Eugene C. Bwalya

Prevalence of canine gastrointestinal helminths in urban Lusaka and rural Katete Districts of Zambia

Faecal samples were collected from January 2010 through September 2010 to determine the prevalence of gastrointestinal (GI) helminths infestation in dogs in urban Lusaka and rural Katete Districts of Zambia. A total of 452 faecal samples (n=160 Katete, n=292 Lusaka) were examined by faecal flotation for the presence of helminth eggs and 82.5% of dogs were positive for GI helminths in Katete compared to 76% for Lusaka. Positive results with the presence of at least one parasite corresponded to 72.9% Ancylostoma caninum, 11% Toxocara canis, 4.8% Toxascaris leonina, 2.4% Dipylidium caninum, 0.7% Taeniidae and 0.3% T. vulpis, species for Lusaka while Katete recorded 70.6% A. caninum, 18.1% T. vulpis, 11.1% T. canis, 13.1% D. caninum, 3.8% T. leonina, and 0.6% Taeniidae. Except for T. vulpis and D. caninum (p<0.05) the results indicated no significant difference in the prevalence of the identified GI helminth between Lusaka and Katete. There was no significant difference in the prevalence between genders of GI helminth infestation demonstrated in this study and only A. caninum showed significant difference in prevalence by age category. The study also showed the presence of zoonotic intestinal helminths A. caninum, T. canis and D. caninum. The study highlights that there was no significant difference in spectrum and prevalence of GI helminths between urban and rural areas in Zambia. It further brings to light the importance of educating owners of dogs on the importance of regular deworming of dogs and control of ectoparasites in order to minimise the risk that these dogs pose to them and the public.

A Study of Naturally Acquired Canine Babesiosis Caused by Single and Mixed Babesia Species in Zambia: Clinicopathological Findings and Case Management

A retrospective and prospective analysis of clinical records of dogs diagnosed with Babesia infections was carried out for the years 2000 to 2013 from practices in Lusaka, Zambia. Records of 363 dogs with confirmed Babesia infections were analysed using demographic factors including sex, breed, age, and clinical signs in relation to haematological findings and Babesia species. The clinical and laboratory findings observed are described as well as Babesia species identification. The study included 18 breeds and the highest proportion were mongrels (32.2%), males representing 64.5% of the population. The most common presenting problems were anorexia (65.3%) and lethargy/weakness (65.3%). The most common clinical signs were fever (87.3%), pallor (52.3%), lymphadenopathy (47.4%), and presence of ticks (44.9%). Anaemia (96.4%) and nucleated erythrocytes (42.2%) were the most common laboratory findings. A mixed infection of Babesia rossi and Babesia gibsoni was present in 59.7% of dogs, whilst 8% and 32.2% had B. rossi and B. gibsoni as a single infection, respectively. Case management mainly involved therapy with tetracyclines and imidocarb and was usually accompanied by clinical improvement. This study highlights, for the first time, the presence of B. gibsoni in natural dog populations in Zambia, where previously only B. rossi was reported.

Seasonal variations in health indices of free-ranging asymptomatic guinea fowls (Numida meleagris) in Zambia

OBJECTIVEnTo determine the impact of seasonal variations on health indices of free-ranging asymptomatic guinea fowls (Numida meleagris) in Zambia.nnnMETHODSnA time series analysis was carried out on a prospective cohort study over a 12 month period between March 2010 and February 2011 by examining a total 147 guinea fowls for haematological and morphometric data of selected organs.nnnRESULTSnThere was a strong correlation in erythrocytic indices between packed cell volume and red blood cell counts (=0.824, P<0.001) as well as between packed cell volume and hemoglobulin (Hb) counts (r=0.648, P<0.001). Seasonal differences showed that erythrocytic indices were higher in the males than the females and that the difference was significantly higher (P<0.001) during the rainy season, which coincided with the breeding period when females were laying eggs. Increase in total plasma protein was positively correlated with overall body weight. Generally, females had higher body weights and total plasma protein levels than the males in the rain season. Of the 147 birds examined, 51% (n=147) had the bursa of Fabricius. For birds that had the bursa of Fabricius, the weights of bursae were higher (P<0.05) in the cold-dry season than the other seasons and no sex differences were observed. Spleen morphometric data did not show any seasonal nor sex differences.nnnCONCLUSIONSnOverall, data presented herein demonstrate that seasonal variations have a significant influence on health indices of free-ranging guinea fowls and that these factors could influence the susceptibility of this species of birds to disease infections at different times of the year.

