Phylogenomic evidence for ancient hybridization in the genomes of living cats (Felidae)

Inter-species hybridization has been recently recognized as potentially common in wild animals, but the extent to which it shapes modern genomes is still poorly understood. Distinguishing historical hybridization events from other processes leading to phylogenetic discordance among different markers requires a well-resolved species tree that considers all modes of inheritance and overcomes systematic problems due to rapid lineage diversification by sampling large genomic character sets. Here, we assessed genome-wide phylogenetic variation across a diverse mammalian family, Felidae (cats). We combined genotypes from a genome-wide SNP array with additional autosomal, X- and Y-linked variants to sample ∼150 kb of nuclear sequence, in addition to complete mitochondrial genomes generated using light-coverage Illumina sequencing. We present the first robust felid time tree that accounts for unique maternal, paternal, and biparental evolutionary histories. Signatures of phylogenetic discordance were abundant in the genomes of modern cats, in many cases indicating hybridization as the most likely cause. Comparison of big cat whole-genome sequences revealed a substantial reduction of X-linked divergence times across several large recombination cold spots, which were highly enriched for signatures of selection-driven post-divergence hybridization between the ancestors of the snow leopard and lion lineages. These results highlight the mosaic origin of modern felid genomes and the influence of sex chromosomes and sex-biased dispersal in post-speciation gene flow. A complete resolution of the tree of life will require comprehensive genomic sampling of biparental and sex-limited genetic variation to identify and control for phylogenetic conflict caused by ancient admixture and sex-biased differences in genomic transmission.There is an emerging consensus that gene flow frequently occurs following speciation despite the establishment of reproductive barriers that otherwise maintain species-level distinctiveness (Roca et al. 2005; Good et al. 2008; Ellegren et al. 2012; Garrigan et al. 2012; Toews and Brelsford 2012; Cahill et al. 2013, 2014; Cui et al. 2013; Martin et al. 2013; Kutschera et al. 2014; Sullivan et al. 2014). However, incomplete lineage sorting (ILS) is assumed by default to underpin most cases of phylogenetic discordance. Few studies in the literature account for hybridization by analyzing all inheritance patterns (uniparental, sex-biased, biparental) with high-resolution data, or instead have focused on only a few species (Roca et al. 2005; Cahill et al. 2013, 2014; Trigo et al. 2013; Khan et al. 2014). The cat family Felidae contains 38 recognized species within eight lineages (designated henceforth by a capitalized name, e.g., Puma lineage) that vary in the breadth of their geographic occurrence (Buckley-Beason et al. 2006; Johnson et al. 2006). While the relationships within many felid clades are robust to variation in subgenomic sampling, several inter-generic and inter-specific relationships remain unresolved and have not been assessed genome-wide to determine the specific drivers of discordance observed in previous studies (Johnson et al. 2006; Davis et al. 2010).Although hybrid zones between related cat species have been reported (Schwartz et al. 2004; Homyack et al. 2008; Trigo et al. 2008, 2013), the extent to which ancient and contemporary introgression has occurred is poorly understood on broad geographic and genomic scales. Recent genetic evidence suggests complex patterns of admixture in felids of the Neotropical genus Leopardus, including the presence of cryptic species (Trigo et al. 2008, 2013). These observations are matched by the prevalence of felid hybridization in captivity (Gray 1972), which has generated numerous hybrids of both large cats and medium to small cats. These include the gigantic liger, a hybrid between a male lion and female tiger, as well as domestic cat inter-specific hybrid breeds, including the Bengal and Savannah, which are common household pets worldwide. This proclivity for hybridization is facilitated by the strong colinearity among felid genomes coupled with recent genetic divergence (Wurster-Hill and Centerwall 1982; Davis et al. 2009; Cho et al. 2013). The genomes of modern felids thus present a unique resource to study the dynamics of introgression and the genetic basis of reproductive isolation in both controlled crosses and natural populations (Davis et al. 2015).Previous studies have demonstrated that robust phylogenetic signal can be obtained by querying domestic animal SNP arrays with DNA from related species of the same genus, family, or order, despite having diverged tens of millions of years from the array reference genome (Decker et al. 2009; McCue et al. 2012). Here, we generated genome-wide SNP data from 38 cat species and analyzed these separately and together with Y-linked variation and whole mitogenomes, allowing us to disentangle different maternal, paternal, and biparental histories within a diverse family of mammals. We assessed genome-wide patterns of intra-lineage phylogenetic discordance and identified signals of ancient hybridization throughout the genomes of many cat species. Many of these nuclear signatures were accompanied by patterns of mitonuclear discordance. Our results allow further insight into the evolutionary processes leading to the diversification of extant cats of the world and provide a roadmap for future in-depth population genomics in this group as a model system for better understanding the speciation process.     

