Differentiation of feline coronavirus type I and II infections by virus neutralization test

Feline coronavirus (FCoV) is divided into two types I and II, based on their growth in vitro and antigenicity. In this study, virus neutralization (VN) test was applied for type differentiation of FCoV infections. Sera of cats which were clinically and serologically diagnosed as feline infectious peritonitis (FIP) possessed significantly higher VN titers to type I FCoV, and sera from cats experimentally infected with FIPV type II had high VN titers to type II but not type I viruses. A total of 79 cat sera collected in the years between 2004 and 2005 were examined to evaluate seroprevalence by the VN test, showing the following results: (1) 50 cats (63.3%) were sero-positive to FCoV; (2) of the 50 FCoV positive cat serum samples, 49 (98%) showed significantly higher titers to type I virus and only one (2%) for type II virus. These results indicate that the VN test described here can be used for serological differentiation of FCoV infections of cats, and that FCoV type I is a dominant type in recent years of Japan.     

Quarantine protects Falkland Islands (Malvinas) cats from feline coronavirus infection

Feline coronavirus (FCoV) causes feline infectious peritonitis (FIP). Since 2002, when 20 cats on the Falkland Islands were found to be FCoV seronegative, only seronegative cats could be imported. Between 2005-2007, 95 pet and 10 feral cats tested negative by indirect immunofluorescence antibody (IFA) analysis using two strains of type II FCoV, two transmissible gastroenteritis virus assays, an enzyme-linked immunosorbent assay and rapid immunomigration test. Twenty-four samples (23%) showed non-specific fluorescence, mostly attributable to anti-nuclear antibodies (ANA). The reason for ANA was unclear: reactive samples were negative for Erhlichia canis antibodies; seven were feline immunodeficiency virus positive, but 15 were negative. It was not possible to determine retrospectively whether the cats had autoimmune disease, hyperthyroidism treatment, or recent vaccination which may also cause ANA. The FCoV/ FIP-free status of the Falkland Islands cats should be maintained by FCoV testing incoming cats. However, ANA can complicate interpretation of IFA tests.     

Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

Feline coronaviruses (FCoV) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (FIP). Independent of virulence variations they are separated into two different types, type I, the original FCoV, and type II, which is closely related to canine coronavirus (CCV). The prevalence of FCoV types in Austrian cat populations without FIP has been surveyed recently indicating that type I infections predominate. The distribution of FCoV types in cats, which had succumbed to FIP, however, was fairly unknown. PCR assays have been developed amplifying parts of the spike protein gene. Type-specific primer pairs were designed, generating PCR products of different sizes. A total of 94 organ pools of cats with histopathologically verified FIP was tested. A clear differentiation was achieved in 74 cats, 86% of them were type I positive, 7% type II positive, and 7% were positive for both types. These findings demonstrate that in FIP cases FCoV type I predominates, too, nonetheless, in 14% of the cases FCoV type II was detected, suggesting its causative involvement in cases of FIP.     

Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada

The objectives of this study were (1) to evaluate the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes and (2) to estimate the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada. With fox and coyote reference sera the test performance of the ELISA, IFAT and IB was excellent (100% sensitivity and specificity). NAT showed a low sensitivity (50%). Serum was collected from 201 coyotes and 271 foxes. The seroprevalence observed in the different assays ranged from 0.5 to 14.0% in coyotes and 1.1 to 34.8% in foxes. The seroprevalence, when taking more than one test positive as cut-off value was 3.3 and 1.1% for coyotes and foxes, respectively. From the N. caninum-positive group, all coyotes were older than 3 years. Agreement among assays (measured as prevalence-adjusted bias-adjusted kappa) using the field sera ranged from 0.17 to 0.97. Best agreement was observed between ELISA and IFAT, poor agreement was observed between NAT and the other assays. Positive agreement was moderate to poor among all assays utilized in this study. Although the seroprevalence observed was low, N. caninum antibodies are present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study.     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p≥0.05) and between IFAT and IB was moderate (κ=0.53, p≥0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.     

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.     

