Effect of primary and recurrent infections bovine rhinotracheitis virus infection on the bovine ovary

Six heifers were inoculated IV at estrus with the Iowa or Colorado isolates of infectious bovine rhinotracheitis virus (IBRV). Subsequent measurements of plasma progesterone indicated that corpus luteum function was depressed in all heifers. In the 1st estrous cycle after inoculation, progesterone values did not exceed 2 ng/ml in 3 heifers given the Iowa isolate. Although maximal progesterone values were greater than or equal to 2 ng/ml in 3 heifers given the Colorado isolate, values were lower than those in later cycles. Five heifers had maximal diestrual progesterone values greater than or equal to 5 ng/ml within 5 weeks after inoculation, but in the 6th heifer, this amount of progesterone was not present until 8 weeks after inoculation. Three to 5 months after inoculation, all heifers were given 5 daily injections of dexamethasone, 2 heifers each during metestrus, diestrus, or proestrus. Subsequent recrudescence of IBRV was demonstrated in all heifers by the isolation of virus from vaginal or nasal swab samples. The heifers were killed 10 to 17 days after initiation of dexamethasone treatment and their reproductive organs were examined for lesions and IBRV. Lesions were not seen, and IBRV was isolated only from the corpus luteum of a heifer given dexamethasone during diestrus.     

Ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovine rhinotracheitis virus on the day after breeding

Four commercially available vaccine strains of modified-live infectious bovine rhinotracheitis virus were passaged once in embryonic bovine kidney cells. Heifers were inoculated IV on the day after breeding with 5.0 ml of nondiluted cell culture fluid of each of the 4 strains. Virus was reisolated from nasal swabs and blood collected during the week after inoculation. The heifers were killed 9 to 14 days after inoculation. Mild-to-marked inflammatory and necrotic lesions were seen in the corpora lutea and ovaries of the heifers. The lesions were similar to, and almost as severe as, those resulting from the inoculation of virulent strains of infectious bovine rhinotracheitis virus. Adrenal lesions were also found in all heifers examined. Virus was reisolated from the ovaries of only 4 of the 8 heifers. However, virus was confirmed in the ovaries of all 8 heifers, using immunofluorescent or ultrastructural studies. Heifers with severe luteal damage had abnormally low plasma progesterone concentrations.     

Culture-positive persistence and serum agglutinating antibody response after intrauterine inoculation of Haemophilus somnus in virgin heifers

Viable Haemophilus somnus of reproductive tract origin (OSU-1167) was inoculated transcervically into the uterus of 6 virgin heifers. Five heifers were sham-inoculated (intrauterine) with sterile mycoplasmal medium and served as controls. After inoculation and observation, all heifers had nasal and vaginal vestibular swab specimens and serum obtained periodically for 44 days. Signs of systemic illness were not detected. On the day after inoculation, all inoculated heifers had signs of vulvovaginitis, whereas none of the control heifers had similar signs (P less than 0.002). Haemophilus somnus was not isolated from any nasal or vaginal vestibular swab specimens obtained before inoculation or from any nasal swab specimens obtained after inoculation. During the 44 days after inoculation, H somnus was isolated from 25 of 54 vestibular specimens obtained from inoculated heifers and from 3 of 45 specimens obtained from controls (P less than 0.02). Vulvovaginal lesions were associated with vestibular isolation of H somnus in 23 of 25 (92%) such isolations from inoculated heifers; lesions were never associated with concurrent isolation of H somnus in controls. All heifers had H somnus microagglutination test (MAT) titer less than or equal to 256 against a commercially prepared H somnus antigen at the beginning of the study. Considered as groups, neither inoculated nor control heifers achieved fourfold increases in MAT titer during the 44 days after inoculation. When compared by day of sample collection, inoculated heifers did have significantly (P less than 0.04) lower geometric mean titer at 7 days after inoculation than did control heifers when tested by use of a commercially prepared antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte blastogenesis in bluetongue virus or Mycobacterium bovis-inoculated bovine fetuses

Bovine fetuses were inoculated with Mycobacterium bovis, bluetongue virus or placebo at approximately 125 days of gestation, and blastogenic responses of peripheral blood lymphocytes and lymph node cells were determined at various time intervals after inoculation. Lymphocytes from all fetuses were stimulated by phytohemagglutinin, concanavalin A, and pokeweed mitogen, and peripheral blood lymphocytes gave consistently greater stimulation indices than did prescapular lymph node cells. Bluetongue virus infection did not consistently suppress mitogen induced lymphocyte blastogenesis. Lymphocytes taken from fetuses at 20 or 50 days after Mycobacterium bovis inoculation were not stimulated by purified protein derivative (PPD), whereas lymphocytes taken from adult cattle at similar intervals after Mycobacterium bovis inoculation were stimulated by PPD. Although lymphocytes from bovine fetuses may be stimulated by mitogens, antigen specific blastogenesis to a known inducer of cellular immunity was not detected by 175 days of gestation.     

