b50ff3c47997229021ff6bf2fd0dd7c3 ijms-26-04918-v2.pdf 276c657963c6739b873c024a8492eaa82fa4791a ijms-26-04918-v2.pdf 27e31ebd66ce545f87299ed88356d3b2527711a2f4bdb482ba546e04b5e56d6b ijms-26-04918-v2.pdf Title: Deciphering the Causes of IbfA-Mediated Abortive Infection in the P22-like Phage UAB_Phi20 Subject: The study of bacterial defense mechanisms against phages is becoming increasingly relevant due to their impact on the effectiveness of phage therapy. Employing a multifaceted approach that combines bioinformatics, molecular microbiology, TEM microscopy, and conventional microbiology techniques, here, we identify the ibfA gene as a novel defense factor targeting the virulent phage UAB_Phi20, acquired by Salmonella Typhimurium through lateral transfer on the IncI1 conjugative plasmid pUA1135 after oral phage therapy in broilers. IbfA, a two-domain protein containing ATPase and TOPRIM domains, significantly reduces UAB_Phi20 productivity, as indicated by decreased EOP, ECOI, and a diminished burst size, potentially reducing cellular viability without causing observable lysis. Our results indicate that IbfA enhances the transcription of early genes, including the antirepressor ant, which inhibits the C2 repressor of the lytic cycle. This may cause an imbalance in Cro/C2 concentration, leading to the observed reduction in the transcription of late genes encoding structural and cellular lysis proteins, and resulting in the abortion of UAB_Phi20 infection. Keywords: Salmonella; phage therapy; bacterial defense mechanism; IncI1 conjugative plasmid; ibfA; abortive infection Author: Júlia López-Pérez, Pilar Cortés, Susana Campoy, Ivan Erill and Montserrat Llagostera Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed May 21 09:31:04 2025 CEST ModDate: Wed May 21 09:35:44 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 23 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3750234 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NXQZWG+EURM10 Type 1 Builtin yes yes yes 10 0 WMDAML+VnURWPalladioL Type 1 Custom yes yes yes 15 0 AIVVPP+URWPalladioL-Roma Type 1 Custom yes yes yes 21 0 LFVXTU+URWPalladioL-Bold Type 1 Custom yes yes yes 27 0 ZYAZNU+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 MJSSOL+CMSY10 Type 1 Builtin yes yes yes 37 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 73 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 176 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 179 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 188 0 QWQWCP+MSAM10 Type 1 Builtin yes yes yes 196 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 206 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 209 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 234 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 237 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 262 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 265 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 284 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 287 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 299 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 302 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 316 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 319 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 334 