Shollie M. Falkenberg

Comparison of acute infection of calves exposed to a high-virulence or low-virulence bovine viral diarrhea virus or a HoBi-like virus

OBJECTIVE To compare acute infection of cattle exposed to a high-virulence (HV) bovine viral diarrhea virus (BVDV), low-virulence (LV) BVDV, or HoBi-like virus. ANIMALS 24 Holstein bull calves. PROCEDURES Colostrum-deprived 2- to 4-week-old calves, free of BVDV antigen and antibodies, were allocated into 4 groups (6 calves/group). Calves in 3 groups were exposed to an LV BVDV strain (BVDV2-RS886), an HV BVDV strain (BVDV2-1373), or a HoBi-like virus (D32/00 HoBi), whereas calves in the fourth group were not exposed to a virus but were cohoused with calves exposed to the HoBi-like virus. Circulating WBCs, platelets, rectal temperature, and presence of virus in the blood were monitored. RESULTS Infection of calves with any of the 3 viruses resulted in reduced numbers of circulating WBCs. Pyrexia was detected in all calves exposed to HV BVDV or LV BVDV but in only 3 of 6 calves exposed to the HoBi-like virus. Diarrhea was observed in 0 of 6 calves exposed to the HoBi-like virus, 2 of 6 calves exposed to the LV BVDV, and 6 of 6 calves exposed to the HV BVDV. The HoBi-like virus was transmitted from acutely infected calves to naïve cohorts. CONCLUSIONS AND CLINICAL RELEVANCE The HoBi-like viruses are an emerging species of pestivirus isolated from water buffalo and cattle in South America, Southeast Asia, and Europe but not from cattle in the United States. Understanding the clinical course of disease caused by HoBi-like pestiviruses will be important for the design of surveillance programs for the United States.

Generation of Calves Persistently Infected with HoBi-Like Pestivirus and Comparison of Methods for Detection of These Persistent Infections

ABSTRACT The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV—two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)—and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein Erns detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

Lack of evidence for the presence of emerging HoBi-like viruses in North American fetal bovine serum lots

The detection of an emerging pestivirus species, “HoBi-like virus,” in fetal bovine serum (FBS) labeled as U.S. origin, but packaged in Europe, raised concerns that HoBi-like virus may have entered the United States. In the current study, 90 lots of FBS originating in North America (NA) were screened for pestivirus antigen and antibodies. Lots in group 1 (G1, 72 samples) and group 2 (G2, 9 samples) originated in NA and were packaged in the United States. Group 3 (G3) was composed of 9 lots collected in NA and processed in Europe. Lots in G1 were claimed negative for Bovine viral diarrhea virus (BVDV), while lots in G2 and G3 were claimed positive by the commercial processor. All lots in G1 and G2 tested negative by reverse transcription polymerase chain reaction (RT-PCR) using HoBi-like–specific primers. Two G1 lots tested positive by BVDV RT-PCR. One of these was also positive by virus isolation. All G2 lots were positive by BVDV RT-PCR. In addition, four G2 lots were VI positive while 1 lot was antigen-capture enzyme-linked immunosorbent assay (ELISA) positive. Two G3 lots were positive by HoBi-like–specific RT-PCR tests. All lots were negative for HoBi_D32/00 neutralizing antibodies. Seven lots (4 G1; 1 G2; 2 G3) had antibodies against BVDV by virus neutralization and/or antigen-capture ELISA. While there is no evidence of HoBi-like viruses in NA based on tested samples, further studies are required to validate HoBi-like virus–free status and develop means to prevent the spread of HoBi-like virus into NA.

Experimental infection of calves, sheep, goats and pigs with HoBi-like viruses by direct inoculation or exposure to persistently infected calves.

HoBi-like viruses are an emerging species of pestiviruses associated with respiratory and reproductive disease in cattle and in water buffaloes. Although cattle appear to be the main natural hosts, little is know about the potential for HoBi-like viruses to be transmitted to other livestock. In this study, seronegative calves, goats and pigs, and sheep harboring pestivirus antibodies (probably due to previous exposure to BVDV) were exposed to HoBi-like viruses either by direct inoculation (GIn) or by contact with calves persistently infected with HoBi-like viruses (GEx). Both GIn and GEx groups were monitored for clinical signs, lymphocyte count, virus in buffy coats and nasal swabs up to day 18 post-inoculation (pi). Evidence of transmission of HoBi-like virus by PI calves was observed in all studied species. No difference in clinical presentation was observed between animals in the GIn or GEx groups. Evidence of infection, depending on the species included lymphocyte depletion, fever, viral RNA detection, and/or seroconversion. Depletion of lymphocytes was observed in calves and goats (35% and 50%, respectively) but not in pigs. Seroconversion was observed in at least one animal of each group and for all exposed species. The rate of seroconversion was higher in animals in the GIn experimental groups. In sheep, pre-existing moderate to high neutralizing titers against BVDV did not prevent viral replication and shed. The study demonstrated that naive cattle, goats and pigs, in addition to antibody positive sheep, can be infected by HoBi-like virus via persistently infected calf and potentially transmit the virus.

