Feline IgG Research Articles

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications.

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.

Serodiagnosis of toxoplasma gondii, associated risk factors in domesticated cats: preventing zoonosis in humans and implications for livestock extension

Toxoplasmosis, an important zoonotic parasitic disease-causing infection in humans and contaminating the environment. Cats are the sole source of spreading infection. Current study was designed to serodiagnoses in the flooded area, Jhang, Pakistan. Using rapid identification pen side strip test 316 pet cats were examined at CVAS, Jhang and other private clinics in that area. Rapid ID Chromatographic immune assay was performed to detect the antibodies for Feline toxoplasma IgM and IgG in serum. Related associated risk factors like breed, sex, contact with other livestock animals, deworming, location type (urban or rural area), diet (nature of food either cooked or uncooked meat), and outdoor access for wandering were also interviewed by owner. Total seroprevalence of cats was 10.4% (33/316). IgG antibodies were found 9.09 (29/316) while IgM antibodies were 2.21% (7/316) in cats. Seroprevalence was significantly high in cats older than one year. No significant difference was recorded between males and females. Cats from peri urban areas showed higher prevalence. Cats having access to outside, contact with other animals and eating uncooked food showed high seroprevalence. The current study confirms that Toxoplasma gondii is widespread in pet animals in district Jhang, Pakistan. This study urges pets lower to be aware about the Toxoplasmosis diseases. Whereas, the livestock extension sector should disseminate the precautionary awareness among the public

Serodiagnosis of toxoplasma gondii, associated risk factors in domesticated cats: preventing zoonosis in humans and implications for livestock extension

Toxoplasmosis, an important zoonotic parasitic disease-causing infection in humans and contaminating the environment. Cats are the sole source of spreading infection. Current study was designed to serodiagnoses in the flooded area, Jhang, Pakistan. Using rapid identification pen side strip test 316 pet cats were examined at CVAS, Jhang and other private clinics in that area. Rapid ID Chromatographic immune assay was performed to detect the antibodies for Feline toxoplasma IgM and IgG in serum. Related associated risk factors like breed, sex, contact with other livestock animals, deworming, location type (urban or rural area), diet (nature of food either cooked or uncooked meat), and outdoor access for wandering were also interviewed by owner. Total seroprevalence of cats was 10.4% (33/316). IgG antibodies were found 9.09 (29/316) while IgM antibodies were 2.21% (7/316) in cats. Seroprevalence was significantly high in cats older than one year. No significant difference was recorded between males and females. Cats from peri urban areas showed higher prevalence. Cats having access to outside, contact with other animals and eating uncooked food showed high seroprevalence. The current study confirms that Toxoplasma gondii is widespread in pet animals in district Jhang, Pakistan. This study urges pets lower to be aware about the Toxoplasmosis diseases. Whereas, the livestock extension sector should disseminate the precautionary awareness among the public

A lineage-specific rapid diagnostic test (Chagas Sero K-SeT) identifies Brazilian Trypanosoma cruzi II/V/VI reservoir hosts among diverse mammalian orders.

Trypanosoma cruzi, the protozoan agent of Chagas disease in the Americas, is comprised of six genetic lineages (TcI-TcVI) and a possible seventh (TcBat, related to TcI). Identification of T. cruzi lineages infecting reservoir mammalian species is fundamental to resolving transmission cycles. However, this is hindered by the limited sensitivity and technical complexity of parasite isolation and genotyping. An alternative approach is serology using T. cruzi lineage-specific epitopes, such as those of the trypomastigote small surface antigen (TSSA). For surveillance of T. cruzi lineage infections in mammal species from diverse Brazilian regions, we apply a novel rapid diagnostic test (RDT, Chagas Sero K-SeT), which incorporates the TSSA peptide epitope specific to TcII/V/VI (TSSApep-II/V/VI) and Protein G detection of antibodies. Chagas Sero K-SeT RDT results with sera from experimentally infected mice, from tamarin primates (Leontopithecus spp.) and from canines (Canis familiaris) were concordant with corresponding TSSApep-II/V/VI ELISAs. The Chagas Sero K-Set detected TcII/V/VI infections in Leontopithecus spp. from the Atlantic forest (n = 46), in C. familiaris (n = 16) and Thrichomys laurentius (n = 2) from Caatinga biome and Chiroptera (n = 1) from Acre, Amazonia. The Chagas Sero K-SeT RDT is directly applicable to TcII/V/VI-specific serological surveillance of T. cruzi infection in several different mammalian Orders. It can replace ELISAs and provides efficient, point-of-sampling, low-cost detection of TcII/V/VI infections, with at least equivalent sensitivity, although some mammals may be difficult to trap, and, not unexpectedly, Chagas Sero K-SeT could not recognise feline IgG. Knowledge of sylvatic hosts of T. cruzi can be expanded, new reservoir species discovered, and the ecology of transmission cycles clarified, particularly with adaptation to further mammalian Orders.

