Magnitude and Kinetics of T Cell and Antibody Responses During H1N1pdm09 Infection in Inbred Babraham Pigs and Outbred Pigs.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αß, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to days 4 to 5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from days 5 to 6 post infection reaching a peak at 9 to 14 days. γδ T cells produced cytokines ex vivo at day 2 post infection, while virus reactive IFNγ producing γδ T cells were detected from day 7 post infection. Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes, and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4, and γδ T cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyze immune responses to influenza.

Aerosol Delivery of a Candidate Universal Influenza Vaccine Reduces Viral Load in Pigs Challenged with Pandemic H1N1 Virus.

Influenza A viruses are a major health threat to livestock and humans, causing considerable mortality, morbidity, and economic loss. Current inactivated influenza vaccines are strain specific and new vaccines need to be produced at frequent intervals to combat newly arising influenza virus strains, so that a universal vaccine is highly desirable. We show that pandemic H1N1 influenza virus in which the hemagglutinin signal sequence has been suppressed (S-FLU), when administered to pigs by aerosol can induce CD4 and CD8 T cell immune responses in blood, bronchoalveolar lavage (BAL), and tracheobronchial lymph nodes. Neutralizing Ab was not produced. Detection of a BAL response correlated with a reduction in viral titer in nasal swabs and lungs, following challenge with H1N1 pandemic virus. Intratracheal immunization with a higher dose of a heterologous H5N1 S-FLU vaccine induced weaker BAL and stronger tracheobronchial lymph node responses and a lesser reduction in viral titer. We conclude that local cellular immune responses are important for protection against influenza A virus infection, that these can be most efficiently induced by aerosol immunization targeting the lower respiratory tract, and that S-FLU is a promising universal influenza vaccine candidate.

Genome-Wide Analysis in Swine Associates Corneal Graft Rejection with Donor-Recipient Mismatches in Three Novel Histocompatibility Regions and One Locus Homologous to the Mouse H-3 Locus.

In rodents, immune responses to minor histocompatibility antigens are the most important drivers of corneal graft rejection. However, this has not been confirmed in humans or in a large animal model and the genetic loci are poorly characterised, even in mice. The gene sequence data now available for a range of relevant species permits the use of genome-wide association (GWA) techniques to identify minor antigens associated with transplant rejection. We have used this technique in a pre-clinical model of corneal transplantation in semi-inbred NIH minipigs and Babraham swine to search for novel minor histocompatibility loci and to determine whether rodent findings have wider applicability. DNA from a cohort of MHC-matched and MHC-mismatched donors and recipients was analysed for single nucleotide polymorphisms (SNPs). The level of SNP homozygosity for each line was assessed. Genome-wide analysis of the association of SNP disparities with rejection was performed using log-likelihood ratios. Four genomic blocks containing four or more SNPs significantly linked to rejection were identified (on chromosomes 1, 4, 6 and 9), none at the location of the MHC. One block of 36 SNPs spanned a region that exhibits conservation of synteny with the mouse H-3 histocompatibility locus and contains the pig homologue of the mouse Zfp106 gene, which encodes peptide epitopes known to mediate corneal graft rejection. The other three regions are novel minor histocompatibility loci. The results suggest that rejection can be predicted from SNP analysis prior to transplant in this model and that a similar GWA analysis is merited in humans.

Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis.

Recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (FCoV), which results in an amino acid change from methionine to leucine at position 1058, may be associated with feline infectious peritonitis (FIP). Tissue and faecal samples collected post mortem from cats diagnosed with or without FIP were subjected to RNA extraction and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect FCoV RNA. In cats with FIP, 95% of tissue, and 81% of faecal samples were PCR-positive, as opposed to 22% of tissue, and 60% of faecal samples in cats without FIP. Relative FCoV copy numbers were significantly higher in the cats with FIP, both in tissues (P < 0.001) and faeces (P = 0.02). PCR-positive samples underwent pyrosequencing encompassing position 1058 of the FCoV spike protein. This identified a methionine codon at position 1058, consistent with the shedding of an enteric form of FCoV, in 77% of the faecal samples from cats with FIP, and in 100% of the samples from cats without FIP. In contrast, 91% of the tissue samples from cats with FIP and 89% from cats without FIP had a leucine codon at position 1058, consistent with a systemic form of FCoV. These results suggest that the methionine to leucine substitution at position 1058 in the FCoV spike protein is indicative of systemic spread of FCoV from the intestine, rather than a virus with the potential to cause FIP.

Ischaemic distal limb necrosis and Klebsiella pneumoniae infection associated with arterial catheterisation in a cat.

This case report describes dorsal pedal arterial thrombosis and infection with Klebsiella pneumoniae subsequent to arterial catheter placement in a cat. The complication led to avascular necrosis of the metatarsal and pedal soft tissue. The catheter was placed for blood pressure monitoring during surgery for correction of a peritoneopericardial diaphragmatic hernia. The exact mechanism of thrombosis was unclear. Amputation of the limb was required and the histopathological findings are presented. This is the first report of such a complication.