Efficacy of dry-cleaning in removing Fel d 1 allergen from wool fabric exposed to cats.

BACKGROUND: The main cat allergen (Fel d 1) is ubiquitous, having been found even in indoor environments and public places where a cat has never been kept. Clothes of cat owners constitute a carrier for the distribution of Fel d 1 allergen in these environments. Schools, for example, may be a site of indirect exposure to cat allergens. OBJECTIVE: Our goal was to investigate the efficacy of commercial dry-cleaning in removing cat allergens from wool fabrics that had been exposed to cats to evaluate a possible preventive procedure. METHODS: Twenty-six identical wool "squares" (80 x 100 cm) were put in cat baskets for 1 week. In our laboratory, the squares were cut in half (40 x 50 cm), and one half was subjected to high-volume sampling for 5 minutes in a cat-free room. The other half was subjected to commercial dry-cleaning and then the high-volume sampling. Five wool squares not exposed to cats served as controls. Dust was collected from the wool squares with a high-volume air sampler. Particulate material was harvested onto glass fiber filters (AP 20 Millipore, Milan, Italy) with 25-mm diameter and 2-microm pore size. Each dust sample was assayed by affinity-purified monoclonal antibody against purified Fel d 1. The results were expressed as micrograms per filter. Statistical analysis was done by using the paired t test. RESULTS: Before dry-cleaning, Fel d 1 allergen was detected on all cat-exposed wool squares. No appreciable cat allergen was detected on control materials. After commercial dry-cleaning, the amounts of Fel d 1 extracted from cat-exposed squares were significantly reduced (t = 14.63; P < 0.001) but not abolished. Three of the five control squares were contaminated by Fel d 1. CONCLUSIONS: Commercial dry-cleaning effectively removes large amounts of cat allergen from wool materials exposed to cats but does not completely abolish this protein. Further, low Fel d 1 contamination may occur during this procedure.     

Evidence for a new malaria vector species, species E, within the Anopheles culicifacies complex (Diptera: Culicidae).

Female Anopheles culicifacies Giles from Ramanathapuram district, Tamil Nadu state, India, were examined for oocysts and sporozoites and their larval progeny for mitotic karyotype. Collections were made from Mandapam and Uchipuli on the mainland, and Thangachimadam and Pamban on Rameshwaram Island. Of the 451 An. culicifacies females that were collected and dissected, 24 were found positive for Plasmodia (21 for sporozoites and 3 for oocysts). Both acrocentric and submetacentric Y-chromosome karyotypes were observed among the progeny of females from all villages. All 11 iso-female lines whose parental females were positive for sporozoites or oocysts had progeny with submetacentric Y-chromosomes. Total absence of sporozoite-positives among mothers of acrocentric males was evidence of assortative mating between these 2 sympatric populations (i.e., 2 species). We propose that the nonvector population with acrocentric Y-chromosome sons retain the original designation of species B and that the vector population with the submetacentric Y-chromosome sons be designated as species E, a new species.     

