Myat Kyaw-Tanner

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis.

Equine laminitis: increased transcription of matrix metalloproteinase‐2 (MMP‐2) occurs during the developmental phase

REASONS FOR PERFORMING STUDY The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. OBJECTIVES To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. METHODS Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression. RESULTS Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P < 0.01) elevated in laminitis affected tissues. CONCLUSION The lamellar pathology of laminitis is associated with increased transcription of MMP-2. POTENTIAL RELEVANCE Real-time PCR analysis of lamellar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies. Controlling increased MMP-2 transcription may ameliorate or prevent laminitis in high risk clinical situations. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before clinical signs are apparent.

Morphological and molecular characterisation of Echinococcus granulosus in livestock and humans in Punjab, Pakistan

Cystic echinococcus poses an important economic and public health problem to Pakistan. Our study determined the prevalence and genotypes of Echinococcus present in domestic livestock and humans in Punjab, Pakistan. Out of 39,738 animals examined, 6.67% of animals were found infected. The prevalence and fertility of hydatid cysts was highest in camels (prevalence 17.29%; proportion fertile 95%), followed by sheep (prevalence 7.52%; proportion fertile 86.4%), buffalo (prevalence 7.19%; proportion fertile 84.3%), goats (prevalence 5.48%; proportion fertile 79.09%) and cattle (prevalence 5.18%; proportion fertile 75.25%). Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the common sheep strain (G1) and buffalo strain (G3) are cycling among livestock in Punjab and that these strains are highly adapted to goats, camels and cattle. Both human cysts were found to belong to the common sheep strain (G1) of E. granulosus, reinforcing this strain has the most potential for zoonotic transfer. Both morphological and molecular results support earlier studies suggesting that Echinococcus of sheep and buffalo origin is phenotypically and genetically similar which adds further evidence to support its recognition as one species viz, Echinococcus granulosus sensu stricto.

Equine laminitis: Membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset

REASONS FOR PERFORMING STUDY Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. OBJECTIVES To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. METHODS Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. RESULTS Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. CONCLUSION MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. POTENTIAL RELEVANCE Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity.

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n=4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n=4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6h (n=4), 12h (n=4), 24h (n=4) and 48 h (n=4), and in normal control horses (n=4). The only change in gene expression observed was an upregulation of MMP-9 (p<0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p<0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

The induction of in vivo superinfection and recombination using feline immunodeficiency virus as the model

SummaryThis study investigated the hypothesis that under certain conditions, superinfection of cats with feline immunodeficiency virus (FIV), may occur. One FIV isolate (T91) was used to inoculate three FIV and FeLV-free cats. Blood from an FIV-infected cat (N), which contained two variants and differed from T91 by at least 5% in nucleotide sequence in theenv gene, was inoculated into a fourth cat. Both T91 and blood from N were inoculated simultaneously into a fifth cat. After 22 weeks, two of the three cats initially infected with T91 were challenged with blood from N. At 30 weeks following initial infection, peripheral blood mononuclear cells were obtained from all cats, DNA was extracted, and a segment of theenv gene was PCR amplified, cloned and sequenced. Nucleotide sequence analysis of the cloned PCR product showed that virus strains used in initial infection were recovered from cats not challenged with a second variant. Challenge of cats with the blood of N following initial infection with T91 resulted in superinfection occurring in one cat and recombination occurring in the other. Furthermore, the use of blood as a source of challenge, in cats where superinfection and simultaneous infections were attempted, may have induced the appearance of variants which more closely resembled the most heterologous strain present in the infectious source.

Fluoroquinolone resistance mechanisms in multidrug-resistant Escherichia coli isolated from extraintestinal infections in dogs.

Fluoroquinolone resistance is an emerging problem in companion animal practice. The present study aimed to determine comparative fluoroquinolone minimum inhibitory concentrations (MICs) for enrofloxacin, marbofloxacin and pradofloxacin and identify plasmid-mediated quinolone resistance (PMQR) mechanisms in 41 multidrug-resistant (MDR) Escherichia coli isolates representing three main clonal groups (CGs) cultured from extraintestinal infections in dogs. All isolates were resistant to fluoroquinolones and the PMQR genes qnrA1, qnrB2, qnrS1 and qepA were identified in isolates from each CG. For a subset of 13 representative isolates, fluoroquinolone chromosomal resistance mechanisms were characterized. CG1 isolates had three mutations in the quinolone resistance determining region (QRDR), two in gyrA (Ser TCG-83→Leu TTG and Asp GAC-87→Asn AAC) and one in parC (Ser AGC-80→Ile ATT), whilst CG2 and CG3 isolates also possessed an additional mutation in parC (Glu GAA-84→Gly GGA) which was reflected in higher fluoroquinolone MICs compared to CG1. Organic solvent tolerance was demonstrated in 8 of the 13 isolates, and all 13 isolates demonstrated enhanced efflux on the basis of a 4-fold decrease or greater in the MIC of enrofloxacin when incubated with an efflux pump inhibitor. A mutation in acrR which can cause overexpression of the AcrAB multidrug efflux pump was detected in CG1 strains. These findings indicate that fluoroquinolone resistance in MDR E. coli isolated from extraintestinal infections in dogs is associated with a combination of target mutations in the QRDRs, transferable PMQR mechanisms and enhanced efflux.

