Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Dogs
2.2. Quantitative ELISA for the Detection of L. infantum-Specific Antibodies
2.3. Blood DNA Extraction and Leishmania quantitative PCR
2.4. IFN-γ Release Whole Blood Stimulation Assay (WBA)
2.5. Statistical Analysis
3. Results
3.1. Signalment
3.2. Clinical Data
3.3. Clinicopathological Findings in Seropositive Sick Dogs
3.4. Relationship between Clinicopathological Findings, Signalment, Antibody Levels, Leishmania Quantitative PCR, IFN-γ Concentration and Clinical Staging
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Qualitative Characteristics | Total (n = 212) % (95% CI) | Seropositive Healthy (n = 105) % (95% CI) | Seropositive Sick (n = 107) % (95% CI) | p-Value (Fisher’s Exact Test) | |
---|---|---|---|---|---|
Breed | Crossbreed | 43.9 (37.1–50.8) | 46.7 (36.9–56.7) | 41.1 (31.7–51) | 0.49 |
Purebred | 56.1 (49.2–62.9) | 53.3 (43.3–63.1) | 58.9 (48.9–68.3) | ||
Sex | Female | 43.4 (36.6–50.4) | 41 (31.5–51) | 45.8 (36.1–55.7) | 0.49 |
Male | 56.6 (49.6–63.4) | 59 (49–68.5) | 54.2 (44.3–63.9) | ||
ELISA positivity | High or medium | 44.8 (38–51.8) | 19 (12–27.9) | 70.1 (60.5–78.6) | <0.0001 |
Low | 55.2 (48.2–62) | 81 (72.1–88) | 29.9 (21.4–39.5) | ||
Leishmania qPCR (n = 147) | Positive | 44.9 (36.7–53.3) | 28.4 (18.5–40.1) | 61.6 (49.5–72.8) | <0.0001 |
Negative | 55.1 (46.7–63.3) | 71.6 (60–81.5) | 38.4 (27.2–50.5) | ||
LSA IFN-γ production (n = 141) | Producer | 46.8 (38.4–55.4) | 58.3 (44.9–70.9) | 38.3 (27.7–49.7) | 0.026 |
Non-producer | 53.2 (44.6–61.6) | 41.7 (29.1–55.1) | 61.7 (50.3–72.3) | ||
LeishVet stage * | II ** | NA | NA | 19.6 (12.6–28.4) | NA |
IIa | 55.1 (45.2–64.8) | ||||
IIb | 9.4 (4.6–16.5) | ||||
III | 15 (8.8–23.1) | ||||
IV | 0.9 (0–5.1) | ||||
Quantitative Characteristics | Total (n = 212) Median (min–max) | Seropositive Healthy (n = 105) Median (min–max) | Seropositive Sick (n = 107) Median (min–max) | p-Value (Mann-Whitney U test) | |
Age (years) | 5 (0.5–14) | 4 (1–14) | 5 (0.5–12) | 0.09 | |
Weight (kg) | 22 (3–62) | 23 (6–62) | 20 (3–58) | 0.45 | |
Endpoint ELISA (EU) | 247 (51–61286) | 137 (51–1181) | 789 (75–61286) | <0.0001 | |
Leishmania qPCR (parasites/mL) (n = 147) | 0.1 (0.003–886.6) | 0.01 (0.004–5.86) | 0.48 (0.003–886.6) | 0.002 | |
IFN-γ concentration (pg/mL) (n = 141) | Medium with LSA | 80 (0–14,190) | 203 (0–14,190) | 20 (0–8001) | 0.0008 |
Medium with ConA | 5985 (805–43,290) | 6929 (1147–43,290) | 5447 (805–38,198) | 0.04 |
CBC Parameters (Units) | Reference Intervals * [32,40] | Total (n = 212) Median (Min–Max) | Seropositive Healthy (n = 105) Median (Min–Max) | Seropositive Sick (n = 107) Median (Min–Max) | p-Value |
---|---|---|---|---|---|
RBC (106/µL) | 5.1–7.6 | 6.6 (4–9.1) | 6.8 (5–9.1) | 6.1 (4–8.3) | <0.0001 a |
