Anti-histone antibodies in dogs with leishmaniasis and glomerulonephritis

The association between serum anti-histone antibodies and glomerulonephritis was studied in 43 dogs with leishmaniasis (Leishmania infantum). Dogs with increased serum creatinine levels and urine protein-creatinine ratio >1 were considered to have glomerulonephritis. Moderately elevated anti-histone antibodies were found in 38.89% (7/18) of infected dogs without glomerulonephritis, whereas 88% of dogs with glomerulonephritis (22/25) showed moderate or strongly elevated anti-histone antibodies. Prevalence of positive anti-histone antibodies reactions and mean serum concentration was significantly higher (P < 0.001; P < 0.0001) in infected dogs with glomerulonephritis. Correlation between anti-histone antibodies and urine protein-creatinine ratio was significant when groups were analysed together (P < 0.046). Positive predictive value for glomerulonephritis of positive anti-histone antibodies was 88%. In conclusion, high anti-histone antibodies are significantly associated with glomerulonephritis. Although other factors must be involved, dogs with moderate or strong positive anti-histone antibodies reactions may have a higher probability to develop glomerular lesions in canine leishmaniasis.     

Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs

Latent canine herpesvirus-1 (CHV-1) infection is common in domestic dogs, but recrudescent CHV-1 diseases are poorly characterized. To determine if administration of an immunosuppressive dosage of prednisolone to adult dogs latently infected with CHV-1 results in recurrent ocular disease, adult beagles with and without experimentally induced CHV-1 latent infection were divided into groups: group 1 latently infected and administered prednisolone, group 2 latently infected and administered placebo, and group 3 not latently infected and administered prednisolone. Prednisolone (3.0 mg/kg/day) was administered to dogs in groups 1 and 3 for seven consecutive days beginning on study day 1. Samples for CHV-1 polymerase chain reaction and serum neutralization (SN) assays were collected, and physical, ophthalmologic, and in vivo ocular confocal microscopic examinations were performed at intervals for 42 days. Bilateral ocular disease (i.e., conjunctivitis or keratitis) was detected in 83% of group 1 dogs between study days 3 and 18. In vivo confocal microscopic abnormalities included conjunctival leukocyte infiltration and corneal leukocyte infiltration, abnormal epithelial cell morphology, and Langerhans cell infiltration. Ocular viral shedding was detected in 50% of group 1 dogs on study days 10 and 13. Fourfold elevations in CHV-1 SN titers were detected in 100% of group 1 dogs by study day 14. Dogs in control groups did not develop clinical ocular disease (P < 0.05), CHV-1 titer elevations (P < 0.005), or viral shedding. Administration of an immunosuppressive dosage of systemic prednisolone to adult dogs latently infected with CHV-1 may result in viral reactivation and ocular disease recrudescence.     

Results from an indirect fluorescent antibody test using three different strains of Ehrlichia canis

An indirect fluorescent antibody (IFA) test is usually performed to detect antibodies in dogs naturally infected by Ehrlichia canis. In this work, results obtained using three different E. canis strains as antigen (a commercial antigen, the E. canis Oklahoma strain and the E. canis Madrid strain) were compared. One hundred and forty-nine serum samples obtained from dogs living in the centre of Spain were analysed. When qualitative results were evaluated, identical results were detected in 87.2% of samples for the three antigens tested. When comparing antibody titre results, differences between the Madrid strain and the commercial antigen, and between the Madrid and Oklahoma strains were statistically significant (P < 0.0001). No differences were found when comparing the Oklahoma strain with the commercial antigen (P = 0.562). Subtle intra-laboratory variations shown in this study suggest a higher sensitivity of the IFA test when an autochthonous strain is used as antigen.     

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤ 0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.     

