To the Irish March 17 was St. Patrick’s Day, but for me it was a major milestone for VMRD and for me personally. We received a USDA product license for our Caprine Arthritis-Encephalitis Virus Antibody Test Kit, cELISA, the culmination of many years of careful research and development. This assay in my opinion is the best serodiagnostic test in the world today for CAEV infection of goats. If one types the acronym "CAEV" on the search line of PubMed, 251 citations appear going back to 1980. Many of them describe diagnostic tests for CAEV serology. So why do I think VMRD’s new cELISA is the best? It has to do with the long history of the research organizations and people involved in the development of this assay. If one types in names of people I know who were directly involved in development of this assay and CAEV, a total of 89 citations appear. The point is that those involved in the development of this assay have played a major role in what is known and published about CAE. Tim Crawford started the CAE project in the early 1970s when he was alerted to a rheumatoid arthritis-like disease in a herd of Toggenberg goats by the owner, a savvy physician’s wife named Betty Gustafson. Linda Cork, under Tim’s tutelage and whose committee included such distinguished scientists as Bill Hadlow and John Gorham, worked on leukoencephalitis which occurred among young goats in the same herd. It was thought to be viral because lesions could be produced with a filtrate injected intra-cerebrally. However, no virus was isolated. The other clinical presentation in the index herd was chronic arthritis. It was not necessarily thought that the two conditions were related. Tim obtained grants from the Kroc Foundation and NIH to try to determine the etiology of the arthritis in part because it was clinically and histologically quite similar to rheumatoid arthritis. I was his graduate student on the project and Travis McGuire was a member of my graduate committee. After multiple explants of goat synovial membranes from affected animals, syncytia were observed in a flask of cells taken from a Toggenberg Buck named Victory (G63). Cytopathic effect and indirect immunofluorescence indicated that we had isolated a viral agent. Reverse transcriptase assays performed by Phil Cheevers, buoyant density and electron microscopy confirmed that it was a retrovirus. Paula Klevjer-Andersen worked out the in-vitro biology of the isolate. The isolate was called 79-63 virus. In vivo experiments with the isolate confirmed Koch’s Postulates. The pathogenesis and immune responses of experimentally-induced disease were worked out. (3&4) The clinical disease was characterized and an agar gel immunodiffusion assay was developed to determine seroprevalence. Morbidity of clinical arthritis in adult goats was much higher than the leukoencephalitis of kid goats so we changed the name to CAE. All of this happened before 1981. Scrolling forward to 2001-2003, two publications came out of Don Knowles’ laboratory describing reactivity of monoclonal antibodies to the gp135 surface envelope glycoprotein (SU) of CAEV isolate 79-63 (CAEV-63) and use of VMRD’s cELISA which is based upon two of those monoclonal antibodies (7). The cELISA’s performance was compared to gp135/90 SU/TM protein immunoprecipitation (IP) which is considered the gold standard for detection of antibodies to CAEV viral antigens. Two hundred goat sera collected from the United States were tested by IP and the cELISA. Sensitivity and specificity of the cELISA against IP were 100% and 96.4%, respectively. To analyze the possibility that the cELISA was more sensitive and not less specific than IP, sequential sera from eight experimentally-infected goats were tested by both assays. The cELISA detected antibody in seven of the eight goats before IP, indicating that it is more sensitive than IP. A field study was carried out by three independent university-associated veterinary diagnostic laboratories located in the eastern, central and western areas of the United States. Four hundred sixteen samples obtained by the participating laboratories were tested by these laboratories with the cELISA and compared to results from a USDA-licensed agar gel immunodiffusion (AGID). Discorrelates were refereed by IP. Against AGID, the cELISA had 14 false negatives and 20 false positives. Twenty-four of these samples had sufficient volume for IP analysis. Immunoprecipitation results agreed with the cELISA on 23 of the samples. The single sample agreeing with AGID was a negative, but remember that the cELISA was more sensitive than IP on the experimental infections. A panel of 50 samples (24 positive and 26 negative) was also tested by the laboratories to determine tester interdependency. All three laboratories scored 100% correct results, indicating the field robustness of the cELISA. Questions or Comments on this article? Scott Adams, President of VMRD, Inc., may be reached by e-mail at scott@vmrd.com References: 1Cork LC, Hadlow WJ, Crawford TB, Gorham JR, Piper RC. Infectious leukoencephalomyelitis of young goats. J Infect Dis. 1974 Feb;129(2):134-41. 2 Crawford TB, Adams DS, Cheevers WP, Cork LC. Chronic arthritis in goats caused by a retrovirus. Science. 1980 Feb 29;207(4434):997-9. 3 Adams DS, Crawford TB, Klevjer-Anderson P. A pathogenetic study of the early connective tissue lesions of viral caprine arthritis-encephalitis. Am J Pathol. 1980 May;99(2):257-78. 4 Adams DS, Crawford TB, Banks KL, McGuire TC, Perryman LE. Immune responses of goats persistently infected with caprine arthritis-encephalitis virus. Infect Immun. 1980 May;28(2):421-7. 5 Crawford TB, Adams DS. Caprine arthritis-encephalitis: clinical features and presence of antibody in selected goat populations. J Am Vet Med Assoc. 1981 Apr 1;178(7):713-9. 6 Ozyoruk F, Cheevers WP, Hullinger GA, McGuire TC, Hutton M, Knowles DP. Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera. Clin Diagn Lab Immunol. 2001 Jan;8(1):44-51. 7 Herrmann LM, Cheevers WP, McGuire TC, Adams DS, Hutton MM, Gavin WG, Knowles DP. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication. Clin Diagn Lab Immunol. 2003 Mar;10(2):267-71.

