Effects of passive transfer of immunity on results of diagnostic tests for antibodies against feline immunodeficiency virus in kittens born to vaccinated queens

OBJECTIVE: To determine whether passive transfer of immunity affects results of diagnostic tests for antibodies against FIV in kittens born to vaccinated queens. DESIGN: Experimental trial. ANIMALS: 12 specific-pathogen-free queens and their 55 kittens. PROCEDURE: Queens were vaccinated with a whole-virus FIV vaccine prior to breeding. Serum was obtained from the queens on the day of parturition and from the kittens on days 2 and 7, then weekly until results of tests for antibodies against FIV were negative for 2 consecutive weeks. Milk was collected from the queens daily for the first week and then weekly. Serum and milk were tested for antibodies against FIV with 2 commercial assays. RESULTS: Antibodies against FIV were detected in serum obtained from the queens on the day of parturition and in the milk throughout lactation. All kittens tested positive for antibodies against FIV at 2 days of age. At 8 weeks of age, 30 (55%) kittens tested positive with 1 of the commercial assays, and 35 (64%) tested positive with the other. All kittens tested negative for antibodies against FIV by 12 weeks of age. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that kittens readily absorb antibodies against FIV in colostrum from vaccinated queens and that these antibodies may interfere with results of commercially available tests for FIV infection past the age of weaning. Currently licensed diagnostic tests for FIV infection are unable to distinguish among kittens with antibodies against FIV as a result of infection, passive transfer from infected queens, and passive transfer from vaccinated queens.     

No evidence of vertical transmission of naturally acquired feline immunodeficiency virus infection.

A naturally occurring feline immunodeficiency virus (FIV) infection in a closed breeding colony of cats, was studied for a period of 9 months. The colony consisted of 25 adult cats, of which six proved to be infected with FIV as judged by serological examination and virus isolation. In all, 48 kittens were monitored for levels of antibodies against FIV during their first 6 months of life. All the kittens (n = 30) born of FIV-infected queens showed maternal antibodies against FIV, although these declined to undetectable levels by the age of 5 months. Antibodies against FIV were not shown in any of 18 kittens born of FIV-negative queens. An attempt to isolate the virus from 12 kittens between 2 and 6 weeks of age did not succeed. None of the cats in the colony seroconverted during the observation period. In conclusion, neither vertical nor horizontal transmission of FIV infection were demonstrated in the colony during the 9-month investigation period.     

Efficacy and safety of a feline immunodeficiency virus vaccine

Fel-O-Vax FIV is an inactivated virus vaccine designed as an aid in the prevention of infection of cats, 8 weeks or older, by feline immunodeficiency virus (FIV). It contains two genetically distinct FIV strains. The efficacy of this vaccine was demonstrated in a vaccination-challenge study designed to meet various regulatory requirements for registering the vaccine. Eight-week-old kittens were vaccinated with an immunogenicity vaccine which contained minimal release levels of FIV antigens formulated with a proprietary adjuvant system. Twelve months later, all vaccinates and controls were challenged with a heterologous FIV strain. Following the vigorous challenge exposure, cats were monitored for FIV viremia. It was found that 16% of the vaccinated cats developed viremia while 90% of the controls became persistently infected with FIV, which demonstrated that the vaccine was efficacious and the protective immunity lasted for at least 12 months. The safety of the vaccine was demonstrated by a field safety trial in which only 22 mild reactions of short duration were observed following administering 2051 doses of two pre-licensing serials of Fel-O-Vax FIV to cats of various breeds, ages and vaccination histories. Thus, Fel-O-Vax FIV is safe and efficacious for the prevention of FIV infection in cats.     

Transmission of feline leukaemia virus in the milk of a non-viraemic cat

The possibility of the transmission of feline leukaemia virus (FeLV) from latently infected cats was studied. Five female cats with latent infections were examined for evidence of transmission of the virus to their kittens. One of the cats infected members of four consecutive litters of kittens which subsequently became persistently viraemic and transmitted the virus to other susceptible kittens by contact. Shortly after birth its kittens were apparently FeLV-free since neither viral antigen nor infectious virus was detected in their blood and no virus was found in cell cultures made from aspirates of bone marrow. The kittens became viraemic from 45 days of age onwards at a time when their passively acquired colostral FeLV neutralising antibodies were no longer detectable. Transmission of the virus occurred via the milk since both FeLV antigen and infectious virus were found in milk samples taken six weeks after kittening and the virus was transmitted to a fostered kitten. Eleven weeks after the birth of the fourth litter the cat became viraemic. The intermittent presence of FeLV antigens detected by the Leukassay F test, but not infectious virus, in the plasma of this cat over the previous months and a low level of serum neutralising antibodies distinguished it from four other latently infected queens which did not transmit infection to their kittens. These factors may indicate a risk of milk transmission and reactivation of latent virus.     