Hemoglobin (g/dL) | 12.4–19.2 | 16.2 (10.4–22.5) | 17.1 (13.4–22.5) | 15.2 (10.4–19.8) | <0.0001 b |
Hematocrit (%) | 35–52 | 47 (29–62) | 49 (36–62) | 44 (29–59) | <0.0001 c |
MCV (fL) | 60–77 | 71 (59–81) | 71 (59–80) | 71 (61–81) | 0.27 |
MCH (pg) | 21.9–26.3 | 24.6 (20.2–33.3) | 24.9 (20.2–33.3) | 24.2 (21.5–28.6) | 0.02 d |
MCHC (g/dL) | 34.4–38.1 | 34.4 (29.5–45.8) | 35.2 (29.9–45.8) | 34 (29.5–39.7) | <0.0001 e |
WBC (109/L) | 5.6–20.4 | 9.5 (3.4–23.5) | 9.4 (4.8–23.5) | 9.6 (3.4–22.7) | 0.91 |
Neutrophils (109/L) | 2.9–13.6 | 6.1 (2.4–20) | 6 (2.7–17.2) | 6.2 (2.4–20) | 0.52 |
Lymphocytes (109/L) | 1.1–5.3 | 1.9 (0.2–4.7) | 2.1 (0.4–4.5) | 1.7 (0.2–4.7) | <0.0001 f |
Monocytes (109/L) | 0.4–1.6 | 0.4 (0–2) | 0.4 (0.1–1.6) | 0.4 (0–2) | 0.49 |
Eosinophils (109/L) | 0.1–3.1 | 0.4 (0–4) | 0.5 (0–4) | 0.4 (0–3.3) | 0.47 |
Platelets (103/µL) ** | 200–500 | Adequate | Adequate | Adequate | - |
Parameters (Units) | Reference Intervals [41,42,43] | Total (n = 212) Median (Min–Max) | Seropositive Healthy (n = 105) Median (Min–Max) | Seropositive Sick (N = 107) Median (Min–Max) | p-Value |
---|---|---|---|---|---|
Total protein (g/L) | 54–71 | 71 (54–117) | 67 (54–83) | 75 (59–117) | <0.0001 a |
Albumin (g/L) | 26–33 | 33 (24–56) | 34 (26–52) | 33 (24–56) | 0.002 b |
Globulin (g/L) | 27–44 | 37 (26–81) | 32 (26–41) | 42 (29–81) | <0.0001 c |
A/G ratio | 0.86–1.93 | 0.9 (0.4–1.7) | 1.1 (0.7–1.7) | 0.8 (0.4–1.5) | <0.0001 d |
ALT (U/L) | 21–102 | 43 (12–1037) | 44 (18–132) | 42 (12–1037) | 0.19 |
ALP (U/L) | 20–156 | 47 (14–1271) | 44 (14–208) | 55 (20–1271) | 0.001 e |
Creatinine (mg/dL) | 0.5–1.5 | 0.9 (0.4–3.9) | 0.9 (0.5–1.4) | 0.8 (0.4–3.9) | 0.01 f |
Urea (mg/dL) | 21.4–59.9 | 35 (14–155) | 35 (14–77) | 34 (14–155) | 0.81 |
Serum electrophoresis (g/L) | |||||
Albumin | 24.4–49.6 | 34.5 (19.1–49.1) | 36.7 (25.5–45.4) | 31.5 (19.1–49.1) | <0.0001 g |
Alpha-1 globulin | 1.7–4.5 | 3.5 (1.7–8.4) | 3.4 (1.7–4.8) | 3.7 (2–8.4) | 0.008 h |
Alpha-2 globulin | 3.8–10.2 | 7.3 (2.9–18.2) | 6.5 (2.9–12.7) | 8.1 (4.3–18.2) | <0.0001 i |
Beta globulin | 8–18 | 13.2 (7.5–37.9) | 12.3 (7.5–17.9) | 14.7 (9.2–37.9) | <0.0001 j |
Gamma globulin | 2.6–11.7 | 10.1 (4.4–60.6) | 7.9 (4.4–11.9) | 15.1 (5.3–60.6) | <0.0001 k |
UPCR | <0.5 | 0.1 (0–101.8) | 0.1 (0.02–0.44) | 0.2 (0–101.8) | 0.007 l |
USG (g/L) | 1006–1050 | 1034 (1007–1058) | 1036 (1016–1058) | 1031 (1007–1056) | 0.06 |
Clinicopathological Findings | Number of Dogs (%; 95% CI) |
---|---|
Hematological alterations (n = 106) | 42 (39.6;30.3–49.6) |
Anemia | 7 (6.6;2.7–13.3) |
Lymphopenia | 23 (21.7;14.3–30.8) |
Leukopenia | 8 (7.6;3.3–14.3) |
Lymphopenic leukopenia | 4 (3.8;1–9.4) |
Neutropenic leukopenia | 1 (0.9;0.02–5.1) |
