Are stray dogs confined in animal shelters at increased risk of seropositivity to Leishmania infantum? A case control study

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1 12 SARIDOMICHELAKIS (M.N.) AND COLLABORATORS Are stry dogs confined in niml shelters t incresed risk of seropositivity to Leishmni infntum? A cse control study M.N. SARIDOMICHELAKIS 1 *, K.N. APOSTOLIDIS 1, M.K. CHATZIS 1, T. PETANIDES 1, K. KOKKINAKI 1, L.V. ATHANASIOU 1, D. KASAMBALIS 1, L.S. LEONTIDES 2 1 Clinic of Medicine, Fculty of Veterinry Science, University of Thessly, Triklon Str. 224, GR-43132, Krdits, Greece 2 Lbortory of Epidemiology, Biosttistics nd Animl Helth Economics, Fculty of Veterinry Science, University of Thessly, Triklon Str. 224, GR-43132, Krdits, Greece *Corresponding uthor: msrido@vet.uth.gr SUMMARY The im of this study ws to compre the risk of seropositivity between stry dogs nd privtely-owned dogs living in the sme endemic re. Sixty-seven stry (group A) nd 115 privtely-owned (group B) dogs were enrolled. History, physicl exmintion findings nd results of serology, hemtology nd totl solids were compred between groups nd between seropositive group A nd seropositive group B dogs. Vribles with p vlue 0.2 were used to build two multivrible models using results of serology s the dependent vrible: the first model included the group nd historicl informtion, wheres the second included the group, physicl exmintion nd results of hemtology nd totl solid mesurement. Stry dogs hd 4.59-fold nd 3.72-fold higher risk of seropositivity compred with privtelyowned dogs in the first nd second model, respectively. Additionl fctors ssocited with positive serology included femle sex, not smpling during trnsmission period, mstictory muscle trophy, footpd hyperkertosis, low hemtocrit nd low pltelet count. In conclusion, stry dogs were t higher risk of seropositivity. Keywords: Cnine, kennel, Leishmni infntum, stry, serology RÉSUMÉ Les chiens errnts confinés dns des refuges pour nimux ont-ils un risque ccru de séropositivité à Leishmni infntum? Une étude cstémoin L objectif de cette étude étit de comprer le risque de séropositivité entre chiens errnts et chiens vivnts en hbittion dns l même zone endémique. Soixnte-sept chiens errnts (groupe A) et 115 chiens pprtennt à des prticuliers (groupe B) ont été suivis. Les ntécédents, les résultts de l exmen physique et les résultts de sérologie, hémtologie, solides totux ont été comprés entre groupes. Les vribles ynt une vleur p 0,2 ont été utilisées pour construire deux modèles multivribles utilisnt les résultts de l sérologie comme vrible dépendnte: le premier modèle comprenit le groupe et l informtion historique, tndis que le second incluit le groupe, l exmen physique et les résultts de l hémtologie et des solides totux. Les chiens errnts présentient un risque de séropositivité respectivement 4,59 fois et 3,72 fois plus élevé que chez les chiens de propriétire dns le premier et deuxième modèle. Prmi les utres fcteurs ssociés à une sérologie positive, mentionnons le sexe femelle, l bsence d échntillonnge pendnt l période de trnsmission, l trophie musculire mstictoire, l hyperkértose du pvé uditif, l hémtocrite fible et l fible numértion plquettire. En conclusion, les chiens errnts présentient un risque plus élevé de séropositivité. Mots-clés : Chien errnt, chenil, Leishmniose, sérologie, épidémiologie Introduction Most dogs tht live in res where Leishmni infntum is endemic re exposed to the prsite, but only some infected dogs become seropositive nd even fewer develop clinicl signs nd/or lbortory bnormlities of cnine leishmniosis (CnL) [10, 17]. With some notble exceptions, like hres in the Mdrid region, infected dogs re the primry reservoir of L. infntum which they trnsmit to snd flies nd it hs been repetedly shown tht seropositive dogs re the most infectious for the vectors [7, 21]. In some endemic countries, including Greece, there is lrge popultion of stry dogs which re free-roming nd/ or confined in niml shelters. Seroprevlence (0-37.5%) nd prevlence of CnL in these dogs is highly vrible ccording to published studies [9, 20]. World Helth Orgniztion considers stry dogs to be very importnt for the infection of vectors [26], minly due to their outdoor lifestyle, lck of prevention ginst snd fly bites nd suboptiml nutrition nd helth sttus. However, in mny published studies the seroprevlence nd/or the prevlence of CnL ws similr or even lower in stry thn in privtely-owned dogs living in the sme re: for exmple, in two different studies from Georgi, seroprevlence rtes were 16% nd 15.3% in stry dogs, respectively nd 23.7% nd 18.2% in pet dogs, respectively [3, 13], in Irn the relevnt figures were 1.6% in stry dogs nd 10% in pet dogs [14], nd in Portugl 15.4% nd 18.5%, respectively [18].

