Primate specific sialoglycoprotein of sperm head plasma membrane defined by an anti-carbohydrate monoclonal antibody

A sialoglycoprotein, an integral component of the head plasma membrane of human spermatozoa, is recognized by the a-HS 1A.1 monoclonal antibody. The antigenicity is associated with the sugar moiety since: a) trypsin digestion did not affect the antigenic determinant; b) pretreatment of the cells with beta-glucosidase, alpha-mannosidase and neuraminidase completely abolished antibody binding. Endoglycosidase D and glycopeptidase F were inactive. The a-HS 1A.1 did not recognize a variety of blood-group related synthetic oligosaccharides. The species specificity was studied by indirect immunofluorescence assay. The antibody also recognized an antigen on Macaca fascicularis sperm, but failed to bind to spermatozoa of boar, bull, goat, ram, stallion, dog, rabbit, rooster, carp and eel.     

Induced homocytotropic antibody to bovine serum albumin in nonatopic dogs

Six dogs with no skin reactivity to bovine serum albumin (BSA) were used to determine the antibody response to that antigen. The animals were divided into 3 equal groups given 5, 10, or 25 mg. BSA in Freund's incomplete adjuvant weekly, until a definite skin reaction could be detected. All sera were studied by passive transfer, double-gel diffusion, tanned sheep red blood cell hemagglutination, co-precipitation, and passive cutaneous anaphylaxis (PCA). Serum from one dog contained passively transferable antibody and was studied further by autoradiography and fractionation on Sephadex G-200. Skin reactivity to BSA could be detected up to 27 weeks in 5 of the 6 dogs. Hemagglutinating antibody was inhibited by the addition of 2-mercaptoethanol, and coprecipitation demonstrated that the antigenbinding capacity could be correlated with hemagglutinating antibody. Serum from the dog with passive transfer activity also contained precipitating antibody, and PCA activity could be demonstrated in guinea pigs. Autoradiography of the serum demonstrated 2 distinct arcs of anti-BSA activity. Homocytotropic antibody activity could be detected on the ascending slope of the second peak by Sephadex G-200 filtration, while hemagglutination antibody was localized to the first peak. These results indicate that antibodies with different activities can be induced in the dog following injection of BSA. The induced homocytotropic antibody has characteristics similar to spontaneous canine homocytotropic antibody.     

Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen.

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, greater than 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.     

Value of cytokeratin 5/6 immunostaining using D5/16 B4 antibody in the spectrum of proliferative intraepithelial lesions of the breast. A comparative study with 34βE12 antibody

Previous studies have shown that basal-type cytokeratins (CKs) can distinguish usual ductal hyperplasia (UDH) from the spectrum of atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Indeed, expression of these CKs is weak or absent in ADH, DCIS and LCIS. However, the diagnostic usefulness of D5/16B4 antibody (anti-CK5/6) has never been compared with that of 34betaE12 antibody (anti-CK1/5/10/14). We performed immunostaining of CK 5/6 and CK1/5/10/14 on 100 breast lesions, including UDH ( n=31), ADH ( n=5), DCIS ( n=54) and LCIS ( n=10). Abundant immunostaining was observed in all UDH using both antibodies. Four of five of the ADH cases showed less than 5% of CK5/6 stained cells, the remaining case showed 30% of labeled cells. With 34betaE12 antibody, three of five of the ADH cases showed less than 5% labeled cells, while two cases showed more than 30% of stained cells. None of the 54 DCIS or the 10 LCIS was labeled by D5/16B4, while a lack of 34betaE12 immunostaining was observed in only 15 of 54 DCIS and 2 of 10 LCIS. We confirmed that D5/16B4 antibody directed against CK5/6 is useful in distinguishing UDH from the spectrum of ADH/DCIS/LCIS. We also demonstrated that D5/16B4 is far a more specific marker than 34betaE12 antibody.     

A new Francisella (Beggiatiales: Francisellaceae) inquiline within Dermacentor variabilis say (Acari: Ixodidae)

While estimating the prevalence of the Dermacentor variabilis (Say) symbiont (DVS) in dog ticks on Martha's Vineyard, MA, we identified DNA that may represent a heretofore unrecognized Francisella sp. Polymerase chain reaction targeting a portion of the 16S rDNA specific for DVS yielded an amplicon that was only 96.6% similar to that of DVS accessioned in GenBank. Phylogenetic analysis of the 16S and 23S rDNA genes suggests the presence of a distinct bacterium closely related to the other endosymbionts of Dermacentor spp. Fifty-five percent of dog ticks tested from three sites in Massachusetts showed evidence of infection with this new agent, called Dermacentor variabilis francisella (DVF), whereas 100% tested positive for DVS. All larval progeny of dog ticks known to contain DVF also showed evidence of colonization, demonstrating that this agent may be maintained by transovarial transmission. Coinfection of ticks with both Francisella species did not seem to interfere with transmission.     