Neutropenic and lymphopenic leukopenia | 3 (2.8;0.6–8.1) |
Neutrophilic leukocytosis | 1 (0.9;0.02–5.1) |
Neutrophilia | 3 (2.8;0.6–8.1) |
Neutrophilia and lymphopenia | 1 (0.9;0.02–5.1) |
Eosinophilia | 3 (2.8;0.6–8.1) |
Eosinophilia and monocytosis | 1 (0.9;0.02–5.1) |
Thrombocytopenia | 3 (2.8;0.6–8.1) |
Thrombocytosis | 2 (1.9;0.2–6.7) |
Biochemical alterations (n = 107) | 105 (98.1;93.4–99.8) |
Serum protein alterations (n = 106) | 87 (82.1;73.4–88.9) |
Hyperproteinemia | 76 (71.7;62.1–80) |
Hypoalbuminemia | 12 (11.3;6–18.9) |
Hyperglobulinemia | 48 (45.3;35.6–55.3) |
Decreased A/G ratio | 68 (64.1;54.3–73.2) |
Polyclonal hypergammaglobulinemia | 79 (74.5;65.1–82.5) |
Polyclonal hyperbetaglobulinemia | 26 (24.5;16.7–33.8) |
Polyclonal hypergammaglobulinemia and hyperbetaglobulinemia | 18 (17.9;11.1–26.6) |
Protein electrophoretic patterns (n = 106) | 87 (82.1;73.4–88.9) |
Chronic antigenic stimulation | 69 (65.1;55.2–74.1) |
Chronic antigenic stimulation and acute phase response | 18 (17;10.4–25.5) |
Normal | 19 (17.9;11.2–26.6) |
Renal alterations | 40 (46;35.2–57) |
Proteinuria (n = 87) | 31 (35.6;25.7–46.6) a |
Inadequate USG | 40 (46;35.2–57) |
Isosthenuria (n = 87) | 10 (11.5;5.7–20.1) a |
Renal azotemia (n = 106) | 2 (1.9;0.2–6.7) |
Increased hepatic enzymes (n = 106) | 14 (13.2;7.4–21.2) |
Others (n = 106) | 23 (21.7;14.3–30.8) |
Low urea | 13 (12.3;6.7–20.1) |
High urea | 6 (5.7;2.1–11.9) a |
Low creatinine | 5 (4.7;1.6–10.7) |
High creatinine | 3 (2.8;0.6–8.1) a |
CBC Parameters | Age | Serology ELISA Units | IFN-γ Concentration (LSA Stimulation) | IFN-γ Concentration (ConA Stimulation) | ||||
---|---|---|---|---|---|---|---|---|
rs | p-Value | rs | p-Value | rs | p-Value | rs | p-Value | |
RBC | 0.01947 | 0.7786 | −0.3382 | <0.0001 | 0.2742 | 0.001 | 0.1746 | 0.0391 |
Hemoglobin | 0.08224 | 0.2342 | −0.3831 | <0.0001 | 0.3046 | 0.0003 | 0.2039 | 0.0157 |
Hematocrit | 0.0547 | 0.4293 | −0.4288 | <0.0001 | 0.2801 | 0.0008 | 0.09803 | 0.2492 |
Reticulocytes | −0.02686 | 0.8332 | −0.5704 | <0.0001 | −0.1212 | 0.5816 | 0.02174 | 0.9216 |
MCV | 0.0464 | 0.5026 | −0.1664 | 0.0155 | −0.07767 | 0.3617 | −0.1441 | 0.0894 |
MCH | 0.08829 | 0.2015 | −0.2123 | 0.0019 | 0.1062 | 0.2118 | 0.07653 | 0.3688 |
MCHC | 0.05852 | 0.3977 | −0.07718 | 0.2643 | 0.2278 | 0.0068 | 0.2848 | 0.0006 |
WBC | −0.1429 | 0.0390 | −0.08484 | 0.222 | 0.07476 | 0.3818 | 0.02388 | 0.7803 |
Neutrophils conc | −0.09672 | 0.1646 | −0.05535 | 0.4272 | 0.04511 | 0.5993 | −0.04691 | 0.5849 |
Band Neutrophils conc | −0.1475 | 0.0322 | 0.0853 | 0.2172 | 0.00141 | 0.9868 | 0.00279 | 0.9739 |
Lymphocytes conc | −0.1089 | 0.1174 | −0.161 | 0.0202 | 0.08133 | 0.343 | 0.1589 | 0.0626 |
Monocytes conc | −0.1048 | 0.1291 | 0.1221 | 0.0767 | −0.04173 | 0.6245 | 0.03668 | 0.667 |