2 LEISHMANIA SEROPOSITIVITY IN STRAY DOGS 13 nd thus during the trnsmission period of L. infntum. The personnel of the shelter were not ble to provide informtion on the dte or on the helth sttus of the dogs when they first entered the shelter. Group B dogs were enrolled retrospectively nd they included ll the non-pregnnt, non-lctting, privtely-owned dogs living in the prefectures of Krdits, Trikl, Lriss or Mgnesi tht hd been dmitted to the Clinic of Medicine, Fculty of Veterinry Science, University of Thessly between Jnury 2012 nd Jnury 2013 for vrious medicl resons or for wellness check-up nd hd been tested serologiclly for Leishmni spp., ccording to the recommendtion of the ttending clinicin nd/or their owner s request. None of the group B dogs hd been previously dignosed with CnL, hd received nti-leishmni mediction or hd been vccinted ginst CnL. HISTORICAL INFORMATION, PHYSICAL EXAMINATION AND BLOOD SAMPLING Figure 1: Mp of the study re. Stry (group A) dogs were confined in n niml shelter pproximtely 5 Km from the center of the city of Krdits, wheres privtely-owned (group B) dogs originted from the shded re The im of the present study ws to compre the risk of seropositivity to L. infntum between stry dogs confined in n niml shelter nd privtely-owned dogs, while ccounting for multiple confounding fctors tht my be ssocited with positive serology. Mterils nd methods DOGS Two groups of dogs were used: group A included 67 stry dogs tht were confined in Krdits s Animl Welfre Society shelter nd group B included 115 privtely-owned dogs tht were dmitted to the Clinic of Medicine, Fculty of Veterinry Science, University of Thessly. The niml shelter tht is locted in suburbn re, pproximtely 5 Km from the centre of the city (ltitude: 39 o 21 N; longitude: 21 o 55 E), homes stry dogs which re cptured in the city of Krdits nd in neighbouring res nd hs non-killing policy. Group B dogs lived in the prefectures of Krdits, Trikl, Lriss nd Mgnesi (ltitude rnge of prefecture cpitls: 39 o o 38 N; longitude rnge of prefecture cpitls: 21 o o 56 E) (Figure 1). Group A dogs were enrolled between Jnury 2012 nd Jnury 2013 nd they were rndomly selected mong those tht were not pregnnt or lctting, did not pper likely to bite nd redily ccepted physicl restrin. The 67 dogs represented pproximtely one third of the totl cnine popultion of the shelter where they were living in open runs, receiving bsic medicl cre not including insect repellents. None of them ws smpled more thn once nd ll dogs tht were younger thn 1 yer were smpled on July 2012 Historicl informtion tht were recorded for group A nd group B dogs include: i) sex (intct mle or intct femle; missing for one group A dog); ii) breed (purebred or not); iii) if they belonged to breed with incresed seroprevlence nd/or with susceptibility to CnL, including Boxer, Cocker Spniel, Dobermn Pincher, Germn Shepherd, Pit Bull Terrier, Rottweiler, English nd Irish Setter [11, 13, 19], or not; iv) ge in yers (missing for 8 group A dogs, wheres for the remining group A dogs it ws estimted bsed on informtion from shelter s stff nd on their teeth eruption pttern nd wer); v) lifestyle (indoors, outdoors or both, with ll group A dogs recorded s hving outdoors lifestyle); nd vi) smpling during the trnsmission period of L. infntum in Greece (April-November) or not. Physicl exmintion findings tht were recorded for group A nd group B dogs include: i) presence of clinicl signs or mnifesttions tht hve been ssocited with CnL: poor body condition, mucosl pllor, peripherl lymphdenomegly, heptomegly, splenomegly, blephritis, conjunctivitis, kertitis, uveitis, mstictory muscle trophy, generlized muscle trophy, rthritis, epistxis, exfolitive dermtitis, ulcertive dermtitis, pustulr dermtitis, nsl hyperkertosis, footpd hyperkertosis, onychogryphosis [16]; nd ii) presence or not of t lest one of the bove clinicl signs. Approximtely 5 ml of blood were obtined from either jugulr or sphenous vein, 2 ml were immeditely trnsferred to n EDTA-contining tube nd were used for hemtology, wheres the remining blood ws left to clot t room temperture nd centrifuged (3.000 rpm for 20 min) to seprte serum tht ws used for mesurement of totl solids nd for serology. No more thn 5 h elpsed from smpling to hemtology, mesurement of totl solids nd freezing of serum. Frozen serum smples were defrosted t lter dte for serology.