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 337 0 PACEOD+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 348 0 PACEOF+PalatinoLinotype TrueType WinAnsi yes yes no 351 0 PACEOH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 354 0 QDAZKD+URWPalladioL-BoldItal Type 1 Custom yes yes yes 368 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-05-30 02:12:06 CEST RepresentationInformation: ijms-26-04918-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-05-29 06:48:14 CEST Size: 3750234 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 730 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Unravelling the Plasmid Region That Disrupts the UAB_Phi20 Multiplicative Cycle Destination: subsection.2.1 Item: Title: ibfA Impacts Phage Productivity Without Impairing Bacterial Growth Kinetics Destination: subsection.2.2 Item: Title: Impact of the ibfA Gene on the Multiplicative Cycle of UAB_Phi20 Bacteriophage Destination: subsection.2.3 Item: Title: Visualization of UAB_Phi20 Virions by Electron Microscopy Destination: subsection.2.4 Item: Title: Distribution of ibfA-Related Genes in Prokaryotic Genomes Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Bacterial Strains Destination: subsection.4.1 Item: Title: Phage Lysate Production Destination: subsection.4.2 Item: Title: Bacterial Growth Kinetics Destination: subsection.4.3 Item: Title: EOP, Phage Adsorption Constant, Efficiency of Center of Infection (ECOI), and Burst Size Determination Destination: subsection.4.4 Item: Title: One-Step Growth Destination: subsection.4.5 Item: Title: Phage Infection Kinetics Destination: subsection.4.6 Item: Title: Deletion of ibfA Through One-Step Inactivation Destination: subsection.4.7 Item: Title: Transcriptional Analysis of ibfA Operon Destination: subsection.4.8 Item: Title: Cloning of ibfA onto the Vector pBAD33 Destination: subsection.4.9 Item: Title: Replication of UAB_Phi20 Genome Destination: subsection.4.10 Item: Title: Gene Expression Quantification Destination: subsection.4.11 Item: Title: Transmission Electron Microscopy (TEM) of Infected Salmonella Cells Destination: subsection.4.12 Item: Title: Bioinformatic Studies Destination: subsection.4.13 Item: Title: Quantification and Statistical Analysis Destination: subsection.4.14 Item: Title: References Destination: appendix.A. Info: Title: Deciphering the Causes of IbfA-Mediated Abortive Infection in the P22-like Phage UAB_Phi20 Author: Júlia López-Pérez, Pilar Cortés, Susana Campoy, Ivan Erill and Montserrat Llagostera Subject: The study of bacterial defense mechanisms against phages is becoming increasingly relevant due to their impact on the effectiveness of phage therapy. Employing a multifaceted approach that combines bioinformatics, molecular microbiology, TEM microscopy, and conventional microbiology techniques, here, we identify the ibfA gene as a novel defense factor targeting the virulent phage UAB_Phi20, acquired by Salmonella Typhimurium through lateral transfer on the IncI1 conjugative plasmid pUA1135 after oral phage therapy in broilers. IbfA, a two-domain protein containing ATPase and TOPRIM domains, significantly reduces UAB_Phi20 productivity, as indicated by decreased EOP, ECOI, and a diminished burst size, potentially reducing cellular viability without causing observable lysis. Our results indicate