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I.
Two monoclonal antibodies directed against reduced/alkylated chain I bound to the overlapping peptides 53–66 and 60–70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36–49 and 43–56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies.
Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies (27). It bound to peptides 1–16 and 60–70 of chain 1 and peptides 1–14 and 50–63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies.
A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60–70 of chain 1 and peptides 1–14 and 43–56 of chain 2. For two monoclonal antibodies, binding to each peptide was investigated and shown to be inhibitable by native Fel d I. These antibodies are therefore probably directed against a conformational epitope formed by these four regions.
These studies give us substantial information about the quaternary structure of Fel d I.     

The scaling of postcranial muscles in cats (Felidae) II: hindlimb and lumbosacral muscles

In quadrupeds the musculature of the hindlimbs is expected to be responsible for generating most of the propulsive locomotory forces, as well as contributing to body support by generating vertical forces. In supporting the body, postural changes from crouched to upright limbs are often associated with an increase of body mass in terrestrial tetrapods. However, felids do not change their crouched limb posture despite undergoing a 300‐fold size increase between the smallest and largest extant species. Here, we test how changes in the muscle architecture (masses and lengths of components of the muscle‐tendon units) of the hindlimbs and lumbosacral region are related to body mass, to assess whether there are muscular compensations for the maintenance of a crouched limb posture at larger body sizes. We use regression and principal component analyses to detect allometries in muscle architecture, with and without phylogenetic correction. Of the muscle lengths that scale allometrically, all scale with negative allometry (i.e. relative shortening with increasing body mass), whereas all tendon lengths scale isometrically. Only two muscles' belly masses and two tendons' masses scale with positive allometry (i.e. relatively more massive with increasing body mass). Of the muscles that scale allometrically for physiological cross‐sectional area, all scale positively (i.e. relatively greater area with increasing body mass). These muscles are mostly linked to control of hip and thigh movements. When the architecture data are phylogenetically corrected, there are few significant results, and only the strongest signals remain. None of the vertebral muscles scaled significantly differently from isometry. Principal component analysis and manova s showed that neither body size nor locomotor mode separate the felid species in morphospace. Our results support the inference that, despite some positively allometric trends in muscle areas related to thigh movement, larger cats have relatively weaker hindlimb and lumbosacral muscles in general. This decrease in power may be reflected in relative decreases in running speeds and is consistent with prevailing evidence that behavioural changes may be the primary mode of compensation for a consistently crouched limb posture in larger cats.     

Influence of cat characteristics on Fel d 1 levels in the home

BACKGROUND: Previous studies investigating cat characteristics and cat allergen production focused on clinical experiments that quantified allergen from either the shaved skin or the fur of the animal; however, these studies did not address these experimental relationships in the home. OBJECTIVE: To determine the relationships between cat characteristics and cat allergen isolated from household dust. METHODS: Fel d 1 allergen levels in dust from homes participating in a population-based study of environmental effect on allergy development were analyzed using a standard monoclonal antibody-based assay. Cat characteristics were based on interviews conducted during home visits by study personnel. RESULTS: Households with any cats had higher geometric mean Fel d 1 levels than households without cats (32.88 vs 0.43; P < .01), and cat allergen levels increased with increasing numbers of cats in the home (P < .01). Length of cat hair, cat sex, reproductive status, and time spent indoors were analyzed; the only characteristic associated with higher levels of Fel d 1 was whether the cat had been neutered or spayed. CONCLUSIONS: Having cats in the home is significantly associated with increased Fel d 1 levels, and having more cats in the home is correlated with more cat allergen. Cat reproductive characteristics may be associated with measurable differences in cat allergen levels.     