3 14 LABORATORY ANALYSES Hemtology ws performed in poch-100iv (Sysmex Europe GmbH, Hmburg, Germny) veterinry hemtology nlyser nd differentil cell count ws clculted mnully in Diff-Quik (Merck, Germny) stined blood smers. Dt tht were recorded include: i) hemtocrit; ii) hemoglobin concentrtion; iii) men corpusculr hemoglobin concentrtion (MCHC-missing for 3 group B dogs); iv) white blood cell count; v) neutrophil, bnd neutrophil, lymphocyte, monocyte nd eosinophil counts (missing for 2 group A dogs); vi) pltelet count; vii) presence of nemi or not; viii) incresed, within reference rnge or decresed white blood cell count; ix) incresed, within reference rnge or decresed neutrophil, bnd neutrophil, lymphocyte, monocyte nd eosinophil counts (missing for 2 group A dogs); nd x) presence of thrombocytopeni or not. Totl solids were mesured with hndheld clinicl refrctometer (Americn Opticl, U.S.A.). Dt tht were recorded include: i) totl solid concentrtion, nd ii) incresed, within reference rnge or decresed totl solid concentrtion (both missing for 4 group A dogs). Indirect immunofluorescence ntibody testing (IFAT) ws used for the detection of nti-leishmni IgG. Commercilly vilble slides nd conjugte (Fluoleish, BVT, Frnce) were used nd serum smples were tested t twofold dilutions in phosphte buffered sline, strting from 1:50, until reching the end point titre. For the purposes of this study, dogs with titre 50 were considered seropositive. Dt tht were recorded include: i) seropositivity or not, nd ii) the end point titre. STATISTICAL ANALYSIS All sttisticl nlyses were performed using Stt 13.1 (Stt Sttisticl Softwre. College Sttion, TX). In the initil univrible nlysis, dt regrding historicl informtion, physicl exmintion, hemtology, totl solid mesurement nd serology were compred between group A nd B dogs by either Person s χ 2 or Fisher s exct test (ctegoricl vribles) nd t-test (continuous vribles). The sme tests were used to compre historicl informtion, physicl exmintion, hemtology nd totl solid mesurement between seropositive group A nd seropositive group B dogs. Vribles with p vlue 0.2 in the univrible nlysis, were subsequently used to build two multivrible models with the results of serology s the dependent vrible: the first model included the dog group nd historicl informtion (i.e. possible risk fctors for infection by L. infntum), wheres the second model included the dog group, physicl exmintion findings nd the results of hemtology nd totl solid mesurement (i.e. possible consequences of infection by L. infntum). The models were subsequently reduced by bckwrd elimintion until ll remining vribles were significnt t P<0.05. SARIDOMICHELAKIS (M.N.) AND COLLABORATORS Results COMPARISONS BETWEEN GROUP A AND GROUP B DOGS Of the historicl informtion, group A included significntly fewer purebred dogs, fewer dogs belonging to breeds with incresed seroprevlence nd/or with susceptibility to CnL nd more dogs living outdoors compred with group B (Tble I). Of the physicl exmintion findings, the only significnt difference ws the incresed frequency of splenomegly in group A dogs (Tble II). Significnt differences of the results of hemtology included lower hemoglobin concentrtion, lower MCHC, higher white blood cell, neutrophil, bnd neutrophil, lymphocyte nd eosinophil counts, lower monocyte count, lower pltelet count, incresed frequency of leucocytosis, neutrophili nd left shift nd incresed frequency of monocyte count within reference rnge in group A dogs (Tble III). Totl solids were less frequently within reference rnge in group A thn in group B dogs (Tble III). Additionl vribles tht were included in the multivrible models (p vlue 0.2) included sex, ge, smpling or not during the trnsmission period of L. infntum (Tble I), heptomegly, onychogryphosis (Tble II), hemtocrit nd frequency of eosinophili (Tble III). COMPARISONS BETWEEN SEROPOSITIVE GROUP A AND SEROPOSITIVE GROUP B DOGS Seroprevlence ws significntly (p = 0.004) higher in group A (18/ %) thn in group B (12/ %) dogs (Tble IV). IFAT end point titres rnged between 1/50 nd 1/800 nd did not differ between the two groups. Comprison of historicl informtion between the 18 seropositive group A nd the 12 seropositive group B dogs showed tht the former included significntly fewer purebred dogs, fewer dogs belonging to breeds with incresed seroprevlence nd/or with susceptibility to CnL nd they were more frequently smpled during the trnsmission period of L. infntum (Tbles IV & V). There were no significnt differences of the physicl exmintion findings between seropositive group A nd seropositive group B dogs (Tble VI). Significnt differences of the results of hemtology included lower MCHC nd incresed frequency of monocyte count within reference rnge in seropositive group A thn seropositive group B dogs (Tbles IV & VII). Additionl vribles tht were included in the multivrible models (p vlue 0.2) included splenomegly, mstictory muscle trophy, rthritis, nsl hyperkertosis, footpd hyperkertosis, onychogryphosis (Tble VI), white blood cell count, neutrophil count, monocyte count, frequency of neutrophili nd frequency of incresed totl solid concentrtion (Tble VII).

