Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.     

Histopathologic changes in bovine fetuses after repeated reintroduction of a spirochete-like agent into pregnant heifers: association with epizootic bovine abortion

A spirochete-like organism was found in the plasma of bovine fetuses affected with epizootic bovine abortion (EBA). The spirochete-like organism was frequently found in abattoir-collected fetuses as an inapparent infection, and EBA was found in cattle on foothill rangeland where the vector tick Ornithodorus coriaceus could repeatedly reintroduce the infectious agent into pregnant cattle (superinfection). Epizootic bovine abortion resembled a naturally acquired superinfection in circumstances where the agent was frequently present in the environment under conditions favoring transmission. Therefore, to determine whether fetal lesions could be experimentally induced in utero, spirochete-like organisms collected from clinically normal fetuses at an abattoir were inoculated IV and subcutaneously into 2 pregnant heifers 5 times over a 4-month period to mimic repeated tick transmission in the field. Macroscopic and microscopic examinations of tissues from 2 cesarean-collected fetuses and from 3 calves born at term with the naturally acquired spirochete infection indicated that the calves had evidence of an infection that caused morphologic changes compatible with immunologic stimulation and mild reticuloendothelial hyperplasia. Compared with findings in the calves, lesions in the superinfected fetuses were more severe, and the lesion distribution in various organs was more extensive. The spirochete-like organism appeared to be a mild pathogen because of its persistence in the host. Clinical disease from the infection may only develop with repeated superinfections. Therefore, a relationship between this microorganism and EBA is probable.     

Cellular and humoral responses of Brucella abortus-infected bovine fetuses

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.     

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.     

Experimental transmission of epizootic bovine abortion (foothill abortion)

Advances in defining the biology of epizootic bovine abortion (EBA), including identification of the etiologic agent, have been hampered by the inability to reproduce the disease with confidence. Experimental reproduction of EBA, by feeding the tick vector Ornithodoros coriaceus on susceptible pregnant heifers, is not reliable. The primary objectives of this study were to identify specific tissue(s) obtained from EBA-infected fetuses that could transmit the disease, and then utilize such an infectious challenge system to better define the pathogen, host immunity and geographic distribution of the agent. Described here is the ability to routinely reproduce EBA following inoculation of cryopreserved suspensions of homogenized thymus into susceptible pregnant heifers. This challenge system permitted experiments demonstrating the agent was non-filterable, inactivated upon sonication and susceptible to antibiotics. These findings suggest a prokaryotic microbe and represent a major advance in EBA research. Additional experiments demonstrated that inoculation of the cryopreserved EBA-infectious tissue into heifers, prior to breeding, conferred immunity. Furthermore, such immunized heifers were resistant to challenge with heterologous sources of infectious tissue, suggesting monovalent vaccine development might be feasible. Lastly, challenge studies employing animals from Central Nevada, an area considered free of EBA, demonstrated partial immunity, suggesting the pathogen, and possibly the disease, enjoy a broader distribution than previously thought.     

Comparison of antigenic structure and pathogenicity of bovine intestinal Chlamydia isolate with an agent of epizootic bovine abortion.

A bovine fecal Chlamydia, isolated from a clinically normal cow, had pathogenic capabilities and antigenic structure similar to those of a chlamydial agent of epizootic bovine abortion (EBA). Intravenous inoculation of the fecal chlamydia into a pregnant heifer caused abortion and lesions in the fetus indistinguishable from those of experimentally induced EBA. In serotests with species-specific complement fixation antigens, the intestinal agent was similar to the EBA agent.     

Association of bovine viral diarrhea virus with multiple viral infections in bovine respiratory disease outbreaks

We investigated eleven outbreaks of naturally occurring bovine respiratory diseases in calves and adult animals in the St-Hyacinthe area of Quebec. Specific antibodies to bovine herpesvirus-1, bovine viral diarrhea virus, respiratory syncytial virus, parainfluenza type 3 virus, reovirus type 3, and serotypes 1 to 7 of bovine adenovirus were found in paired sera from diseased animals. Several bovine viruses with respiratory tropism were involved concomitantly in herds during an outbreak of bovine respiratory disease. In addition, concomitant fourfold rises of antibody titers were frequently observed to two or more viral agents in seroconverted calves (61%) or adult animals (38%). Bovine viral diarrhea virus was found to be the most frequent viral agent associated with multiple viral infection in calves only (92%).     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.     

New concepts in the pathogenesis, diagnosis and control of diseases caused by the bovine viral diarrhea virus

The new information on the pathogenesis and epidemiology of mucosal disease of cattle is reviewed. It is now known that clinical mucosal disease occurs only in cattle which were infected with a pestivirus in early gestation and were born with persistent viral infection and specific immunotolerance. These animals may be clinically normal at birth but may develop fatal mucosal disease, perhaps following superinfection with another pestivirus, usually between 6 and 24 months of age. They may also remain clinically normal indefinitely and breed successfully. The progeny from persistently infected females will similarly be persistently viremic, and maternal families of such animals may be established.