Fel d 1 production in the cat skin varies according to anatomical sites

BACKGROUND: Fel d 1 is the major cat allergen, inducing asthma in sensitized individuals. It is produced by the skin and lies on fur. Recently, it was established that the amount of Fel d 1 on fur varies among anatomical sites. However, it is not known whether the allergen production by skin varies in parallel. The objective was to compare the Fel d 1 production by male cat skin in two anatomical sites, the face and the chest, in order to correlate it with Fel d 1 amounts on fur, and to assess the reaccumulation of Fel d 1 after washing. METHODS: Ten intact male cats were shaved under general anesthesia at both areas, and the fur was collected. The skin was washed and the washing fluid collected for Fel d 1 assays. Fel d 1 levels were measured in microg/g fur and ng/cm2 skin by ELISA before and after washing and 48 h later. RESULTS: In skin washing, the mean Fel d 1 level was significantly higher in the face (1015.2 +/- 821.6 ng/cm2) than the chest (115.2 +/- 66.8 ng/cm2). In the fur, the respective levels were 63.6 +/- 34 and 29.6 +/- 13.6 microg/g. In the skin sample taken after skin washing, the level of Fel d 1 dropped to 25.1 +/- 25.7 ng/cm2 on the face and to 22 +/- 17.4 ng/cm2 on the chest. After 2 days, skin Fel d 1 levels returned to basal values, with higher values on the face than the chest. CONCLUSIONS: This study shows that Fel d 1 levels on the skin are dramatically higher on the facial area than chest. This anatomical variation is concordant with the levels of Fel d 1 found on fur. Washing reduces levels of major allergen on cat skin and fur, but the accumulation on skin is restored within 2 days.     

Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE.

In this paper we show the reactivity of monoclonal antibodies (mAbs) and human IgE with Fel d I from different allergen sources in reduced SDS-PAGE immunoblots. By SDS-PAGE analysis of affinity-purified 125I-Fel d I, a 14- to 20-kD band was found, which dissociated under reducing conditions into a 4- to 5-kD chain (chain 1) and a 11- to 15-kD chain (chain 2). In initial immunoblotting experiments with mAbs against Fel d I however, only chain 1 was detected, while the mAbs lost activity upon reduction of Fel d I. Therefore mAbs were raised against reduced and alkylated Fel d I. Two of the four mAbs to 'denatured' Fel d I that were obtained did react with chain 2 on an immunoblot under reducing conditions; the other two reacted with chain 1. The mAbs did not react with native Fel d I. With these mAbs and human IgE, differences between allergen source materials in blot patterns of Fel d I were detected. A variable molecular weight for the protein stained with mAb antichain 2 was found, and occasionally the presence of a 12-kD band stained with mAb antichain 1. Human IgE strongly bound to chain 1 of Fel d I, while only 2 out of 6 sera gave a strong reaction with chain 2. The additional 12-kD band was also recognized by human IgE. In a competitive radioimmunoassay with mAb antichain 1, differences in levels of 'denatured' Fel d I between commercial extracts were quantitated. In vitro 'denatured' Fel d I was generated under high pH conditions. The reactivity of human IgE with this 'denatured' Fel d I was demonstrated in indirect RAST experiments with mAb antichain 1. We conclude that mAb antichain 1 recognizes a form of Fel d I that is not detected by mAb antinative Fel d I, but does react with human IgE.     

Monoclonal antibody based radioimmunoassay for the quantitation of the main cat allergen (Fel d I or Cat-1)

A two-site solid-phase radioimmunoassay has been developed for the quantitation of the main cat allergen, Fel d I or cat allergen 1. The assay is based on two different monoclonal antibodies which recognize different epitopes on the Fel d I molecule; one antibody (C5/24) was immobilized on the solid phase and the other (C5/8) was labeled with 125I, being the allergen molecule sandwiched between them. The assay is specific for the Fel d I molecule and sensitive enough to detect as little as 0.25 ng/ml of allergen. The Fel d I RIA was compared with a radial immunodiffusion technique for the determination of allergen levels in several cat extracts and a good quantitative correlation was found. The same good correlation was found when the results obtained with the Fel d I RIA were compared with the determination of the total allergenic activity by RAST inhibition. The results indicate that the MAb RIA could be very useful in the standardization of allergenic extracts from feline origin.     