4 LEISHMANIA SEROPOSITIVITY IN STRAY DOGS 15 Sex Breed Vrible Group A Group B P vlue Mle 25/66 (37.9%) 59/115 (51.3%) Femle 41/66 (62.1%) 56/115 (48.7%) Purebred 5/67 (7.5%) 98/115 (85.2%) Not purebred 62/67 (92.5%) 17/115 (14.8%) < Predisposed breed Age (yers) Lifestyle Yes 2/67 (3%) 30/115 (26.1%) No 65/67 (97%) 85/115 (73.9%) < Rnge Medin Indoors - 17/115 (14.8%) Outdoors 67/67 (100%) 94/115 (81.7%) Indoors nd outdoors - 4/115 (3.5%) < Smpling during trnsmission period Yes 61/67 (91%) 96/115 (83.5%) No 6/67 (9%) 19/115 (16.5%) Tble 1: Comprison of historicl informtion between stry (group A; n=67) nd privtely-owned (group B; n=115) dogs Vrible Group A (n=67) Group B (n=115) P vlue Poor body condition 11 (16.4%) 14 (12.2%) NS Mucosl pllor 6 (9%) 14 (12.2%) NS Peripherl lymphdenomegly 33 (49.3%) 48 (41.7%) NS Heptomegly 2 (3%) Splenomegly 10 (14.9%) 1 (0.9%) < Blephritis 5 (7.5%) 5 (4.3%) NS Conjunctivitis 10 (14.9%) 19 (16.5%) NS Kertitis 1 (1.5%) 3 (2.6%) NS Uveitis 1 (1.5%) - NS Mstictory muscle trophy 21 (31.3%) 40 (34.8%) NS Generlized muscle trophy 5 (7.5%) 9 (7.8%) NS Arthritis - 2 (1.7%) NS Epistxis - 1 (0.9%) NS Exfolitive dermtitis 3 (4.5%) 10 (8.7%) NS Ulcertive dermtitis 3 (4.5%) 8 (7%) NS Pustulr dermtitis - 1 (0.9%) NS Nsl hyperkertosis 14 (20.9%) 20 (17.4%) NS Footpd hyperkertosis 11 (16.4%) 20 (17.4%) NS Onychogryphosis 13 (19.4%) 12 (10.4) 0.09 At lest one clinicl sign 40 (59.7%) 61 (53%) NS NS: non-significnt with P vlue >0.2 Tble II: Comprison of physicl exmintion findings between stry (group A; n=67) nd privtely-owned (group B; n=115) dogs

5 16 SARIDOMICHELAKIS (M.N.) AND COLLABORATORS Vrible Group A Group B P vlue Hemtocrit (%) Rnge Medin Hemoglobin concentrtion (g/dl) Rnge Medin Anemi 26/67 (38.8%) 37/115 (32.2%) NS c MCHC (g/dl) Rnge Medin < White blood cell count (/μl) Rnge 4,900-32,400 4,400-33,700 Medin 14,600 11,200 < White blood cell count Incresed 52/67 (77.6%) 48/115 (41.7%) WRR b 13/67 (19.4%) 63/115 (54.8%) Decresed 2/67 (3%) 4/115 (3.5%) < Neutrophil count (/μl) Rnge 2,989-26,010 2,904-32,689 Medin 10,540 7,452 < Neutrophil count Incresed 24/65 (36.9%) 12/115 (10.4%) WRR 40/65 (61.5%) 102/115 (88.7%) Decresed 1/65 (1.5%) 1/115 (0.9%) < Bnd neutrophil count (/μl) Rnge 0-1, ,274 Medin Bnd neutrophil count Incresed 26/65 (40%) 17/115 (14.8%) WRR 39/65 (60%) 98/115 (85.2%) < Lymphocyte count (/μl) Rnge , ,432 Medin 2,800 2, Lymphocyte count Incresed 7/65 (10.8%) 5/115 (4.3%) WRR 52/65 (80%) 98/115 (85.2%) Decresed 6/65 (9.2%) 12/115 (10.4%) NS Monocyte count (/μl) Rnge 0-1, ,170 Medin Monocyte count Incresed 1/65 (1.5%) 9/115 (7.8%) WRR 32/65 (49.2%) 35/115 (30.4%) Decresed 32/65 (49.2%) 71/115 (61.7%) Eosinophil count (/μl) Rnge 0-9, ,115 Medin Eosinophil count Incresed 33/65 (50.8%) 41/115 (35.7%) WRR 23/65 (35.4%) 57/115 (49.6%) Decresed 9/65 (13.8%) 17/115 (14.8%) Pltelet count (/μl) Rnge 83, ,000 49, ,000 Medin 274, , Thrombocytopeni 7/67 (10.4%) 9/115 (7.8%) NS Totl solids (g/dl) Rnge Medin NS Totl solids Incresed 5/63 (7.9%) - WRR 37/63 (58.7%) 90/115 (78.3%) Decresed 21/63 (33.3%) 25/115 (21.7%) MCHC: men corpusculr hemoglobin concentrtion b WRR: within reference rnge c NS: non-significnt with P vlue >0.2 Tble III: Comprison of the results of hemtology nd totl solid mesurement between stry (group A; n=67) nd privtely-owned (group B; n=115) dogs

6 LEISHMANIA SEROPOSITIVITY IN STRAY DOGS 17 Vrible All dogs Group A > or < group B (P vlue) Seropositive dogs Seropositivity Group A > B (p = 0.004) NS e Purebred Group A < B (p < 0.001) Group A < B (p < 0.001) Predisposed breed b Group A < B (p < 0.001) Group A < B (p = 0.006) Outdoor lifestyle Group A > B (p < 0.001) NS Smpling during trnsmission period NS Group A > B (p = 0.009) Splenomegly Group A > B (p < 0.001) NS Hemoglobin concentrtion (g/dl) Group A < B (p = 0.01) NS MCHC c (g/dl) Group A < B (p < 0.001) Group A < B (p = 0.007) White blood cell count (/μl) Group A > B (p < 0.001) NS Leucocytosis Group A > B (p < 0.001) NS Neutrophil count (/μl) Group A > B (p < 0.001) NS Neutrophili Group A > B (p < 0.001) NS Bnd neutrophil count (/μl) Group A > B (p = 0.001) NS Incresed bnd neutrophil count Group A > B (p < 0.001) NS Lymphocyte count (/μl) Group A > B (p = 0.005) NS Monocyte count (/μl) Group A < B (p = 0.001) NS Monocyte count WRR d Group A > B (p = 0.018) Group A > B (p = 0.021) Eosinophil count (/μl) Group A > B (p = 0.044) NS Pltelet count (/μl) Group A < B (p = 0.013) NS Totl solids WRR Group A < B (p = 0.001) NS Purebred dogs belonged to the following breeds (in lphbeticl order): Boxer, Bulgrin Hound, Dlmtin, Dobermn Pincher, English Pointer, English Setter, Epgneul Breton, French Bulldog, Germn Shepherd, Golden Retriever, Greek Hound, Greek Sheepdog, Griffon Terrier, Jur Hound, Lbrdor Retriever, Pit Bull Terrier, Pres Cnrio, Rottweiler, Shih-Tzu, Siberin Husky nd Turkish Hound b Predisposed breed included Boxer, Dobermn Pincher, English Setter, Germn Shepherd, Pit Bull Terrier nd Rottweiler c MCHC: men corpusculr hemoglobin concentrtion d WRR: within reference rnge e NS: non-significnt (P vlue 0.05) Tble IV: Significnt differences (p<0.05) of historicl informtion, physicl exmintion nd lbortory vribles between stry (group A; n=67) nd privtely-owned (group B; n=115) dogs s well s between seropositive group A (n=18) nd seropositive group B (n=12) dogs Vrible Group A Group B P vlue Sex Mle 10 (55.6%) 8 (66.7%) Femle 8 (44.4%) 4 (33.3%) NS Breed Purebred - 12 (100%) Not purebred 18 (100%) - < Predisposed breed Yes - 5 (41.7%) No 18 (100%) 7 (58.3%) Age (yers) Rnge Medin NS Lifestyle Indoors - 1 (8.3%) Outdoors 18 (100%) 11 (91.7%) Indoors nd outdoors - - NS Smpling during trnsmission period Yes 17 (94.4%) 6 (50%) No 1 (5.6%) 6 (50%) NS: non-significnt with P vlue >0.2 Tble V: Comprison of historicl informtion between seropositive stry (group A; n=18) nd seropositive privtely-owned (group B; n=12) dogs

7 18 SARIDOMICHELAKIS (M.N.) AND COLLABORATORS Vrible Group A (n=18) Group B (n=12) P vlue Poor body condition 5 (27.8%) 4 (33.4%) NS Mucosl pllor 5 (27.8%) 3 (25%) NS Peripherl lymphdenomegly 16 (88.9%) 12 (100%) NS Heptomegly 2 (11.1%) - NS Splenomegly 6 (33.3%) 1 (8.3%) Blephritis 3 (16.7%) 2 (16.7%) NS Conjunctivitis 4 (22.2%) 4 (33.3%) NS Kertitis 1 (5.6%) - NS Uveitis 1 (5.6%) - NS Mstictory muscle trophy 9 (50%) 10 (83.3%) Generlized muscle trophy 3 (16.7%) 4 (33.3%) NS Arthritis - 2 (16.7%) Epistxis - 1 (8.3%) NS Exfolitive dermtitis 2 (11.1%) 3 (25%) NS Ulcertive dermtitis 3 (16.7%) 4 (33.3%) NS Pustulr dermtitis - - Nsl hyperkertosis 7 (38.9%) 8 (66.7%) Footpd hyperkertosis 6 (33.3%) 7 (58.3%) Onychogryphosis 9 (50%) 2 (16.7%) At lest one clinicl sign 16 (88.9%) 12 (100%) NS NS: non-significnt with P vlue >0.2 Tble VI: Comprison of physicl exmintion findings between seropositive stry (group A; n=18) nd seropositive privtely-owned (group B; n=12) dogs Vrible OR SE b z P vlue 95% CI c Dog group nd historicl informtion Dog group Sex Smpling period Dog group, physicl exmintion nd lbortory dt Dog group Mstictory muscle trophy Footpd hyperkertosis Hemtocrit < Pltelet count (x 10 6 /μl) OR: odds rtio b SE: stndrd error c CI: confidence intervl Tble VIII: Results of multiple logistic regression nlysis for possible fctors ssocited with seropositivity to L. infntum

8 LEISHMANIA SEROPOSITIVITY IN STRAY DOGS 19 Vrible Group A (n=18) Group B (n=12) P vlue Hemtocrit (%) Rnge Medin NS c Hemoglobin concentrtion (g/dl) Rnge Medin NS Anemi 11 (61.1%) 8 (66.7%) NS MCHC (g/dl) Rnge Medin White blood cell count (/μl) Rnge 4,900-22,800 4,800-16,500 Medin 13,750 10, White blood cell count Incresed 10 (55.6%) 4 (33.3%) WRR b 7 (38.9%) 7 (58.3%) Decresed 1 (5.6%) 1 (8.3%) NS Neutrophil count (/μl) Rnge 2,989-15,089 3,744-10,792 Medin 7, , Neutrophil count Incresed 4 (22.2%) - WRR 13 (72.2%) 12 (100%) Decresed 1 (5.6%) Bnd neutrophil count (/μl) Rnge 0-1, Medin 0 24 NS Bnd neutrophil count Incresed 3 (16.7%) 1 (8.3%) WRR 15 (83.3%) 11 (91.7%) NS Lymphocyte count (/μl) Rnge 264-9, ,635 Medin 2, ,955 NS Lymphocyte count Incresed 1 (5.6%) 1 (8.3%) WRR 15 (83.3%) 9 (75%) Decresed 2 (11.1%) 2 (16.7%) NS Monocyte count (/μl) Rnge ,415 Medin Monocyte count Incresed - 2 (16.7%) WRR 13 (72.2%) 3 (25%) Decresed 