Infertility in heifers inoculated with modified-live bovine herpesvirus-1 vaccinal strains against infectious bovine rhinotracheitis on postbreeding day 14

Heifers were inoculated IV with 1 of 4 modified-live bovine herpesvirus-1 vaccinal strains against infectious bovine rhinotracheitis (2 heifers/strain) on postbreeding day (PBD) 14. The effect of infection on fertility was monitored by plasma progesterone assay at 1- to 3-day intervals from the time of virus exposure until PBD 60. Infertility was detected in 4 of 8 inoculated heifers. In 2 heifers, progesterone concentrations decreased to values indicative of estrus within 10 days after inoculation (PBD 24). The 2 other heifers had evidence of embryonic death on PBD 40 and 42. Two control heifers inoculated with culture medium from noninfected cells maintained their pregnancies.     

Experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus

Pairs of heifers were inoculated IV with infectious bovine rhinotracheitis virus on postbreeding days (PBD) 7, 14, 21, or 28, and were euthanatized 13 to 15 days after inoculation. Reproductive tracts were examined for cytopathologic changes (light microscopy), virus (cell culture), and viral antigen (immunohistochemical evaluation). Heifers inoculated on PBD 7 or 14 had mild oophoritis characterized by foci of necrosis and mononuclear cell accumulations in the corpus luteum. Most of these heifers also had a few necrotic follicles in at least one ovary. Heifers inoculated on PBD 21 or 28 did not have corpus luteum lesions, but necrotic follicles were numerous in both ovaries. Viral antigen was observed in all ovarian lesions, and infectious virus was isolated from a few of the affected tissues. The uteri of all heifers inoculated on PBD 21 or 28 and 1 heifer inoculated on PBD 7 contained normal-appearing concepti. The uterus of the other PBD 7 heifer contained a degenerating conceptus that was infected with infectious bovine rhinotracheitis virus, as determined by viral isolation, immunohistochemical evaluation, and electron microscopy. Heifers inoculated on PBD 14 were not pregnant at necropsy, but histologic evidence was found that the postbreeding estrous cycle had been longer than normal, indicating that early embryonic death had occurred.     

Ovarian lesions in heifers exposed to infectious bovine rhinotracheitis virus by non-genital routes on the day after breeding

Twelve heifers were exposed to either a Colorado infectious bovine rhinotracheitis (IBR) virus isolate or an Iowa IBR isolate obtained from a bovine respiratory disease outbreak. All inoculations were made on the day after the heifers had been in estrus and bred by an IBR virus-negative bull. Pairs of heifers were inoculated with each virus isolate intravenously, intramuscularly or exposed by aerosol. The heifers were killed 11-15 days after inoculation and their reproductive tracts and ovaries subjected to virological and pathological study. Virus was isolated from the ovaries of all 4 heifers inoculated intravenously and from 3 of the 4 heifers inoculated intramuscularly, but not from the ovaries of heifers exposed by aerosol. Virus isolations and lesions were, with only 1 exception, confined to the ovary containing the corpus luteum. In ovaries from which IBR virus was isolated, lesions in the corpus luteum ranged from focal necrosis and infiltration of mononuclear cells to diffuse hemorrhage and necrosis. Most of these ovaries also had necrotic follicles and a diffuse mononuclear cell accumulation in the stroma. Lesions were not found in ovaries from which IBR virus was not isolated. It was concluded that lesions are readily induced in the ovaries of post-estrus heifers as a result of hematogenous spread of IBR virus and suggest that the differences in lesion development observed with the 3 routes are related to whether or not a viremia occurred.     

Feline Panleukipenia. I. Pathogenesis in germfree and specific pathogen-free cats.

Germfree and specific pathogen-free cats were inoculated panleukopenia vivus. Total leucocyte counts decreased significantly in both germfree and specific pathogen-free cats. Clinical illness was not seen in any germfree cat. Specific pathogen-free cats had anorexia and slight diarrhea 5-6 days after inoculation. None of the cats died. Both germfree and specific pathogen-free cats had thymic involution. No other gross lesions were seen. Tissues for histological virus isolation and immunofluorescence studies were taken daily from days 2 through 6 after inoculation. Virus-infected cells and lesions of panleukopenia were seen in the small intestine of both germfree and specific pathogen-free cats. The incidence of virus-infected cells and lesions was greater in specific pathogen-free cats than in germfree cats.     

