Effect of vaccination with a pentavalent leptospiral vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis on type hardjo-bovis infection of cattle

Effectiveness of 2 pentavalent leptospiral vaccines containing Leptospira interrogans serovar hardjo was evaluated for protection of steers from infection with serovar hardjo type hardjo-bovis. The hardjo component of 1 vaccine was prepared from serovar hardjo type hardjoprajitno. The hardjo component of the other vaccine was prepared from serovar hardjo type hardjo-bovis. Two steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjoprajitno. Four steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjo-bovis. Four steers were maintained as non-vaccinated controls. Steers given vaccine containing type hardjo-bovis developed higher mean serum microscopic agglutination titers against serovar hardjo than steers given vaccine containing hardjoprajitno. Six months after the first vaccination, all steers were challenge-exposed on 3 occasions by conjunctival instillation of 10(7) serovar hardjo type hardjo-bovis organisms, and on 1 occasion by conjunctival instillation of urine from a steer shedding hardjo-bovis. All control and all vaccinated steers became infected and shed serovar hardjo type hardjo-bovis in the urine. Lesions were detected in kidneys of 3 of 4 nonvaccinated control steers, 5 of 6 steers given hardjoprajitno vaccine, and 6 of 8 steers given hardjo-bovis vaccine. Leptospires were detected in kidneys of 4 of 4 control steers and 13 of 14 vaccinated steers.     

Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type hardjo-bovis infection of pregnant cattle

Effectiveness of a pentavalent leptospiral vaccine to protect cattle from infection and reproductive problems caused by Leptospira interrogans serovar hardjo type hardjo-bovis was evaluated. Seven cows were vaccinated once and 8 cows were vaccinated twice with a USDA-licensed pentavalent leptospiral vaccine. Five cows were maintained as nonvaccinated controls. Cows were bred 1 to 2 months after the last vaccination. During the 4th to 6th month of gestation, all cows were challenge exposed on 4 occasions by conjunctival instillation of 10(8) serovar hardjo type hardjo-bovis organisms and on 3 occasions by conjunctival instillation of urine from a cow shedding hardjo-bovis. All control cows and 13 of 15 vaccinated cows became infected and shed leptospires in the urine. Leptospires were detected in fewer urine samples collected from vaccinated cows, compared with those collected from control cows. Four stillborn calves and 3 weak calves were born to control and vaccinated cows. Leptospires were detected in the kidneys of 11 apparently healthy calves born to vaccinated and control cows. Agglutinating antibodies were not detected in the precolostral serum of these calves.     

Use of a monovalent leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo.

OBJECTIVE: To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. ANIMALS: 24 Hereford heifers that lacked detectable antibodies against serovar hardjo. PROCEDURE: Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. RESULTS: All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urine and had evidence of renal colonization. All 4 control heifers challenged by conjunctival instillation and 2 of 4 control heifers challenged by intraperitoneal inoculation shed leptospires in their urine, and all 8 had evidence of renal colonization. In contrast, leptospires were not detected in the urine or tissues of any of the 8 heifers that received the commercial hardjo vaccine. Heifers that received the commercial hardjo vaccine had significantly higher antibody titers than did heifers that received the reference vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle that received 2 doses of the commercial hardjo vaccine were protected against renal colonization and urinary shedding when challenged with L borgpetersenii serovar hardjo strain 203 four months after vaccination.     

Comparison of three techniques to detect Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine

Nucleic acid hybridization, bacteriologic culture, and a fluorescent antibody test were compared for detection of Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine. Seventy-five urine samples were collected from pregnant cows challenge exposed with type hardjo-bovis. Twenty samples were collected from steers not exposed to hardjo-bovis. Sediments from each sample were examined, using fluorescent antibodies and a repetitive sequence element nucleic acid probe, to detect the presence of leptospires. Urine samples were processed for bacteriologic culture, using standard techniques. Under laboratory conditions typically used for these techniques, leptospires were detected in 60 of 75 urine samples from challenge exposed cows by nucleic acid hybridization, in 24 samples by fluorescent antibody test, and in 13 samples by bacteriologic culture. Leptospires were not detected in the urine of steers not exposed to hardjo-bovis.     