that IbfA enhances the transcription of early genes, including the antirepressor ant, which inhibits the C2 repressor of the lytic cycle. This may cause an imbalance in Cro/C2 concentration, leading to the observed reduction in the transcription of late genes encoding structural and cellular lysis proteins, and resulting in the abortion of UAB_Phi20 infection. Keywords: Salmonella; phage therapy; bacterial defense mechanism; IncI1 conjugative plasmid; ibfA; abortive infection Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed May 21 09:31:04 CEST 2025 ModDate: Wed May 21 09:35:44 CEST 2025 ID: 0xa3e233eec795e36b15515fc9b328222f, 0xa3e233eec795e36b15515fc9b328222f Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ZYAZNU+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: ZYAZNU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: QDAZKD+URWPalladioL-BoldItal FontSubset: true FirstChar: 65 LastChar: 105 FontDescriptor: FontName: QDAZKD+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: QWQWCP+MSAM10 FontSubset: true FirstChar: 3 LastChar: 78 FontDescriptor: FontName: QWQWCP+MSAM10 Flags: Symbolic FontBBox: 8, -463, 1331, 1003 FontFile: true ToUnicode: true Font: BaseFont: AIVVPP+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 250 FontDescriptor: FontName: AIVVPP+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MJSSOL+CMSY10 FontSubset: true FirstChar: 0 LastChar: 15 FontDescriptor: FontName: MJSSOL+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: NXQZWG+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: NXQZWG+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: LFVXTU+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 250 FontDescriptor: FontName: LFVXTU+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true ToUnicode: true Font: BaseFont: WMDAML+VnURWPalladioL FontSubset: true FirstChar: 128 LastChar: 225 FontDescriptor: FontName: WMDAML+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 108, 177, 152, 186 Annotation: Subtype: Link Rect: 34, 165, 130, 174 Annotation: Subtype: Link Rect: 36, 75, 152, 84 Annotation: Subtype: Link Rect: 34, 63, 89, 72 Annotation: Subtype: Link Rect: 34, 773, 180, 809 Annotation: Subtype: Link Rect: 515, 773, 560, 809 Annotation: Subtype: Link Rect: 193, 663, 204, 677 Annotation: Subtype: Link Rect: 342, 663, 353, 677 Annotation: Subtype: Link Rect: 295, 238, 302, 246 ActionDest: 398 Annotation: Subtype: Link Rect: 513, 238, 520, 246 ActionDest: 398 Annotation: Subtype: Link Rect: 421, 27, 560, 37 Annotation: Subtype: Link Rect: 34, 320, 87, 339 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 254, 703, 261, 712 ActionDest: 398 Annotation: Subtype: Link Rect: 262, 703, 269, 712 ActionDest: 398 Annotation: Subtype: Link Rect: 252, 661, 259, 670 ActionDest: 398 Annotation: Subtype: Link Rect: 235, 647, 242, 656 ActionDest: 398 Annotation: Subtype: Link Rect: 548, 647, 555, 656 ActionDest: 398 Annotation: Subtype: Link Rect: 309, 633, 316, 642 ActionDest: 398 Annotation: Subtype: Link Rect: 477, 633, 484, 642 ActionDest: 398 Annotation: Subtype: Link Rect: 305, 619, 317, 628 ActionDest: 401 Annotation: Subtype: Link Rect: 469, 619, 481, 628 ActionDest: 401 Annotation: Subtype: Link Rect: 380, 605, 392, 614 ActionDest: 401 Annotation: Subtype: Link Rect: 393, 605, 405, 614 ActionDest: 401 Annotation: Subtype: Link Rect: 452, 591, 464, 600 ActionDest: 401 Annotation: Subtype: Link Rect: 544, 563, 556, 572 ActionDest: 401 Annotation: Subtype: Link Rect: 228, 480, 240, 489 ActionDest: 401 Annotation: Subtype: Link Rect: 240, 480, 252, 489 ActionDest: 401 Annotation: Subtype: Link Rect: 361, 466, 373, 475 ActionDest: 401 Annotation: Subtype: Link Rect: 417, 466, 429, 475 ActionDest: 401 Annotation: Subtype: Link Rect: 430, 466, 442, 475 ActionDest: 401 Annotation: Subtype: Link Rect: 543, 466, 555, 475 ActionDest: 401 Annotation: Subtype: Link Rect: 221, 452, 233, 461 ActionDest: 401 Annotation: Subtype: Link Rect: 222, 424, 234, 433 ActionDest: 401 Annotation: Subtype: Link Rect: 234, 424, 246, 433 ActionDest: 401 Annotation: Subtype: Link Rect: 331, 410, 343, 419 ActionDest: 401 Annotation: Subtype: Link Rect: 219, 285, 231, 294 ActionDest: 401 Annotation: Subtype: Link Rect: 340, 229, 352, 238 ActionDest: 401 Annotation: Subtype: Link Rect: 230, 201, 242, 210 ActionDest: 401 Annotation: Subtype: Link Rect: 543, 201, 555, 210 ActionDest: 401 Annotation: Subtype: Link Rect: 267, 174, 279, 182 ActionDest: 401 Annotation: Subtype: Link Rect: 525, 132, 537, 141 ActionDest: 401 Annotation: Subtype: Link Rect: 531, 118, 543, 127 ActionDest: 401 Annotation: Subtype: Link Rect: 544, 118, 556, 127 ActionDest: 401 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 349, 630, 361, 639 ActionDest: 401 Annotation: Subtype: Link Rect: 364, 630, 376, 639 ActionDest: 401 Annotation: Subtype: Link Rect: 278, 602, 290, 611 ActionDest: 401 Annotation: Subtype: Link Rect: 348, 530, 355, 542 ActionDest: 63 Annotation: Subtype: Link Rect: 270, 185, 277, 197 ActionDest: 168 Annotation: Subtype: Link Rect: 353, 146, 365, 155 ActionDest: 401 Annotation: Subtype: Link Rect: 504, 132, 516, 141 ActionDest: 401 Annotation: Subtype: Link Rect: 270, 118, 282, 127 ActionDest: 401 Annotation: Subtype: Link Rect: 217, 90, 229, 99 ActionDest: 405 Annotation: Subtype: Link Rect: 203, 76, 215, 85 ActionDest: 401 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 540, 742, 547, 754 ActionDest: 168 Annotation: Subtype: Link Rect: 437, 254, 449, 263 ActionDest: 405 Annotation: Subtype: Link Rect: 295, 126, 302, 138 ActionDest: 168 Annotation: Subtype: Link Rect: 531, 73, 543, 82 ActionDest: 405 Annotation: Subtype: Link Rect: 543, 73, 555, 82 ActionDest: 405 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 362, 758, 374, 767 ActionDest: 405 Annotation: Subtype: Link Rect: 311, 689, 323, 698 ActionDest: 405 Annotation: Subtype: Link Rect: 324, 689, 336, 698 ActionDest: 405 Annotation: Subtype: Link Rect: 314, 563, 326, 572 ActionDest: 405 Annotation: Subtype: Link Rect: 439, 561, 446, 573 ActionDest: 185 Annotation: Subtype: Link Rect: 255, 477, 262, 489 ActionDest: 185 Annotation: Subtype: Link Rect: 368, 450, 375, 461 ActionDest: 185 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 242, 700, 249, 712 ActionDest: 193 Annotation: Subtype: Link Rect: 267, 658, 274, 670 ActionDest: 193 Annotation: Subtype: Link Rect: 419, 616, 426, 628 ActionDest: 193 Annotation: Subtype: Link Rect: 418, 82, 425, 94 ActionDest: 254 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 545, 686, 552, 698 ActionDest: 254 Annotation: Subtype: Link Rect: 348, 146, 355, 158 ActionDest: 276 Annotation: Subtype: Link