5 (27.8%) 7 (58.3%) Eosinophil count (/μl) Rnge 0-2, ,115 Medin NS Eosinophil count Incresed 8 (44.4%) 5 (41.7%) WRR 6 (33.3%) 3 (25%) Decresed 4 (22.2%) 4 (33.3%) NS Pltelet count (/μl) Rnge 86, ,000 87, ,000 Medin 237, ,000 NS Thrombocytopeni 3 (16.7%) 2 (16.7%) NS Totl solids (g/dl) Rnge Medin NS Totl solids Incresed 5 (27.8%) - WRR 10 (55.6%) 11 (91.7%) Decresed 3 (16.7%) 1 (8.3%) 0.08 MCHC: men corpusculr hemoglobin concentrtion b WRR: within reference rnge c NS: non-significnt with P vlue >0.2 Tble VII: Comprison of the results of hemtology nd totl solid mesurement between seropositive stry (group A; n=18) nd seropositive privtelyowned (group B; n=12) dogs

9 20 MULTIPLE LOGISTIC REGRESSION ANALYSIS The first model included the dog group nd historicl informtion. Of the seven vribles with p vlue 0.2, only six were used; the vribles purebred or not nd breed with incresed seroprevlence nd/or with susceptibility to CnL were highly correlted nd the former ws not entered into the model. The results showed tht being stry (group A) dog ws n independent, highly significnt (p = 0.001), risk fctor for seropositivity (Tble VIII). Additionl fctors ssocited with positive serology included femle sex nd not smpling during the trnsmission period of L. infntum. The second model included the dog group, physicl exmintion, hemtology nd totl solid mesurement. Of the 24 vribles with p vlue 0.2, only 18 were used. Highly correlted pirs of vribles, where only the first vrible ws entered into the model, included: i) hemtocrit nd hemoglobin concentrtion ; ii) incresed, within reference rnge or decresed white blood cell count nd white blood cell count ; iii) incresed, within reference rnge or decresed neutrophil count nd neutrophil count ; iv) incresed, within reference rnge or decresed bnd neutrophil count nd bnd neutrophil count ; v) incresed, within reference rnge or decresed monocyte count nd monocyte count ; nd vi) incresed, within reference rnge or decresed eosinophil count nd eosinophil count. The results showed tht being stry (group A) dog ws n independent, significnt (p = 0.014), risk fctor for seropositivity (Tble VIII). Additionl fctors ssocited with positive serology included mstictory muscle trophy, footpd hyperkertosis, low hemtocrit nd low pltelet count. Discussion In the univrible nlysis, certin historicl informtion differed between stry (group A) nd privtely-owned (group B) dogs. Due to the uncontrolled breeding of sexully intct stry dogs, there were less purebred nd, more importntly, less dogs belonging to predisposed breeds in group A thn in group B (Tble I) nd this difference would fvour seropositivity in the ltter. Indeed, ll (12/12) seropositive group B dogs were purebreds nd 41.7% of them belonged to predisposed breeds (Tble V). On the other hnd, stry dogs lived outdoors more frequently thn group B dogs nd thus they my hve hd incresed chnces of becoming seropositive through repeted exposure to infected snd fly bites. Also, smpling period my hve influenced the results becuse n lmost 2-fold higher percentge of seropositive stry dogs were smpled during the period of snd fly ctivity compred with seropositive privtely-owned dogs (Tble V). The first multivrible model ws designed to overcome the confounding effects on the min reserch inquiry (dog being stry or not) of the bove fctors nd lso of the sex nd the ge of the dogs. In multivrite nlysis, the dog group ws the strongest independent predictor of seropositivity SARIDOMICHELAKIS (M.N.) AND COLLABORATORS with stry dogs hving 4.59-fold higher risk of being seropositive compred with privtely-owned dogs. Two dditionl fctors ssocited with positive serology, femle sex nd not smpling during the trnsmission period, were lso found. Incresed seropositivity in femle dogs hs been reported [29] but in the mjority of seroepidemiologicl surveys no sex predisposition mong privtely-owned nd/or stry dogs hs been found [2, 5, 20]. The higher susceptibility of mle dogs to CnL [19], which is lmost lwys ccompnied by positive ntibody titre [23], my hve reduced the chnces of seropositive mle dog enrolment in the