Immunohistochemical characterisation of the lymph node reaction in pig post-weaning multisystemic wasting syndrome (PMWS)

The goal of this study was to identify a strain of feline immunodeficiency virus (FIV) that would be more virulent for adult cats than the prototype FIV-APetaluma and, thereby, enhance the FIV infection model for HIV-1 related research. Diehl et al. reported that one clade C strain of FIV, FIV-CPGammar, was more virulent than other known FIV isolates. Mortalities from 58 to 100% were reported for kittens 12 weeks of age and less following intravenous inoculation. A more variable and somewhat less virulent disease course was observed in neonatal to 8-10-week-old kittens infected orally, intravaginally or intrarectally with this same isolate (Obert and Hoover, 2000). However, no studies have been done with FIV-CPGammar in adult cats. Therefore, the virulence of FIV-CPGammar for young adult cats was compared to that of FIV-APetalulma, the original FIV isolate. One group of five cats were inoculated intraperitoneally with 470 TCID(50) of FIV-CPGammar in the form of pooled plasma from acutely infected cats, while a second group was infected with plasma containing the 750 TCID50 of FIV-APetaluma. The cats were observed for 20 weeks for gross signs of disease, hematologic abnormalities, time of antibody appearance, and plasma and peripheral blood mononuclear cell (PBMC) associated virus levels. Viral RNA and proviral DNA were measured by a real-time PCR, sensitive to 50 copies per milliliter. The only outward sign of disease was lymphadenopathy, which occurred at a similar time and intensity in both groups of cats. Cats infected with FIV-CPGammar were more likely to be neutropenic and lymphopenic during the first 10-12 weeks of infection than cats infected with FIV-APetaluma. Both groups of cats showed similar overall declines in absolute mean CD4 cell counts and identical concomitant increases in CD8 cells. CD4/CD8 cell ratios were also similar. Antibody, as measured by an ELISA against recombinant FIV-TM antigen, appeared in all cats by 4 weeks post-infection. The most significant differences were in plasma viral RNA and PBMC proviral DNA levels. Cats infected with FIV-CPGammar had up to 100 times higher mean levels of viral RNA during the first few weeks of infection than cats infected with FIV-APetaluma. This difference was also mirrored in levels of proviral DNA in PBMC, which were significantly higher in the FIV-CPGammar infected cats. Plasma viral RNA and PBMC proviral DNA levels were virtually identical in both groups of cats at 20 weeks post-infection. However, proviral DNA in tissues such as thymus and popliteal lymph nodes was 10-fold or so higher in FIV-CPGammar infected cats at 20 weeks and histopathologic lesions were more severe. Based on these various parameters, we concluded that FIV-CPGammar was more virulent than FIV-APetaluma in young adult cats during the 20-week study period. However, we were not able to recreate the severe and rapidly progressive disease previously reported for kittens, suggesting an age-related resistance similar to that observed previously for FIV-APetaluma (George et al., 1993).     

Pathogenesis of feline enteric coronavirus infection

Fifty-one specific pathogen-free (SPF) cats 10 weeks to 13 years of age were infected with a cat-to-cat fecal-oral passed strain of feline enteric coronavirus (FECV). Clinical signs ranged from unapparent to a mild and self-limiting diarrhea. Twenty-nine of these cats were FECV na?ve before infection and followed sequentially for fecal virus shedding and antibody responses over a period of 8-48 months. Fecal shedding, as determined by real-time polymerase chain reaction (RT-PCR) from rectal swabs, appeared within a week and was significantly higher in kittens than older cats. FECV shedding remained at high levels for 2-10 months before eventually evolving into one of three excretion patterns. Eleven cats shed the virus persistently at varying levels over an observation period of 9-24 months. Eleven cats appeared to have periods of virus shedding interlaced with periods of non-shedding (intermittent or recurrent shedders), and seven cats ceased shedding after 5-19 months (average 12 months). There was no change in the patterns of virus shedding among cats that were excreting FECV at the time of a secondary challenge exposure. Four cats, which had ceased shedding, re-manifested a primary type infection when secondarily infected. Cats with higher feline coronavirus (FCoV) antibody titers were significantly more likely to shed virus, while cats with lower titers were significantly less likely to be shedding. Twenty-two kittens born to experimentally infected project queens began shedding virus spontaneously, but never before 9-10 weeks of age. Natural kittenhood infections appeared to be low grade and abortive. However, a characteristic primary type infection occurred following experimental infection with FECV at 12-15 weeks of age. Pregnancy, parturition and lactation had no influence on fecal shedding by queens. Methylprednisolone acetate treatment did not induce non-shedders to shed and shedders to increase shedding.     