Rect: 253, 76, 260, 88 ActionDest: 276 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 235, 759, 247, 767 ActionDest: 401 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 361, 742, 368, 754 ActionDest: 308 Annotation: Subtype: Link Rect: 371, 310, 378, 322 ActionDest: 326 Annotation: Subtype: Link Rect: 197, 227, 209, 239 ActionDest: 326 Annotation: Subtype: Link Rect: 307, 172, 314, 184 ActionDest: 365 Annotation: Subtype: Link Rect: 251, 144, 258, 156 ActionDest: 365 Annotation: Subtype: Link Rect: 193, 116, 200, 128 ActionDest: 365 Annotation: Subtype: Link Rect: 516, 102, 528, 114 ActionDest: 326 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 275, 497, 287, 506 ActionDest: 401 Annotation: Subtype: Link Rect: 264, 330, 275, 339 ActionDest: 405 Annotation: Subtype: Link Rect: 276, 329, 288, 339 ActionDest: 405 Annotation: Subtype: Link Rect: 522, 329, 534, 339 ActionDest: 401 Annotation: Subtype: Link Rect: 370, 299, 377, 311 ActionDest: 168 Annotation: Subtype: Link Rect: 225, 243, 232, 255 ActionDest: 168 Annotation: Subtype: Link Rect: 269, 229, 276, 241 ActionDest: 185 Annotation: Subtype: Link Rect: 245, 201, 252, 213 ActionDest: 185 Annotation: Subtype: Link Rect: 206, 134, 218, 144 ActionDest: 401 Annotation: Subtype: Link Rect: 450, 120, 462, 130 ActionDest: 401 Annotation: Subtype: Link Rect: 462, 120, 474, 130 ActionDest: 401 Annotation: Subtype: Link Rect: 475, 120, 487, 130 ActionDest: 405 Annotation: Subtype: Link Rect: 429, 107, 441, 116 ActionDest: 401 Annotation: Subtype: Link Rect: 397, 93, 409, 102 ActionDest: 405 Annotation: Subtype: Link Rect: 392, 65, 404, 74 ActionDest: 405 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 484, 744, 496, 754 ActionDest: 405 Annotation: Subtype: Link Rect: 341, 661, 353, 670 ActionDest: 405 Annotation: Subtype: Link Rect: 411, 661, 423, 670 ActionDest: 405 Annotation: Subtype: Link Rect: 479, 647, 491, 656 ActionDest: 405 Annotation: Subtype: Link Rect: 412, 605, 424, 614 ActionDest: 405 Annotation: Subtype: Link Rect: 459, 575, 465, 587 ActionDest: 193 Annotation: Subtype: Link Rect: 350, 522, 361, 531 ActionDest: 405 Annotation: Subtype: Link Rect: 492, 505, 499, 517 ActionDest: 193 Annotation: Subtype: Link Rect: 464, 491, 471, 503 ActionDest: 193 Annotation: Subtype: Link Rect: 348, 449, 360, 461 ActionDest: 326 Annotation: Subtype: Link Rect: 409, 449, 416, 461 ActionDest: 365 Annotation: Subtype: Link Rect: 412, 408, 419, 420 ActionDest: 193 Annotation: Subtype: Link Rect: 437, 338, 444, 350 ActionDest: 254 Annotation: Subtype: Link Rect: 377, 324, 384, 336 ActionDest: 254 Annotation: Subtype: Link Rect: 288, 310, 295, 322 ActionDest: 193 Annotation: Subtype: Link Rect: 421, 296, 428, 308 ActionDest: 254 Annotation: Subtype: Link Rect: 539, 171, 546, 183 ActionDest: 276 Annotation: Subtype: Link Rect: 548, 171, 555, 183 ActionDest: 308 Annotation: Subtype: Link Rect: 524, 157, 531, 169 ActionDest: 276 Annotation: Subtype: Link Rect: 369, 132, 381, 141 ActionDest: 405 Annotation: Subtype: Link Rect: 184, 90, 196, 99 ActionDest: 405 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 329, 728, 336, 740 ActionDest: 326 Annotation: Subtype: Link Rect: 319, 714, 326, 726 ActionDest: 193 Annotation: Subtype: Link Rect: 282, 700, 294, 712 ActionDest: 326 Annotation: Subtype: Link Rect: 437, 689, 