Evaluation of an enzyme immunoassay for diagnosis of bovine leptospirosis caused by Leptospira interrogans serovar hardjo type hardjo-bovis

Sensitivity and specificity of 4 different antigen preparations from Leptospira interrogans serovar hardjo were compared in an enzyme immunoassay for detection of antibodies against serovar hardjo type hardjo-bovis in serum. Two antigens prepared using detergents showed serogroup cross-reactivity. A mechanically extracted membrane and a lipopolysaccharide antigen showed a high degree of leptospiral serogroup specificity. The lipopolysaccharide antigen was the most suitable antigen for detection of anti-hardjo antibodies. Enzyme immunoassay was more sensitive than the microscopic agglutination test for detecting antibodies in serum from experimentally and naturally infected cattle. It was not possible to differentiate vaccinated from infected animals or to detect a secondary immune response in vaccinated animals that were subsequently infected.     

Evaluation of two antimicrobial therapies in the treatment of Leptospira borgpetersenii serovar hardjo infection in experimentally infected cattle

This study demonstrated the ability of the antimicrobials tulathromycin (Draxxin) and ceftiofur crystalline free acid sterile suspension (Excede) to clear the spirochete Leptospira borgpetersenii serovar hardjo type hardjo-bovis (L. hardjo-bovis) from experimentally infected cattle. Treatment with tulathromycin resulted in clearance of L. hardjo-bovis organisms from the urine and kidney tissue of all animals (9 of 9), and treatment with ceftiofur crystalline free acid resulted in clearance of the organisms from the urine of 8 of 10 heifers and the kidney tissue of all 10 animals. In contrast, 10 of 10 placebo-treated cattle had L. hardjo-bovis organisms in their urine and 8 of 10 had the organisms in kidney tissue.     

Survey to estimate prevalence of Leptospira interrogans infection in mature cattle in the United States.

A total of 5,142 kidney tissue samples and 5,111 serum samples from mature cattle in 49 states and Puerto Rico were collected at slaughter. Age of cattle ranged from 1 to 16 years (mean, 6.6 years). Leptospires were isolated from 88 (1.7%) kidney tissues, and 2,493 (49%) sera contained antibodies against 1 or more of 12 Leptospira interrogans serovars. Leptospires were observed by immunofluorescence in 41 (0.8%) kidney tissues. Using agglutinin-absorption tests, 73 (83%) isolates were identified as serovar hardjo, 11 (12.5%) as serovar pomona, and 4 (4.5%) as serovar grippotyphosa. By use of restriction endonuclease analysis studies of chromosomal DNA, all isolates differed from reference serovars but were identical to strains previously isolated from cattle or swine in the United States. Of the serovar hardjo isolates, 85% were identical to restriction endonuclease analysis type (genotype) hardjo-bovis A and 11 (15%) were identical to genotype hardjo-bovis B. Serovar pomona isolates were identical to genotypes kennewicki A (64%) or kennewicki B (36%), and serovar grippotyphosa isolates were identical to the RM 52 strain. Isolation rates were significantly (P less than 0.001) higher for beef cattle than for dairy cattle and were higher (P less than 0.001) for bulls than for cows. Combined culture and immunofluorescence results indicated that 2% of mature cattle were renal carriers of leptospires.     

Comparison of polymerase chain reaction assays with bacteriologic culture, immunofluorescence, and nucleic acid hybridization for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle

OBJECTIVE: To compare sensitivity and specificity of various polymerase chain reaction (PCR) assays for detection of Leptospira borgpetersenii serovar hardjo in bovine urine and to compare results of the optimal PCR assay with results of immunofluorescence, nucleic acid hybridization, and bacteriologic culture. ANIMALS: 6 heifers. PROCEDURE: Heifers were exposed to serovar hardjo type hardjo-bovis by conjunctival instillation of 10(6) leptospires on 3 successive days. Urine samples were collected before and after infection. Sensitivity and specificity of 5 PCR assays were compared, to determine the optimal assay for use with bovine urine samples. The optimal PCR assay was then compared with results of bacteriologic culture, nucleic acid hybridization, and immunofluorescence. RESULTS: A PCR assay with the best combination of specificity (100%) and sensitivity (91%) was selected for comparison with the other diagnostic tests. Sensitivity for nucleic acid hybridization was 55%, whereas sensitivity for bacteriologic culture and immunofluorescence was 89 to 93%. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture, PCR, and immunofluorescence were sensitive for detection of L borgpetersenii serovar hardjo type hardjo-bovis in urine specimens of cattle, but a single technique was not the most sensitive for each animal tested. Therefore, the use of 2 techniques in combination is warranted for maximal sensitivity for diagnosis.     