Assessment of Safety and the Immune Response to the CD4 “Internal Antigen” Mouse Anti-Idiotypic MAb 16D7 in Four Patients with SLE

We have analyzed the immune response elicited with the human CD4 internal antigen anti-idiotypic monoclonal antibody 16D7 in four patients with active systemic lupus erythematosus and assessed the safety of the treatment. Patients 1 and 2 received three 2-mg 16D7 subcutaneous (SC) injections at 3-week intervals and mainly developed IgG1, whereas IgG1, IgG3, and IgG4 were detected in the sera of the other two patients (3 and 4) who received the same amount of 16D7 on days 0, 28, and 70. 16D7-F(ab`)<sub>2</sub> isotypic determinant-specific antibodies levels, evaluated by sera reactivity with the 16D7-isotype matched anti-idiotypic monoclonal antibody 14D6-F(ab`)₂, were low or undetectable in patient 1 and became detectable following the first and second boosters in patient 3 and patients 2 and 4, respectively. The highest level was seen in patients 3 and 4. The focusing pattern (spectrotype) of 16D7 idiotypic-specific antibodies suggested that multiple V genes are involved or many somatic mutations occur. Once established, each patients spectrotype remained stable. Although spectrotype were individually distinct, all four patients produced CD4-specific antibodies, indicating that this response crosses the genetic barrier. Disease relapsed after 11 (patient 2), 40 (patients 3 and 4), and 125 (patient 1) weeks. The lack of side effects and the prolonged periods of disease control (33 and 103 weeks after the last booster) warrants an extension of this study.     

Predominance of Ehrlichia ewingii in Missouri dogs

To investigate the species distribution of Ehrlichia present in Missouri dogs, we tested 78 dogs suspected of having acute ehrlichiosis and 10 healthy dogs. Blood from each dog was screened with a broad-range 16S rRNA gene PCR assay that detects known pathogenic species of Ehrlichia and ANAPLASMA: The species was determined by using species-specific PCR assays and nucleotide sequencing. Ehrlichia antibody testing was performed by using an indirect immunofluorescence assay with Ehrlichia chaffeensis as the antigenic substrate. The broad-range assay detected Ehrlichia or Anaplasma DNA in 20 (26%) of the symptomatic dogs and 2 (20%) of the asymptomatic dogs. E. ewingii accounted for 20 (91%), and E. chaffeensis accounted for 1 (5%) of the positives. Anaplasma phagocytophilum DNA was detected in one dog, and the sequences of regions of the 16S rRNA gene and the groESL operon amplified from the blood of this dog matched the published sequences of this organism. Antibodies reactive with E. chaffeensis were detected in 14 (67%) of the 21 PCR-positive dogs and in 12 (19%) of the 64 PCR-negative dogs. Combining the results of PCR and serology indicated that 33 (39%) of 85 evaluable dogs had evidence of past or current Ehrlichia infection. We conclude that E. ewingii is the predominant etiologic agent of canine ehrlichiosis in the areas of Missouri included in this survey. E. canis, a widely recognized agent of canine ehrlichiosis, was not detected in any animal. The finding of E. ewingii in asymptomatic dogs suggests that dogs could be a reservoir for this Ehrlichia species.     

Canine reaginic antibody. Characterization of the spontaneous anti-ragweed and induced anti-dinitrophenyl reaginic antibodies of the atopic dog

Anti-2,4-dinitrophenyl reaginic antibody has been induced in the atopic dog and compared with anti-ragweed reaginic antibody of spontaneous canine atopic hypersensitivity. Immunization of an atopic dog with ragweed hypersensitivity with dinitrophenylated ragweed pollen produced a high and sustained level of canine reaginic anti-DNP antibody. The induced and spontaneous forms of canine reaginic antibodies were shown to be identical in heat lability, sulfhydryl sensitivity, molecular size, charge, in their ability to remain fixed at passively-sensitized dermal sites for a prolonged period, and in their capacity to sensitized homologous white blood cells from normal donors for an immunospecific release of histamine. The induced anti-DNP and spontaneous anti-ragweed reaginic antibodies were physically separated and immunochemically differentiated from six other classes of canine immunoglobulins: γ2a, γ2b, γ2c, γ1, γA and γM, and shown to be members of a distinct class of canine immunoglobulins analogous to human γE (IgE).A high incidence of spontaneous atopic hypersensitivity has been observed in the progeny of the atopic dog.     