449, 698 ActionDest: 405 Annotation: Subtype: Link Rect: 452, 689, 464, 698 ActionDest: 405 Annotation: Subtype: Link Rect: 464, 633, 476, 642 ActionDest: 405 Annotation: Subtype: Link Rect: 337, 605, 349, 614 ActionDest: 405 Annotation: Subtype: Link Rect: 214, 549, 226, 559 ActionDest: 405 Annotation: Subtype: Link Rect: 227, 549, 239, 559 ActionDest: 405 Annotation: Subtype: Link Rect: 348, 480, 360, 489 ActionDest: 405 Annotation: Subtype: Link Rect: 297, 354, 309, 364 ActionDest: 408 Annotation: Subtype: Link Rect: 381, 257, 393, 266 ActionDest: 408 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 191, 758, 198, 767 ActionDest: 398 Annotation: Subtype: Link Rect: 198, 758, 205, 767 ActionDest: 398 Annotation: Subtype: Link Rect: 206, 758, 218, 767 ActionDest: 401 Annotation: Subtype: Link Rect: 218, 758, 230, 767 ActionDest: 401 Annotation: Subtype: Link Rect: 231, 758, 243, 767 ActionDest: 401 Annotation: Subtype: Link Rect: 243, 759, 255, 767 ActionDest: 405 Annotation: Subtype: Link Rect: 255, 758, 267, 767 ActionDest: 405 Annotation: Subtype: Link Rect: 471, 602, 483, 611 ActionDest: 401 Annotation: Subtype: Link Rect: 265, 560, 277, 569 ActionDest: 408 Annotation: Subtype: Link Rect: 507, 490, 519, 499 ActionDest: 401 Annotation: Subtype: Link Rect: 520, 491, 532, 499 ActionDest: 401 Annotation: Subtype: Link Rect: 493, 450, 505, 459 ActionDest: 408 Annotation: Subtype: Link Rect: 394, 408, 406, 417 ActionDest: 408 Annotation: Subtype: Link Rect: 205, 190, 216, 199 ActionDest: 408 Annotation: Subtype: Link Rect: 443, 176, 455, 185 ActionDest: 405 Annotation: Subtype: Link Rect: 254, 134, 266, 144 ActionDest: 408 Annotation: Subtype: Link Rect: 340, 121, 352, 130 ActionDest: 408 Annotation: Subtype: Link Rect: 428, 121, 440, 130 ActionDest: 408 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 346, 730, 358, 740 ActionDest: 408 Annotation: Subtype: Link Rect: 332, 647, 344, 656 ActionDest: 408 Annotation: Subtype: Link Rect: 205, 591, 217, 600 ActionDest: 408 Annotation: Subtype: Link Rect: 273, 563, 284, 572 ActionDest: 408 Annotation: Subtype: Link Rect: 285, 563, 297, 572 ActionDest: 408 Annotation: Subtype: Link Rect: 470, 509, 482, 518 ActionDest: 408 Annotation: Subtype: Link Rect: 408, 356, 420, 365 ActionDest: 408 Annotation: Subtype: Link Rect: 496, 204, 508, 213 ActionDest: 408 Annotation: Subtype: Link Rect: 322, 80, 333, 89 ActionDest: 408 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 525, 759, 537, 767 ActionDest: 408 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 495, 384, 507, 393 ActionDest: 408 Annotation: Subtype: Link Rect: 261, 300, 273, 309 ActionDest: 408 Annotation: Subtype: Link Rect: 543, 286, 555, 295 ActionDest: 408 Annotation: Subtype: Link Rect: 219, 120, 231, 129 ActionDest: 408 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 417, 577, 429, 586 ActionDest: 408 Annotation: Subtype: Link Rect: 259, 120, 271, 129 ActionDest: 408 Annotation: Subtype: Link Rect: 324, 120, 336, 129 ActionDest: 401 Annotation: Subtype: Link Rect: 428, 120, 440, 129 ActionDest: 408 Annotation: Subtype: Link Rect: 440, 120, 452, 129 ActionDest: 408 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 472, 758, 484, 767 ActionDest: 408 Annotation: Subtype: Link Rect: 254, 717, 266, 726 ActionDest: 408 Annotation: Subtype: Link