Amoxycillin as an alternative to dihydrostreptomycin sulphate for treating cattle infected with Leptospira borgpetersenii serovar hardjo

Objective To assess the effect of amoxycillin treatment on urinary excretion of leptospires from cattle infected with Leptospira borgpetersenii serovar hardjo .
Design A chemotherapy trial with controls.
Procedure Fourteen heifers serologically negative to L hardjo were inoculated with L hardjo via the conjunctival route and assessed for evidence of infection by serological, fluorescent antibody and microbiological tests. Two injections (48 h apart) of amoxycillin at a dose of 15 mg/kg were administered intramuscularly to seven heifers 6.5 weeks after infection; the remaining heifers acted as untreated controls. Later, these seven control group heifers were treated with a single dose of amoxycillin (15 mg/kg). Samples of urine were collected before and after amoxycillin treatments; kidneys were collected at slaughter, and examined by fluorescent antibody test and microbiological culture.
Results Leptospires were isolated from the urine of 11 of 14 heifers inoculated with L hardjo . After treatment of six of these with two injections of amoxycillin, leptospires were not isolated. Of the controls, four of the five initially leptospiruric heifers continued to shed leptospires; after a single injection of amoxycillin, no leptospires were detected in the kidneys of these four.
Conclusion Amoxycillin may be an acceptable alternative to dihydrostreptomycin sulphate for the treatment of cattle infected with L hardjo .     

Bovine leptospirosis: experimental serovar hardjo infection

Almost the full range of clinical signs observed in pregnant cattle naturally infected with Leptospira interrogans serovar hardjo was observed in this experimental study in which 22 heifers were infected by intraplacentome inoculation with serovar hardjo strains. These features included abortion, mummification, stillbirth, premature and term birth of weak calves and full-term birth of live apparently healthy calves. Leptospires were demonstrated in all but three calves by culture and or immunofluorescence.     

Leptospiral vaccines: immunogenicity of protein-free medium cultivated whole cell bacterins in swine

Swine serologically negative for anti-Leptospira antibodies were given 2 doses of a pentavalent vaccine (3 weeks between doses) prepared from Leptospira serovars canicola, icterohaemorrhagiae, hardjo, pomona, and grip-potyphosa (0.2 mg/serovar/dose). Leptospires used for vaccinal production were cultivated in a protein-free medium or in a bovine albumin-containing medium. All vaccinated swine had demonstrable antibody titers within 1 week of the initial vaccination. Peak microscopic agglutination titers were between 256 and 1,024 after the 2nd vaccinal dose was given. After challenge exposure with serovar canicola, control swine had titers of at least 13,653 and the vaccinated swine had titers of 3,403 to 8,192, depending on the vaccine. Leptospiremia and kidney infections were not detected in any canicola Moulton immunized swine, but did appear in control swine. The Al(OH)3 adjuvant had no obvious influence of any of the vaccinal titers.     

The long term efficacy of a Hardjo-pomona vaccine in preventing leptospiruria in cattle exposed to natural challenge with Leptospira interrogans serovar hardjo