16alpha-Bromo-epiandrosterone therapy modulates experimental feline immunodeficiency virus viremia: initial enhancement leading to long-term suppression

16alpha-Bromo-epiandrosterone (epiBr), a synthetic derivative of the natural hormone dehyroepiandrosterone (DHEA), was evaluated for its effects on feline immunodeficiency virus (FIV) infection in experimental cats. The rationale for this study was based on the ability of DHEA to significantly reduce the mortality to viral infections in mice. DHEA and epiBr also have demonstrable in vitro anti-viral activity for both HIV-1 and FIV. Preliminary pharmacokinetic studies in cats demonstrated that subcutaneously injected epiBr was rapidly absorbed, completely metabolized, and nontoxic. Metabolites were excreted in both urine and feces, with the latter having the most complex pattern of breakdown products. Cats were then divided into four groups; two groups were infected with FIV and two uninfected. Two groups, one infected and one uninfected were treated on 5 consecutive days of weeks 0, 4, 8, 12 and 16 with epiBr. The remaining two groups were mock treated with the drug vehicle alone. Treatment started 1 week prior to infection and extended for 4 weeks after infection. Cats were observed for 20 weeks post-FIV infection. Infected cats had identical decreases in blood neutrophil and lymphocyte counts following, regardless of whether they were treated with epiBr or vehicle alone. The CD4/CD8 T-cell ratio was decreased following FIV exposure, but was significantly more decreased for the epiBr treated animals from week 2 post-infection onward. CD4+ T cells were decreased in FIV-infected cats treated with epiBr compared to their untreated cohort, while CD8+ T cells tended to be higher in treated animals. FIV infected cats that were treated with epiBr had over one-log higher virus loads at week 2 post-infection than non-epiBr treated cohorts. In spite of this enhanced initial viremia, the subsequent levels of virus in the blood were significantly lower in epiBr treated versus untreated animals. EpiBr treated cats had significantly higher FIV-p24 antibody responses than control cats receiving vehicle alone, although primary and secondary antibody responses to a T-cell dependent non-FIV antigen, keyhole limpet hemocyanin (KLH), were unaffected. EpiBr treatment significantly decreased the expected FIV-induced suppression of IL-12 p40 mRNA levels in peripheral blood mononuclear cells (PBMCs) observed at weeks 4, 5, 8, 9 and 16 post-infection, but had no influence on FIV-induced changes in IL-4, IL-6, IL-10, IFN-gamma, MIP-1alpha and RANTES.     

Immunoglobulin concentrations in feline colostrum and milk, and the requirement of colostrum for passive transfer of immunity to neonatal kittens

The purpose of this study was to clarify whether cats have a colostral and milk phase of lactation differentiated by concentrations of immunoglobulins, and whether colostrum ingestion by newborn kittens is essential for optimal transfer of passive immunity. Milk from specific pathogen-free queens was analyzed for IgG and IgA concentrations from parturition through 6 weeks of lactation. Serum IgG and IgA concentrations from birth through 8 weeks of age were determined for colostrum-fed kittens, colostrum-deprived kittens that were fed a milk replacer, and colostrum-deprived kittens that were fostered onto queens in the milk phase of lactation. The total IgG and IgA concentrations in milk were significantly higher on the day of parturition than on day 7 of lactation, indicating cats do have a colostral phase of lactation. The predominant immunoglobulin in both colostrum and milk was IgG. The serum IgG concentrations in colostrum-deprived kittens fostered on queens in the milk phase of lactation were similar to colostrum-deprived kittens fed a milk replacer, and the concentrations were significantly lower than in colostrum-fed kittens for the first 4 weeks of life. The serum IgA concentrations in both colostrum-deprived groups were significantly lower than colostrum-fed kittens on day 2 after parturition, but were similar thereafter. Colostrum-deprived kittens fostered onto queens in the milk phase of lactation had failure of passive transfer of maternal antibodies. Protective concentrations of immunoglobulins can be restored in kittens with failure of passive transfer of immunity by parenteral administration of adult cat serum, but not by fostering on queens in mid-lactation.     

CD4+CD25+ regulatory T cells are infected and activated during acute FIV infection

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.     

Macrophage functions in cats experimentally infected with feline immunodeficiency virus and Toxoplasma gondii.

