive correlations were found between IgG levels in the SGH and rSP03B tests and between concentrations of SGH IFN- and rSP03B IFN-. While concentrations of SGH IFN- and rSP03B IFN- were low and produced only by a minority of dogs (less than 20%), high levels and frequencies of LSA IFN- as well as anti-saliva IgG for SGH and rSP03B were detected in a majority of dogs (61% and 75%, respectively). LSA IFN- levels were positively correlated with age and Leishmania-specific antibodies. In conclusion, dogs from a leishmaniosis-endemic area presented high humoral immunity against P. perniciosus salivary proteins, but their cellular immunity to these proteins was low and less frequent. Keywords: anti-saliva antibodies; canine; Leishmania infantum; recombinant salivary proteins; specific P. perniciosus saliva IFN- Author: Núria Balsells-Aguilar, Maria Magdalena Alcover, Marta Baxarias, Alejandra Álvarez-Fernández, Lourdes Alarcón, Petra Sumova, Petr Volf and Laia Solano-Gallego Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Jun 10 12:06:04 2025 CEST ModDate: Tue Jun 10 12:20:31 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 346724 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- RRLNOU+EURM10 Type 1 Builtin yes yes yes 10 0 ICDSLH+VnURWPalladioL Type 1 Custom yes yes yes 15 0 MTJOWC+URWPalladioL-Roma Type 1 Custom yes yes yes 21 0 HWHUJB+URWPalladioL-Bold Type 1 Custom yes yes yes 27 0 OVBKVM+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 UDONDW+URWPalladioL-BoldItal Type 1 Custom yes yes yes 37 0 COGLGI+CMSY10 Type 1 Builtin yes yes yes 69 0 GEWEQC+PazoMath-Italic Type 1 Builtin yes yes yes 74 0 DJCELH+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 88 0 DJCGGH+Cambria-Bold CID TrueType Identity-H yes yes yes 94 0 DJCEMH+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 100 0 DJCENI+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 106 0 AKXRSZ+EURB10 Type 1 Builtin yes yes yes 115 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-18 03:44:39 CEST RepresentationInformation: pathogens-14-00576.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-17 04:37:31 CEST Size: 346724 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 322 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Data of Ibizan Hounds and Dogs of Other Breeds Destination: subsection.2.1 Item: Title: Cytokine Release Whole Blood Assay Destination: subsection.2.2 Item: Title: Sandwich ELISAs to Determine IFN- Concentration Destination: subsection.2.3 Item: Title: ELISA to Determine Anti-L. infantum Antibodies Destination: subsection.2.4 Item: Title: ELISA to Determine Anti-P. perniciosus IgG Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: Agreement Between Tests Destination: subsection.3.1 Item: Title: Spearman’s Rho Correlation and Linear Regression Destination: subsection.3.2 Item: Title: Proportion of Positive Diagnostic Tests and Median Results for All Dogs Studied Destination: subsection.3.3 Item: Title: Relationship Between Demographic Parameters and Parasite-Specific Immunological Parameters, P. perniciosus Salivary Antibodies and IFN- Concentrations Destination: subsection.3.4 Item: Title: SGH IFN- and rSP03B IFN- Destination: subsection.3.5 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Humoral and T-Cell-Mediated Immunity Against Phlebotomus perniciosus Salivary Proteins in Dogs from a Leishmaniosis-Endemic Area Author: Núria Balsells-Aguilar, Maria Magdalena Alcover, Marta Baxarias, Alejandra Álvarez-Fernández, Lourdes Alarcón, Petra Sumova, Petr Volf and Laia Solano-Gallego Subject: Compounds in sand fly saliva elicit specific immune responses that may play a role in the establishment of canine Leishmania infection. Although canine antibodies to anti-sand fly saliva antigens have been extensively studied, little is known about cellular immune responses against Phlebotomus perniciosus salivary proteins. This study aimed to explore humoral and T-cell-mediated immunity against P. perniciosus salivary proteins in dogs (n = 85) from Mallorca (Spain), a leishmaniosis-endemic area, and find correlations with demographic (age, sex, and breed) and