The long term efficacy of a commercially prepared bivalent vaccine against Leptospira interrogans serovar hardjo and L. interrogans serovar pomona was evaluated in a group of 82 dairy heifers exposed to natural challenge with L. interrogans serovar hardjo for up to 55 weeks after calfhood vaccination. Nineteen heifers were vaccinated twice with a one-month interval, at calfhood (9 to 10 months). A further 18 heifers received a similar calfhood vaccination regimen plus a third injection at adulthood (22 to 23 months), while 19 heifers were vaccinated twice at adulthood only and finally 22 heifers were used as unvaccinated controls. At 55 weeks after calfhood vaccination and prior to adulthood vaccination, only 2.7% of the vaccinated heifers were found to have leptospiruria compared with 58.5% of the unvaccinated heifers (p less than 0.0001). Microscopic agglutination (MA) titres at the same time in the unvaccinated heifers ranged from 32 to 4096 while those vaccinated at calfhood ranged from 32 to 64. Adult vaccination of infected animals did not significantly reduce leptospiruria . Prior to adulthood vaccination, 9 of 19 heifers had leptospiruria , in comparison to 4 of 15 after adulthood vaccination. At the same sampling periods 15 of 22 controls had leptospiruria in comparison to 4 of 9 subsequently tested.     

Protection of cattle against natural challenge with Leptospira interrogans serovar hardjo using a hardjo-pomona vaccine

The hardjo component of a bivalent hardjo/pomona vaccine prepared from cultures grown in a protein-free medium was tested in calves which experienced a natural challenge about 7 months after vaccination. A significant degree of protection was obtained using either seroconversion or positive urine cultures as in-dicators of infection. Seroconversion occurred in 10/10 non-vaccinates and 2/9 vaccinates (p <0.01), Leptospires of serovar hardjo were isolated from the urine of 6/10 non-vaccinated calves and 0/9 vaccinated animals (p <0.05).     

Evaluation of the immune response of cattle to leptospiral bacterins.

Cattle were vaccinated against leptospirosis with 3 bacterin preparations: (a) trivalent (serotypes grippotyphosa, pomona, and hardjo) whole cell bacterin; (b) bivalent pomona and hardjo whole cell bacterin; and (c) pentavalent (canicola, grippotyphosa, icterohaemorrhagiae, pomona, and hardjo) cell wall bacterin. Microscopic agglutinating antibody responses in cattle given the last-named bacterin were higher than those in cattle vaccinated with the 2 whole cell bacterins (trivalent and bivalent). However, microscopic agglutinating antibody responses occurred in all vaccinated cattle after they were given a challenge inoculation of serotype hardjo. Leptospires were isolated from 2 of 4 challenge controls (i.e., not given any bacterin), but none of the 15 vaccinated cattle given any one of the bacterins and then challenge inoculated with hardjo became culturally positive. It was concluded that the 3 multivalent bacterins were protective against experimental challenge inoculation of hardjo.     

Experimental infection of calves with Leptospira interrogans serovar hardjo: conjunctival versus intravenous route of exposure

Eight-month-old calves, housed under maximum isolation, were exposed to pathogenic Leptospira interrogans serovar hardjo by the conjunctival route or IV. One calf served as an unexposed control. Infection was monitored serologically (microscopic agglutination test and enzyme-linked immunosorbent assay; ELISA) and by leptospiral culture isolation from periodic urine samples and from the kidneys, epididymides, and aqueous humor collected at slaughter. Microscopic agglutination test titers of greater than or equal to 1:40 were detected among all IV exposed calves at postinoculation day (PID) 7 and among conjunctival exposed calves at PID 14. By ELISA, all IV exposed calves were positive by PID 3, whereas conjunctival exposed calves were positive at PID 14. The ELISA was more sensitive for the detection of antibodies against leptospires in cattle. Leptospires were isolated from the urine of 4 calves and from the kidney of 3 calves exposed by the conjunctival route, but not from IV exposed calves. The results indicated that the conjunctival route of exposure was a more natural and successful route for experimental infection of cattle with serovar hardjo.     