Eosinophils conc | −0.1263 | 0.0677 | −0.1483 | 0.0317 | 0.063 | 0.4596 | 0.03614 | 0.6717 |
Basophils conc | −0.07155 | 0.3009 | −0.2184 | 0.0014 | 0.0234 | 0.7837 | −0.00702 | 0.9344 |
Platelets conc * | - | - | - | - | - | - | - | - |
Parameters (Reference Intervals, Units) | Stage II (n = 21) Median (Min–Max) | Stage IIa (n = 59) Median (Min–Max) | Stage IIb (n = 10) Median (Min–Max) | Stage III (n = 16) Median (Min–Max) | p-Value (Kruskal-Wallis H Test) |
---|---|---|---|---|---|
Total protein (54–71, g/L) | 81.5 (66–117) | 74 (59 -103) | 68 (60–89) | 75.5 (71–110) | 0.01697 a |
Albumin (26–33, g/L) | 33 (24–47) | 34 (26–56) | 31 (26–35) | 30 (25–39) | 0.03142 b |
Globulin (27–44, g/L) | 45.5 (37–81) | 40 (29–71) | 36 (30–63) | 46 (37–75) | 0.001 c |
A/G ratio (0.86–1.93) | 0.68 (0.27–0.89) | 0.82 (0.26–1.33) | 0.86 (0.35–1.34) | 0.52 (0.27–1.52) | 0.04389 d |
ALT (21–102, U/L) | 31.5 (12–278) | 44 (17–1037) | 38 (18–88) | 52 (21–228) | 0.0239 e |
ALP (20–156, U/L) | 63.5 (20–107) | 55 (20–1271) | 48.5 (20–105) | 51.5 (20–861) | 0.71 |
Creatinine (0.5–1.5, mg/dL) | 0.94 (0.6–1.22) | 0.8 (0.43–1.39) | 0.77 (0.62–1.21) | 0.81 (0.47–1.9) | 0.42 |
Urea (21.4–59.9, mg/dL) | 35 (14–54) | 32 (14–62) | 37.5 (24–80) | 46.5 (19–150) | 0.046 f |
Serum electrophoresis (g/L)
|
|
|
|
|
|
UPCR (<0.5) | 1.1 (0.2–1.92) | 0.11 (0.02–0.58) | 0.96 (0.61–1.84) | 1.93 (0.13–101.75) | <0.0001 j |
USG (1006–1050, g/L) | 1033.5 (1026–1041) | 1034.5 (1007–1056) | 1020.5 (1008–1042) | 1024 (1008–1046) | 0.0194 k |
Parameters (Units) | Age | Serology ELISA Units | IFN-γ Concentration (LSA Stimulation, pg/mL) | IFN-γ Concentration (ConA Stimulation, pg/mL) | ||||
---|---|---|---|---|---|---|---|---|
rs | p-Value | rs | p-Value | rs | p-Value | rs | p-Value | |
Total protein (g/L) | −0.071 | 0.31 | 0.4 | <0.0001 | −0.2109 | 0.0124 | −0.1507 | 0.0756 |
Albumin (g/L) | −0.06 | 0.39 | −0.223 | 0.001 | 0.1381 | 0.1037 | 0.1109 | 0.1921 |
Globulin (g/L) | −0.008 | 0.91 | 0.51 | <0.0001 | −0.2593 | 0.002 | −0.2206 | 0.0088 |
A/G ratio | −0.01 | 0.13 | −0.55 | <0.0001 | 0.2522 | 0.0027 | 0.2261 | 0.0072 |
ALT (U/L) | 0.24 | 0.0004 | −0.095 | 0.18 | 0.1345 | 0.1185 | 0.1032 | 0.2319 |
ALP (U/L) | 0.13 | 0.07 | 0.14 | 0.041 | −0.05758 | 0.4992 | −0.03891 | 0.6481 |
Creatinine (mg/dL) | −0.18 | 0.007 | −0.079 | 0.26 | −0.01492 | 0.8611 | 0.1262 | 0.1373 |
Urea (mg/dL) | −0.091 | 0.19 | −0.06 | 0.4 | −0.06855 | 0.4210 | 0.06555 | 0.4416 |
Serum electrophoresis (g/L) | ||||||||
Albumin | −0.13 | 0.06 | −0.46 | <0.0001 | 0.1792 | 0.0342 | 0.1899 | 0.0246 |
Globulins | 0.032 | 0.64 | 0.53 | <0.0001 | −0.2499 | 0.0029 | −0.2407 | 0.0042 |
Alpha-1 globulin | −0.03 | 0.69 | 0.11 | 0.12 | −0.08755 | 0.3036 | −0.1649 | 0.0516 |