parasite-specific immunological parameters. Anti-sand fly saliva IgG was examined using a P. perniciosus whole salivary gland homogenate (SGH) ELISA and recombinant salivary protein rSP03B ELISA. Interferon gamma (IFN-) release whole blood assays with L. infantum soluble antigen (LSA), SGH, and rSP03B were also performed. Positive correlations were found between IgG levels in the SGH and rSP03B tests and between concentrations of SGH IFN- and rSP03B IFN-. While concentrations of SGH IFN- and rSP03B IFN- were low and produced only by a minority of dogs (less than 20%), high levels and frequencies of LSA IFN- as well as anti-saliva IgG for SGH and rSP03B were detected in a majority of dogs (61% and 75%, respectively). LSA IFN- levels were positively correlated with age and Leishmania-specific antibodies. In conclusion, dogs from a leishmaniosis-endemic area presented high humoral immunity against P. perniciosus salivary proteins, but their cellular immunity to these proteins was low and less frequent. Keywords: anti-saliva antibodies; canine; Leishmania infantum; recombinant salivary proteins; specific P. perniciosus saliva IFN- Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue Jun 10 12:06:04 CEST 2025 ModDate: Tue Jun 10 12:20:31 CEST 2025 ID: 0xa88e84a0a6a13ac6fcf443dac478e91c, 0xa88e84a0a6a13ac6fcf443dac478e91c Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OVBKVM+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 150 FontDescriptor: FontName: OVBKVM+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: AKXRSZ+EURB10 FontSubset: true FirstChar: 13 LastChar: 13 FontDescriptor: FontName: AKXRSZ+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true ToUnicode: true Font: BaseFont: UDONDW+URWPalladioL-BoldItal FontSubset: true FirstChar: 45 LastChar: 117 FontDescriptor: FontName: UDONDW+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MTJOWC+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 243 FontDescriptor: FontName: MTJOWC+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: COGLGI+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: COGLGI+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: RRLNOU+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: RRLNOU+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: GEWEQC+PazoMath-Italic FontSubset: true FirstChar: 103 LastChar: 103 FontDescriptor: FontName: GEWEQC+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true ToUnicode: true Font: BaseFont: HWHUJB+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 250 FontDescriptor: FontName: HWHUJB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: ICDSLH+VnURWPalladioL FontSubset: true FirstChar: 133 LastChar: 254 FontDescriptor: FontName: ICDSLH+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 165, 96, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 152, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 294, 643, 305, 656 Annotation: Subtype: Link Rect: 383, 643, 394, 656 Annotation: Subtype: Link Rect: 243, 629, 254, 642 Annotation: Subtype: Link Rect: 381, 629, 392, 642 Annotation: Subtype: Link Rect: 438, 84, 445, 93 ActionDest: 142 Annotation: Subtype: Link Rect: 445, 84, 452, 93 ActionDest: 142 Annotation: Subtype: Link Rect: 399, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 367, 87, 386 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 454, 758, 461, 767 ActionDest: 142 Annotation: Subtype: Link Rect: 439, 745, 446, 754 ActionDest: 142 Annotation: Subtype: Link Rect: 267, 730, 274, 740 ActionDest: 142 Annotation: Subtype: Link Rect: 274, 730, 281, 740 ActionDest: 142 Annotation: Subtype: Link Rect: 430, 675, 437, 684 ActionDest: 142 Annotation: Subtype: Link Rect: 518, 619, 525, 628 ActionDest: 142 Annotation: Subtype: Link Rect: 304, 591, 311, 600 ActionDest: 142 Annotation: Subtype: Link Rect: 298, 549, 305, 559 ActionDest: 142 Annotation: Subtype: Link Rect: 194, 536, 206, 545 ActionDest: 142 Annotation: Subtype: Link Rect: 297, 536, 304, 545 ActionDest: 142 Annotation: Subtype: Link Rect: 273, 494, 285, 503 ActionDest: 142 Annotation: Subtype: Link Rect: 225, 452, 237, 461 ActionDest: 142 