Multilocus variable-number tandem-repeat analysis scheme for chlamydia felis genotyping: comparison with multilocus sequence typing.

Chlamydia felis is an important ocular pathogen in cats worldwide. A multilocus variable-number tandem-repeat analysis (MLVA) system for the detection of tandem repeats across the whole genome of C. felis strain Fe/C-56 was developed. Nine selected genetic loci were tested by MLVA in 17 C. felis isolates, including the C. felis Baker vaccine strain, and 122 clinical samples from different geographic origins. Analysis of the results identified 25 distinct C. felis MLVA patterns. In parallel, a recently described multilocus sequence typing scheme for the typing of Chlamydia was applied to 13 clinical samples with 12 different C. felis MLVA patterns. Rare sequence differences were observed. Thus, the newly developed MLVA system provides a highly sensitive high-resolution test for the differentiation of C. felis isolates from different origins that is suitable for molecular epidemiological studies.

A model of corneal graft rejection in semi-inbred NIH miniature swine: significant T-cell infiltration of clinically accepted allografts.

PURPOSE: The purpose of our study is to develop a pre-clinical model of corneal graft rejection in the semi-inbred NIH minipig as a model of human rejection. METHODS: NIH minipigs received corneal allografts with MHC and minor mismatches, or minor mismatches alone. Clinical rejection was monitored, and major subsets of leukocytes and ingress of vessels were quantified post-mortem by automated digital methods. Spectratypes of recipient T-cell receptor ß-subunit variable region (TRßV) were analyzed. The capacity of pig corneal endothelial cells to proliferate in vivo was assessed. RESULTS: Autografts (n = 5) and SLA(cc) to SLA(cc) allografts (minor mismatches, n = 5) were not rejected. Median graft survival of SLA(dd) and SLA(bb) allografts in SLA(cc) strain recipients (major and minor mismatches) was 57 (n = 10) and 67 (n = 6) days, respectively. Rejected grafts did not recover clarity in vivo, and corneal endothelial cells did not proliferate in organ culture after cryo-injury. There were significantly more leukocytes in clinically rejected versus accepted grafts (P < 0.0001) and in transplanted versus contralateral eyes (P < 0.0001). Numbers of T-cells were significantly greater in clinically accepted grafts versus autografts and in rejected grafts versus accepted (P < 0.005 for most subsets). There were significant differences in TRßV spectratype between graft groups in cornea, but not in draining lymph node or blood (P < 0.05). CONCLUSIONS: The NIH minipig offers a robust model of human rejection suitable for immunological or therapeutic studies. In particular, there is limited capacity for corneal endothelial repair in vivo, and histological evidence suggests that allosensitization of the recipient may develop in the absence of clinical rejection.

Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+)) antigen-presenting cell subset, whilst SIRPα(-)CD11R1(+) antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+) antigen-presenting cells as orchestrators of early-life mucosal immune development.

The Chlamydophila felis plasmid is highly conserved.

The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

Polymorphic membrane proteins 1 and 7 from Chlamydophila felis are significant immunodominant proteins.

Chlamydophila felis is a common cause of conjunctivitis in cats. Greater understanding of C. felis infection and immunity and identification of protective antigens will facilitate improved vaccine design. Chlamydial polymorphic membrane proteins (Pmps) represent a family of homologous proteins of likely importance in chlamydial infection and immunity. To identify immunogenic C. felis Pmps, we generated recombinant C. felis Pmps (rPmps) and used these to detect serum antibody reactivity against Pmps arising during C. felis infection in cats. Sequencing of Pmp genes 1, 7, 13, 18, 19 and 20 from 3 laboratory strains of C. felis (K2487, 1497V and Cello) and alignment with the Fe/C-56 genome revealed high genetic identity in Pmp genes between strains. PCR products lacking the predicted N-terminal signal sequence peptide and C-terminal domain were generated, cloned and expressed in Escherichia coli prior to purification by nickel-agarose affinity chromatography. Serum samples from 4 cats collected up to 55 days post-inoculation with C. felis (K2487) were analysed by western blotting and rPmp-specific ELISAs for evidence of serum antibody reactivity against each rPmp. Strong serum antibody reactivity against rPmps 1 and 7, and weak heterogeneous serum immunoreactivity against rPmps 13, 19 and 20, were detected from 14 to 21 days post-infection (dpi), peaking at 28-35 dpi and tending to plateau thereafter. No significant serum antibody reactivity was detected against rPmp18. This study provides the first evidence that C. felis Pmps 1 and 7 are likely to represent immunodominant proteins and recommends investigation of their potential as serodiagnostic antigens and novel vaccine candidates.

Correlation of the feline PKD1 genetic mutation with cases of PKD diagnosed by pathological examination.