Early Detection of Brucella Canis via Quantitative Polymerase Chain Reaction Analysis

Canine brucellosis is a reportable zoonotic disease that can lead to canine reproductive losses and human infection through contact with infected urine or other genitourinary secretions. Although many locations require testing and euthanasia of positive dogs, current diagnosis is limited by the time required for seroconversion, for example, presence of B. canis‐specific antibodies. The goal of this study was to determine the diagnostic ability of Brucella canis‐specific quantitative polymerase chain reaction (qPCR) assay to detect B. canis in field samples prior to serological positivity for faster diagnosis and prevention of transmission within kennels or in households. Two kennels, one of which was located in the owner's home, were sampled following observation of suggestive clinical signs and positive serology of at least one dog. Specimens obtained were comparatively analysed via serology and qPCR analysis. 107 dogs were analysed for B. canis infection via qPCR: 105 via whole‐blood samples, 65 via vaginal swab, six via urine and seven via genitourinary tract tissue taken at necropsy. Forty‐five dogs were found to be infected with canine brucellosis via qPCR, of which 22 (48.89%) were seropositive. A statistically significant number (P = 0.0228) of qPCR‐positive dogs, 5/25 (20.00%), seroconverted within a 30‐day interval after initial serologic testing. As compared to serology, qPCR analysis of DNA from vaginal swabs had a sensitivity of 92.31% and specificity of 51.92%, and qPCR analysis of DNA from whole‐blood samples had a sensitivity of 16.67% and specificity of 100%. B. canis outer membrane protein 25 DNA qPCR from non‐invasive vaginal swab and urine samples provided early detection of B. canis infection in dogs prior to detection of antibodies. This assay provides a critical tool to decrease zoonotic spread of canine brucellosis, its associated clinical presentation(s), and emotional and economic repercussions.     

A study of dendritic cell and MHC class II expression in dogs with immunomodulatory-responsive lymphocytic-plasmacytic pododermatitis

The term immunomodulatory-responsive lymphocytic-plasmacytic pododermatitis (ImR-LPP) has previously been proposed to denote a sub-population of dogs with idiopathic pododermatitis. The objective of this study was to investigate dendritic cell (DC) and MHC class II antigen expression in lesional skin of dogs with ImR-LPP (n = 47). Median epidermal CD1c⁺ cell counts were 37.8 and 12.5 mm⁻¹ in ImR-LPP dogs and healthy controls (n = 27), respectively (P < 0.01), while the corresponding dermal cell counts were 180.9 and 45.0 mm⁻², respectively (P < 0.01).Intra-epidermal clusters of DCs were observed in 18/47 dogs with ImR-LPP. Median epidermal MHC class II⁺ cell counts were 32.5 and 10.5 mm⁻¹ in ImR-LPP dogs and healthy controls, respectively (P < 0.01), while the corresponding dermal cell counts were 216.9 and 46.9 mm⁻², respectively (P < 0.01). Dermal MHC class II⁺ staining was primarily associated with DCs (47/47 dogs), mononuclear inflammatory cells (45/47), fibroblast-like cells (19/47) and vascular endothelium (14/47). The DC hyperplasia and increased MHC class II expression in lesional ImR-LPP skin are consistent with enhanced antigen presentation, and suggest that both parameters may contribute to the pathogenesis of ImR-LPP through the priming and activation of CD4⁺ T cells. Equally, it is possible that the enhanced DC numbers observed in this study may contribute to the immunoregulation of steady-state pathology in lesional ImR-LPP skin through additional expanded, although as yet unresolved, mechanisms.     

Description of outcomes of experimental infection with feline haemoplasmas: Copy numbers, haematology, Coombs’ testing and blood glucose concentrations

The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), ‘Candidatus M. haemominutum’ (Group HM: 3 cats) or ‘Candidatus M. turicensis’ (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs’ testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P < 0.05. Cats in Group TU had significantly lower blood copy numbers than cats in Group HF (P < 0.001) and HM (P < 0.001). All Group HF cats developed anaemia (often severe), macrocytosis and evidence of erythrocyte-bound antibodies whereas Groups HM and TU cats did not. Group HF had significantly lower PCVs, haemoglobin concentrations and red blood cell counts, and significantly higher mean cell volumes, than Groups HM and TU. In Group HF, erythrocyte-bound antibodies reactive at 4 °C (both IgM and IgG) appeared between 8 and 22 DPI and persisted for two to four weeks, whereas those reactive at 37 °C (primarily IgG) appeared between 22 and 29 DPI and persisted for one to five weeks. In most cats antibodies appeared after the fall in haemoglobin started. Although Group TU had significantly lower glucose concentrations than Groups HF (P = 0.006) and HM (P = 0.027), mean blood glucose concentrations remained within the reference range in all groups. This study demonstrates that M. haemofelis infection, in contrast to ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’ infection, can result in a severe macrocytic anaemia and the development of cold and warm reactive erythrocyte-bound antibodies.     