Annotation: Subtype: Link Rect: 389, 410, 396, 419 ActionDest: 142 Annotation: Subtype: Link Rect: 450, 382, 457, 391 ActionDest: 142 Annotation: Subtype: Link Rect: 458, 382, 470, 391 ActionDest: 142 Annotation: Subtype: Link Rect: 250, 354, 262, 364 ActionDest: 142 Annotation: Subtype: Link Rect: 262, 355, 274, 364 ActionDest: 142 Annotation: Subtype: Link Rect: 412, 341, 424, 350 ActionDest: 142 Annotation: Subtype: Link Rect: 544, 299, 556, 308 ActionDest: 142 Annotation: Subtype: Link Rect: 351, 271, 363, 280 ActionDest: 142 Annotation: Subtype: Link Rect: 544, 257, 556, 266 ActionDest: 142 Annotation: Subtype: Link Rect: 544, 201, 556, 210 ActionDest: 142 Annotation: Subtype: Link Rect: 443, 174, 450, 182 ActionDest: 142 Annotation: Subtype: Link Rect: 451, 173, 463, 182 ActionDest: 145 Annotation: Subtype: Link Rect: 463, 173, 475, 182 ActionDest: 145 Annotation: Subtype: Link Rect: 489, 159, 501, 169 ActionDest: 145 Annotation: Subtype: Link Rect: 190, 118, 202, 127 ActionDest: 142 Annotation: Subtype: Link Rect: 202, 118, 214, 127 ActionDest: 145 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 294, 730, 305, 740 ActionDest: 142 Annotation: Subtype: Link Rect: 319, 717, 331, 726 ActionDest: 142 Annotation: Subtype: Link Rect: 419, 675, 426, 684 ActionDest: 142 Annotation: Subtype: Link Rect: 408, 490, 415, 499 ActionDest: 142 Annotation: Subtype: Link Rect: 327, 462, 334, 472 ActionDest: 142 Annotation: Subtype: Link Rect: 489, 198, 501, 207 ActionDest: 145 Annotation: Subtype: Link Rect: 465, 184, 477, 193 ActionDest: 142 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 428, 302, 562, 314 Annotation: Subtype: Link Rect: 524, 236, 536, 244 ActionDest: 145 Annotation: Subtype: Link Rect: 441, 181, 453, 190 ActionDest: 145 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 397, 605, 409, 614 ActionDest: 145 Annotation: Subtype: Link Rect: 544, 467, 556, 476 ActionDest: 142 Annotation: Subtype: Link Rect: 433, 453, 444, 462 ActionDest: 145 Annotation: Subtype: Link Rect: 269, 147, 281, 156 ActionDest: 142 Annotation: Subtype: Link Rect: 207, 105, 219, 114 ActionDest: 142 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 165, 673, 284, 686 Annotation: Subtype: Link Rect: 222, 660, 376, 672 Annotation: Subtype: Link Rect: 423, 419, 430, 431 ActionDest: 82 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 549, 542, 556, 554 ActionDest: 112 Annotation: Subtype: Link Rect: 488, 113, 495, 125 ActionDest: 112 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 456, 697, 463, 709 ActionDest: 120 Annotation: Subtype: Link Rect: 482, 697, 489, 709 ActionDest: 120 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 549, 685, 556, 697 ActionDest: 123 Annotation: Subtype: Link Rect: 373, 140, 380, 152 ActionDest: 126 Annotation: Subtype: Link Rect: 364, 84, 371, 96 ActionDest: 126 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 194, 742, 200, 754 ActionDest: 129 Annotation: Subtype: Link Rect: 248, 700, 255, 712 ActionDest: 129 Annotation: Subtype: Link Rect: 549, 658, 556, 670 ActionDest: 129 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 194, 714, 201, 723 ActionDest: 142 Annotation: Subtype: Link Rect: 202, 714, 214, 723 ActionDest: 145 Annotation: Subtype: Link Rect: 449, 686, 456, 695 ActionDest: 142 Annotation: Subtype: Link Rect: 456, 686, 468, 695 ActionDest: 145 Annotation: Subtype: Link Rect: 235, 658, 247, 667 ActionDest: 145 Annotation: Subtype: Link Rect: 545, 644, 556, 653 ActionDest: 145 Annotation: Subtype: Link Rect: 223, 630, 235, 639 ActionDest: 145 Annotation: Subtype: Link Rect: 262, 533, 274, 542 ActionDest: 145 Annotation: Subtype: Link Rect: 435, 505, 447, 514 ActionDest: 145 Annotation: Subtype: Link Rect: 544, 491, 556, 500 ActionDest: 145 Annotation: Subtype: Link Rect: 523, 463, 534, 472 ActionDest: 145 Annotation: Subtype: Link Rect: 201, 407, 213, 416 ActionDest: 145 Annotation: Subtype: Link Rect: 249, 379, 261, 388 ActionDest: 145 Annotation: Subtype: Link Rect: 392, 379, 404, 388 ActionDest: 145 Annotation: Subtype: Link Rect: 354, 240, 366, 249 ActionDest: 145 Annotation: Subtype: Link Rect: 366, 240, 