Fel d 1 and Can f 1 in settled dust and airborne Fel d 1 in allergen avoidance day-care centres for atopic children in relation to number of pet-owners, ventilation and general cleaning

BACKGROUND: Special day-care centres for atopic children have been established in Sweden. OBJECTIVE: To study concentrations of cat (Fel d 1) and dog (Can f 1) allergens in settled dust and airborne cat allergen in day-care centres in relation to pet ownership among children and staff, ventilation and general cleaning. METHODS: Twelve allergen avoidance day-care centres and 22 conventional day-care centres were included in the study. Settled dust was collected and analysed with ELISA. Airborne cat allergen levels were measured in eight allergen avoidance and seven conventional centres with a personal air sampler and analysed with an amplified ELISA. Air change rate per hour (ACH) was measured. A questionnaire which focused on keeping of cat and dog among staff and children and frequency of general cleaning was used. RESULTS: In the allergen avoidance day-care centres neither children nor staff reported ownership of cats or dogs, compared with 21/22 of the conventional centres in which children and staff kept furred animals. Fel d 1 and Can f 1 were found in settled dust in all day-care centres. In the allergen avoidance compared with the conventional centres the concentrations of Fel d 1 and Can f 1 were lower, Fel d 1: median 0. 64 microg/g vs 5.45 microg/g and Can f 1: 0.39 microg/g vs 2.51, both P < 0.001, and airborne Fel d 1 was also lower in the allergen avoidance centres compared with the control centres, 1.51 ng/m3 vs 15.8 ng/m3, P = 0.002. A correlation was found between airborne and settled Fel d 1, rs = 0.75, P < 0.001. Furthermore, a correlation was found between increased ACH and decreased levels of Fel d 1 in the air in the day-care centres with no cat-owners, rs = - 0.86, P = 0.007. No relation was found between levels of cat or dog allergen and amount of general cleaning. CONCLUSION: Not keeping pets seems to reduce children's exposure to pet-allergen in their 'working environment'. Additionally, appropriate ventilation seems to reduce Fel d 1 in the air in day-care centres.     

The scaling of postcranial muscles in cats (Felidae) I: forelimb,cervical, and thoracic muscles

The body masses of cats (Mammalia, Carnivora, Felidae) span a ~300‐fold range from the smallest to largest species. Despite this range, felid musculoskeletal anatomy remains remarkably conservative, including the maintenance of a crouched limb posture at unusually large sizes. The forelimbs in felids are important for body support and other aspects of locomotion, as well as climbing and prey capture, with the assistance of the vertebral (and hindlimb) muscles. Here, we examine the scaling of the anterior postcranial musculature across felids to assess scaling patterns between different species spanning the range of felid body sizes. The muscle architecture (lengths and masses of the muscle‐tendon unit components) for the forelimb, cervical and thoracic muscles was quantified to analyse how the muscles scale with body mass. Our results demonstrate that physiological cross‐sectional areas of the forelimb muscles scale positively with increasing body mass (i.e. becoming relatively larger). Many significantly allometric variables pertain to shoulder support, whereas the rest of the limb muscles become relatively weaker in larger felid species. However, when phylogenetic relationships were corrected for, most of these significant relationships disappeared, leaving no significantly allometric muscle metrics. The majority of cervical and thoracic muscle metrics are not significantly allometric, despite there being many allometric skeletal elements in these regions. When forelimb muscle data were considered in isolation or in combination with those of the vertebral muscles in principal components analyses and MANOVAs, there was no significant discrimination among species by either size or locomotory mode. Our results support the inference that larger felid species have relatively weaker anterior postcranial musculature compared with smaller species, due to an absence of significant positive allometry of forelimb or vertebral muscle architecture. This difference in strength is consistent with behavioural changes in larger felids, such as a reduction of maximal speed and other aspects of locomotor abilities.     

Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor

BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures. CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.     