Changes observed in the thymus and lymph nodes 14 days after exposure to BVDV field strains of enhanced or typical virulence in neonatal calves.

Clinical presentation following uncomplicated acute infection with bovine viral diarrhea viruses (BVDV) ranges from clinically unapparent to severe (including hemorrhagic disease and death) depending on the strain virulence. Regardless of clinical presentation, BVDV infection of cattle results in a generalized immunosuppression. BVDV immunosuppression is characterized by a reduction of circulating white blood cells (WBC) that is typically evident by day 3 post infection (PI). In infections with typical BVDV field strains WBC counts decrease until days 6-9 PI and then return to baseline values. In infections with enhanced virulence BVDV, WBC counts may continue to decline through day 14. In this study, the lymph nodes and thymus of non-infected neonatal calves and neonatal calves infected 14 days previously with either a BVDV of typical virulence or one of enhanced virulence were compared. It was found that while calves, infected with the typical virulence BVDV, had cleared BVDV, and WBC counts had returned to near baseline, the number of B-B7(+) cells in lymph nodes were reduced whereas numbers of CD4(+) cells were increased as compared to control calves. In contrast, calves infected with the high virulence strain, had not cleared the virus by day 14 and WBC counts had not returned to pre-exposure levels. Furthermore, these calves had more substantial deficits of B-B7(+) and CD4(+) cell subpopulations, compared to calves infected with a typical virulence strain. There were also an increased number of macrophages observed in both lymphoid tissues examined. The thymuses from both groups of BVDV-infected calves were significantly smaller than non-infected age matched calves. The reduction in size was accompanied by a significant depletion of the thymic cortex. These results indicate that regardless of the virulence of the infecting BVDV, infection leaves neonatal calves with deficits in specific lymphocyte subsets and lymphoid tissues that could have long-term immunosuppressive implications.

An inactivated influenza D virus vaccine partially protects cattle from respiratory disease caused by homologous challenge.

Abstract Originally isolated from swine, the proposed influenza D virus has since been shown to be common in cattle. Inoculation of IDV to naïve calves resulted in mild respiratory disease histologically characterized by tracheitis. As several studies have associated the presence of IDV with acute bovine respiratory disease (BRD), we sought to investigate the efficacy of an inactivated IDV vaccine. Vaccinated calves seroconverted with hemagglutination inhibition titers 137–169 following two doses. Non-vaccinated calves challenged with a homologous virus exhibited signs of mild respiratory disease from days four to ten post challenge which was significantly different than negative controls at days five and nine post challenge. Peak viral shedding of approximately 5 TCID50/mL was measured in nasal and tracheal swabs and bronchoalveolar lavage fluids four to six days post challenge. Viral titers were significantly (P <0.05) decreased 1.4 TCID50/mL, 3.6 TCID50/mL and 5.0 TCID50/mL, respectively, in the aforementioned samples collected from vaccinated animals compared to non-vaccinated controls at peak shedding. Viral antigen was detected in the respiratory epithelium of the nasal turbinates and trachea by immunohistochemistry from all unvaccinated calves but in significantly fewer vaccinates. Inflammation characterized by neutrophils was observed in the nasal turbinate and trachea but not appreciably in lungs. Together these results support an etiologic role for IDV in BRD and demonstrate that partial protection is afforded by an inactivated vaccine.

Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. Although, BVDV can be identified readily by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely difficult. Detection at the single lymphoid cell level is important due to the immunomodulation that accompanies BVDV infection. A novel PrimeFlow RNA assay using in-situ detection of BVDV was evaluated. The model used to develop this technique included three BL-3 cell lines with different infection statuses, one not infected with BVDV, one infected with BVDV and one dual infected with BVDV and bovine leukosis virus. Using RNA probes specific for the BVDV-2a Npro-Erns coding region, BVDV RNA was detected from both contaminated BL-3 cell lines by flow cytometry and fluorescent microscopy. This is the first report on in-situ detection of BVDV at the single-cell level.