Evaluation of dose-volume metrics for microbeam radiation therapy dose distributions in head phantoms of various sizes using Monte Carlo simulations

This work evaluates four dose-volume metrics applied to microbeam radiation therapy (MRT) using simulated dosimetric data as input. We seek to improve upon the most frequently used MRT metric, the peak-to-valley dose ratio (PVDR), by analyzing MRT dose distributions from a more volumetric perspective. Monte Carlo simulations were used to calculate dose distributions in three cubic head phantoms: a 2 cm mouse head, an 8 cm cat head and a 16 cm dog head. The dose distribution was calculated for a 4 × 4 mm2 microbeam array in each phantom, as well as a 16 × 16 mm2 array in the 8 cm cat head, and a 32 × 32 mm2 array in the 16 cm dog head. Microbeam widths of 25, 50 and 75 μm and center-to-center spacings of 100, 200 and 400 μm were considered. The metrics calculated for each simulation were the conventional PVDR, the peak-to-mean valley dose ratio (PMVDR), the mean dose and the percentage volume below a threshold dose. The PVDR ranged between 3 and 230 for the 2 cm mouse phantom, and between 2 and 186 for the 16 cm dog phantom depending on geometry. The corresponding ranges for the PMVDR were much smaller, being 2-49 (mouse) and 2-46 (dog), and showed a slightly weaker dependence on phantom size and array size. The ratio of the PMVDR to the PVDR varied from 0.21 to 0.79 for the different collimation configurations, indicating a difference between the geometric dependence on outcome that would be predicted by these two metrics. For unidirectional irradiation, the mean lesion dose was 102%, 79% and 42% of the mean skin dose for the 2 cm mouse, 8 cm cat and 16 cm dog head phantoms, respectively. However, the mean lesion dose recovered to 83% of the mean skin dose in the 16 cm dog phantom in intersecting cross-firing regions. The percentage volume below a 10% dose threshold was highly dependent on geometry, with ranges for the different collimation configurations of 2-87% and 33-96% for the 2 cm mouse and 16 cm dog heads, respectively. The results of this study illustrate that different dose-volume metrics exhibit different functional dependences on MRT geometry parameters, and suggest that reliance on the PVDR as a predictor of therapeutic outcome may be insufficient.     

Improved sectioned images and surface models of the whole dog body

The objective of this research was to produce high-quality sectioned images of a whole dog which can be used to create sectional anatomy atlases and three-dimensional (3D) models. A year old female beagle was sacrificed by potassium chloride injection and frozen. The frozen dog was then serially ground using a cryomacrotome. Sectioned surfaces were photographed using a digital camera to create 3555 sectioned images of whole dog body (intervals, 0.2 mm; pixel size, 0.1 mm; 48 bit color). In a sectioned image, structures of dimension greater than 0.1 mm could be identified in detail. Photoshop was used to make segmented images of 16 structures. Sectioned and segmented images were stored in browsing software to allow easy access. Segmented images were reconstructed to make surface models of 16 structures using Mimics software and stored in portable document format (PDF) using Adobe 3D Reviewer software. In this research, state-of-art sectioned images and surface models were produced for the dog. The authors hope that the sectioned images produced will become a useful source of software for basic and clinical veterinary medicine, and therefore, are distributing the sectioned images and surface models through browsing software and PDF file available free of charge.     

Characterization of neutralizing monoclonal antibody escape mutants of Hantaan virus 76118

Summary.  Neutralizing monoclonal antibody (MAb) escape mutants of Hantaan virus were generated using MAbs to envelope protein G1 (16D2) and G2 (11E10). The mutant viruses (mu16D2 and mu11E10), lacked reactivity only to the selecting MAb, or a MAb belonging to the same antigenic site. Both mutants had a single amino acid (a.a.) substitution. The a.a. substitution, found in mu16D2, was different from that found in another mutant selected with the same MAb (16D2). Although MAb 11E10 immunoprecipitated G2 protein, a deduced a.a. substitution was located in the G1 region. These results suggest that antigenic sites defined by neutralizing MAbs are composed of discontinuous epitopes over the G1 and G2 proteins. Mutant 11E10 showed a significant decrease in virulence in suckling mice. A virulence revertant of mu11E10, selected through passages in suckling mice brain, showed exactly the same deduced a.a. sequence as mu11E10 and still was not neutralized by MAb 11E10. Since mutant 16D2 was virulent for suckling mice, neutralization related epitopes found with MAbs 11E10 and 16D2 were independent of pathogenicity in BALB/c mice. Accepted August 13, 1997 Received March 17, 1997     