Differentiation potential of synoviocytes derived from joints with cranial cruciate ligament rupture and medial patella luxation in dogs

The objective of this study was to assess the differentiation capability of synoviocytes derived from dogs with inflammatory joint conditions. Cranial cruciate ligament ruptured (CCLr) (n=12) and medial patella luxated (MPL) (n=10) knee joints of the dogs were used to collect the synovial membrane (SM). Synoviocytes were enzymatically released from the SM and analyzed by flow cytometry for specific cellular markers (CD44 and CD90) of mesenchymal stem cells (MSCs), while doing histopathology from another part of SM sections. Under specific culture conditions, synoviocytes were forced to differentiate into chondrogenesis, adipogenesis, osteogenesis and osteoclastogenesis to investigate the multipotency. Upon treatments phenotypes of cell cultures were analyzed by histopathology and by semi-quantitative reverse transcriptase polymerase chain reaction for the expression of each differentiation marker genes. Although flow cytometry showing similar MSCs populations in CCLr and MPL synovium, synovial cells derived from CCLr showed higher multipotency compared to MPL-derived samples. Further, synovial changes such as vascularity, mononuclear cell infiltration and cellular hypertrophy were more pronounced in CCLr-derived synovial tissue than in MPL. Taken together, these findings suggested that the differentiation capability of SM-derived multipotent stem cells varies with inflammatory severity occurring in different joint conditions.

Pentosan polysulfate inhibits IL-1β-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes

Osteoarthritic (OA) chondrocytes are shown to express inducible nitric oxide synthase (iNOS) which produces high concentrations of nitric oxide (NO), particularly when stimulated with proinflammatory cytokines. NO is involved in OA cartilage degradation. On the other hand, c-Jun N-terminal Kinase (JNK) pathway mediates the activation and transcription of c-Jun, which is required for interleukin-1 (IL-1)-induction of matrix metalloproteinases-13 (MMP-13) in OA pathogenesis. Therefore, the selective inhibition of iNOS and c-Jun is a promising target for treatment and prevention of OA. The purpose of the study was to investigate the inhibitory effects of pentosan polysulfate (PPS) on IL-1β-induced iNOS, c-Jun and HIF-α isoforms upregulation in canine articular chondrocytes (CACs). Primary (P0) chondrocytes were isolated and cultured from femoral head cartilages of three (3) dogs. First passage (P1) chondrocytes were preincubated with 0, 1, 5, 15 and 40 μg/mL of PPS for 4 hr before treatment with 10 ng/mL rhIL-1β for a further 8 hr. In addition, we evaluated the effects of single and multiple cytokine with or without LPS on iNOS protein induction. PPS significantly inhibited (P < 0.05) IL-1β-induced iNOS, c-Jun and HIF-1α mRNA upregulation in a dose-dependent pattern. iNOS mRNA was significantly inhibited at 15 and 40 μg/mL whereas c-Jun and HIF-1α were significantly downregulated at 5, 15 and 40 μg/mL of PPS compared to chondrocytes treated with only rhIL-1β. Intriguingly, CACs were recalcitrant to single IL-1β, TNF-α or LPS-induction of iNOS protein including to a combination of IL-1β+TNF-α, IL-1β+LPS except to TNF-α+LPS and IL-1β+TNF-α+LPS suggestive of a protective mechanism from iNOS detrimental effects on perpetuating OA. IL-1β+TNF-α+LPS-induced iNOS protein expression was significantly abrogated by PPS. We demonstrate for the first time that PPS is a novel inhibitor of IL-1β-induced iNOS, c-Jun, and HIF-1α mRNA upregulation and iNOS protein induction which may be beneficial for prevention and treatment OA.