Congenital defects may occur when infection of the fetus occurs in mid-gestation. Although fetuses may be infected in utero in late gestation, the infections do not persist, the fetuses develop antibodies, and they appear to suffer no ill-effects. Postnatal infection can result in subclinical disease (bovine viral diarrhea) with a normal immune response; the virus may also be responsible for enhanced susceptibility to other infections, diarrhea in newborn calves, and reproductive failure.

Prevention of the economically important diseases caused by the virus is dependent upon the identification and elimination of persistently viremic animals, which are reservoirs of infection, and the vaccination of immunocompetent females at least three weeks before breeding. However, because of serotypic differences between strains, there is some doubt whether vaccination will reliably provide protection against the transplacental fetal infections that are important in the pathogenesis of this disease. There is no substantial evidence to warrant the vaccination of feedlot cattle.

     

Radiographic and radionuclide lung perfusion imaging in healthy calves and calves naturally infected with bovine respiratory syncytial virus.

Nine calves between three and 18 weeks old with serologically confirmed natural bovine respiratory syncytial virus infection were examined clinically, radiographically and by radionuclide lung perfusion imaging. The results were compared with those from seven healthy calves. The diseased calves were euthanased and examined pathologically, virologically and bacteriologically. The clinical signs indicated that the disease was in an acute stage. Radiography of the diseased animals revealed cysts, corresponding morphologically with bullous emphysema, and infiltrations roughly corresponding in distribution with atelectatic and, or, pneumonic areas. Radionuclide lung perfusion imaging revealed no perfusion shifts between the left and right lungs and a normal perfusion pattern in five of the nine diseased calves. The abnormalities in the perfusion patterns of three calves were probably caused by anatomical disorders such as cysts and pleural adhesions, but no cause of the abnormality could be found in one calf. These findings suggest that in calves infected with bovine respiratory syncytial virus, the normal perfusion pattern is maintained until anatomical disorders occur. The pathological examination and radiography revealed that the cranioventral lung fields were particularly poorly ventilated. This finding and the normal perfusion pattern indicate that these parts of the lungs are probably the sites where shuntings and perfusion-ventilation mismatchings occur.     

Hereditary bovine syndactyly: diagnosis in bovine fetuses

Diagnostic guidelines were established for progeny testing of hereditary bovine syndactyly. Through the use of superovulation and embryo transfer, 139 fetuses were recovered at 50 to 77 days gestation. Normal (+/+, +/sy) and syndactylous (sy/sy) anatomy of Holstein fetuses was defined, and the accuracy of macroscopic versus microscopic limb diagnosis was assessed. Chondrification and ossification differences between normal (+/+, +/sy) and syndactylous (sy/sy) fetuses were only age-related. Normal (+/+, +/sy) fetal limbs differed from normal (+/+, +/sy) adult bovine limbs in two ways. Fetal metacarpal and metatarsal III and IV bones were not fused, and fetal metacarpal and metatarsal II and V bones often extended up to three-fourths the length of metacarpal and metatarsal III and IV bones. In syndactylous (sy/sy) fetuses, synostosis asymmetries occurred within and between fetal limbs, and between fetuses, representing variable gene expressivity. Synostosis pattern within limbs did not correspond with those of the adult bovine; the second phalangeal pair was synostotic most frequently in the fetus, followed by the first, and then the third pair. Synostosis patterns between fetal limbs agreed with those of the adult; there was a right-left and front-rear limb gradient. Partial synostoses occurred sporadically in all three paired phalanges. Those of the first and third pair always involved the tip closest to the second phalangeal pair. A unique example of variable gene expressivity occurred in one syndactylous fetus. Both front limbs were syndactylous, while both rear limbs were normal grossly. Microscopically the right rear limb was normal while the left rear limb consisted of closely apposed phalangeal blastemata without coalescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of cattle immunotolerant to bovine viral diarrhea virus.

Inoculation of bovine virus diarrhea virus into 58 to 125 day old fetuses of bovine virus diarrhea virus seropositive pregnant cows, or inoculation of bovine virus diarrhea virus into seronegative cows 42 to 114 days pregnant, may produce clinically normal calves which are persistently infected with the specific isolate of bovine virus diarrhea virus yet seronegative to the homologous and heterologous isolates. Reinoculation of these persistently infected cattle with their homologous isolate produced no neutralizing antibody response to bovine virus diarrhea virus. These persistently infected cattle were immunocompetent as they developed neutralizing serotiters to infectious bovine rhinotracheitis, parainfluenza-3 viruses and agglutinating serotiters to Pasteurella hemolytica .     

Detection of bluetongue virus in bovine fetuses using the avidin-biotin complex immunoperoxidase method

The avidin-biotin complex immunoperoxidase technique was adapted for use in detecting bluetongue virus (BTV) antigens in BTV serotype 11-infected bovine fetuses. Fetuses were infected with BTV serotype 11 at 120 days of gestation and then removed 20 days later by Cesarean section. Blood and tissue samples were collected from each animal and used for virus isolation in embryonated chicken eggs, the immunofluorescent antibody test, and the avidin-biotin complex test. The avidin-biotin complex method successfully identified BTV antigens in both fresh and autolyzed fetal brains. Thus, the avidin-biotin complex immunoperoxidase method has potential as a possible procedure for diagnosing bluetongue disease in aborted bovine fetuses.     