Association of feline morbillivirus infection with defined pathological changes in cat kidney tissues

Feline morbillivirus (FeMV) is an emerging member of morbillivirus discovered in 2012. Although association of FeMV infection with kidney diseases in cats has been suggested, the pathogenicity of the virus has not been clear to date. To study the association between FeMV infection and pathological changes in kidney tissues of infected cats, we performed immunohistochemistry and immunofluorescent assays to detect FeMV antigens and analyzed the effect of FeMV infection on the pathological changes in the kidney tissues. In 38 kidney tissue samples from cats, some tissue injury scores were significantly higher when the FeMV antigens were detected, especially those for the tubular tissues in which the FeMV antigens were mostly localized. Pathological changes associated with the FeMV antigens included the ones typically found in chronic kidney diseases, such as interstitial cell infiltration, glomerulosclerosis, tubular atrophy and fibrosis. We also detected some feline IgG localizations in glomerular tissues, though co-localization or significant association with the FeMV antigens were not found. Our study confirms the association of FeMV infection with some kidney tissue injuries and provides additional information about roles of FeMV infection in chronic kidney diseases.

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)–UPLC, reversed phase (RP)–UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC–UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.

In Vitro functional characterization of feline IgGs

Very little is known about the functional properties of feline IgGs. Here we report the in vitro characterization of cloned feline IgGs. Rapid amplification of cDNA ends (RACE) and full-length PCR of cat splenic cDNA were used to identify feline sequences encoding IgG heavy chain constant regions (IGHC). Two of the sequences are possibly allelic and have been previously reported in the literature as the only feline IgG, IgG1. Although we confirmed these alleles to be highly abundant (∼98%), analysis of numerous amplification products revealed an additional sequence (∼2%). We cloned and characterized chimeric monoclonal antibodies with each of these heavy chains. Using RACE we revealed the sequences for feline Fc gamma receptor I (FcγRI) and feline Fc neonatal receptor (FcRn). We constructed these recombinant receptors as well as fFcγRIII and determined their binding affinities to the chimeras. All of the chimeras bound to Protein A but not to Protein G, and bound tightly to fFcRn (KD=2–5nM). Both IgG1 alleles have a high affinity for fFcγRI (KD=10–20nM), they bind to the low-affinity fFcγRIII receptor (2–4μM), and also bind to human complement C1q. Thus, feline IgG1a and 1b are expected to induce strong effector function in vivo. The additional IgG detected does not bind to recombinant fFcγRI or fFcγRIII and has negligible binding to hC1q. Consequently, although this putative subclass is projected to have a similar serum half-life as the IgG1 alleles based on comparable in vitro affinity to FcRn, it may not elicit the effector responses mediated by fFcγRI or fFcγRIII. Further testing with native receptors and functional cell-based assays would confirm effector function capabilities of feline IgG subclasses; however this is the first report characterizing affinities of feline IgGs to their Fc receptors and helps pave the way for construction of feline-specific IgGs for therapeutic use.

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, alpha-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH(4))(2)SO(4) fractionation (33-50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS-PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.

In vivo depletion of CD4+CD25+ regulatory T cells in cats

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.

Antibodies against Gag are diagnostic markers for feline foamy virus infections while Env and Bet reactivity is undetectable in a substantial fraction of infected cats

Spumaretroviruses or foamy viruses constitute a distinct subfamily of retroviruses. The biology of foamy viruses within the authentic host, their mode of transmission, and disease potential in the authentic host or after zoonotic transmission into human or other species are almost unknown. Using feline foamy virus (FFV) as model system, we established modular enzyme-linked immunosorbent assays (ELISA) suited to determine feline IgG and IgM antibody responses against structural and non-structural FFV proteins. We validated the ELISAs with standard reference sera. In 99 cats admitted to a Swiss veterinary hospital, overall FFV Gag antibody prevalence was 36%, reactivity against Env and the non-structural protein Bet each was about 25%, and 19% of the sera were directed against all three FFV antigens. With one exception, all Bet- and/or Env-positive sera were also positive for Gag. In this small epidemiological pilot study, FFV antibodies were not significantly associated with clinical disease.

Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived neonatal kittens.

To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. 22 kittens from 6 specific pathogen-free queens. Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow cytometry assay providing simultaneous measurement of bacteria-induced phagocytosis and oxidative burst. The opsonic capacity of kitten plasma was determined in an opsonophagocytosis assay with bacteria incubated in untreated or heat-inactivated plasma. Among treatment groups, there were no significant differences in neutrophil phagocytic and oxidative burst responses to bacteria or opsonic capacity of plasma. In all samples of plasma, inactivation of complement and other heat-labile opsonins significantly reduced the opsonic capacity. Plasma IgG concentrations in kittens did not correlate with neutrophil function or plasma opsonic capacity before or after inactivation of complement. The plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses in vitro of kittens receiving passive transfer of IgG via colostrum intake or IgG supplementation and those deprived of colostrum were similar. The alternate complement pathway or other heat-labile opsonins may be more important than IgG in bacterial opsonization and phagocytosis.

Densitometric analysis of Western blot assays for feline immunodeficiency virus antibodies

Western blot (WB) strips for antibodies directed to feline immunodeficiency virus (FIV) were analysed using reflectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a first format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values ( P=0.001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunodeficiency virus-1 (HIV-1) infections.

Immunohistochemical characterization of the lesions of feline progressive lymphocytic cholangitis/cholangiohepatitis

The histopathological features of liver biopsies from 20 cats with progressive lymphocytic cholangitis/cholangiohepatitis are reported. These biopsies were subject to immunohistochemical investigation for expression of CD3, CD79. Major Histocompatibility Complex (MHC) Class II molecules, and feline IgG, IgM and IgA. Livers from five normal cats, which were also examined showed constitutive expression of MHC Class II by sinusoidal Kupffer cells and bile duct epithelium, in addition to a population of portal, and bile duct inter-epithelial, CD3+ T lymphocytes. In liver biopsies from cats with the active phase of lymphocytic cholangitis/cholangiohepatitis (n = 11), the portal lymphocytes were predominantly CD3+ T cells that infiltrated bile duct epithelium and periportal hepatic parenchyma, CD79+ B lymphocytes formed distinct aggregates or follicles within the regions of T-cell infiltration. Low numbers of plasma cells were present, and these predominantly expressed IgA. MHC Class II was expressed by Kupffer cells, infiltrating T and B lymphocytes and macrophages. There was membrane and cytoplasmic Class II expression by bile duct epithelium, some vascular endothelium, and fibroblasts within areas of fibrosis. In liver biopsies from cats with chronic lymphocytic cholangitis/cholangiohepatitis (n = 9), there was less in flammation, but the composition of the infiltrates was similar to that in the active phase of disease. The findings provide further evidence for an immune mediated pathogenesis in progressive lymphocytic cholangitis/cholangiohepatitis.

Isolation and characterisation of immunoglobulin g and IgG subclasses of the African elephant ( Loxodonta africana)

Immunoglobulins were precipitated from pooled African elephant sera with ammonium sulphate and separated by gel filtration and fast protein liquid chromatography (ion exchange). Analysis of the fractions by SDS–PAGE showed IgG of 150 kDa with up to five subclasses, each having heavy chains of 57 kDa and light chains of 27 kDa. Three monoclonal antibodies against human IgG and polyclonal antibodies against canine, bovine, cameline, equine, phocine and feline IgG showed strong cross-reactivity with the African elephant IgG subclasses. No serum molecules corresponding to IgM or IgA could be detected, even when ammonium sulphate precipitation was used at 50% saturation.

Selective thymocyte depletion and immunoglobulin coating in the thymus of cats infected with feline immunodeficiency virus.