Quasispecies and naturally occurring superinfection in feline immunodeficiency virus infection

SummaryAnalysis of individual clones containing the V1 and V2 domains of the segment of the FIVenv gene present in a naturally infected cat (T) was carried out. The polymerase chain reaction (PCR) was used to amplify proviral FIV DNA extracted from peripheral blood mononuclear cells (PBMCs) obtained in October 1994 from this cat. The PCR products were cloned and the DNA sequences determined for 11 clones. Sequences obtained were aligned with sequences corresponding to FIV isolates (T90, T91, T92) previously obtained from the same cat in 1990, 1991 and 1992. Phylogenetic analysis was performed which included consensus sequences of another Australian isolate, N91, as well as UK, US, Swiss and Japanese isolates of FIV.All clones varied from each other, and none of these clones was identical to the consensus sequences of the isolates obtained previously from the same cat (the T-series). However, most of these clones appeared to have originated from the ancestor of the most recent isolate (T92). In addition, 2 of the clones (7&11) are closely related to another Australian isolate N91, obtained from a different cat (N) in 1991. Because these two cats (T and N) were housed together for at least 3 years (1990–1993) it is suggested that the first cat (T) has become superinfected with an isolate from a second cat (N) under natural conditions.The identification of clones of differing sequences, which were not identical to each other nor to their ancestors, emphasises the rapid mutation of lentiviruses within theenv region, and the difficulty of developing an effective FIV vaccine. More importantly, the possibility of natural superinfection with FIV in cats has implications for the development of a successful lentiviral vaccine.

High allergen-specific serum immunoglobulin E levels in nonatopic West Highland white terriers.

Human and canine atopic dermatitis (AD) share an association with IgE specific to environmental allergens, but few studies have evaluated serum allergen-specific IgE in nonatopic dogs. This study compared serum allergen-specific IgE levels in 30 atopic and 18 nonatopic West Highland white terriers. Atopic dermatitis was confirmed using standard criteria. Nonatopic dogs were over 5 years of age and had no clinical signs or history of AD. Serum allergen-specific IgE levels were measured with Allercept(®) IgE ELISAs using a 48-allergen Australian panel. Positive reactions were defined as ≥150 ELISA absorbance units. Intradermal tests were performed in 16 atopic dogs, either at the time of or at various times prior to serum collection. In atopic dogs, the most common positive ELISA and intradermal test results were to Dermatophagoides farinae (11 of 30 dogs), but there were no statistically significant correlations between results from the two methods for any allergen. In nonatopic dogs, multiple high-positive ELISA reactions were reported to 45 of 48 allergens, most commonly D. farinae and Tyrophagus putrescentiae (17 of 18 dogs each). Positive ELISA results in nonatopic dogs were statistically significantly higher than those in atopic dogs for 44 of 48 allergens, including two allergens (D. farinae and Dermatophagoides pteronyssinus) commonly regarded as significant in canine AD. In conclusion, positive allergen-specific IgE ELISAs were not specific for canine AD, and high allergen-specific IgE levels were seen in nonatopic dogs. The clinical significance of this and whether it characterizes a protective phenotype is unclear.

IgE Responsiveness to Dermatophagoides farinae in West Highland White Terrier Dogs Is Associated with Region on CFA35

Immunoglobulin E (IgE)-mediated hypersensitivity against environmental allergens, commonly including Dermatophagoides farinae, is associated with atopic diseases in both humans and dogs. We have recently identified a family of clinically healthy West Highland white terriers (WHWTs) with high-serum D. farinae-IgE levels. In this study, we investigated the genetic mechanism controlling IgE responsiveness in dogs by performing a genome-wide association study (GWAS) using the Affymetrix V2 Dog SNP array in 31 high-IgE and 24 low-IgE responder WHWTs. A gene-dropping simulation method, using SIB-PAIR software, showed significant allelic association between serum D. farinae-specific IgE levels and a 2.3-Mb area on CFA35 (best empirical P = 1 × 10(-5)). A nearby candidate gene, CD83, encodes a protein which has important immunological functions in antigen presentation and regulation of humoral immune responses. We sequenced this gene in 2 high-IgE responders and 2 low-IgE responders and identified an intronic polymorphic repeat sequence with a predicted functional effect, but the association was insufficient to explain the GWAS association signal in this population (P = 1 × 10(-3)). Further studies are necessary to investigate the significance of these findings for IgE responsiveness and atopic disease in the dog.