Serologic examination of aborted ovine and bovine fetal fluids for the diagnosis of border disease, bluetongue, bovine viral diarrhea, and leptospiral infections

Fetal serum from most of 994 bovine and 553 ovine aborted fetuses was tested serologically for antibodies to border disease (BD), bovine viral diarrhea (BVD), and bluetongue (BT) viruses, and to Leptospira sp., and the results were compared with the results of isolation procedures, fluorescent antibody tests (FAT), and histologic examinations of the same fetuses. Antibodies to BT virus were not found in any of the 994 bovine and 553 ovine fetuses. Antibody titers to BVD virus were present in 39 of 966 bovine fetuses tested, and BVD virus was detected in 4 of the 39. Four of 74 fetuses in which the BVD virus was detected by FAT or isolation had titers to BVD virus. Microagglutination (MAT) titers to 1 or more of 5 serovars of leptospires were present in 52 of 773 bovine fetal sera tested. Leptospires were not detected by FAT in any bovine fetuses that had leptospiral antibody titers. Leptospires were detected by FAT in 15 aborted calves, and none of these had MAT titers. Antibody titers to BD virus were present in 80 of 486 fetal lamb sera tested, and the virus was detected by FAT or isolation in 3 of the 80 fetuses. Border disease virus was detected in 14 of 486 fetal lambs tested. Twelve of the 14 were tested serologically and 3 had titers to BD virus. Leptospiral antibody titers were present in 27 of 326 ovine fetal sera tested. Leptospires were not detected in any of the 326 ovine fetuses tested by FAT.     

Congenital spirochetosis in calves: association with epizootic bovine abortion

Congenital spirochetosis was encountered as a newly recognized infection of cattle. The spirochete was seen in blood of fetuses with lesions of epizootic bovine abortion. A spirochete with morphologic features similar to those found in the fetuses was detected in Ornithodoros coriaceus ticks. Ticks collected from rangelands were allowed to feed on cows that then produced epizootic bovine abortion-affected fetuses, and the fetuses had spirochetosis. Inapparent spirochetosis also was found in fetuses in clinically normal cattle sent to slaughter. Only a few lesions were seen in abattoir-collected fetuses. Fetal spirochetosis was common in the bovine population studied, and it appeared that infection may be limited only by the availability of the tick vector.     

Genetic comparison of ovine and bovine pestiviruses

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.     

Viral and bacterial pathogens in bovine respiratory disease in Finland

Pathogens causing bovine respiratory tract disease in Finland were investigated. Eighteen cattle herds with bovine respiratory disease were included. Five diseased calves from each farm were chosen for closer examination and tracheobronchial lavage. Blood samples were taken from the calves at the time of the investigation and from 86 calves 3-4 weeks later. In addition, 6-10 blood samples from animals of different ages were collected from each herd, resulting in 169 samples. Serum samples were tested for antibodies to bovine parainfluenza virus-3 (PIV-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine adenovirus-3 (BAV-3) and bovine adenovirus-7 (BAV-7). About one third of the samples were also tested for antibodies to bovine virus diarrhoea virus (BVDV) with negative results. Bacteria were cultured from lavage fluid and in vitro susceptibility to selected antimicrobials was tested. According to serological findings, PIV-3, BAV-7, BAV-3, BCV and BRSV are common pathogens in Finnish cattle with respiratory problems. A titre rise especially for BAV-7 and BAV-3, the dual growth of Mycoplasma dispar and Pasteurella multocida, were typical findings in diseased calves. Pasteurella sp. strains showed no resistance to tested antimicrobials. Mycoplasma bovis and Mannheimia haemolytica were not found.     

Natural bovine lentiviral type 1 infection in Holstein dairy cattle. I. Clinical,serological, and pathological observations

Clinical, serological, and pathological abnormalities observed in Holstein cows naturally infected with bovine lentivirus 1 bovine immunodeficiency virus (BIV) and other infections were progressive and most commonly associated with weight loss, lymphoid system deficiency, and behavioral changes. Clinical evidence of meningoencephalitis was dullness, stupor, and occasional head or nose pressing postures. The polymerase chain reactions associated the BIV provirus with the lesions in the central nervous system and lymphoid tissues. Multiple concurrent infections developed in retrovirally infected cows undergoing normal stresses associated with parturition and lactation. A major functional correlate of the lymphoreticular alterations was the development of multiple secondary infections which failed to resolve after appropriate antibacterial therapy. The chronic disease syndrome in dairy cows associated with BIV may be useful as a model system for investigation of the pathogenesis of the nervous system lesions and lymphoid organ changes that occur in humans with lentiviral infection.