Seroprevalence study of feline coronavirus in owned and feral cats in Sydney, Australia

OBJECTIVES: i) To establish the seroprevalence of Feline Coronavirus (FCoV) infection in two defined groups of cats in Sydney: owned and feral cats; ii) to identify factors associated with an increased risk of infection with FCoV; and iii) to establish the seroprevalence and FCoV antibody titres of owned cats with immunohistochemically confirmed feline infectious peritonitis (FIP). DESIGN: Prospective multi-institutional cross sectional study. Procedure Serum samples from owned cats presented to three inner city veterinary clinics in Sydney and feral cats from a colony in South Western Sydney over an 11-month period were tested for FCoV antibodies using the Immunocomb test kit. The relationship between serological score and six major factors (breed, age, gender, number of cats per household, living environment and health status) in the owned cat sample population was analysed and compared to cats with FIR RESULTS: The seroprevalence of FCoV infection in the sample population of owned and feral cats was 34% and 0%, respectively. The median Immunocomb scores of DSH, Persian, Siamese and Devon Rex cats were significantly lower than that of Burmese, BSH, Abyssinian, Birman, Ragdoll and Russian Blue. The median lmmunocomb score of pedigree cats less than 2 years-of-age was significantly higher than for pedigree cats greater than 2 years-of-age. This distinction was not evident in DSH cats in these age groups. The number of cats per household at the time of blood collection had a strong positive association with Immunocomb score. The median Immunocomb score of cats with immunohistochemically confirmed FIP was significantly higher than cats in the sample population of owned cats but there was sufficient overlap between these two groups to make definitive diagnosis of FIP by serology impossible. CONCLUSION: This represents the first seroprevalence study of FCoV in Australia. The major determinants of antibody score of owned cats identified in this study were breed, age and the number of cats per household. The significant relationship between the breed of the cat and the FCoV antibody titre further supports the notion, proposed previously by the authors, that breed related differences exist in the immunological response to FCoV infection.     

Detection of antibodies against infectious bursal disease virus: a comparison of three serological methods

Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.818) was between VN and QAGP and the lowest (0.573) between QAGP and ELISA. Generally the ELISA detected positive sera earlier than the VN test which in turn was more sensitive than the QAGP test. The ELISA and QAGP test were less variable, more reproducible and easier to perform than the VN test.     

Elaboration of a crude antigen ELISA for serodiagnosis of caprine neosporosis: validation of the test by detection of Neospora caninum-specific antibodies in goats from Sri Lanka

The protozoan parasite N. caninum is a major pathogen in cattle and dogs. However, clinical symptoms are occasionally described for other potential hosts. Natural abortion in goats due to N. caninum has been rarely reported and only little data is available on the seroprevalence of N. caninum in this species. In the present study, 486 goats from Sri Lanka were tested in a crude antigen ELISA for the presence of serum antibodies against N. caninum. Additionally, the sera were analysed by N. caninum-Western blot and indirect fluorescence antibody test (IFAT). In all three tests applied, only three sera (0.7%) were scored clearly positive for anti-N. caninum antibodies. The optimal correlation between ELISA, IFAT, and Western blot confirms the suitability of the ELISA for large-scale seroepidemiologic studies, not only in cattle but also in goats.     

Comparison of three techniques for the serological diagnosis of Neospora caninum in the dog and their use for epidemiological studies

An enzyme-linked immunosorbent assay (ELISA) was developed in our laboratory and used to determine the seroprevalence of Neospora caninum in three different dog populations in Belgium: healthy dogs from cattle farms and urban dogs with or without various neurological disorders. The test was validated and compared with two other tests: an indirect fluorescent antibody test (IFAT) and a commercially available competitive enzyme-linked immunosorbent assay (C-ELISA). The study showed a good correlation between the IFAT and the ELISA developed. When the two tests were compared with the C-ELISA, moderate positive and negative agreement indices were observed. Using our ELISA and the IFAT techniques, a high prevalence was found in farm dogs. This result showed that the neurological symptoms are not usually associated with the Neospora infection. In conclusion, the ELISA developed in our laboratory could replace the IFAT for the screening of a large number of dogs' sera.     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in Venezuela (Apure State)

Six hundred and fifteen serum samples obtained from cows in five districts of Apure State, Venezuela, were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). The same samples were also ELISA-tested for antibodies to bovine herpesvirus type 1 (BHV-1) and bovine respiratory syncytial virus (BRSV). Additionally, the haemagglutination-inhibition (HI) test was used for detecting antibodies to parainfluenza virus type 3 (PIV-3). Overall, seroprevalence to BVDV was 36±7% (SE); seroprevalence varied by district (19–42%). BHV-1 seroprevalence was 67±4%; variation by district was similar to that of BVDV. However, the first 80 serum samples tested by BHV-1 ELISA all had a strong background reaction with the control antigen. Therefore, these sera were adsorbed to a homogenate of non-infected bovine kidney cell line (MDBK) and re-tested by ELISA. The non-specific reactivity was significantly reduced (p < 0.001 by Wilcoxon's signed-rank test). Compared to the virus-neutralisation (VN) test, the adsorbed BHV-1 ELISA showed 94% agreement and gave a κ value of 0.84, indicating that the adsorption did not interfere with test accuracy. Seroprevalence against BRSV was 85±3%, and showed differences across districts. Most of the cows (94±2%) were seropositive to PIV-3, and there were no significant differences among districts.     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

High viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (FCoV) type I and in naturally FCoV-infected cats

Specified pathogen-free cats were naturally infected with FCoV or experimentally infected with FCoV type I. Seroconversion was determined and the course of infection was monitored by measuring the FCoV loads in faeces, whole blood, plasma and/or monocytes. Tissue samples collected at necropsy were examined for viral load and histopathological changes. Experimentally infected animals started shedding virus as soon as 2 days after infection. They generally displayed the highest viral loads in colon, ileum and mesenteric lymph nodes. Seroconversion occurred 3-4 weeks post infection. Naturally infected cats were positive for FCoV antibodies and monocyte-associated FCoV viraemia prior to death. At necropsy, most animals tested positive for viral shedding and FCoV RNA was found in spleen, mesenteric lymph nodes and bone marrow. Both experimentally and naturally infected cats remained clinically healthy. Pathological findings were restricted to generalized lymphatic hyperplasia. These findings demonstrate the presence of systemic FCoV infection with high viral loads in the absence of clinical and pathological signs.     

Evaluation of an in-practice test for feline coronavirus antibodies

A commercially available in-practice test for feline coronavirus (FCoV) antibodies (FCoV Immunocomb, Biogal Galed Laboratories) was evaluated by comparison with the gold standard FCoV immunofluorescent antibody (IFA) test. One hundred and three serum or plasma samples were selected and tested: 70 were positive by both tests, 24 were negative by both tests. The in-practice test produced five false positive and four false negative results. The sensitivity of the in-practice test was 95% and the specificity was 83%. When the titres were compared it was found that the in-practice test results were significantly correlated with IFA titres but the degree of correlation was not likely to be clinically useful. The IFA titres of the four false negative samples were found to be low (less than 40) which suggests that even a cat with a false negative result is still unlikely to be excreting FCoV. A negative result with the in-practice assay is likely to be reliable for screening cats prior to entry into an FCoV-free cattery or stud. It would also be useful in the investigation of suspected FIP as most cats with this condition have high IFA titres of antibodies. A strong positive result would be useful in the diagnosis of FIP (in conjunction with other biochemical and cytological testing), but positive results would be of limited value in monitoring FCoV infection in healthy cats as the antibody titre could not be reliably compared with those obtained with IFA. All positive results obtained using the in-practice kit should be confirmed and titrated by IFA. The kit also appeared to work efficiently with ascites samples (n=6) but too few samples were analysed to draw firm conclusions.     

Blocking ELISA to distinguish pseudorabies virus-infected pigs from those vaccinated with a glycoprotein gIII deletion mutant

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was greater than 0.7 for all sera. No false positives were identified. Likewise, the S/N values were greater than 0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1-4 times with the standard dose (2 x 10(5) TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII:HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values less than 0.7 and more than 175 had S/N values less than 0.1. Sixteen sera from fetal pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers less than 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4-1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit.     

Prevalence of antibodies to Neospora caninum in Korean native beef cattle

A total of 438 sera from Korean native beef cattle in 9 provinces were tested for Neospora caninum antibodies using an immunofluorescent antibody test (IFAT). Eighteen (4.1%) cattle were positive by IFAT. The titers ranged from 1:200 (10 animals), 1:400 (5 animals), 1:800 (2 animals) to 1:1,600 (1 animal). Although the seroprevalence was slightly higher in Chungnam (8.9%), this was not significantly different from those noted in Kyunggi, Kangwon, Kyungbuk, Kyungnam, and Cheju provinces. Sera obtained from beef cattle in the provinces of Chungbuk, Jeonbuk and Jeonnam were all negative. Neospora positive sera were also tested for anti-Toxoplasma gondii antibodies using a commercial latex agglutination test (LAT). Antibody to T. gondii was detected in only 1 (5.6%) of 18 N. caninum positive sera. These results indicate that N. caninum and T. gondii infection are present at a low level in the Korean native beef cattle.