FC‐14 Clonality studies of feline cutaneous lymphocytosis

A previous study described cutaneous lymphocytosis (CL) in 23 cats. The process resembles cutaneous pseudolymphoma in humans, a heterogeneous group of benign reactive proliferations of well‐differentiated lymphocytes in the skin of humans. Morphological and immunophenotypic characteristics do not offer reliable criteria to accurately predict the clinical outcome of feline CL or pseudolymphoma in humans. Presence of clonal cell populations is more consistent with a neoplastic process. In a previous study, feline CL lesions (20 cats) were evaluated for clonality using PCR, and only two cats had monoclonal T‐cell populations. Because false‐negative results may occur, the purpose of this study was to repeat the PCR using a revised primer set based on analysis of additional feline T‐cell receptor γ (TCRγ) sequences. DNA was isolated from 29 skin lesions and six internal organs of 20 cats. DNA integrity was assessed by glyceraldehyde‐3‐phosphate dehydrogenase PCR. Polymerase chain reaction clonality was performed using the revised primer set specific for feline TCRγ, and duplicate samples were evaluated. The PCR products were assessed by heteroduplex analysis. Clonal rearrangement of TCRγ was detected in 14 cats (24 of 35 tissues: 21 of 29 skin lesions and three of six internal organs); eight of these cats are still alive and six were euthanized. Monoclonal populations were seen in three of five cats that had involvement of internal organs. These findings indicate that feline CL is best considered as a slowly progressive process which may be reactive, but often evolves into a low‐grade indolent lymphoma. Funding: George H. Muller Fund for Research in Dermatology.     

Organic zinc and copper supplementation on antioxidant protective mechanism and their correlation with sperm functional characteristics in goats

Trace minerals feeding had significant effects on sperm production and fertility with better absorption and proper utilization within the body for optimum reproductive function. Several studies have shown that more influenced trace elements in the diets of animals are copper (Cu) and zinc (Zn). Bucks showing deficiency of this mineral might affect the quality of semen production which in turn would affect the fertility. This experiment was thus designed to test the effects of organic Cu and Zn supplementation on antioxidants enzyme activities and sperm functional attributes in fresh semen of bucks. Forty bucks (n = 40, Aged 5 months) were assigned to ten groups of four animals in each group, supplemented (for a period of 8 months) with different levels of organic Zn: 20 mg (T2), 40 mg (T3) and 60 mg (T4), organic Cu: 12.5 mg (T5), 25 mg (T6), 37.5 mg (T7) and combined organic Zn and Cu: 20 + 12.5 mg (T8), 40 + 25 mg (T9), 60 + 37.5 mg (T10), respectively, per kg dry matter and no additional mineral diet (control; T1). One hundred and sixty semen samples were collected through electro‐ejaculator and analysed for sperm quantity, quality, acrosome intactness and plasma membrane integrity and correlated with the catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzyme activities in seminal plasma. The results indicated organic Cu and zinc supplemented bucks produced more sperm cells, had higher sperm concentrations, maintained higher (p < .01) sperm livability, plasma membrane and acrosome integrities, more motility and velocity. The increased antioxidant enzyme activities, reduced oxidative stress and lowered lipid peroxidation were positively correlated (p < .05) with the sperm functional attributes. In conclusion, organic Cu and Zn supplement to male goats showed protective roles against oxidative damage and maintained better fresh semen characteristics.     