present study: privtely-owned mle dogs with signs of CnL would be more likely to hve been previously dignosed nd thus not be eligible nd stry mle dogs with CnL would hve higher chnces to hve died of the disese. An dditionl explntion for the ssocition between femle dogs (especilly those of group A) nd seropositivity is tht they my hd become infected nd seropositive fter repeted copultion with infected mles [28], wheres the reverse hs not been demonstrted. There is no cler evidence for the influence of the smpling period on the incidence of seropositivity becuse the ltter hs been reported to be incresed [1], similr [10] or even decresed [22] during the trnsmission period of the prsite compred with the rest of the yer. These conflicting results re probbly due to: ) the vrible time period between the repeted exposures to the prsite through multiple bites from infected snd flies during the trnsmission period nd seroconversion nd b) of the vrible time period tht Leishmni-specific IgG remin bove the cut-off titre fter their initil rise. Mny fctors, relted to the frequency of snd fly bites nd to the immunologicl responses of the dogs, re expected to influence the durtion of these time periods [23]. For this reson our finding of 3.29-fold higher risk of seropositivity when smpling ws not done during the trnsmission period should be interpreted crefully tking into considertion the study re which is highly endemic [17, 25] nd the study popultion which consisted by more thn one third of stry dogs. It is lso conceivble tht tretment of the smpling period s binry vrible, lthough convenient for sttisticl purposes, is rtificil (e.g. no ctul difference is expected between smpling few dys before the end of the trnsmission period nd smpling few dys fter its end). In the univrible nlysis, splenomegly ws the only physicl exmintion finding tht differed between the two groups, being more frequent in stry thn in privtelyowned dogs. An incresed prevlence of CnL in group A is not likely explntion for this difference becuse the other clinicl signs s well s the presence of t lest one CnLssocited clinicl sign were not more frequent mong stry thn privtely-owned dogs. Similrly, n incresed severity of CnL in group A dogs does not seem to be responsible becuse there were no differences in the comprison of

10 LEISHMANIA SEROPOSITIVITY IN STRAY DOGS 21 physicl exmintion findings (including splenomegly) between seropositive stry nd seropositive privtely-owned dogs. Detection of splenomegly on bdominl plption is subjective [16] nd this is exemplified by the highly vrible prevlence of this finding in CnL which hs been reported to rnge from less thn 10% [16] up to more thn 50% [4]. The dogs of the present study were exmined by different clinicins nd visul exmintion of the dt clerly showed tht the prevlence of splenomegly depended on the exminer. In ddition, due to the lck of regulr prevention ginst ectoprsites, stry dogs my hve been predisposed to other vector-borne diseses, like monocytic ehrlichiosis nd bbesiosis, which re lso ssocited with splenomegly. Similrly, concurrent diseses nd infections re the most plusible explntion for the significnt differences of lbortory dt between group A nd group B dogs. In the multivrible nlysis, the dog group ws gin the strongest independent predictor of seropositivity with stry dogs hving 3.72-fold higher risk of being seropositive compred with privtely-owned dogs. Interestingly, dditionl fctors ssocited with positive serology in the second multivrible model included two clinicl signs tht re reltively specific for CnL nd the two more frequent hemtologicl bnormlities of the disese. Mstictory muscle trophy occurs due to CnL-triggered immunemedited inflmmtion of mstictory muscles [30], footpd hyperkertosis is mnifesttion of loclized defect of kertiniztion [24] nd both signs hve reltively few differentils tht do not include other vector-borne diseses. On the contrry, reduced hemtocrit nd low pltelet counts re frequent in mny vector-borne diseses but they re lso seen in 50% or more of dogs with CnL [16]. There re mny, non-mutully exclusive, explntions