It was suspected that feline immunodeficiency virus (FIV) infection would affect the function of feline macrophages, and that the concomitant infection of cats with FIV and Toxoplasma gondii would cause even greater changes in macrophage function. Sixteen specific-pathogen-free kittens, four per group, were infected either with FIV, T. gondii, both pathogens, or neither pathogen. After the cats had been infected with FIV for 14 weeks (8 weeks after T. gondii infection), peritoneal macrophages were collected. Some macrophages were stimulated with lipopolysaccharide and supernatants were collected for the measurement of interleukin-1 production. Other macrophages were infected with T. gondii in a microbiocidal assay. Peritoneal macrophages from cats infected with FIV had decreased interleukin-1 secretion and increased antimicrobial activity. Co-infection with T. gondii apparently had no effect on these modifications of macrophage activity. Thus, acute FIV infection alone caused significant changes in macrophage functions that were not affected by concomitant T. gondii infection.     

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD4+/CD8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.     

Detection of Toxoplasma gondii in the milk of experimentally infected lactating cats.

Tachyzoites of Toxoplasma gondii have been found in the milk of sheep, goats, cows and mice and infection by ingestion of raw goat milk has been documented in humans. Lactational transmission from infected cats to their kittens is suspected but the organism has not been detected in the milk. The purpose of this study was to demonstrate the presence of T. gondii in the milk of experimentally infected cats. Pregnant specific pathogen free cats were inoculated orally with T. gondii at various times prior to parturition. Feces were examined for oocyst shedding after sugar solution centrifugation. Milk was collected for polymerase chain reaction (PCR) and bioassay in mice. T. gondii was detected in the milk of five of six cats by either bioassay or PCR.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Optimization of the use of C57BL/6 mice as a laboratory animal model for Neospora caninum vaccine studies

The development and testing of vaccines for Neospora caninum in mice require challenge studies to demonstrate a reduction in clinical signs or prevention of vertical transmission of the parasite after vaccination. Genetic susceptibility to N. caninum varies with the strain of mice. In this study, C57BL/6 mice were evaluated as a model for Neospora vaccine studies. A lethal challenge model was developed and the LD(50) was determined to be 1.5 x 10(7)N. caninum tachyzoites/mouse, delivered intraperitoneally. Brain lesions encountered in sections from sub-lethally challenged mice were scored on the basis of severity and total number of lesions to develop a histopathological scoring system for vaccine efficacy. A vertical transmission model for N. caninum vaccine studies was developed by studying mice that were infected either 2 weeks prior to mating or between days 12 and 14 of pregnancy. It was found that infection prior to mating reduced the average number of pups per litter. DNA extracted from fetal tissue was examined by a N. caninum specific polymerase chain reaction (PCR). The rate of vertical transmission was 0, 100 and 90.5% for the uninfected controls, mice infected during pregnancy and mice infected before mating, respectively. This study demonstrates that the C57BL/6 strain of mice is a good model for N. caninum vaccine studies because it is possible to establish a clear-cut lethal challenge model in C57BL/6 mice and they transmit the disease to their offspring efficiently.     

Early events in the immunopathogenesis of feline retrovirus infections.

Feline leukemia virus and feline immunodeficiency virus (FIV) are lymphotropic retroviruses that cause a wide range of diseases in domestic cats. Although it is known that both viruses are capable of infecting T lymphocytes and that infected cats are lymphopenic, it was not known how infection with either virus might alter specific lymphocyte subpopulations. Using a panel of monoclonal antibodies to feline lymphocyte subpopulations, we examined, by use of flow cytometric analysis, lymphocyte changes in cats naturally infected with FeLV or FIV and explored the early stages in the immunopathogenesis of experimentally induced infection with these viruses. Both groups of naturally infected cats had T-cell lymphopenia. In the FIV-infected cats, the T-cell decrease was principally attributable to loss of CD4+ cells, whereas CD8+ and B-cell numbers remained normal. This led to inversion of the CD4+ to CD8+ ratio in these cats. In contrast, the T-cell lymphopenia in FeLV-infected cats resulted from decrease in CD4+ and CD8+ cells, which led to a CD4+ to CD8+ ratio within normal limits. Experimentally induced infection with these 2 viruses supported these findings. Infection with FIV induced early (10 weeks after infection), chronic inversion of the CD4+ to CD8+ ratio. In contrast, infection with FeLV did not alter CD4+ to CD8+ ratio in the first 20 weeks after infection.     

Polymorphic expression in the CD8alpha chain surface receptor of African lions (Panthera leo).

Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).