Abortion in sheep caused by a nonclassified, anaerobic, flagellated bacterium

Twenty-eight pregnant ewes were inoculated IV with approximately 6 X 10(8) nonclassified, anaerobic, flagellated bacteria (NAFB) that had been isolated from an aborted lamb. Abortion occurred in 3 of the ewes and 1 ewe gave birth to a weak lamb. The remaining 24 ewes and 3 other ewes inoculated orally with NAFB did not develop clinical signs of illness. Suppuration and vasculitis were seen in the placentas of the 3 aborted lambs, 1 of which had necropurulent hepatitis indistinguishable from that usually attributed to Campylobacter fetus infection. The NAFB was isolated from fetal placenta, abomasal content, or internal organs of 2 aborted lambs and the weak lamb. A morphologically similar organism was seen in the abomasal content of the other aborted lamb, but the organism did not grow on bacteriologic culture medium. Therefore, in susceptible pregnant ewes, NAFB can cause fetal placentitis and hepatitis and subsequent birth of weak lambs or abortion.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

ISOLATION OF MYCOPLASMA HYOPNEUMONIAE AND ITS ASSOCIATION WITH PNEUMONIA OF PIGS IN AUSTRALIA

Nine strains of mycoplasmas were isolated from the lungs of 5 pigs with clinical signs of naturally acquired enzootic pneumonia. Mycoplasma hyopneumoniae was isolated from the lungs of 1 pig and M. hyorhinis from the lungs of 4. An unidentified mycoplasma, which utilized arginine, grew rapidly in broth culture and produced centred colonies on solid medium, was isolated from the lungs of 4 pigs. The pathogenicity of the isolated strain of M. hyopneumoniae was determined by inoculation of pigs from an enzootic pneumonia-free herd. Enzootic pneumonia was produced in the lungs of all 5 pigs inoculated intranasally and intratracheally with broth cultures of the organism isolatied by limit dilution techniques. Enzootic pneumonia was produced in 3 of 6 pigs inoculated intranasally and intratracheally with M. hyopneumoniae purified by the passage of colonies on agar blocks. M. hyopneumoniae was isolated in pure culture from the lungs of all pigs with induced pneumonic lesions.     

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.     

Effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, Bordetella bronchiseptica, or a combination of both organisms in pigs

OBJECTIVE: To examine effects of co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Bordetella bronchiseptica in pigs. ANIMALS: Forty 3-week-old pigs. Procedure-30 pigs (10 pigs/group) were inoculated with PRRSV, B bronchiseptica, or both. Ten noninoculated pigs were control animals. RESULTS: Clinical signs, febrile response, and decreased weight gain were most severe in the group inoculated with both organisms. The PRRSV was isolated from all pigs in both groups inoculated with virus. All pigs in both groups that received PRRSV had gross and microscopic lesions consistent with interstitial pneumonia. Bordetella bronchiseptica was cultured from all pigs in both groups inoculated with that bacterium. Colonization of anatomic sites by B bronchiseptica was comparable between both groups. Pigs in the group that received only B bronchiseptica lacked gross or microscopic lung lesions, and B bronchiseptica was not isolated from lung tissue. In the group inoculated with B bronchiseptica and PRRSV, 3 of 5 pigs 10 days after inoculation and 5 of 5 pigs 21 days after inoculation had gross and microscopic lesions consistent with bacterial bronchopneumonia, and B bronchiseptica was isolated from the lungs of 7 of those 10 pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical disease was exacerbated in co-infected pigs, including an increased febrile response, decreased weight gain, and B bronchiseptica-induced pneumonia. Bordetella bronchiseptica and PRRSV may circulate in a herd and cause subclinical infections. Therefore, co-infection with these organisms may cause clinical respiratory tract disease and leave pigs more susceptible to subsequent infection with opportunistic bacteria.     

Isolation of cilia-associated respiratory (CAR) bacillus from pigs and calves and experimental infection of gnotobiotic pigs and rodents.

Filamentous, gram-negative bacteria morphologically similar to cilia-associated respiratory (CAR) bacillus of rodents and rabbits were isolated from the tracheas of 5 pigs and 4 calves. All pigs but none of the calves had histologic lesions of chronic tracheitis. In silver-stained histologic sections, CAR bacilli were adhered to the tracheal epithelium of each pig but were not found in the calves. Like CAR bacillus of rats, the bacteria displayed gliding motility and grew only in cell culture or cell culture medium supplemented with fetal serum. Initially, all isolates were contaminated by Mycoplasma spp. This contamination was eliminated from 4 pig isolates by limiting dilutions, and mycoplasma-free isolates were used to intranasally inoculate gnotobiotic pigs and CAR bacillus-free mice and rats and to immunize guinea pigs. The gnotobiotic pigs remained healthy, and when they were necropsied 4 and 7 weeks after infection no macroscopic or microscopic lesions were found in the respiratory tract. However, CAR bacillus was isolated at both times from the nasal cavities and tracheas of inoculated pigs, and the ciliated tracheal epithelium of infected pigs necropsied 7 weeks after infection was colonized by low numbers of CAR bacillus-like bacteria. The rats and mice remained healthy through week 12 postinoculation, and evidence of short- or long-term colonization was not detected by histologic examination or culture. When used as primary antibody for immunohistochemical staining, sera from guinea pigs immunized with pig CAR bacillus specifically stained CAR bacilli colonizing the respiratory epithelium of naturally infected pigs, whereas sera collected prior to immunization failed to react with the bacteria. These results indicate that CAR bacilli are unlikely to be primary pathogens of pigs or cattle and that rodents do not act as reservoirs.     