Alpha-2 globulin | 0.13 | 0.07 | 0.31 | <0.0001 | −0.2067 | 0.0146 | −0.2083 | 0.0139 |
Beta globulin | 0.003 | 0.96 | 0.16 | 0.02 | −0.06619 | 0.4371 | −0.0768 | 0.3671 |
Gamma globulin | 0.05 | 0.49 | 0.6 | <0.0001 | −0.2295 | 0.0064 | −0.2273 | 0.0069 |
UPCR | 0.15 | 0.04 | 0.2 | 0.007 | 0.1315 | 0.2888 | −0.08458 | 0.4962 |
USG (g/L) | −0.075 | 0.32 | −0.054 | 0.48 | −0.0269 | 0.7715 | 0.1352 | 0.1426 |
Categorical Alterations (Number of Dogs) | Stage II (n = 21) N (%) | Stage IIa (n = 59) N (%) | Stage IIb (n = 10) N (%) | Stage III (n = 16) N (%) | Stage IV (n = 1) N (%) | p-Value (Chi-Squared; df) |
---|---|---|---|---|---|---|
Thrombocytosis (n = 2) | 0 (0) | 2 (3.4) | 0 (0) | 0 (0) | 0 (0) | <0.001 (37.8; 8) |
SPE pattern: chronic antigenic stimulation (n = 69) | 15 (75) | 40 (67.8) | 4 (40) | 10 (62.5) | 0 (0) | 0.004 (22.9; 8) |
Hyperglobulinemia (n = 48) | 12 (60) | 21 (35.6) | 3 (30) | 12 (75) | 0 (0) | 0.022 (11.5; 4) |
Increased alpha-2-globulins (n = 14) | 3 (15) | 5 (8.5) | 0 (0) | 6 (37.5) | 0 (0) | 0.025 (11.1; 4) |
Hypergammaglobulinemia (n = 79) | 19 (95) | 44 (74.6) | 4 (40) | 12 (75) | 0 (0) | 0.009 (13.6; 4) |
Decreased A/G ratio (n = 68) | 16 (80) | 33 (55.9) | 4 (40) | 14 (87.5) | 1 (100) | 0.029 (10.81; 4) |
Renal azotemia (n = 2) | 0 (0) | 0 (0) | 0 (0) | 1 (6.3) | 1 (100) | <0.001 (55.4; 4) |
Increased creatinine (n = 3) | 0 (0) | 0 (0) | 0 (0) | 2 (12.5) | 1 (100) | <0.001 (46.4; 8) |
Increased urea (n = 6) | 0 (0) | 1 (1.7) | 1 (10) | 3 (18.8) | 1 (100) | 0.001 (27.8; 8) |
Proteinuria (n = 31) | 1/2 (50) | 4/58 (6.9) | 10/10 (100) | 15/16 (93.8) | 1/1 (100) | <0.001 (64.5; 4) |
Isosthenuria (n = 10) | 0/2 (0) | 4/58 (6.9) | 2/10 (20) | 3/16 (18.8) | 1/1 (100) | 0.03 (10.7; 4) |
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Baxarias, M.; Jornet-Rius, O.; Donato, G.; Mateu, C.; Alcover, M.M.; Pennisi, M.G.; Solano-Gallego, L. Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs. Animals 2023, 13, 1649. https://doi.org/10.3390/ani13101649
Baxarias M, Jornet-Rius O, Donato G, Mateu C, Alcover MM, Pennisi MG, Solano-Gallego L. Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs. Animals. 2023; 13(10):1649. https://doi.org/10.3390/ani13101649
Chicago/Turabian StyleBaxarias, Marta, Oriol Jornet-Rius, Giulia Donato, Cristina Mateu, Mª Magdalena Alcover, Maria Grazia Pennisi, and Laia Solano-Gallego. 2023. "Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs" Animals 13, no. 10: 1649. https://doi.org/10.3390/ani13101649
APA StyleBaxarias, M., Jornet-Rius, O., Donato, G., Mateu, C., Alcover, M. M., Pennisi, M. G., & Solano-Gallego, L. (2023). Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs. Animals, 13(10), 1649. https://doi.org/10.3390/ani13101649