Purpura haemorrhagica in 53 horses

The medical records of 53 horses with purpura haemorrhagica were reviewed. Seventeen of them had been exposed to or infected with Streptococcus equi, nine had been infected with Corynebacterium pseudotuberculosis, five had been vaccinated with S. equi M protein, five had had a respiratory infection of unknown aetiology, and two had open wounds; the other 15 cases had no history of recent viral or bacterial infection. The horses were between six months and 19 years of age (mean 8.4 years). The predominant clinical signs were well demarcated subcutaneous oedema of all four limbs and haemorrhages on the visible mucous membranes; other signs included depression, anorexia, fever, tachycardia, tachypnoea, reluctance to move, drainage from lymph nodes, exudation of serum from the skin, colic, epistaxis and weight loss. Haematological and biochemical abnormalities commonly detected were anaemia, neutrophilia, hyperproteinaemia, hyperfibrinogenaemia, hyperglobulinaemia and high activities of muscle enzymes. All of the horses were treated with corticosteroids; 42 also received non-steroidal anti-inflammatory drugs and 26 received antimicrobial drugs. Selected cases received special nursing care, including hydrotherapy and bandaging of the limbs. Most of the horses were treated for more than seven days and none of them relapsed. Forty-nine of the horses survived, one died and three were euthanased, either because their severe clinical disease failed to respond to treatment or because they developed secondary complications. Two of the four non-survivors had been vaccinated against S. equi with a product containing the M protein, one had a S. equi infection and the other had a respiratory infection of undetermined aetiology.     

Equine bullous pemphigoid IgG autoantibodies target linear epitopes in the NC16A ectodomain of collagen XVII (BP180, BPAG2)

Bullous pemphigoid (BP) is an autoimmune subepithelial blistering dermatosis of humans, dogs, cats and pigs. It is characterized by skin-fixed and circulating IgG autoantibodies that target one or both BP antigens. An immunological homologue of BP in humans was diagnosed in two horses with cutaneous and mucosal ulcerations as well as microscopic subepithelial vesiculation. Immunological investigations revealed similar findings for both the horses. Direct immunofluorescence demonstrated the presence of IgG deposited linearly at the dermoepidermal junction in mucosal and skin biopsy specimens. Indirect immunofluorescence testing confirmed the existence of circulating basement membrane-specific IgG autoantibodies. Using intact and salt-split epithelial substrates, serum IgG were shown to target antigens situated not only at the basal, but also at the lateral and apical aspects of stratum basale keratinocytes. Immunoblotting and ELISA corroborated that the IgG from affected horses, but not those from normal controls, exhibited high immunoreactivity against the NC16A extracellular domain of type XVII collagen (BPAG2, BP180). Equine BP could be proposed, therefore, as another spontaneous model of this most common basement membrane autoimmune dermatosis of humans.     

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.     

Combined decongestive therapy including equine manual lymph drainage to assist management of chronic progressive lymphoedema in draught horses

Equine chronic progressive lymphoedema (CPL) is a disabling disorder of draught horse breeds. Combined decongestive therapy (CDT) is the treatment of choice for lymphoedema in man and has been adapted for use in horses. Equine CDT, which includes manual lymph drainage (MLD) and subsequent bandaging with short stretch bandages, was expected to improve the signs of CPL in draught horses because CPL resembles primary lymphoedema in man. Five affected horses ‐ Gypsy cob (n = 1), Clydesdale (n = 1), Shires (n = 3) ‐ were included. Lesions were documented pre‐ and post treatment. Percentage volume loss of the distal legs was calculated using the disc model. Initial plans for daily CDT had to be adapted; intermittent treatment of Chorioptes infections required alternating between CDT and MLD in 4/5 horses. Concurrent pyoderma (1/5 horses) was treated throughout the study. Development of unrelated lameness (hoof abscess) allowed limited CDT treatment only in one horse. Marked softening of previously firm tissue indicated the change from ‘brawny’ to pitting oedema in 2/5 horses. Fibrotic nodules and folds in the pasterns became markedly softened and smaller in 2/5 horses. Skin surface notably improved in all horses: hyperkeratosis decreased, erosions and ulcerations healed completely and crusts disappeared. After 2 weeks, a mean volume reduction of 11.25% was seen, ranging from 4.75–21.74% and quality of movement improved. This pilot study documents evidence that CDT assists management of CPL. Current CPL management is limited to palliative treatments of secondary infections. Whilst not a permanent treatment, CDT offers a promising tool to manage horses with CPL, improving their quality of life and potential usefulness. More extensive and prolonged studies with a larger number of horses are warranted to evaluate the full potential of CDT.     

Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.