Immunohistochemical study of lung adenocarcinoma using monoclonal antibody for 60-kilodalton antigen in type II pneumocytes and nonciliated bronchiolar epithelial cells. Comparison with two antisurfactant apoprotein antibodies

Monoclonal antibody KP16D3 was produced by immunizing mice with monkey bronchoalveolar lavage. KP16D3 revealed the immunohistochemical reactivity in the cytoplasm of some nonciliated bronchiolar epithelial cells and type II pneumocytes and thereby recognized specifically a protein with an apparent molecular weight of 60 kD with the use of Western blotting and immunoaffinity column chromatography followed by SDS-PAGE. Examination of 76 primary and 4 metastatic lung carcinomas in primary lung carcinoma KP16D3 showed immunohistochemical positivity only to mucin-nonproducing papillary adenocarcinoma (27/28) and bronchioloalveolar carcinoma (2/2), except for one case of large cell carcinoma. All other primary lung carcinomas such as squamous cell carcinoma, acinar adenocarcinoma, and small cell carcinoma had negative results. From these findings, KP16D3 seems to be an effective immunohistochemical marker of mucin-nonproducing papillary adenocarcinoma and bronchioloalveolar carcinoma of the lung and it appears to be useful to investigate both the histogenesis and functional expression of primary lung adenocarcinoma.     

Lysis of a broad range of epithelial tumour cells by human gamma delta T cells: involvement of NKG2D ligands and T-cell receptor- versus NKG2D-dependent recognition

Human gammadelta T cells expressing a V gamma 9V delta 2 T-cell receptor (TCR) kill various tumour cells including autologous tumours. In addition to TCR-dependent recognition, activation of NKG2D-positive gammadelta T cells by tumour cell-expressed NKG2D ligands can also trigger cytotoxic effector function. In this study, we investigated the involvement of TCR versus NKG2D in tumour cell recognition as a prerequisite to identify tumour types suitable for gammadelta T-cell-based immunotherapy. We have characterized epithelial tumour cells of different origin with respect to cell surface expression of the known NKG2D ligands MHC class I-chain-related antigens (MIC) A/B and UL16-binding proteins (ULBP), and susceptibility to gammadelta T-cell killing. Most tumour cells expressed comparable levels of MICA and MICB as well as ULBP with the exception of ULBP-1 which was absent or only weakly expressed. Most epithelial tumours were susceptible to allogeneic gammadelta T-cell lysis and in the case of an established ovarian carcinoma to autologous gammadelta T-cell killing. Lysis of resistant cells was enhanced by pre-treatment of tumour cells with aminobisphosphonates or pre-activation of gammadelta T cells with phosphoantigens. A potential involvement of TCR and/or NKG2D was investigated by antibody blockade. These experiments revealed three patterns of inhibition, i.e. preferential inhibition by anti-TCR antibody, preferential inhibition by anti-NKG2D antibody, or additive blockade by anti-TCR plus anti-NKG2D antibodies. Our results indicate for the first time that the NKG2D pathway is involved in the lysis of different melanomas, pancreatic adenocarcinomas, squamous cell carcinomas of the head and neck, and lung carcinoma.     

Specific suppression of antibody rebound after extracorporeal immunoadsorption. I. Comparison of single vs combination chemotherapeutic agents

