Synovial fluid and radiographic assessment of joints in dogs with visceral leishmaniasis | Parasites & Vectors

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animals and groups

The dogs included in this study were referred to the Municipal Control Center for Zoonoses due to a diagnosis of CanVL when euthanasia of these animals was recommended in Brazil; consequently it was not possible to obtain her medical history. However, all dogs showed multiple clinical signs of CanVL at the time of sampling, such as weight loss, skin lesions, onychogryphosis, epistaxis, lymphadenopathy, and diarrhea.

Of the 46 G1 dogs, 21 (45.6%) were male and 25 (54.3%) were female. There were 32 (69.5%) mixed breeds and 14 (30.4%) pedigree dogs (Doberman, n = 3; Poodle, n = 2; Cocker spaniel, n = 2; Boxer, n = 2; Rottweiler, n = 2; bull terrier, n = 1; yorkshire terrier, n = 1; labrador retriever, n = 1). The ages of the G1 dogs ranged from 1 to 12 (median: 3) years, with 32 dogs (69.5%) being 1 to 3 years old, 12 (26.0%) being 4 to 7 years old and two (4.3%) > 8 years.

Of the 43 G2 dogs, 28 (65.1%) were male and 15 (34.8%) were female. There were 28 (65.1%) mixed breed and 15 (34.8%) purebred dogs (poodle, n = 5; Dachshund, n = 5; Brazilian Terrier, n= 3; Cocker spaniel, n= 1; Maltese, n= 1). The age of the G2 dogs ranged from 1 to 12 (median: 3) years, with 24 (55.8%) dogs being 1 to 3 years old, 17 (39.5%) dogs being 4 to 7 years old and two (4.6%) dogs age > 8 years.

X-ray examination

Overall, 74 (83.1%, 95% CI: 74.0-89.5) of the 89 dogs examined in the study had joint abnormalities on radiographs suggestive of osteoarthritis: 40/46 (86.9%; 95% CI = 74.3-93.8) in G1 and 34/43 (79.0%; 95% CI = 64.7-88.5) in G2. There was no difference between the groups (Fisher’s exact test, P= 0.320, OR = 1.7, 95% CI = 0.5-5.4). All 74 dogs (G1 and G2) with abnormal joint radiographs had bilateral radiographic lesions regardless of lesion characteristics, with 65 (87.8%, 95% CI = 78.4-93.4) ​​dogs affecting the tarsal joints were carpal joints in 51 (68.9%, 95% CI = 57.6-78.3) dogs, elbows in 25 (33.7%, 95% CI = 24.0-45.1) dogs and knee joints in 10 (13.5%, 95% CI = 7.5-23.1) dogs.

In G1 dogs, radiographic findings with abnormalities suggestive of osteoarthritis were observed at 162 joints: (i) apparent trabecular pattern in 118 (72.8%; 95% CI = 65.5-79.1) joints; (ii) subchondral bone sclerosis in 91 (56.1%; 95% CI = 48.4-63.5) joints; (iii) osteolysis in 89 (54.9%; 95% CI = 47.2-62.4) joints; (iv) soft tissue swelling around the joint (edema) in 77 (47.5%; 95% CI = 39.9-55.1) joints; (v) joint space narrowing in 58 (35.8%; 95% CI = 28.8-43.4) joints; (vi) mixed bone lesions (osteolytic-proliferative lesions) in 15 (9.2%; 95% CI = 5.6-14.7) joints; and (vi) bone proliferation in 10 (6.1%; 95% CI = 3.3-10.9) joints (Fig. 1).

In G2 dogs, radiographic findings with abnormalities suggestive of osteoarthritis were observed at the 140 joints: (i) apparent trabecular pattern in 112 (80.0%; 95% CI = 72.6-85.7) joints; (ii) subchondral bone sclerosis in 73 (52.1%; 95% CI = 43.9-60.2) joints; (iii) osteolysis in 62 (44.2%; 95% CI 36.3-52.5) ​​joints; (iv) joint space narrowing in 40 (28.5%; 95% CI = 21.7-36.5) joints; (v) soft tissue swelling around the joint (edema) in 34 (24.2%; 95% CI = 17.9-32.0) joints; (vi) mixed bone lesions (osteolytic-proliferative lesions) in 13 (9.2%; 95% CI = 5.5-15.2) joints; (vii) bone proliferation in nine (6.4%; 95% CI = 3.4-11.7) joints; and (viii) increased medullary opacity in one (0.7%; 95% CI = 00.1-03.9) joint.

Soft tissue swelling around the joint (Fisher’s exact test, PP= 0.029, OR = 1.7, 95% CI = 1.0-2.7) were observed more frequently in G1 than in G2 dogs. There was no difference between the groups with regard to other abnormalities indicative of osteoarthritis, such as B. An obvious trabecular pattern (Fisher’s exact testP= 0.145, OR = 0.6, 95% CI = 0.3-1.1), subchondral bone sclerosis (Fisher’s exact test, P= 0.483, OR = 1.1, 95% CI = 0.7-1.8), osteolysis (Fisher’s exact test, P= 0.064, OR = 1.5, 95% CI = 0.9-2.4), osteolytic-proliferative lesions (Fisher’s exact test, P= 0.993, OR = 0.9, 95% CI = 0.4-2.1) or bone proliferation (Fisher’s exact testP= 0.927, OR = 0.9, 95% CI = 0.3-2.4).