Effects of pentosan polysulfate and polysulfated glycosaminoglycan on chondrogenesis of canine bone marrow-derived mesenchymal stem cells in alginate and micromass culture

Mesenchymal stem cells (MSC) are a potential alternative source of differentiated chondrocytes for cartilage tissue regeneration and repair of osteoarthritic (OA) joints. We investigated the effects of pentosan polysulfate (PPS) and polysulfated glycosaminoglycan (PSGAG) on chondrogenesis of canine bone marrow-derived mesenchymal stem cells (cBMSC) in alginate and micromass cultures (MMC). Chondrogenic differentiation medium (CDM) was supplemented with PPS or PSGAG at concentrations of 0 (positive control; PC), 1, 3 and 5 µg/ml. 10% DMEM was used as negative control. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) for alginate cultures and Alcian blue staining for proteoglycan (PG) synthesis for MMC. In alginate culture, PPS and PSGAG showed no significant effect on type II collagen, aggrecan and HIF-2α mRNA expression. PPS had no significant effect on type I collagen whereas PSGAG significantly upregulated (P<0.05) it at all concentrations relative to other treatments. PPS demonstrated a dose-dependent inhibitory effect on type X collagen mRNA with significant inhibition observed at 5 µg/ml compared to the NC. PSGAG showed an inverse effect on type X collagen with 1 µg/ml significantly inhibiting its expression while increase in the concentration correspondingly increased type X collagen expression. In MMC, PPS significantly enhanced chondrogenesis and PG deposition whereas PSGAG inhibited chondrogenesis and promoted a fibrocartilage-like phenotype with reduced PG deposition. While PPS enhances chondrogenesis of cBMSC in MMC, the response of MSC to chondroinductive factors is culture system-dependent and varies significantly between alginate and MMC.

The Effects of Gastrointestinal Helminths on packed Cell Volume (PCV), Eosinophil Count and Total Plasma Protein (TPP) in Local Breed Dogs in Zambia

The Effects of Gastrointestinal Helminths on packed Cell Volume (PCV), Eosinophil Count and Total Plasma Protein (TPP) in Local Breed Dogs in Zambia nBlood samples were concurrently collected from 269 local breed dogs during a study to determine the prevelance of gastrointestinal (GI) helminths and the results matched with those of faecal samples. The aim of the present study was to determine the effects of GI helminth infection on the clinical health indices and evaluate the ability of age and GI infection status to predict these parameters for local breed dogs in Zambia. Two hundred and sixty nine dogs were sampled and of these 211 (78.4%) were parasitized with Ancylostoma caninum, Toxocara canis, Dipylidium caninum, Trichuris vulpis or Toxascaris leonine while 21.56% (58/269) were non-parasitized.

Independent chondrogenic potential of canine bone marrow-derived mesenchymal stem cells in monolayer expansion cultures decreases in a passage-dependent pattern

Although chondroinductive growth factors are considered necessary for chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSC), independent and spontaneous chondrogenesis has been previously demonstrated in adult horses, bovine calves and adult human BMSC. Surprisingly, adult canine BMSC under similar culture conditions previously failed to demonstrate chondrogenesis. The present study evaluated independent chondrogenic potential of BMSC sourced from three young dogs in the absence of known chondroinductive factors. BMSC were culture expanded in 10% DMEM up to third passage (P3). At each passage, the phenotype of BMSC was evaluated by RT-PCR gel electrophoresis and qPCR. BMSC exhibited a chondrogenic phenotype in the absence of dexamethasone and TGF-β1 as verified by the expression of Sox-9, type II collagen and aggrecan. Sox-9 was significantly downregulated (P<0.05) from P1−P3 compared to P0 while type II and X collagen, and aggrecan were significantly downregulated at P3 compared to P0. There was a significant (P<0.01) negative correlation between passaging and Sox-9, type II collagen and aggrecan gene expression. These results indicate that independent chondrogenic potential and phenotype retention of BMSC decreases in a passage-dependent pattern. Therefore, caution should be exercised for future experiments evaluating the chondrogenic potential of BMSC after extensive expansion cultures in 10% DMEM.