Thymus alterations associated with feline immunodeficiency virus (FIV), an AIDS animal model, were investigated by measuring phenotypic composition of thymocytes, structure of thymic epithelial cells, and transcription of viral RNA in the thymus of FIV-infected juvenile kittens. These kittens either acquired infection by natural vertical transmission or were experimentally inoculated with the virus at defined times of fetal or neonatal life. Thymocytes from FIV-infected cats were analyzed by flow cytometry for the differential expression of CD4, CD8, Pan T, and IgG and subpopulation percentages were compared to values from uninfected littermates. Infected cats demonstrated a decrease in the percentage of CD4+/CD8+ lymphocytes and a concurrent increase in the percentage of CD4-/CD8-, CD4-/CD8+, and IgG+ lymphocytes. Absolute numbers of IgG+ cells were increased with FIV infection. On bivariate distribution scatter plots generated by two-color flow cytometry, this population of IgG+ cells overlapped extensively with cells having low to minimally detectable levels of a pan-T lymphocyte marker, suggesting that thymocytes were coated with IgG. Immunohistochemical detection of feline IgG defined a broad zone of IgG+ cells within the residual cortex but outside lymphoid follicles. However, cells stained with B5, a feline B lymphocyte marker, localized almost exclusively to the centers of lymphoid follicles that were also characterized by a lack of internal cytokeratin staining. FIV RNA transcripts detected by in situ hybridization using an FIVgag RNA probe were evenly distributed throughout the thymic parenchyma except in lymphoid follicles, which were generally devoid of FIV expression. Despite these phenotypic and structural changes, thymus weight, expressed as a percentage of body weight, was not significantly reduced. From these data, we conclude that the clinically asymptomatic stage of FIV infection is associated with two distinct B cell-related phenomena within the thymus-the formation of germinal centers and the coating of thymocytes with IgG. These changes accompany a distorted thymocyte distribution characterized by a reduced percentage of CD4+/CD8+ lymphocytes and a relative increase in CD4-/CD8+ and CD4-/CD8- lymphocytes. Together, these findings suggest that degenerative thymic changes after lentivirus infection may involve humoral immune mechanisms.

Serum Concentration of Circulating Immune Complexes in Cats Infected with Feline Immunodeficiency Virus Detected by Immune Adherence Hemagglutination Method.

Circulating immune complexes (CIC) in the sera from 45 clinically healthy cats and 23 feline immunodeficiency virus (FIV) infected cats were measured by an immune adherence hemagglutination (IAHA) method. The level of CIC in the sera from FIV sera-negative healthy cats was 47.2 +/- 47.3 micrograms/ml when expressed as heat aggregated feline IgG equivalent value in IAHA reactivity. On the other hand, the level of CIC in the sera from FIV infected cats was 757.4 +/- 910.5 micrograms/ml, which was significantly higher than that of healthy ones. CIC levels of 11 symptomatic cats and 12 asymptomatic ones were 837.8 +/- 1138.2 micrograms/ml and 683.0 +/- 684.2 micrograms/ml, respectively. These results showed that IAHA method was reliable to detect CIC levels of cat sera and that CIC levels in the sera of cats infected with FIV were higher than those of healthy ones.

Comparison of methods for estimation of Toxoplasma gondii-specific antibody production in the aqueous humor of cats

SUMMARY Intraocular production of Toxoplasma gondii-specific antibody in cats has been estimated by comparing the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of total immunoglobulins in serum and aqueous humor (Goldmann-Witmer coefficient; aqueous antibody coefficient; C value). It has been proposed that in human beings, comparison of the ratio of T gondii-specific antibody in aqueous humor and serum with the ratio of antibodies against a nonocular pathogen in serum and aqueous humor is more accurate than methods using total immunoglobulin quantification. We developed an elisa for detection of calicivirus-specific antibodies in the serum and aqueous humor of cats. By evaluating calicivirus-specific antibody concentrations in the aqueous humor of healthy and diseased cats, calicivirus was assessed as a nonintraocular pathogen. The ratio of T gondii-specific antibodies in the aqueous humor and serum and the ratio of calicivirus-specific antibodies in serum and aqueous humor were evaluated as a means of estimating intraocular T gondii-specific antibody production. A field strain of feline calicivirus was isolated, cultured, and purified. A calicivirus-specific IgG elisa was developed for detection of feline calicivirus-specific IgG in serum and aqueous humor. Calicivirus-specific IgG was measured in the serum and aqueous humor from 3 groups of control cats. Results suggested that calicivirus is a nonintraocular pathogen in cats and that calicivirus IgG detected in aqueous humor is attributable to leakage across a damaged blood-ocular barrier. Intraocular production of T gondii-specific antibodies was estimated, using 2 formulas. The C value was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of total immunoglobulins (using the corresponding IgM or IgG class) in serum and aqueous humor. The Ctc value (Toxoplasma-calicivirus Goldmann-Witmer coefficient) was calculated by multiplying the ratio of T gondii-specific IgM or IgG in aqueous humor and serum by the ratio of calicivirus-specific IgG in serum and aqueous humor. Serum and aqueous humor samples were obtained from 41 client-owned cats with uveitis, and T gondii-specific C values and Ctc values were calculated. Toxoplasma gondii-specific IgM or IgG C values of 10 or greater or T gondii-specific IgM or IgG Ctc values of 1 or greater were considered to be suggestive of intraocular T gondii-specific antibody production. Of the 41 cats, 20 (48.7%) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG C value of 10 or greater. A Ctc value could not be calculated in 3 cats because calicivirus-specific IgG was not present in aqueous humor. Of the 38 cats for which Ctc values could be calculated, 25 (65.8 %) had evidence of intraocular production of T gondii-specific antibody on the basis of either an IgM or IgG Ctc value of 1 or greater. The C values and Ctc values were in agreement for 75.9 % of IgM containing samples and 75% of IgG containing samples. Sensitivity, specificity, predictive value of a positive test result, and predictive value of a negative test result for an IgM or IgG C value, when compared with the corresponding IgM or IgG Ctc value were determined. The results indicate that use of the C value for estimation of intraocular T gondii-specific antibody production will result in 28.6 (IgM) to 50 % (IgG) false-negative results and 12.5% (IgM and IgG) false-positive results, when compared with the Ctc value.