Fig. 1

Fig. 1. Mediolateral radiograph of the carpus (A), elbow (B) and tarsus (D) and dorsoplantar of the tarsal (C) joints in a dog with visceral leishmaniasis. Observe collapse of the joint space [arrow – (A)]bone sclerosis [arrow – (C)]distinct trabecular bone [asterisk – (D)]irregular periosteal reaction [arrow – (D)]and osteolysis [arrow head – (B and C)]. 1. radius; 2. cubit; 2a. olecranon; 3. carpal bones; 4. Accessory wrist; 5. Metacarpal; 6. humerus; 7. condyle of the humerus; 8. tibia; 9. heel bone; 10. talus; 11. tarsal bones; 12. Metatarsal bones.

SF exam

The SF was obtained without complications (sufficient volume and no blood in the sample) from 315 of 368 (85.5%) joints of G1 dogs and from 307 of 344 (89.2%) joints of G2 dogs. SF derived from 174 of 315 (55.2%; 95% CI = 49.7-60.6) joints of G1 dogs and from 152 of 307 (49.5%; 95% CI = 43.9-55 ,0) joints collected from G2 dogs showed an inflammatory infiltrate with no significant association between the presence of inflammatory infiltrate and the groups (neutrophils: Mann-Whitney U test, u(18)= 12,969, Z= 1.71, P= 0.083; mononuclear cells: Mann-Whitney U test, u(18)= 12,909, Z= − 1.78, P= 0.072). The inflammatory infiltrate consisted of non-degenerate and degenerate neutrophils and mononuclear cells (small lymphocytes, monocytes and macrophages), with rare observations of erythrocytes in some specimens. In none of the evaluated joints was there a statistical difference between the groups in the proportion of neutrophils (Tarsal: Mann-Whitney-U-Test, u(18)= 880, Z= 1.57, P= 0.112; Carpal: Mann-Whitney U test, u(18)= 921, Z= 0.12, P= 0.899; Elbow: Mann-Whitney U test, u(18)= 686, Z= 1.20, P= 0.223; Knee joints: Mann-Whitney U test, u(18)= 757, Z= 0.49, P= 0.194) or mononuclear cells (tarsal: Mann-Whitney U test, u(18)= 880, Z= − 1.57, P= 0.112; Carpal: Mann-Whitney U test, u(18)= 921, Z=−0.12, P= 0.899; Elbow: Mann-Whitney U test, u(18)= 674.5, Z= − 1.31, P= 0.184; Knee joints: Mann-Whitney U test, u(18)= 757, Z= − 0.49, P= 0.613). The proportion of neutrophils varied from 15% to 100% (mean: 45.89 ± 22.02) in G1 dogs and from 15% to 90% (mean: 41.7 ± 20.8) in G2 dogs (male -Whitney U test, u(18)= 12,969, Z= 1.71, P= 0.083). The proportion of mononuclear cells varied from 0 to 85% in G1 dogs (mean: 53.9 ± 22.0) and from 10% to 85% (mean: 58.2 ± 20.8) in G2 dogs (male Whitney U test, u(18)= 12,909, Z= − 1.78, P= 0.072).

All joints showing SF with an inflammatory infiltrate also had radiographic evidence of osteoarthritis. Many macrophages contained magenta-staining organisms 2–3 µm in length and a kinetoplast compatible with Leishmaniasp. Amastigotes were also visualized (Fig. 2). Leishmaniasp. Amastigotes were observed free and within macrophages.

Fig. 2
figure 2

Photomicrograph of a swab of synovial fluid from a dog with visceral leishmaniasis Leishmaniasp. Amastigotes (arrows) in macrophages (×100). The picture shows a mixed inflammatory infiltrate of mononuclear cells (mainly macrophages and lymphocytes) and polymorphonuclear cells (mainly segmented neutrophils)

Leishmaniasp. Amastigotes were found in the joints of 29 of the 46 (63.0%; 95% CI = 48.6-75.4) G1 dogs and 21 of the 43 (48.8%, 95% CI = 34.6-63 ,2) G2 dogs found, with no statistical significance -group difference (Fisher’s exact test, P= 0.177, OR = 1.7, 95% CI = 0.7-4.1); this corresponds to 169 of the 622 (27.1%; 95% CI = 23.8-30.8) joints evaluated. Amastigotes were found in 69 of 315 (21.9%; 95% CI = 17.6-26.8) joints of G1 dogs and in 100 of 307 (32.5%; 95% CI = 27.5-38, 0) joints of G2 dogs observed (Fisher’s exact test, P= 0.002, OR = 0.5, 95% CI = 0.4-0.8) (Table 1). Per dog is the spectrum of infected joints Leishmaniasp. Amastigotes varied from one to eight in both groups, and the percentage of infected joints among the joints evaluated was similar (tarsal: 28.6%; carpus: 28.0%; elbow: 26.8%; knee: 24.8% ). The neutrophil infiltrate was significantly higher in joints with Leishmaniasp. Amastigot forms in both G1 (Mann-Whitney U-Test, u(18)= 8174, Z= -3.76, P= 0.0001) and G2 dogs (Mann-Whitney U test, u(18)= 6543, Z= − 5.06, P

Table 1 Absolute and relative number of joints with LeishmaniaAmastigote forms in the synovial fluid of group 1 and group 2 dogs
Fig. 3
figure 3

Boxplots showing the percentage of neutrophils in cytological evaluation of canine synovial fluid from G1 (a) and G2 (b) dogs according to the presence or absence of amastigote forms Leishmaniasp. in the synovial fluid. Bars indicate minimum and maximum values, boxes represent the first and third quartiles. Asterisks above bar indicate statistically significant difference at ***PPEhrlichiaspp.); G2, Group 2 dogs (CanVL and seropositive for Ehrlichiaspp.)

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