In utero infection of swine fetuses with infectious bovine rhinotracheitis virus (bovine herpesvirus-1)

The pathogenesis of infectious bovine rhinotracheitis (IBR) virus (bovine herpesvirus-1) was studied in porcine fetuses after in utero inoculation. Laparotomies were performed on 8 seronegative pregnant sows at 34 to 86 days of gestation, and all fetuses in 1 uterine horn of each sow were exposed to IBR virus via inoculation into the amniotic sacs. Fetuses in the other horn served as controls. Clinical signs of infection were not observed in the sows, except for 2 sows that aborted at postinoculation days (PID) 11 and 15. Fetuses of the remaining 6 sows were collected at slaughter on PID 15 to 28. Fetuses were examined for gross abnormalities, presence of IBR virus in tissues, and the formation of neutralizing antibodies to IBR virus. Of 33 inoculated fetuses from 6 sows, 10 were mummified, 11 were hemorrhagic and/or edematous, and 12 were alive. Necrotic lesions were observed on the skin and in the liver of dead and live fetuses. Virus was recovered from 29 of 33 inoculated fetuses. Infectious bovine rhinotracheitis virus was isolated from fetal skin, liver, lungs, kidney, spleen, stomach contents, brain, amniotic fluid, and placenta. Virus was isolated from 4 of 11 fetuses recovered from 1 aborting sow. Antibodies to IBR virus were not detected in sera from the sows. However, antibodies were detected in 6 of 15 fetuses inoculated at 63 to 86 days of gestation and collected at slaughter at 86 to 112 days of gestation. The youngest fetus with detectable IBR antibody was estimated to be 74 days of gestation by measuring crown-rump length of the fetus.     

Determination of ability of a thymidine kinase-negative deletion mutant of bovine herpesvirus-1 to cause abortion in cattle.

The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase-negative (TK-) deletion mutant virus. Twelve heifers were inoculated IV at 25 to 29 weeks of pregnancy with either TK- or thymidine kinase-positive (TK+) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of TK+ inoculates were slightly higher and remained above normal a few days longer than in TK- inoculates. Viremia was detected in 5 of 6 TK+ inoculates and in all 6 TK- inoculates. More virus isolations were made from nasal and vaginal swab specimens of TK+ inoculates than from swab specimens of TK- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the TK- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given TK+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the TK gene reduces abortifacient activity of bovine herpesvirus-1.     

Reproductive tract lesions in heifers after intrauterine inoculation with infectious bovine rhinotracheitis virus

Cross-breed heifers given infectious bovine rhinotracheitis virus by intrauterine inoculation 1 day after natural mating with a noninfected bull were killed on postinoculation days 4 to 14. When reproductive organs were examined for gross and microscopic lesions and for virus infection, the most severe uterine lesions were found in the body and caudal portions of the uterine horns of heifers killed between postinoculation days 4 and 9. Primary pathologic features were necrosis, edema, hemorrhage, and a diffuse accumulation of mononuclear cells, mostly lymphocytes; numerous lymphocytes were in mitosis. In cranial parts of uterine horns, the only lesions observed were a few small lymphocytic foci in the endometrial lamina propria. Lesions were not seen in the oviducts. In many heifers, the ovarian corpus luteum (CL) was cystic. In a few of these heifers, the cyst had a necrotic wall that was bordered by a zone of proliferating mononuclear cells. Focal necrosis and lymphoid proliferation were common in the parenchyma of cystic and noncystic CL. Similar necrotizing lesions were sometimes present in non-CL ovarian tissue. Infectious bovine rhinotracheitis virus was most frequently isolated from the uterine body, the internal os of the cervix, and the CL. Isolations were not made from blood samples taken at the time of necropsy. Isolation of virus from the CL correlated with the detection of luteal inflammation by light microscopy, but did not correlate with the presence of cysts. There also was no correlation between cystic CL and the severity of uterine lesions.     

Skin testing, fecal culture, and lymphocyte immunostimulation in cattle inoculated with Mycobacterium paratuberculosis.