A MUCOSAL DISEASE VIRUS AS A CAUSE OF ABORTION HAIRY BIRTH COAT AND UNTHRIFTINESS IN SHEEP

A condition resembling border disease has been transmitted by the inoculation of pregnant ewes with material from affected lambs. Forty-nine merino ewes, mated to merino rams 7 to 87 days previously, were inoculated with an homogenate of brain, spinal cord and spleen from affected lambs. Mummified foetuses, abortions and stillbirths were observed, and lambs with hairy birth coats were born to ewes inoculated between days 12 to 70 of gestation. A mucosal disease virus (MDV), present in the original material, was recovered from the aborted foetuses and lambs. Attempts to induce passive protection using bovine anti-serum to the C24V strain of MDV were not successful. The condition was also transmitted by inoculation of pregnant ewes with a cell culture supernatant prepared from tissue cultures that had been inoculated with an organ homogenate pool. MDV was present in the supernatant and was recovered from aborted foetuses and lambs. It is suggested that a condition in sheep in Australia resembling border disease is due to the infection of the pregnant ewe by a mucosal disease virus.     

Cystine aminopeptidase activity (oxytocinase) in pregnant guinea pigs: normal and infected with Campylobacter fetus.

The cystine aminopeptidase (CAP) activity was determined in plasma from guinea pigs at various stages of gestation and in pregnant guinea pigs infected with Campylobacter fetus subspecies intestinalis. The CAP activity decreased with length of gestation, which is in contrast to the changes in activity in persons. Infected guinea pigs near the time of abortion can be distinguished from normal guinea pigs on the basis of plasma CAP activity, if the plasma samples are taken 1 to 2 days before abortion. Among pregnant guinea pigs at the same stage of gestation, CAP activity was lower for infected than for normal pregnant animals. This test might be used to indicate probable disruption of placentation and the onset of abortion in the guinea pig.     

Pathology of Campylobacter jejuni abortion in sheep

Campylobacter jejuni was inoculated intravenously into pregnant ewes on gestation days 114 and 123 to reproduce ovine abortion. All ewes aborted 7-12 days post-inoculation. High numbers of C. jejuni were isolated from ewe tissues (caruncle, bile, cecal feces), fetal tissues, and placenta. C. jejuni colonies were identified in caruncles and placenta by light microscopy and immunoperoxidase techniques. Histologically, inoculated ewes had a severe purulent endometritis with vasculitis. Placentas from inoculated ewes and field cases showed necrosis and purulent inflammation; however, placentas from inoculated ewes had large numbers of bacterial colonies compared to few bacteria found in field cases. Histologically, only one fetus from the inoculated ewes showed lesions (purulent bronchopneumonia), whereas all fetuses from field cases had a distinct bronchopneumonia, and one fetus showed multifocal hepatic necrosis. These results suggest that C. jejuni (serotypes Penner 1 and Lior 2) is an important abortifacient organism for sheep.     

Vesicular Lesions Produced in Guinea Pigs by a Staphylococcus aureus Strain

A bacterium isolated from vesicular lesions on foot pads of guinea pigs reproduced lesions similar to those seen in experimental infections of guinea pigs with foot-and-mouth disease virus. (FMDV). These bacterial lesions were produced with an inactivated FMDV suspension. Identification as Staphylococcus aureus was determined by growth characteristics on nutrient and blood agar, Gram staining, fermentation of mannitol and coagulase positive reactions. In addition, the organism was sensitive to concentrations of penicillin and streptomycin commonly used in laboratory diluents.     

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.     

Pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.     

Swine dysentery: pathogenicity of Treponema hyodysenteriae.

When pure cultures of Treponema hyodysenteriae were orally inoculated into pigs, severe disease characteristic of swine dysentery developed. Less severe lesions were produced by oral inoculation of infective minced colon. Noninoculated pigs were used as controls. Inoculations of surgically isolated porcine colonic loops with either pure cultures or infective minced colon produced lesions only in the injected loop; the adjacent noninjected colon remained normal. Pigs and other experimental animals, including rabbits, guinea pigs, and mice, inoculated with washed cultures by various parenteral routes remained normal.     