Serum profile of metabolites and hormones in obese (Iberian) and lean (Landrace) growing gilts fed balanced or lysine deficient diets

Serum hormones and metabolites of lean and obese gilts were determined. Fifteen Iberian and fifteen Landrace gilts, of approximately 20 kg body weight, were fed isoenergetic (13.6 kJ ME/g DM, semisynthetic diets either with equilibrated amino acid profile at two crude protein (CP) levels (12% (0.81% lysine (Lys)) and 16% CP (1.08% Lys) as-fed basis) or lysine deficient (12% (0.30% Lys) or 16% CP (0.36% Lys) as-fed basis for Iberian and Landrace, respectively). Lysine deficient diets were offered only at the optimal protein level for maximum protein accretion for each breed. Each dietary treatment was assayed in 5 animals. Gilts were allocated in metabolic cages at 21 ± 1.5 °C with free access to water and fed four times daily at 90% ad libitum during 10 days. On day 11th blood samples were taken 5–5.5 h postprandial and serum obtained and frozen at − 20 °C. Growth hormone (GH), insulin like growth factor I (IGF-1), insulin, leptin, glucose, urea, creatinine, cholesterol and triglycerides were determined. Landrace gilts had higher serum glucose (P < 0.05) and creatinine (P < 0.001) than Iberian while no differences (P = 0.122–0.494) were found for the rest of the biochemical variables. Higher serum levels of insulin, IGF-1 and leptin (60, P < 0.05; 79, P < 0.05 and 189%, P < 0.001, respectively) and no differences in GH (P = 0.512) were encountered in Iberian gilts compared to Landrace. Dietary crude protein did not alter the serum hormonal profile (P = 0.110–0.454). Serum cholesterol (total, P < 0.01; HDL, P < 0.05; LDL, P < 0.05) decreased and triglycerides and urea increased (P < 0.05 and P < 0.001, respectively) as dietary crude protein increased. When lysine deficient diets were used, serum glucose decreased (P < 0.05) and urea increased (P < 0.001) compared to equilibrated diets. Serum insulin (P = 0.052), IGF-1 (P < 0.05) and leptin (P = 0.051) concentration decreased when lysine deficient diets were fed to the animals while GH remained unaffected (P = 0.214).These data suggest that Iberian and Landrace growing gilts have distinct hormone and metabolite serum profiles which are altered by a severe dietary lysine deficiency.     

Hepatozoon canis infecting dogs in the State of Espírito Santo, southeastern Brazil

From May 2007 to March 2008, blood samples were collected from 92 healthy dogs living in 21 households (17 farms in rural area, and 4 homes in urban area) in 6 counties of the State of Espírito Santo, southeastern Brazil. In addition, ticks were collected from these dogs. A mean of 4.4 ± 3.0 dogs (range: 1–12) were sampled per household; 78 and 14 dogs were from rural and urban areas, respectively. Polymerase chain reaction (PCR) designed to amplify fragments of the 18S rDNA gene of Babesia spp or Hepatozoon spp revealed amplicons of the expected size in 20 (21.7%) dogs for Babesia, and 54 (58.7%) dogs for Hepatozoon. All Babesia-positive dogs were also Hepatozoon-positive. Among the 21 households, 15 (71.4%) from 3 counties had at least one PCR-positive dog, including 13 farms (rural area) and 2 homes (urban area). A total of 40 PCR products from the Hepatozoon-PCR, and 19 products from the Babesia-PCR were submitted to DNA sequencing. All generated sequences from Hepatozoon-PCR were identical to each other, and to corresponding 18S rDNA sequences of H. canis in GenBank. Surprisingly, all generated sequences from the Babesia PCR were also identical to corresponding 18S rDNA sequences of H. canis in GenBank. Dogs from 10 rural and 2 urban households were found infested by Rhipicephalus sanguineus ticks. Immature of Amblyomma cajennense ticks were found in dogs from only 4 rural households (also infested by R. sanguineus). All but one household with R. sanguineus-infested dogs had at least one Hepatozoon-infected dog. Statistical analysis showed that the presence of ticks (i.e. R. sanguineus) infesting dogs in the households was significantly (P < 0.05) associated with at least one PCR-positive dog. There was no significant association (P > 0.05) between PCR-positive dogs and urban or rural households. Canine hepatozoonosis caused by H. canis is a high frequent infection in Espírito Santo, Brazil, where it is possibly vectored by R. sanguineus. Since all infected dogs were found apparently healthy, the pathogenicity of H. canis for dogs in Espírito Santo is yet to be elucidated.     