Reduced provirus burden and enhanced humoral immune function in AZT-treated SCID-feline mice inoculated with feline immunodeficiency virus

The lack of a safe, economical murine lentivirus model for human immunodeficiency virus type 1 (HIV-1) infection of humans has hampered the preclinical evaluation of potential antiviral compounds, vaccines, and biological response modifiers. A small animal model that does not employ HIV-1 is needed to minimize risk of accidental human exposure, enhance efficient use of scarce experimental compounds, and reduce laboratory space necessary to conduct statistically significant in vivo trials. Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus of domestic cats, has been used extensively as an animal model for the pathogenesis and therapy of human HIV-1 infection. Cats, however, are not amenable to large-scale efficacy trials because of their relatively large size, high cost, and limited degree of physiologic characterization, particularly with regard to drug metabolism. To adapt the feline immune system to a small laboratory animal host, severe combined immunodeficient mice (SCID mice) were engrafted with feline lymphoid tissues (forming the SCID-fe mouse) and inoculated with FIV. Two quantitative parameters, the incidence of provirus detection in feline tissue grafts and the level of feline IgG in plasma, were used to demonstrate the antiviral efficacy of 3′-azido-3′-deoxythymidine (AZT, azidothymidine, Retrovir ®, zidovudine) in the SCID-fe system. Of 17 SCID-fe mice inoculated with 7 × 10 6 peripheral blood mononuclear cells (PBMC) from an FIV-infected cat, eight had detectable FIV provirus in both the feline thymus and feline lymph node implants, as measured by polymerase chain reaction (PCR)/Southern blot analysis. Treatment of these mice with AZT at a dose of 125 mg kg −1 day −1 in drinking water beginning 1 day prior to FIV inoculation and continuing throughout the study interval prevented the dual detection of provirus in feline lymph node and thymus grafts of all mice tested. In a separate experiment, the level of spontaneous feline IgG production was quantified by ELISA 2 weeks after FIV inoculation with and without AZT treatment. Mean plasma feline IgG level of five SCID-fe mice inoculated with 10 3 TCID 50 cell-free FIV was 2.23 mg ml −1. Mean feline IgG level of five mice inoculated with the same quantity of FIV and treated with AZT beginning 1 day prior to virus inoculation and continuing for 2 weeks thereafter was 14.98 mg ml −1. AZT significantly ( P < 0.05) enhanced feline humoral immune function at a virus inoculum titer of 10 3 TCID 50. These data indicate that a 4 week preclinical study using the FIV-infected SCID-fe model can generate quantitative indicators of provirus burden and immunologic function following treatment with an efficacious human antiretroviral compound.

Microfilariae of Brugia pahangi in the Blood of Cats Have Variable Levels of Feline IgG on Their Sheaths

Feline IgG was found on the sheath of microfilariae (mf) of Brugia pahangi isolated from the blood of cats. Gamma heavy chains, both light chains, and the Fc and F(ab')2 portions of IgG were present, suggesting that the molecule is intact. The amount of IgG on the sheath was variable and the younger the mf the less IgG was present on their sheaths. Possible functions of this adherent layer of host protein are considered.

Isolation of porcine, canine and feline IgG by affinity chromatography using protein A

Isolation of porcine, canine and feline IgG has hitherto been achieved usually by DEAE-ion exchange chromatography and gel filtration. These procedures, however, are rather time-consuming, as they involve purification of IgG. Isolation of IgG by affinity chromatography on a column of protein A-Sepharose was attempted. IgG of all three animal species was very easily isolated with a high yield. In the absence of any other method that allows isolation of IgG of three animal species from a single column, the procedure proposed would be very useful.