Pentosan Polysulfate Sodium Restores the Phenotype of Dedifferentiated Monolayer Canine Articular Chondrocytes Cultured in Alginate Beads

Autologous chondrocyte transplantation is a promising option for the repair of isolated osteoarthritic cartilage lesions that requires isolation and expansion of chondrocytes from a small cartilage biopsy prior to implantation. However, when cultured in vitro, chondrocytes lose their stable phenotype and dedifferentiate to fibroblastic-like cells. The study investigated the potential of pentosan polysulfate (PPS) sodium to restore the phenotype of dedifferentiated monolayer articular chondrocytes. Canine articular chondrocytes isolated from four cartilage samples were culture expanded to establish primary culture. First passage chondrocytes were cultured as alginate beads for 18 days under normoxia in PPS concentrations of 0, 1, 5, 15 and 40 μg/mL in 20% DMEM. Effect of PPS on type I, II and X collagen, aggrecan and Runx2 gene expression were evaluated by real-time PCR. Runx2, HIF-1α and HIF-2α protein expression were evaluated by Western blot and proteoglycan deposition was determined by Alcian blue stain. Dedifferentiated chondrocytes fully retained their phenotype as evidenced by increased synthesis of cartilage-specific genes, type II collagen and aggrecan mRNA with complete suppression of type I and X collagen at PPS concentrations of 15 and 40 μg/mL. Compared to the control, type II collagen and aggrecan mRNA were significantly upregulated (P<0.05) at 5, 15 and 40 μg/mL and 5 and 15 μg/mL PPS, respectively. PPS significantly enhanced proteoglycan with peak deposition at 5 μg/mL compared to control. HIF-1α and HIF-2α proteins were detectable at protein level for the first time under normoxia condition in alginate culture. The study demonstrates for the first time the restoration of dedifferentiated canine articular chondrocytes phenotype by combining alginate encapsulation with culture in PPS without the addition of known chondrocytic growth factors. The study confirms PPS as novel chondroinductive factor with potential to offer a solution to the major challenges that exist in cartilage tissue engineering.

Inhibitory effects of sodium pentosan polysulfate on formation and function of osteoclasts derived from canine bone marrow

BackgroundSodium pentosan polysulfate (NaPPS) was testified as a chondroprotective drug in with a detailed rationale of the disease-modifying activity. This study was undertaken to determine whether anti-osteoarthritis drug, NaPPS inhibited osteoclasts (OC) differentiation and function. Canine bone marrow mononuclear cells (nu2009=u20096) were differentiated to OC by maintaining with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for up to 7xa0days with the treatment of NaPPS at concentration of 0, 0.2, 1 and 5xa0μg/mL. Differentiation and function of OC were accessed using tartrate-resistant acid phosphate (TRAP) staining and bone resorption assay, while monitoring actin ring formation. Invasion and colocalization patterns of fluorescence-labeled NaPPS with transcribed gene in OC were monitored. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, activator protein-1(AP-1) and carbonic anhydrase II was examined using real-time PCR.ResultsSignificant inhibition of OC differentiation was evident at NaPPS concentration of 1 and 5xa0μg/mL (pu2009<u20090.05). In the presence of 0.2 to 5xa0μg/mL NaPPS, bone resorption was attenuated (pu2009<u20090.05), while 1 and 5xa0μg/mL NaPPS achieved significant reduction of actin ring formation. Intriguingly, fluorescence-labeled NaPPS invaded in to cytoplasm and nucleus while colocalizing with actively transcribed gene. Gene expression of CTK, MMP-9 and NFATc1 were significantly inhibited at 1 and 5xa0μg/mL (pu2009<u20090.05) of NaPPS whereas inhibition of c-Fos and AP-1 was identified only at concentration of 5xa0μg/mL (pu2009<u20090.05).ConclusionsTaken together, all the results suggest that NaPPS is a novel inhibitor of RANKL and M-CSF-induced CTK, MMP-9, NFATc1, c-Fos, AP-1 upregulation, OC differentiation and bone resorption which might be a beneficial for treatment of inflammatory joint diseases and other bone diseases associated with excessive bone resorption.