The aim of this experimental trial was to study viraemia and immune response in local breeds of sheep and goat, in order to evaluate their possible role in the epidemiology of bluetongue outbreak in Italy, and to assess the diagnostic value of commonly used antigen and antibody detection procedures. Materials & Methods A group of 5 ewes and 3 goats was challenged with 1 ml of tissue culture supernatant containing 105.6 TCID50/ml bluetongue virus (BTV) serotype 2 by the subcutaneous route. One ewe was inoculated with VERO cell suspension and was used as the control. The BTV-2 suspension used in this study was isolated from sheep during the 2000 Italian outbreak in Sardinia. Intravenous inoculation of embryonated chicken eggs followed by passages in VERO or BHK21 cells (3 and 4) was the isolation system used. Immunofluorescence using BTV monoclonal antibodies and electron microscopy was used as confirming assays. Immunofluorescence, electron microscopy, and ELISA (5,6, and 7) were also used to detect BTV antigen in infected embryo and in blood samples. A polymerase chain reaction (PCR) using group-specific and type-specific primers was used for detection of BTV nucleic acid in blood samples (8). The duration and level of viraemia in the challenged animals were also determined. The antibody responses were monitored by ELISA (Bluetongue Virus Antibody Test Kit, c-ELISA - VMRD, Inc., USA), agar gel immunodiffusion AGID, VMRD, Inc., USA, and serum neutralization (SN) assays (9). Sensitivity values of tests employed were estimated and compared using a bayesian approach (10). Probability of the various possible sensitivity values were estimated using the Beta distribution. Different Beta distributions were calculated on the basis of different periods on infection. Results The inoculated ewes and goats suffered mild clinical symptoms of bluetongue disease whereas the control animal did not show any clinical sign. Starting from the third day post-inoculation (d.p.i.), BTV was isolated from the blood of challenged animals by intravenous inoculation of embryonated chicken eggs followed by two passages in cell culture (BHK 21 or VERO cells) while a PCR technique was able to detect the virus only from the fifth d.p.i. (fig. 2). The sensitivity distributions of the in vitro isolation system was higher than those of the PCR technique (fig. 2 and 4). BTV was recovered from sheep blood samples up to 45 d.p.i. in contrast, in goat blood samples BTV was not more recovered after 24 d.p.i (fig. 5 and 6). In many animals the viraemic titres were often above 103DI50/ml which is the minimum level necessary to infect feeding Culicoides spp. The c-ELISA and SN’s detected BTV antibodies in the inoculated animals starting from the 7th d.p.i. whereas the sera tested positive on AGID only on 9 d.p.i (Fig. 1). The c-ELISA showed the highest sensitivity distributions in both the periods considered in this study (Fig. 1 and 3). Conclusions According to this study, the level of viraemia was earlier and higher in goats although it lasted longer in sheep. For a period of 42 days, at least one infected animal would have been able to infect feeing Culicoides spp. The VMRD c-ELISA was the test which showed the highest sensitivity distribution.

Bovine Virus Diarrhea Virus (BVDV) is not as easily detected by FA in chronic cell culture infections as it is in acute infections. One example is observed during 9CFR 113.51-2 testing, which requires an FA test after 3-4 weeks and 1-2 passages of indicator cells. A BVDV infection that might have been a 3-4+ by direct FA in nearly every cell as an acute infection before the first passage may only show a 2+ fluorescence in one out of 20 cells at the end of the required 21-28 days. For maximum sensitivity when testing biological fluids for infection by BVDV, we recommend that FA test be performed at each cell culture passage. Furthermore, indicator cells must be grown on glass, either in chamber slides or on cover slips, and fixed in pure acetone only.

Bartonella henselae has recently been identified as a major cause of cat scratch disease in humans.(1-3) Seropositivity of affected humans to Bartonella spp. Has been significantly associated with cat contact.(1) Both kittens and adult cats carry IFA antibodies, although it appears that kittens are most often bacteremic.(3-5) There is at least one report of canine Bartonella spp. Infection causing vegetative valvular endocarditis. This dog had a high titer to Bartonella henselae by IFA.(6) 1.     Dalton, M. J., et al. Arch. Intern. Med. 155(15):1670-1676, 1995. 2.     Bergmans, A. M., et al., J. Infect. Dis. 171(4):916-923, 1995. 3.     Demers, D. M., et al., J. Pediatr. 127(1):23-26, 1995. 4.     Ueno, H., et al., Microbiol. Immunol 39(5):339-341, 1995. 5.     Jameson, P., et al., J. Infect. Dis. 172(4):1145-1149, 1995. 6.     Breitschwerdt, E.B., et.al., J. Clin. Micro. 33(1):154-160, 1995.