Autosomal-dominant polycystic kidney disease (AD-PKD) is the most prevalent inherited genetic disease of cats, particularly affecting Persians. Using archived tissue samples from 44 cats a genotype was successfully obtained by real-time PCR for 43 cats. Twenty-five cats (18 Persians, 4 domestic longhair cats and 3 domestic shorthair (DSH) cats) were found to carry the AD-PKD mutation and all of these cats had macroscopic and/or microscopic evidence of renal cysts consistent with PKD. Eighteen cats were found to be wild-type. Twelve of these (all Persians) had no pathological evidence of PKD, but the remaining 6 cats had evidence of renal cystic lesions. On pathological review the cystic lesions in 4 (2 Persians and 2 DSH) of these 6 cats were considered not to be consistent with a primary diagnosis of PKD. Histological evidence of polycystic kidneys was, however, confirmed in the remaining 2 cats (1 DSH and 1 Bengal) and may indicate that other PKD-causing mutations exist in the feline population.

Molecular characterisation of 12 Chlamydophila felis polymorphic membrane protein genes.

A group of genes thought to encode members of the unique chlamydial polymorphic membrane protein (pmp) family were recently described in the Chlamydophila felis genome. This study aimed to commence characterisation of a subset of 12 of these putative pmp genes by developing and using gene-specific real-time (Q)PCR assays to confirm their presence in a wide range of C. felis field isolates and laboratory strains, and to look for pmp mRNA expression during in vitro infection. Sequencing of 525-698 base pair regions of pmp genes 7, 9-11, 13-20 for two laboratory strains of C. felis and alignment with the published Fe/C-56 sequence found only a single nucleotide polymorphism present in pmp9. Following the development of gene-specific (Q)PCR assays, analysis of genomic DNA extracted from 40 C. felis field isolates and 4 laboratory strains found that all 12 pmp genes were represented in all cases. Reverse transcription (RT)-QPCR analysis of RNA extracted from cell cultures at 24 and 48 h post inoculation with 1 of 5 different strains of C. felis detected transcripts for all 12 pmp genes at both time points. Analysis of the relative levels of pmp gene transcription suggested that down-regulation of the expression of multiple C. felis pmp genes occurs between 24 and 48 h post inoculation. This study provides the first evidence that 12 of the putative pmp C. felis genes are transcribed during in vitro infection, and shows that these genes are present in a large range of C. felis field isolates and multiple passage laboratory-grown strains.

Use of quantitative real-time PCR to monitor the response of Chlamydophila felis infection to doxycycline treatment.

Fifteen cats infected with Chlamydophila felis were monitored for the presence of C. felis DNA on ocular swabs by using real-time PCR and for clinical signs of disease. The cats were assigned to three groups: oral doxycycline at 10 mg/kg of body weight/day for 7 days (six cats), oral doxycycline at 10 mg/kg/day for 14 days (five cats), and an untreated control group (four cats). The untreated cats remained positive for C. felis throughout the trial; clinical signs were most severe on days 14 to 21 postinfection, and then they declined. Treatment with 7 and 14 days of doxycycline decreased C. felis relative copy numbers and clinical signs rapidly. C. felis became undetectable in some of the cats during or after treatment. However, after the cessation of treatment, a recurrence of high relative copy numbers of C. felis and severe clinical signs in all cats was seen. Rescue treatment with 21 days of doxycycline was successful at eliminating infection in eight of the cats; a further 28 days of doxycycline was required to eliminate infection in the remaining three cats. It was concluded that 7, 14, and, in some cases, 21 days of treatment with oral doxycycline will not eliminate C. felis infection. At least 28 days of treatment with doxycycline is required to ensure elimination of the organism. Real-time PCR is a sensitive technique for monitoring C. felis infection and the response to antibiotic treatment.

Aortic and cardiac mineralization in the dog.

Aortic and cardiac mineralization was found in 21 of 3443 (0.61%) canine thoracic radiographs. In none of 786 feline thoracic radiographs reviewed were such lesions present. Mineralizations were superimposed on the ascending aorta (19 dogs) or on the caudal cardiac silhouette (2 dogs). In 2 of 4 dogs mineralization was identified echocardiographically dorsal to the aortic valve in close proximity to coronary arteries. Computed tomography confirmed mineralization of the aortic arch and root in 2 of 2 dogs. Necropsy and histopathologic examination in 1 dog revealed multiple nodular aortic tunica media calcifications with adjacent areas of degeneration. Lesions were significantly overrepresented in older dogs and in Rottweilers, and regarded as dystrophic calcification, caused either by age-related degenerative changes or chronic disease-related processes. There was no evidence of clinical significance attributed to the mineralization in any dog. Aortic and cardiac mineralization should be recognized as an incidental, non-significant finding in dogs of advanced age and differentiated from pleural and pulmonary structures.