for the incresed risk of seropositivity in stry dogs tht re confined in niml shelters. These dogs my hve been bndoned by irresponsible owners fter they were found to be seropositive due to misconceptions of the public helth implictions nd/or due to the cost nd the commitment tht re needed for the tretment of CnL. Confinement in n niml shelter is mjor psychogenic stress tht stimultes hypothlmic-pituitry-drenl xis nd the resultnt hypercortisolemi [15] my hve downregulted Leishmni-specific cell-medited immunity leding to prsite multipliction nd ntibody production [23]. Similrly, suboptiml nutrition my lso hve contributed to the suppression of the protective cell-medited immune responses. Not using insect repellents is expected to increse the exposure of stry dogs to L. infntum, wheres their lifestyle nd the lck of prevention ginst ectoprsites my predispose them to numerous co-infections (e.g. by Ehrlichi cnis, Anplsm phgocytophilum, Bbesi spp., Neospor cninum) tht re ssocited with seropositivity [8, 12]. In ny cse, the results of the present study clerly show tht stry dogs tht re confined in niml shelters re t incresed risk of seropositivity nd tht these dogs probbly pose n incresed risk of L. infntum trnsmission to the snd fly vectors [7, 21]. Subsequently, implementtion of preventive mesures (e.g. use of insect repellents, vccintion of seronegtive dogs, nd tretment of dogs with CnL) in stry dogs should become priority in public helth cmpigns ginst leishmniosis. Shortcomings of the present study include the reltively smll number of dogs, filure to exmine for ssocition between biochemicl vribles nd results of serology, lck of dt on possible co-infections, not using other more ccurte predictors of infectiousness to snd flies nd lck of informtion on the density of snd fly vectors in the niml shelter nd the residencies of group B dogs. The number of stry dogs tht were enrolled hd to be restricted due to the limited funding, wheres client-owned dogs included ll eligible dogs tht were dmitted to the Clinic during the sme time period nd hd been tested serologiclly for Leishmni spp. However, in both multivrible models the incresed seroprevlence of stry dogs ws ssocited with highly significnt p vlues nd with lower limits of 95% confidence intervls lying wy from 1. Therefore, the min outcome of the study would probbly remin the sme if more dogs hd been enrolled. Biochemicl vribles were not nlysed becuse there were mny missing dt, but even if this informtion ws vilble, it could only influence the results of the second multivrible model. Investigtion for possible co-infections would hve enlightened one possible reson for the incresed risk of seropositivity mong stry dogs but would not hve chnged the min study outcome. Currently, xenodignosis is the gold stndrd to estimte infectiousness to snd flies nd prsitic density in the er skin, mesured by quntittive PCR, its best surrogte mrker [6]. Nevertheless, serology is less techniclly demnding, invsive nd costly nd ccurte for this purpose. A higher bundnce of infected snd flies in the niml shelter on in the res where sty dogs hd been cptured thn in the residencies of group B dogs could ccount for the incresed seroprevlence of the former. Obviously, it ws not fesible to exmine vector density in the residencies of client-owned dogs or in the res where stry dogs were living before entering the kennel. Nevertheless, the dministrtive region of Thessly (i.e. the prefectures of Krdits, Trikl, Lriss nd Mgnesi), s whole, is considered highly endemic re where cnine infection by L. infntum, seropositivity nd CnL re very frequent in urbn, suburbn nd rurl res [17, 25, 27]. In conclusion, stry dogs confined in niml shelters in highly endemic re hve pproximtely 4-fold higher risk of being seropositive to L. infntum compred with privtely-owned dogs living in the sme re. Additionl fctors ssocited with seropositivity include femle sex, not smpling during the trnsmission period, mstictory muscle trophy, footpd hyperkertosis, low hemtocrit vlue nd low pltelet count.

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