Fourteen calves at 21 days of age were experimentally inoculated with 100 mg (wet weight) of Mycobacterium paratuberculosis. Three calves were inoculated orally, 4 intravenously, and 7 subcutaneously. Lymphocyte immunostimulation, fecal culture, and intradermal tuberculin skin testing were done between 112 to 150 days following exposure. Lymphocyte immunostimulation test results, conducted at 112 days after inoculation, showed all animals positive to Mycobacterium avium purified protein derivative. Fecal culture results, taken at 120 days after inoculation, showed that 2 of 3 animals inoculated intravenously were positive, whereas only 2 of 7 inoculated subcutaneously were positive (8 of 14 total were positive). Intradermal skin testing results at 150 days with M avium purified protein derivative showed 13 of the 14 calves were positive. Calves were examined at necropsy 153 days after inoculation, and M paratuberculosis was isolated from tissues of each of the 14 calves.     

Infection of bovine fetuses at 120 days' gestation with virulent and avirulent strains of bluetongue virus serotype 11.

Bluetongue virus infection in sheep and cattle during fetal development causes neuropathology. Two strains of bluetongue virus serotype 11 designated as UC-2 and UC-8 have different virulence patterns in newborn mice. These viruses have distinctly different electropherotype patterns on polyacrylamide gel electrophoresis indicating a genetic difference in these two viruses of the same serotype. Four bovine fetuses each were inoculated intramuscularly with either UC-2 or UC-8, and one fetus was inoculated with placebo. The inoculation was made intramuscularly through the uterine wall at 120 days' gestation, and the bovine fetuses were recovered by cesarean section 12 or 20 days after inoculation. Fetal blood was collected for virus isolation and serology. Virus was reisolated from brain, blood, lung and liver. Both strains, UC-2 and UC-8, cause severe lesions in the 120 day fetuses. The encephalomalacic lesions occurred earlier and were more severe in fetuses inoculated with UC-8 as compared to those inoculated with UC-2. The subtle differences observed in the fetuses inoculated with the two different strains suggest that there is a difference in pathogenic potential of the two viruses. These differences do not appear to be completely dependent upon the host species.     

STUDIES ON THE EFFECTS OF INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS ON REPRODUCTION IN HEIFERS

Natural venereal infection of heifers with IBR virus resulted in vulvovaginal lesions in 9 of 12 heifers at 3 weeks after introducttion to bulls, and virus was isolated from 5 of the 9 affected animals. The infection was not associated with any effect on conception. Experimental nasal and conjunctival infection of 18 heifers pregnant for 3, 5 or 7 months with a genital strain of IBR virus caused mild rhinitis or conjunctivitis in all inoculated animals and virus was recovered from 14 animals at 8 days after inoculation. No effects on pregnancy were recorded.     

Relationship of fetal age at conjunctival exposure of pregnant heifers and Brucella abortus isolation

Pregnant heifers were exposed by a conjunctival inoculation with 1 X 10(7) colony-forming units of Brucella abortus strain 2308. At parturition, milk and uterine samples from dams plus samples from dead calves were cultured bacteriologically for Brucella. The logistic regression probability of B abortus isolation increased from 0.22 to 0.90, as fetal age at exposure of heifers increased from 60 to 150 gestation days. Strain 2308 was recovered at parturition from 14 (64%) of 22, 17 (71%) of 24, and all 28 (100%) heifers that were at gestation days less than 127, 127 to 157, and greater than 157, respectively, at time of exposure. The number of infected heifers and the number of samples positive for B abortus were significantly increased as fetal age at exposure of heifers increased from gestation days less than 127 to greater than 157 (chi 2 greater than 10, P less than 0.005).     

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.     

Association between route of inoculation with infectious bovine rhinotracheitis virus and site of recrudescence after dexamethasone treatment

Four calves latently infected with infectious bovine rhinotracheitis virus (IBRV) were used to compare the ease of isolation of virus from neuronal ganglia and from mucosal surfaces. Two calves were slaughtered, and neuronal ganglia (cranial cervical, trigeminal, and 3rd and 4th sacral) were cocultivated on bovine fetal kidney cells. Virus was not isolated. Two calves given dexamethasone for 4 days were slaughtered on the 5th day. Virus was not isolated from cocultivated or macerated neuronal ganglia, but virus was isolated from nasal secretions taken from both calves on the day of slaughter. Eleven calves were inoculated with IBRV via different routes and were treated with dexamethasone 3 to 4 months after inoculation. virus was isolated from the nasal cavities, but not the vaginas of 6 heifers inoculated intranasally, and was isolated from the vaginas, but not the nasal cavities of 2 heifers inoculated intravaginally. Of 3 calves inoculated IV, virus was isolated from the nasal cavities of 3, from the oropharynxes of 2, and from the prepuce of 1.     