Improvement in insulin resistance and reduction in plasma inflammatory adipokines after weight loss in obese dogs

Obesity is now a major disease of dogs, predisposing to numerous disorders including diabetes mellitus. Adipocytes are active endocrine cells, and human obesity is characterized by derangements in inflammatory adipokine production. However, it is unclear as to whether similar changes occur in dogs. The purpose of the current study was to assess insulin sensitivity and inflammatory adipokine profiles in dogs with naturally occurring obesity and to investigate the effect of subsequent weight loss. Twenty-six overweight dogs were studied, representing a range of breeds and both sexes. All dogs underwent a weight loss program involving diet and exercise. Body fat mass was measured by dual-energy x-ray absorptiometry; plasma concentrations of insulin, glucose, and a panel of inflammatory adipokines (including acute-phase proteins, cytokines, and chemokines) were also analyzed. Body fat mass before weight loss was positively correlated with both plasma insulin concentrations (Kendall τ = 0.30, P = 0.044) and insulin:glucose ratio (Kendall τ = 0.36, P = 0.022), and both decreased after weight loss (P = 0.0037 and 0.0063, respectively). Weight loss also led to notable decreases in plasma tumor necrosis factor-α (TNF-α), haptoglobin, and C-reactive protein concentrations (P < 0.05 for all), suggesting improvement of a subclinical inflammatory state associated with obesity. This study has demonstrated that in obese dogs, insulin resistance correlates with degree of adiposity, and weight loss improves insulin sensitivity. Concurrent decreases in TNF-α and adipose tissue mass suggest that in dogs, as in humans, this adipokine may be implicated in the insulin resistance of obesity.     

Alfalfa as a supplement of dried cornstalk diets: Associative effects on intake, digestibility, nitrogen metabolisation, rumen environment and hematological parameters in sheep

The aim of the current study was to investigate the associative effects of a cornstalk-based diet supplemented with alfalfa (Medicago sativa) hay on intake, digestibility, nitrogen (N) metabolisation, rumen environment and hematological parameters in Xiaoweihan sheep. We also investigated the optimal range of alfalfa hay to achieve positive associative effects and avoid negative effects. Xiaoweihan sheep (n = 5; fitted with rumen T-cannula) were fed five cornstalk-based diets in a 5 × 5 Latin square design. Diets contained 0, 50, 150, 300, 450 g alfalfa, and were supplemented with 100 g concentrate, respectively. Our results suggested that supplementation of 300 g alfalfa hay reduced (P < 0.05) cornstalk intake, but significantly increased dry matter (DM) intake (P < 0.05). Additionally, DM digestibility of 150 g alfalfa hay supplementation was slightly higher than that noted in other diets. Metabolism studies showed 50–150 g alfalfa hay supplementation had a positive associative effect (P < 0.05) on N utilization, with the greatest benefit noted with 150 g per day (P < 0.05) compared to unsupplemented diets. Alfalfa supplementation (50–450 g per day) resulted in an elevated trend of ammonia nitrogen (NH₃–N) with 50 or 150 g of alfalfa hay more likely to promote sheep rumen environment, with a noticeable increase (P < 0.05) in serum urea nitrogen (UREAN) concentrations observed with 300 g alfalfa hay per day. Our data suggested that the optimal range to achieve beneficial effects and avoid negative effects was 150–300 g per day for cornstalk-based diets for sheep.     