Effects of various vacuum cleaners on the airborne content of major cat allergen (Fel d 1)

Background It has been shown that a vacuum cleaner (VC) can increase airborne cat allergen levels. This study aimed to compare the degree of leakage of airborne Fel d 1 levels among five different VCs, both under laboratory conditions and in an apartment with cats.
Methods Three of the VCs were marketed as antiallergic: a HEPA filter VC (VC A), a water impingement and HEPA filter VC (VC B), and a foam fabric filter VC (VC C). The other two were standard VCs: VC D and VC E. VCs were tested in a 20 m', airtight, experimental room and in a 53 m²* living room in an apartment with three cats. Air was sampled with a glass-fiber filter and an impinger at 20 1/min for 30 min before, during, and after vacuuming. Airborne Fel d 1 was measured with a two-site monoclonal ELISA assay.
Results In the experimental room, no airborne Fel d 1 level was measured before using the VCs. After introducing a dust sample containing Fel d 1 in the VCs. we found that VCs A, B, and E did not provoke any increase in airborne Fel d I. In contrast, VCs C and D significantly increased airborne Fel d 1 levels (GM: 4.9 and 5.3 ng/m, respectively). In the apartment, all VCs induced an increase in airborne Fel d 1, which was carried by particles greater than 5 nm. However, VCs C and D provoked significantly greater increases in airborne Fel d 1 than VCs A, B, and E (P=0.0001).
Conclusions Our results suggest that:

• 1)

  The two VCs with leakage in the experimental room had greater leakages in the apartment.
• 2)

  In the apartment with cats, all VCs provoked increases in airborne Fel d 1, primarily carried by large particles.
• 2)

  Given the increased marketing of "antiallergic" VCs, further studies are needed to standardize methods for testing airborne allergen leakage by VCs.

     

Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

BACKGROUND: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.     

Demonstration of a partially cryptic epitope of the major cat allergen Fel d 1: consequences for mAb-based standardization of cat extracts

BACKGROUND: Antiallergen mAbs that do not recognize clinically important isoforms have been described, raising the question of the selection of mAbs for quantifying major allergens in order to standardize allergenic extracts. This question is even more critical if mAbs can discriminate between different forms of allergen molecules with the same amino acid sequence. OBJECTIVE: We sought to demonstrate that an anti-Fel d 1 mAb was able to discriminate between two forms of the major cat allergen independently of its amino acid sequence and to determine the relative importance and stability of both forms in various cat extracts. METHODS: Anti-Fel d 1 mAbs were raised in mice and characterized. By using two of these mAbs, a two-site ELISA was developed to quantify Fel d 1 in mass units. RESULTS: One of the anti-Fel d 1 mAbs developed was shown to specifically recognize a particular form of Fel d 1. A two-site ELISA with this mAb to capture Fel d 1 was able to quantify the allergen specifically in this form. It was then shown that (1) the quantitative importance of this form of Fel d 1 could vary from one cat extract to another, (2) Fel d 1 was converted into this form under certain conditions, and (3) both converted and unconverted forms of Fel d 1 may bear IgE epitopes that are specific. CONCLUSION: Although the present study emphasizes the issue of selecting mAbs that are not too specific to standardize allergenic extracts, it also demonstrates that very specific mAbs can be of interest, especially to verify the stability of allergens in extracts, since this stability might have clinical implications.     

Evidence for a loss of afferent axons in the visual cortex of monocularly deprived cats

The retrograde transport of horseradish peroxidase (HRP) was used to study the distribution of axonal terminals from the deprived and active laminae of the lateral geniculate nucleus (LGN) to the visual cortex in monocularly deprived cats. In two cats deprived from before natural lid opening, after injection of HRP in the visual cortex far fewer cells were labelled in the deprived than in the normal laminae of the LGN, implying that the distribution of their axons within the cortex had been reduced. One cat, deprived after an initial period in which both eyes were open, had much more equal labelling of deprived and normal laminae. These results suggest that a change in the ocular dominance of cortical neurones is sometimes, but not always, due to an actual loss of afferent fibres.