Relative virulence in bison and cattle of bison-associated genotypes of Mycoplasma bovis

Mycoplasma bovis, a frequent contributor to polymicrobial respiratory disease in cattle, has recently emerged as a major health problem in North American bison. Strong circumstantial evidence suggests it can be the sole pathogen causing disease manifestations in outbreaks of mortality in bison, but direct evidence is lacking. The goal of this study was to compare clinical signs and lesions in bison and cattle experimentally infected with field isolates of M. bovis recovered from bison. Bison (n = 7) and cattle (n = 6), seronegative for anti-M. bovis IgG, were exposed intranasally to M. bovis and necropsied 4-6 weeks later. Blood and nasal swabs were collected on day 0 (before exposure), day 11 and at necropsy. Samples of lung, lymph node, liver and spleen were also collected at necropsy. The only clinical sign observed was an elevation in the core body temperature of bison during the first few weeks post-exposure. Grossly visible lesions were apparent at necropsy in the lungs of five bison and the lymph node of one bison, while none were evident in cattle. Histologic evaluation revealed moderate to severe pulmonary lesions in four bison but none in cattle. M. bovis was recovered from tissues demonstrating gross lesions and from the lymph nodes of one additional bison and two cattle. All animals seroconverted by the time of necropsy. These data provide the first direct evidence that M. bovis can be a sole or primary cause of respiratory disease in healthy bison, although the isolates used were unable to cause disease in healthy cattle.

Evaluation of bovine viral diarrhea virus transmission potential to naïve calves by direct and indirect exposure routes

Bovine viral diarrhea viruses (BVDV) can cause both acute and persistent infections in cattle. Exposure to BVDV persistently infected (PI) animals results in transmission of the virus to a naïve animal which causes a transient acute infection. While it is known that direct exposure to PI animals is a highly efficient means of transmission, less information is available regarding the potential for transmission from acutely infected either by direct or indirect exposure to naïve animals. Therefore, the objective of this study was to evaluate the potential for spread of the virus from calves acutely infected, with typical virulence field viruses know to have minimal shedding and viremia, to naïve contact animals either by direct or indirect exposure. To accomplish this objective, two BVDV isolates belonging to two species of BVDV, type 1 and type 2, were used to inoculate calves. Subsequently on day 2 post-infection, naïve calves were exposed to inoculated calves, either directly or indirectly, over a period of two weeks. All calves were evaluated for the presence of virus in blood samples and nasal swabs, pyrexia, lymphopenia and seroconversion. BVDV was isolated from inoculated calves but not from any of the direct and indirect contact animals or from control calves. Similarly, pyrexia and lymphopenia were observed in the inoculated calves, but not in contact and control calves. Only the inoculated calves seroconverted by day 38 of the study indicating that no transmission had occurred to the naïve contact calves. This data would suggest that there may be an infectious dose needed for transmission of virus for typical virulent isolates.

Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2

Bovine viral diarrhea virus (BVDV) is an important viral cause of reproductive disease, immune suppression and clinical disease in cattle. The objective of this study was to compare reproductive protection in cattle against the impacts of bovine viral diarrhea virus (BVDV) provided by three different multivalent vaccines containing inactivated BVDV. BVDV negative beef heifers and cows (n = 122) were randomly assigned to one of four groups. Groups A-C (n = 34/group) received two pre-breeding doses of one of three commercially available multivalent vaccines containing inactivated fractions of BVDV 1 and BVDV 2, and Group D (n = 20) served as negative control and received two doses of saline prior to breeding. Animals were bred, and following pregnancy diagnosis, 110 cattle [Group A (n = 31); Group B (n = 32); Group C (n = 31); Group D (n = 16)] were subjected to a 28-day exposure to cattle persistently infected (PI) with BVDV (1a, 1b and 2a). Of the 110 pregnancies, 6 pregnancies resulted in fetal resorption with no material for testing. From the resultant 104 pregnancies, BVDV transplacental infections were demonstrated in 73 pregnancies. The BVDV fetal infection rate (FI) was calculated at 13/30 (43%) for Group A cows, 27/29 (93%) for Group B cows, 18/30 (60%) for Group C cows, and 15/15 (100%) for Group D cows. Statistical differences were observed between groups with respect to post-vaccination antibody titers, presence and duration of viremia in pregnant cattle, and fetal infection rates in offspring from BVDV-exposed cows. Group A vaccination resulted in significant protection against BVDV infection as compared to all other groups based upon outcome measurements, while Group B vaccination did not differ in protection against BVDV infection from control Group D. Ability of inactivated BVDV vaccines to provide protection against BVDV fetal infection varies significantly among commercially available products; however, in this challenge model, the inactivated vaccines provided unacceptable levels of BVDV FI protection.