Systemic cytokine profiles of mice vaccinated with naked DNAs encoding six open reading frame antigens of porcine circovirus type 2 (PCV2)

The objective of this study was to characterize the murine immune response to vaccination with DNAs encoding each of six porcine circovirus type 2 (PCV2) open reading frames (ORFs). After intramuscular vaccination with the naked DNAs, blood was taken on days post-infection (DPI) 7 and 35 and subjected to Bio-Plex cytokine assays. ORF3 elicited high levels of the pro-inflammatory cytokine TNF-α (P < 0.001) and decreased IL-12 levels (P < 0.001) on DPI 7, and was lethal for nine of the 15 vaccinated mice, which died on DPIs 4, 6, 9, and 10. The remaining six mice showed general prostration but then recovered. The clinical signs correlated with the systemic TNF-α and IL-12 levels. ORF1 vaccination elevated T-helper (Th)1 (IFN-γ; P < 0.001) and Th2 (IL-13; P < 0.05) cytokine levels on DPI 35, while ORF2 markedly elevated the expression of the humoral immunity- and Th-2-related cytokine IL-10 (P < 0.001) on DPI 35. These observations provide insights into the immune responses generated by each PCV2 ORF.     

Clinical evaluation of urinary liver-type fatty acid-binding protein for the diagnosis of renal diseases in dogs

Liver-type fatty acid-binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. L-FABP is a cytotoxic oxidation product secreted from the proximal tubules under ischemic and oxidative stress conditions. First, L-FABP gene expression in the kidney and liver was evaluated. Next, the urinary L-FABP concentrations in dogs with or without renal diseases were measured using a novel enzyme-linked immunosorbent assay kit. Urinary L-FABP was normalized relative to urinary creatinine (uCre) concentrations (µg/g uCre). Finally, the relationships between urinary L-FABP and renal biomarkers used in canine medicine or serum alanine transaminase (ALT) as an indicator of liver damage were examined. Serum and urine samples from 94 client-owned dogs including 23 dogs with renal diseases and 71 dogs without renal diseases were used for analysis. Relative L-FABP gene expression was confirmed both in the liver and kidney. Dogs with renal diseases had a significantly higher urinary L-FABP than those without, and its predictive cutoff value was 26 µg/g uCre. Urinary L-FABP was significantly correlated with serum creatinine (r=0.4674, P<0.01), urea nitrogen (r=0.4907, P<0.01), urine specific gravity (r=−0.5100, P<0.01), and urine protein/creatinine ratio (r=0.7216, P<0.01), but not with serum ALT. Hence, dogs with a high urinary L-FABP value were more likely to have renal diseases.     

Effects of replacing grass silage harvested at two maturity stages with maize silage in the ration upon the intake, digestibility and N retention in wether sheep

The objective of this experiment was to study the effects of interactions between medium quality grass silage (GS1) and maize silage (MS) as well as between low-quality grass silage (GS2) and MS on ad libitum intake, digestibility and N retention in wether sheep. Two grass silages (GS1 and GS2) were ensiled in round bales, without additives, from the primary growth of orchard grass (Dactylis glomerata L.) harvested at two different maturity stages. The study consisted of seven feeding treatments incorporating GS1, GS2 and MS fed alone and forage mixtures of GS1 and MS as well as GS2 and MS (67:33% and 33:67%, respectively, DM (dry matter) basis).Delayed harvesting lowered (P < 0.05) the crude protein (CP) concentration in GS2 compared to GS1. The DM content (g kg^− 1 fresh sample) and starch concentration (g kg^− 1 DM) of MS were 264 and 211, respectively.Inclusion of MS in the GS1-based ration had positive linear effects on CP and starch digestibility (P < 0.05 and P < 0.01, respectively) and N intake (P < 0.01) while a negative effect on neutral detergent fibre (NDF) and acid detergent fibre (ADF) digestibility (P < 0.05 and P < 0.01, respectively). A positive associative response of GS1 and MS was observed for DM ad libitum intake (g kg^− 1 M^0.75 day^− 1) (quadratic, P < 0.05), CP digestibility (quadratic, P < 0.01), N intake (quadratic, P < 0.01) and N balance (quadratic, P < 0.05). Inclusion of MS into the GS2-based ration had a positive linear effect on the ration fresh matter ad libitum intake (kg day^− 1 and g kg^− 1 M^0.75 day^− 1) (P < 0.01 and P < 0.001, respectively), NDF ad libitum intake (kg day^− 1 and g kg^− 1 M^0.75 day^− 1) (P < 0.01), digestibility of DM (P < 0.01), organic matter (OM) (P < 0.01), ADF (P < 0.05), starch (P < 0.001), digestibility of OM in DM (D-value) (P < 0.001), and N intake (P < 0.01). Positive associative effects of GS2 and MS were observed on all the intake and digestibility parameters measured, N intake (quadratic, P < 0.001) and N balance (quadratic, P < 0.05). It was concluded that, as expected, a positive associative response of GS2 and MS was recorded for all the measured parameters while that of GS1 and MS for a limited number of parameters, probably due to lower quality of MS (lower starch concentration) than required for improved utilization of the GS1-based ration.     