Brucella canis IFA substrate slides were used in parallel with the tube agglutination test (TAT) to test 488 canine samples submitted to the Texas Veterinary Medical Diagnostic Laboratory (TVMDL), Serology Section. Results are listed in the table below. FITC-labeled anti-dog IgG (heavy and light chain) was used as the second antibody. Sera were screened at 1/50 dilution. Sensitivity and specificity compared to the tube agglutination test were 93% and 98%, respectively. Predictive value positive and predictive value negative were 90% and 99%, respectively. Of the seven false positive reactions, five were so at 1/50 dilution but negative at 1/100 dilution. As a hedge against false positive reactions, we recommend the use of IgG-specific FITC-labeled second antibody without light chain activity in order to avoid reactions with potentially spurious IgM. We also recommend a specific confirmatory test, such as AGID, to confirm positive reactions. Se: 93% Sp: 98% Data from 1/50 Screening Dilution: Using IFA, TVMDL considers samples positive at 1/100 to be suspect and at 1/200 to be serologically positive for Canine Brucellosis.

Heavy chain specificity may be confirmed by preincubation of sera in 0.1 M 2-Mercaptoethanol. The procedure is as follows: Dilute sera in PBS (four-fold dilutions) with and without 0.1 M 2-Mercaptoethanol through the endpoint and incubate for 30 minutes at 37°C. Incubate the diluted sera on the substrate slide, wash, add FITC-labeled heavy-chain specific IgG or IgM, incubate, wash, mount and observe. IgM titers in the presence of 2-Mercaptoethanol should be reduced at least two dilutions. IgG titers should be reduced little, if any.

Culture cells to confluency in a 25cm^2 flask. Pour off medium; add back 10 ml of serum-free media. Using a cotton swab, scrape off about half of the cell sheet and triturate with a pipette. Pour off medium; add back 10 ml of serum-free media. Using a cotton swab, scrape off about half of the cell sheet and triturate with a pipette. Pellet cell suspension at low RPM for 5 minutes. Pour off supernatant by quickly inverting, leaving about 0.2 ml of medium to drain back down on the pellet. After letting the tube stand for 30 seconds or so, tap the tube 15-20 times on the bench top to disperse the cells. Using a Pasteur pipette without a bulb, allow the cell suspension to enter the pipette tip via capillary action. Apply the suspension by moving the tip of the pipette around on a glass slide, preferably one masked with Teflon. Dry the suspension in a dry 37°C incubator for 15-30 minutes and fix in pure acetone for 10 minutes at room temperature. Dry in incubator again for 15 minutes to remove any residual water. Then perform the standard direct or indirect FA procedure using BVD-specific reagents. As few as 5% of cells may be expressing antigen. This technique is more sensitive than growing cells directly onto the slide.

Cure for Background with IFA for Dog and Cat Viruses and Other Agents. Because of the presence of bovine origin products in vaccines, dogs and cats develop antibodies that react with cell culture origin substrates.  If sera are diluted in PBS with 10% adult bovine serum, antibodies reacting to bovine constituents will be blocked.  Care should be taken to make sure that the adult bovine serum does not contain antibodies to the agents on the substrate slide, for instance, Neospora spp. VMRD manufactures “Special Serum Diluting Buffer” (catalog no. SSDB-100ML) for this purpose.

Two kinds of antemortem samples are most often examined; blood smears and conjunctival smears. Thin smears of whole blood or (preferably) buffy coat cells should be air dried for 30 minutes and fixed in a mixture of 25% methanol and 75% acetone for 20 minutes at room temperature. Smears should be made with a feathered edge, and the conjugate should be placed at the feathered edge (See Figure). If positive for canine distemper virus antigens, one or more of the following patterns of fluorescence should be observed; lymphocytes with smooth cytoplasmic fluorescence of entire cell; larger blast-like mononuclear cells with one or several sausage-shaped and/or buckshot inclusions and sometimes signet – ring – shaped inclusions; rarely red cells and platelets with particulate inclusions; neutrophils sometimes with dusty fluorescence near the nucleus. Watch for fluorescence of granules in neutrophils and especially eosinophils—this is not distemper specific. Also, nonspecific fluorescence may be seen at the edge where the conjugate dries during incubation. Make certain that the observed fluorescence is uniform throughout the area where the conjugate has been place. Truly positive samples should be visible at 100-200x·and only require confirmation at 400x. This description is valid for mercury vapor and xenon light sources only. Conjunctival smears are made from scrapings of the conjunctival sac, usually with a cotton swab, which are dried and fixed in· the same way. The inclusions, however, are only occasionally seen in the cell types described above for blood smears. Usually most of the antigen is cell-free and appears as "junk” on the slide. This is due to large quantities of cell-free inclusions mixed with various forms of cell debris.