Fetal protection against continual exposure to bovine viral diarrhea virus following administration of a vaccine containing an inactivated bovine viral diarrhea virus fraction to cattle

OBJECTIVE: To evaluate the efficacy of a commercially available killed bovine viral diarrhea virus (BVDV) vaccine to protect against fetal infection in pregnant cattle continually exposed to cattle persistently infected with the BVDV. ANIMALS: 60 crossbred beef heifers and 4 cows persistently infected with BVDV. PROCEDURES: Beef heifers were allocated to 2 groups. One group was vaccinated twice (21-day interval between the initial and booster vaccinations) with a commercially available vaccine against BVDV, and the other group served as nonvaccinated control cattle. Estrus was induced, and the heifers were bred. Pregnancy was confirmed by transrectal palpation. Four cows persistently infected with BVDV were housed with 30 pregnant heifers (15 each from the vaccinated and nonvaccinated groups) from day 52 to 150 of gestation. Fetuses were then harvested by cesarean section and tested for evidence of BVDV infection. RESULTS: 1 control heifer aborted after introduction of the persistently infected cows. Bovine viral diarrhea virus was isolated from 14 of 14 fetuses obtained via cesarean section from control heifers but from only 4 of 15 fetuses obtained via cesarean section from vaccinated heifers; these proportions differed significantly. CONCLUSIONS AND CLINICAL RELEVANCE: A commercially available multivalent vaccine containing an inactivated BVDV fraction significantly reduced the risk of fetal infection with BVDV in heifers continually exposed to cattle persistently infected with BVDV. However, not all vaccinated cattle were protected, which emphasizes the need for biosecurity measures and elimination of cattle persistently infected with BVDV in addition to vaccination within a herd.     

Evaluation of fetal protection against experimental infection with type 1 and type 2 bovine viral diarrhea virus after vaccination of the dam with a bivalent modified-live virus vaccine

OBJECTIVE: To evaluate the efficacy of a modified-live virus (MLV) combination vaccine containing type 1 and type 2 bovine viral diarrhea virus (BVDV) in providing fetal protection against challenge with heterologous type 1 and type 2 BVDV. DESIGN: Prospective study. ANIMALS: 55 heifers. PROCEDURE: Heifers were vaccinated with a commercial MLV combination vaccine or given a sham vaccine (sterile water) and bred 47 to 53 days later. Heifers were challenged with type 1 or type 2 BVDV on days 75 to 79 of gestation. Clinical signs of BVDV infection, presence of viremia, and WBC count were assessed for 14 days after challenge. Fetuses were collected on days 152 to 156 of gestation, and virus isolation was attempted from fetal tissues. RESULTS: Type 1 BVDV was not isolated in any fetuses from vaccinated heifers and was isolated in all fetuses from nonvaccinated heifers challenged with type 1 BVDV. Type 2 BVDV was isolated in 1 fetus from a vaccinated heifer and all fetuses from nonvaccinated heifers challenged with type 2 BVDV. CONCLUSIONS AND CLINICAL RELEVANCE: A commercial MLV combination vaccine containing type 1 and type 2 BVDV given to the dam prior to breeding protected 100% of fetuses against type 1 BVDV infection and 95% of fetuses against type 2 BVDV infection. Use of a bivalent MLV vaccine in combination with a comprehensive BVDV control program should result in decreased incidence of persistent infection in calves and therefore minimize the risk of BVDV infection in the herd.     

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.     

Experimental Leptospira borgpetersenii serovar hardjo infection of pregnant cattle

Objective To observe the effect upon the foetus of experimental infection of pregnant cattle with Leptospira borg-petersenii serovar hardjo .
Design A disease transmission study using pregnant cattle.
Procedure Fourteen heifers serologically negative to L hardjo were artificially inseminated and later challenged with a north-Queensland isolate of L hardjo by conjunctival inoculation. The heifers were serologically monitored and their urine examined for the presence of leptospires using culture and fluorescent-antibody tests at appropriate intervals. Elective caesarean sections were performed on pregnant heifers at 6.5 weeks after the challenge. Foetuses were examined using serological, histopathological, microbiological and fluorescent-antibody tests.
Results Ten of the heifers became pregnant, but three subsequently aborted before challenge. After challenge, all 14 heifers seroconverted and L hardjo was isolated from the urine of 6 of the 7 pregnant heifers. No evidence of foetal L hardjo infection was detected. Two of the foetuses had histopatho-logical lesions consistent with Neospora s p infection.
Conclusion It is likely that the isolate of L hardjo used in this study does not normally infect the foetus. Neospora s p may be a more significant cause of bovine reproductive wastage.     

Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.