378, 249 ActionDest: 145 Annotation: Subtype: Link Rect: 252, 226, 263, 235 ActionDest: 142 Annotation: Subtype: Link Rect: 374, 171, 381, 180 ActionDest: 142 Annotation: Subtype: Link Rect: 381, 171, 393, 180 ActionDest: 145 Annotation: Subtype: Link Rect: 393, 170, 405, 180 ActionDest: 145 Annotation: Subtype: Link Rect: 405, 143, 417, 152 ActionDest: 145 Annotation: Subtype: Link Rect: 500, 101, 512, 110 ActionDest: 145 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 471, 758, 483, 767 ActionDest: 142 Annotation: Subtype: Link Rect: 519, 717, 531, 726 ActionDest: 142 Annotation: Subtype: Link Rect: 194, 633, 206, 642 ActionDest: 142 Annotation: Subtype: Link Rect: 215, 549, 222, 559 ActionDest: 142 Annotation: Subtype: Link Rect: 223, 549, 235, 559 ActionDest: 145 Annotation: Subtype: Link Rect: 426, 508, 433, 517 ActionDest: 142 Annotation: Subtype: Link Rect: 429, 494, 436, 503 ActionDest: 142 Annotation: Subtype: Link Rect: 340, 466, 352, 475 ActionDest: 145 Annotation: Subtype: Link Rect: 290, 396, 302, 405 ActionDest: 145 Annotation: Subtype: Link Rect: 302, 396, 314, 405 ActionDest: 145 Annotation: Subtype: Link Rect: 475, 271, 487, 280 ActionDest: 145 Annotation: Subtype: Link Rect: 490, 271, 502, 280 ActionDest: 145 Annotation: Subtype: Link Rect: 261, 243, 273, 252 ActionDest: 145 Annotation: Subtype: Link Rect: 276, 243, 288, 252 ActionDest: 145 Annotation: Subtype: Link Rect: 527, 229, 539, 238 ActionDest: 145 Annotation: Subtype: Link Rect: 396, 215, 408, 224 ActionDest: 142 Annotation: Subtype: Link Rect: 544, 187, 556, 196 ActionDest: 145 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 299, 661, 306, 670 ActionDest: 142 Annotation: Subtype: Link Rect: 307, 661, 319, 670 ActionDest: 145 Annotation: Subtype: Link Rect: 439, 591, 451, 600 ActionDest: 142 Annotation: Subtype: Link Rect: 454, 591, 466, 600 ActionDest: 142 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 487, 584, 524, 595 Annotation: Subtype: Link Rect: 112, 547, 150, 557 Annotation: Subtype: Link Rect: 141, 521, 179, 531 Annotation: Subtype: Link Rect: 273, 482, 310, 493 Annotation: Subtype: Link Rect: 113, 444, 151, 455 Annotation: Subtype: Link Rect: 171, 405, 209, 416 Annotation: Subtype: Link Rect: 449, 380, 487, 391 Annotation: Subtype: Link Rect: 95, 355, 132, 365 Annotation: Subtype: Link Rect: 208, 329, 245, 340 Annotation: Subtype: Link Rect: 373, 290, 410, 301 Annotation: Subtype: Link Rect: 425, 265, 463, 276 Annotation: Subtype: Link Rect: 104, 227, 141, 237 Annotation: Subtype: Link Rect: 118, 189, 155, 199 Annotation: Subtype: Link Rect: 283, 163, 320, 174 Annotation: Subtype: Link Rect: 463, 124, 501, 135 Annotation: Subtype: Link Rect: 482, 112, 519, 123 Annotation: Subtype: Link Rect: 238, 86, 276, 97 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 255, 731, 293, 742 Annotation: Subtype: Link Rect: 520, 705, 557, 716 Annotation: Subtype: Link Rect: 424, 654, 461, 665 Annotation: Subtype: Link Rect: 202, 616, 240, 627 Annotation: Subtype: Link Rect: 176, 578, 213, 588 Annotation: Subtype: Link Rect: 135, 540, 173, 550 Annotation: Subtype: Link Rect: 478, 513, 516, 524 Annotation: Subtype: Link Rect: 230, 475, 267, 486 Annotation: Subtype: Link Rect: 333, 437, 370, 448 Annotation: Subtype: Link Rect: 297, 399, 334, 410 Annotation: Subtype: Link Rect: 460, 373, 497, 384 Annotation: Subtype: Link Rect: 104, 336, 142, 346 Annotation: Subtype: Link Rect: 333, 296, 371, 307 Annotation: Subtype: Link Rect: 308, 271, 346, 282 Annotation: Subtype: Link Rect: 352, 271, 388, 282 Annotation: Subtype: Link Rect: 59, 233, 96, 244 Annotation: Subtype: Link Rect: 102, 233, 138, 244 Annotation: Subtype: Link Rect: 501, 207, 538, 218 Annotation: Subtype: Link Rect: 477, 182, 515, 193 Annotation: Subtype: Link Rect: 521, 182, 557, 193 Annotation: Subtype: Link Rect: 238, 143, 276, 154 Annotation: Subtype: Link Rect: 109, 119, 146, 129 Checksum: 6df9d76f Type: CRC32 Checksum: 0b2cbeffa80d80718139a1952193e56a Type: MD5 Checksum: 2f26d06c8fbd229a464bfcf74ae869fa6cd787d6 Type: SHA-1 Checksum: 15355534358a709a1ef4b340d80e1a5c2f0a879af2b721cd1e65a9573dddacff Type: SHA-256