The specific removal of circulating bovine serum albumin (BSA) antibodies in canine hosts with a BSA collodion charcoal extracorporeal immunoadsorbent is followed by a rebound and overshoot of specific antibody levels within 7 days after perfusion. Studies were carried out in dogs actively immunized to BSA and human serum albumin (HSA) to determine whether the post-immunoadsorption antibody rebound could be modified by the administration of various chemotherapeutic agents given alone or in combination, at various intervals in the post-perfusion period. Control extracorporeal immunoadsorption was carried out in each dog and its own pattern of post-perfusion antibody rebound was recorded. After a second extracorporeal perfusion, single drug chemotherapy with cyclophosphamide, methotrexate or cytosine arabinoside resulted in a slight delay, or had no effect on the recovery of antibody levels to pre-perfusion values. In contrast, dual (cyclophosphamide and cytosine arabinoside) or triple (cyclophosphamide, cytosine arabinoside and methotrexate) chemotherapy resulted in specific attenuation or arrest of BSA antibody rebound after specific immunoadsorption. BSA binding of serum rebounded to 62% of pre-perfusion values in one dog, but declined 37% and 22% below post-perfusion levels in two others. The immunosuppressive effect of dual or triple chemotherapy on BSA binding was specific, since control levels of HSA binding in sera were unchanged by the extracorporeal immunoadsorption and the chemotherapeutic treatment. Drug toxicity, including myelosuppression, diarrhoea and fever was transient and reversible upon cessation of treatment, but was more severe in dogs given a dual or triple drug treatment. Therefore, chemotherapeutic agents employed after extracorporeal immunoadsorption may result in specific reduction and lasting suppression of circulating antibody levels. Conclusions based on the number of dogs studied must be guarded, but these findings suggest a potentially effective therapeutic approach to many antibody mediated diseases.     

Canine Parvovirus (CPV) Vaccination: Comparison of Neutralizing Antibody Responses in Pups after Inoculation with CPV2 or CPV2b Modified Live Virus Vaccine

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.     

Monoclonal anticardiolipin antibodies derived from mice with experimental lupus erythematosus: Characterization and the induction of a secondary antiphospholipid syndrome

The primary antiphospholipid syndrome and the antiphospholipid syndrome in systemic lupus erythematosus (SLE) patients (defined as secondary antiphospholipid syndrome) are characterized by the presence of anticardiolipin antibodies, thrombosis, thrombocytopenia, and recurrent fetal loss. To determine the role of anticardiolipin antibodies in the pathogenesis of antiphospholipid syndrome, monoclonal anticardiolipin antibodies were derived from mice in which experimental lupus was induced by a murine monoclonal anti-16/6 Id antibody. Two murine monoclonal anticardiolipin antibodies (2C4C2, 2C4D1) were generated and characterized. The 2C4C2, but not the 2C4D1, monoclonal antibody demonstrated remarkable lupus anticoagulant activity. Furthermore, these murine anticardiolipin monoclonal antibodies appear to recognize antigenic epitopes similar to those recognized by anticardiolipin antibodies found in sera of SLE patients. The monoclonal anticardiolipin antibody 2C4C2 was injected into naive female mice. Following immunization, the mice developed high titers of autoantibodies reacting with cardiolipin, DNA, nuclear extract, 16/6 and anti-16/6 Id, and anticardiolipin antibodies. As early as 8 weeks after immunization these mice exhibited significant leukopenia, thrombocytopenia, and proteinuria with immune complex glomerulonephritis. Moreover, mating of 2C4C2-injected mice with allogenic males resulted in low pregnancy rates and a low number of fetuses with a high percentage of fetal loss. These studies provide a new experimental model for secondary antiphospholipid syndrome demonstrating the role of anticardiolipin antibodies in the pathogenesis of this syndrome.     

Parietal cell surface reactive autoantibody in pernicious anaemia demonstrated by indirect membrane immunofluorescence.

We examined, in a 'double blind' study, 60 sera from patients with pernicious anaemia for immunofluorescence reactivity with the surface membranes of viable parietal cells isolated from dog stomachs. Fifty-three sera (88%) gave an IgG autoantibody reaction with the surface membranes of parietal cells. Surface staining was also seen with parietal cells from monkey, pig, rat and mouse. The parietal cell surface reactive autoantibody was not found in any of 14 sera from patients with chronic active hepatitis, 10 from patients with systemic lupus erythematosus and 50 from healthy persons. The surface reactivity autoantibody was present in 13 of 14 sera without parietal cell microsomal antibody, 28 of 31 sera without intrinsic factor antibody and in four of four sera without microsomal and intrinsic factor antibodies. Absorption with parietal cell enriched gastric mucosal cells neutralized the activity of the surface reactive but not the microsomal antibody and cross absorption with gastric microsomes neutralized the activity of the microsomal but not the surface reactive antibody. Surface staining of parietal cells was not abolished by absorption with dog or rat hepatocytes, dog or rat kidney cells, human fibroblasts or human AB red blood cells. The results suggest that the parietal cell surface reactive antibody is probably different from the microsomal antibody. Immune reactions of the cell surface reactive antibody with parietal cell surface antigens may play a role in the pathogenesis of the gastric lesion in pernicious anaemia.     

Canine parvovirus (CPV) vaccination: comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.