Fear-related behaviour of dogs in veterinary practice

To investigate further the occurrence of fear-related behaviour in dogs in veterinary practice and to evaluate associated factors, 135 dogs were observed under practice conditions within the framework of a standardised test examination and the owners interviewed using a questionnaire. Most dogs exhibited fear reactions, particularly on the examination table, with 78.5% (106/135) categorised as ‘fearful’ based on their behaviour. Unlike weight and castration, age, gender and previous experience were significantly (P < 0.05) associated with fearful behaviour. Male dogs were significantly less ‘fearful’ than females and animals under <2 years were significantly less ‘fearful’ compared with older dogs. Those with only positive previous experiences in veterinary surgeries were significantly less ‘fearful’ than dogs that had a previous negative experience. Fear-related behaviour in veterinary practice is an issue of importance.     

Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (ρ = 0.542; P < 0.001) and with serum biochemical parameters, such as γ-globulins, urea and creatinine. Goldmann–Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (ρ = 0.779; P < 0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.     

Effects of Ca salt supplementation on milk yield and composition and on lamb growth rate of Awassi ewes

An experiment was conducted to evaluate the effect of a Ca salt supplement on lactating Awassi ewes and their lambs. Ninety Awassi ewes (average BW = 55 kg ± 1.13) were allocated into three treatment groups of 30 each; 0%, 3%, or 5% Ca salt supplemented groups. The experiment started at lambing and lasted for 60 d. During this period, milk production and composition, final body weights, total feed intake, serum cholesterol and triglycerides, weaning weights of lambs, and milk fatty acid profile were tested. Milk production and energy corrected milk increased (P < 0.05) with Ca salt level in the diet. Milk fat content was higher (P < 0.05) at 3% treatment group compared to 0% and 5% treatment groups. However, no differences were detected in content of crude protein and total solids, and milk energy value. Milk fat yield (g/d) increased (P < 0.05) as Ca salt in the diet increased. Final body weight was higher (P < 0.05) for 5% group when compared to the 0% group with no differences between the 3% group when compared to 0% and 5% groups. No differences were observed in feed intake of ewes. Metabolizable energy intake was greater (P < 0.05) for the 3% and the 5% treatment groups when compared to the 0% treatment group. Feed to milk ratio increased (P < 0.05) when increasing the level of Ca salt in the diet. Serum cholesterol was greater (P < 0.05) in the 5% treatment group than the 0% and 3% treatment groups for ewes and lambs. Serum triglyceride was similar among treatment groups in ewes whereas serum triglyceride was greater (P < 0.05) for lambs in 5% treatment group than 0% and 3% treatment groups. However, serum triglyceride was similar in ewes among treatment groups. Weaning weights and average daily gain of male lambs were higher (P < 0.05) in lambs fed Ca salt (3% and 5%) when compared to 0% treatment group. No differences were detected in weaning weights and average daily gain of female lambs among treatment groups. However, milk conversion ratio was better (P < 0.05) for the 5% group lambs than the 0% and 3% treatment groups. Ca salt reduced (P < 0.05) short and medium-chain milk fatty acids and increased (P < 0.05) content of long chain and unsaturated ones. These results of this indicate that Ca salt supplementation can improve milk production of lactating ewes, the growth rate of their lambs, and produce healthy quality value milk.