Campylobacter jejuni colitis in gnotobiotic dogs.

Campylobacter jejuni of human and canine origin was inoculated orally into six gnotobiotically reared Beagle puppies and reactions were compared with two controls. Inoculated dogs developed transient lassitude, inappetence, mild diarrhea and tenesmus during the period 36-72 hours after inoculation. Pairs of dogs killed 43 hours, and five and seven days after inoculation had lesions limited to typhlitis and colitis. Congestion of colonic mucosa, associated loss of goblet cells, attenuation and exfoliation of surface epithelium with microerosions, hypertrophy of glands and neutrophil infiltration of lamina propria were seen during the acute phase. Less severe surface and inflammatory lesions were evident at five and seven days, with hyperplasia of the proliferative compartment in mucosal glands. Campylobacter established at over 10(10) organisms per gram of colonic content but did not invade the mucosa. It was concluded that the gnotobiotic dog may be a suitable model for investigation of the pathogenesis of Campylobacter colitis.     

Effects of a bovine herpesvirus-1 isolate on reproductive function in heifers: classification as a type-2 (infectious pustular vulvovaginitis) virus by restriction endonuclease analysis of viral DNA

A bovine herpesvirus-1 (BHV-1) isolate (FI) from an aborted fetus was used to infect 9 heifers at various stages of gestation. Two heifers were inoculated IV on postbreeding day (PBD) 1, 7, or 14, and 3 heifers were inoculated in the sixth month of pregnancy. Plasma progesterone assays were used to monitor corpus luteum function in heifers inoculated during early pregnancy. Low progesterone values and infertility were seen in the 2 heifers inoculated on PBD 1. Luteal function remained normal in heifers inoculated on PBD 7 or 14. These 4 heifers inoculated on PBD 7 or 14 carried their fetuses to term, and their calves were free of BHV-1 infection at birth. Three heifers inoculated during the sixth month of pregnancy also carried their fetuses to term. Two calves were born alive, and BHV-1 was not isolated from nasal swab samples of either calf; the third calf was stillborn. Virus was not isolated from the stillborn calf's tissues, but BHV-1 was isolated from the placenta. Lesions were not detected in several tissues examined by light microscopy, and BHV-1 antigen was not detected by immunohistochemical examination of paraffin sections. Restriction endonuclease analysis of viral DNA was used to compare the FI virus to other BHV-1 isolates (Colorado-1, Iowa, and K22). On the basis of restriction endonuclease analysis, the FI isolate should be classified as a type-2 (infectious pustular vulvovaginitis) virus, specifically subtype a.     

Experimental reproduction of respiratory tract disease with bovine respiratory syncytial virus

An experiment was conducted to reproduce respiratory tract disease with bovine respiratory syncytial virus (BRSV) in one-month-old, colostrum-fed calves. The hypothesized role of viral hypersensitivity and persistent infection in the pathogenesis of BRSV pneumonia was also investigated. For BRSV inoculation a field isolate of BRSV, at the fifth passage level in cell culture, was administered by a combined respiratory tract route (intranasal and intratracheal) for four consecutive days. Four groups of calves were utilized as follows: Group I, 6 calves sham inoculated with uninfected tissue culture fluid and necropsied 21 days after the last inoculation; Group II, 6 calves inoculated with BRSV and necropsied at the time of maximal clinical response (4-6 days after the last inoculation); Group III, 6 calves inoculated with BRSV and necropsied at 21 days after the last inoculation; Group IV, 6 calves inoculated with BRSV, rechallenged with BRSV 10 days after initial exposure, and necropsied at 21 days after the initial inoculation. Clinical response was evaluated by daily monitoring of body temperature, heart rate, respiratory rate, arterial blood gas tensions, hematocrit, total protein, white blood cell count, and fibrinogen. Calves were necropsied and pulmonary surface lesions were quantitated by computer digitization. Viral pneumonia was reporduced in each principal group. Lesions were most extensive in Group II. Disease was not apparent in Group I (controls). Significant differences (p less than 0.05) in body temperature, heart rate, respiratory rate, arterial oxygen tension, and pneumonic surface area were demonstrated between control and infected calves. Results indicate that severe disease and lesions can be induced by BRSV in one-month-old calves that were colostrum-fed and seropositive to BRSV. BRSV rechallenge had minimal effect on disease progression. Based on clinical and pathological response, results did not support viral hypersensitivity or persistent infection as pathogenetic mechanisms of BRSV pneumonia.