Rect: 266, 717, 278, 726 ActionDest: 408 Annotation: Subtype: Link Rect: 411, 577, 422, 586 ActionDest: 408 Annotation: Subtype: Link Rect: 488, 577, 500, 586 ActionDest: 408 Annotation: Subtype: Link Rect: 195, 549, 207, 559 ActionDest: 408 Annotation: Subtype: Link Rect: 193, 536, 205, 545 ActionDest: 408 Annotation: Subtype: Link Rect: 206, 508, 218, 517 ActionDest: 405 Annotation: Subtype: Link Rect: 323, 508, 335, 517 ActionDest: 408 Annotation: Subtype: Link Rect: 422, 494, 434, 503 ActionDest: 408 Annotation: Subtype: Link Rect: 388, 466, 400, 475 ActionDest: 405 Annotation: Subtype: Link Rect: 400, 466, 412, 475 ActionDest: 405 Annotation: Subtype: Link Rect: 536, 328, 560, 339 Annotation: Subtype: Link Rect: 165, 315, 380, 326 Annotation: Subtype: Link Rect: 431, 317, 442, 326 ActionDest: 401 Annotation: Subtype: Link Rect: 442, 317, 453, 326 ActionDest: 408 Annotation: Subtype: Link Rect: 454, 317, 465, 326 ActionDest: 408 Annotation: Subtype: Link Rect: 465, 317, 476, 326 ActionDest: 408 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 176, 248, 213, 258 Annotation: Subtype: Link Rect: 219, 248, 255, 258 Annotation: Subtype: Link Rect: 409, 221, 447, 232 Annotation: Subtype: Link Rect: 453, 221, 489, 232 Annotation: Subtype: Link Rect: 59, 197, 96, 207 Annotation: Subtype: Link Rect: 102, 197, 138, 207 Annotation: Subtype: Link Rect: 516, 183, 553, 194 Annotation: Subtype: Link Rect: 59, 159, 96, 169 Annotation: Subtype: Link Rect: 386, 132, 423, 143 Annotation: Subtype: Link Rect: 502, 119, 539, 130 Annotation: Subtype: Link Rect: 404, 68, 441, 79 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 370, 743, 408, 754 Annotation: Subtype: Link Rect: 461, 718, 499, 729 Annotation: Subtype: Link Rect: 505, 718, 541, 729 Annotation: Subtype: Link Rect: 461, 692, 499, 703 Annotation: Subtype: Link Rect: 505, 692, 541, 703 Annotation: Subtype: Link Rect: 133, 668, 170, 678 Annotation: Subtype: Link Rect: 144, 642, 182, 652 Annotation: Subtype: Link Rect: 394, 616, 431, 627 Annotation: Subtype: Link Rect: 436, 590, 473, 601 Annotation: Subtype: Link Rect: 95, 565, 133, 576 Annotation: Subtype: Link Rect: 412, 539, 449, 550 Annotation: Subtype: Link Rect: 350, 513, 388, 524 Annotation: Subtype: Link Rect: 431, 488, 469, 499 Annotation: Subtype: Link Rect: 434, 462, 471, 473 Annotation: Subtype: Link Rect: 477, 462, 513, 473 Annotation: Subtype: Link Rect: 369, 437, 407, 448 Annotation: Subtype: Link Rect: 413, 437, 449, 448 Annotation: Subtype: Link Rect: 519, 411, 557, 422 Annotation: Subtype: Link Rect: 59, 399, 95, 410 Annotation: Subtype: Link Rect: 383, 373, 421, 384 Annotation: Subtype: Link Rect: 59, 336, 96, 346 Annotation: Subtype: Link Rect: 333, 309, 370, 320 Annotation: Subtype: Link Rect: 353, 284, 391, 295 Annotation: Subtype: Link Rect: 336, 258, 373, 269 Annotation: Subtype: Link Rect: 135, 233, 173, 244 Annotation: Subtype: Link Rect: 175, 208, 213, 218 Annotation: Subtype: Link Rect: 107, 182, 145, 193 Annotation: Subtype: Link Rect: 464, 156, 502, 167 Annotation: Subtype: Link Rect: 95, 131, 133, 141 Annotation: Subtype: Link Rect: 312, 105, 349, 116 Annotation: Subtype: Link Rect: 422, 79, 460, 90 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 271, 743, 309, 754 Annotation: Subtype: Link Rect: 131, 680, 168, 690 Annotation: Subtype: Link Rect: 370, 654, 407, 665 Annotation: Subtype: Link Rect: 519, 641, 556, 652 Annotation: Subtype: Link Rect: 308, 603, 345, 614 Annotation: Subtype: Link Rect: 185, 565, 223, 576 Annotation: Subtype: Link Rect: 229, 565, 265, 576 Annotation: Subtype: Link Rect: 392, 539, 430, 550 Annotation: Subtype: Link Rect: 436, 539, 472, 550 Annotation: Subtype: Link Rect: 153, 514, 191, 524 Annotation: Subtype: Link Rect: 197, 514, 233, 524 Annotation: Subtype: Link Rect: 172, 489, 210, 499 Annotation: Subtype: Link Rect: 59, 463, 96, 473 Annotation: Subtype: Link Rect: 304, 437, 341, 448 Annotation: Subtype: Link Rect: 149, 399, 186, 410 Annotation: Subtype: Link Rect: 229, 373, 266, 384 Annotation: Subtype: Link Rect: 410, 347, 447, 358 Annotation: Subtype: Link Rect: 481, 322, 518, 333 Annotation: Subtype: Link Rect: 293, 296, 331, 307 Annotation: Subtype: Link Rect: 337, 296, 373, 307 Annotation: Subtype: Link Rect: 519, 284, 557, 295 Annotation: Subtype: Link Rect: 59, 272, 95, 282 Annotation: Subtype: Link Rect: 469, 258, 506, 269 Annotation: Subtype: Link Rect: 512, 258, 548, 269 Annotation: Subtype: Link Rect: 199, 233, 236, 244 Annotation: Subtype: Link Rect: 256, 207, 293, 218 Annotation: Subtype: Link Rect: 293, 182, 330, 193 Annotation: Subtype: Link Rect: 520, 169, 557, 180 Annotation: Subtype: Link Rect: 391, 143, 429, 154 Annotation: Subtype: Link Rect: 59, 119, 96, 129 Annotation: Subtype: Link Rect: 59, 93, 96, 103 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 95, 731, 133, 742 Annotation: Subtype: Link Rect: 139, 731, 175, 742 Annotation: Subtype: Link Rect: 418, 705, 455, 716 Annotation: Subtype: Link Rect: 313, 679, 350, 690 Annotation: Subtype: Link Rect: 408, 654, 445, 665 Annotation: Subtype: Link Rect: 419, 641, 457, 652 Annotation: Subtype: Link Rect: 520, 628, 557, 639 Annotation: Subtype: Link Rect: 507, 603, 544, 614 Annotation: Subtype: Link Rect: 456, 590, 494, 601 Annotation: Subtype: Link Rect: 337, 565, 375, 576 Annotation: Subtype: Link Rect: 200, 540, 238, 550 Annotation: Subtype: Link Rect: 222, 514, 259, 524 Annotation: Subtype: Link Rect: 265, 514, 301, 524 Annotation: Subtype: Link Rect: 59, 450, 96, 461 Annotation: Subtype: Link Rect: 357, 424, 395, 435 Annotation: Subtype: Link Rect: 401, 424, 437, 435 Annotation: Subtype: Link Rect: 506, 411, 543, 422 Annotation: Subtype: Link Rect: 501, 386, 538, 397 Annotation: Subtype: Link Rect: 346, 360, 384, 371 Annotation: Subtype: Link Rect: 305, 335, 342, 346 Annotation: Subtype: Link Rect: 143, 310, 181, 320 Annotation: Subtype: Link Rect: 498, 296, 536, 307 Annotation: Subtype: Link Rect: 341, 272, 378, 282 Annotation: Subtype: Link Rect: 404, 245, 442, 256 Annotation: Subtype: Link Rect: 448, 245, 484, 256 Annotation: Subtype: Link Rect: 155, 221, 192, 231 Annotation: Subtype: Link Rect: 198, 221, 234, 231 Annotation: Subtype: Link Rect: 473, 194, 510, 205 Annotation: Subtype: Link Rect: 517, 194, 553, 205 Annotation: Subtype: Link Rect: 167, 170, 204, 180 Annotation: Subtype: Link Rect: 404, 143, 442, 154 Annotation: Subtype: Link Rect: 457, 130, 494, 141 Checksum: 65fd368d Type: CRC32 Checksum: b50ff3c47997229021ff6bf2fd0dd7c3 Type: MD5 Checksum: 276c657963c6739b873c024a8492eaa82fa4791a Type: SHA-1 Checksum: 27e31ebd66ce545f87299ed88356d3b2527711a2f4bdb482ba546e04b5e56d6b Type: SHA-256