Evaluation of pregnant rabbits as a laboratory model for bovid herpesvirus-4 infection

A field strain (87-8363) of bovid herpesvirus-4 (BHV-4) isolated from an aborted bovine fetus was used to inoculate pregnant rabbits. Eleven rabbits in midgestation were alloted to 4 groups consisting of 3 infected groups and 1 control group. Rabbits were inoculated with BHV-4 or mock-infected cell culture preparations via IV, intravaginal, and intrauterine routes. Mild vulvovaginitis and endometritis were observed after intravaginal and IV inoculation of BHV-4, whereas intrauterine inoculation of BHV-4 resulted in abortion of hemorrhagic fetuses and nonsuppurative endometritis. Virus was successfully isolated from organ explants of fetal tissues. Rabbits seroconverted 1 week after infection as detected by results of an indirect immunofluorescence assay.     

Early abortion in cattle induced by experimental intrauterine infection with pure cultures of Actinomyces pyogenes

Actinomyces pyogenes from a case of endometritis was used to study the effects of infection of the bovine embryo between days 27 and 41 of pregnancy. From 10(9) to 10(10) washed organisms were introduced into the uterine lumen of four pregnant cows. Two pregnant cows were inoculated with sterile saline and four pregnant cows were treated with cloprostenol. Embryonic death and abortion followed 29 to 144 hours after the inoculation of the live bacteria. The aborted embryos were macerated or clearly degenerating and yielded profuse pure cultures of A pyogenes. Abortion was accompanied by a sustained increase in uterine tone, opening of the cervix, presence of vaginal pus and a vulval discharge and the persistence of the corpus luteum for at least eight days after abortion. Intrauterine inoculation with saline did not affect pregnancy, but embryonic death, abortion and regression of the corpus luteum occurred 66 to 72 hours after the treatment with cloprostenol. The results suggest that A pyogenes is a primary pathogen and is capable of causing embryonic death and abortion.     

Comparative efficacy of ten commercial Campylobacter fetus vaccines in the pregnant guinea pig: challenge with Campylobacter fetus serotype A.

Efficacy of ten commercial Campylobacter fetus vaccines was tested in pregnant guinea pigs and compared with that of an experimental vaccine prepared from the challenge-exposure strain. If the first lot of vaccine failed to protect 50% of the guinea pigs, one or two additional lots of that vaccine were purchased and retested. Three vaccines for cattle, evaluated, as the most effective of those tested, protected 62%, 72%, and 89% of the guinea pigs from abortion; the experimental vaccine protected 98%. The two vaccines for sheep protected 50% and 61% of the guinea pigs from abortion. With the other five vaccines produced for immunizing cattle, protection was from 0% to 36%, with the exception of one lot of a vaccine that protected 74%. Blood infection was found at necropsy in only 6% of the guinea pigs given vaccines that protected 50% or more from abortion, but was found in 66% of those given vaccines that protected less than 50%. Similarly, tissue infection was found at necropsy in only 18% of the guinea pigs given vaccines that protected more than 50%, but was found in 91% of those given vaccines that protected less than 50% from abortion. Oil-emulsion adjuvants appeared to enhance protection from abortion and infection. Nodules persisted at the injection site in most of the guinea pigs immunized with vaccines containing oil-emulsion adjuvants, but rarely persisted in guinea pigs given aqueous-phase adjuvant vaccines. Comparison of efficacy of the vaccines in guinea pigs with efficacy in sheep and cattle remains to be made.     

A preliminary study of the pathogenesis of foot-and-mouth disease virus using in situ hybridization.

Five adult guinea pigs were inoculated intraepithelially in the right hindfoot pad with foot-and-mouth disease virus. Animals were euthanatized with carbon dioxide at 4, 10, 24, 48, and 72 hours post-inoculation. Generalized disease developed in the guinea pigs, as evidenced by depression and inappetance by 24 hours post-inoculation and by the formation of vesicles in the noninoculated hindfoot pad by 48 hours post-inoculation. By in situ hybridization, using a 500 base pair biotinylated RNA probe, viral nucleic acid was detected in the noninoculated fore- and hindfoot pads as early as 10 hours post-inoculation, well before any pathologic changes associated with foot-and-mouth disease virus infection were detected. These tissues remained consistently positive for the presence of viral nucleic acid up to the end of the experiment. At this time, in the forefoot pad, even though virus had first been detected with certainty in that tissue 62 hours previously, there was still no microscopic evidence of foot-and-mouth disease virus-induced damage in the histologic section. Similarly, tongue tissue was positive by in situ hybridization at 4, 48, and 72 hours post-inoculation, yet there was never any microscopic evidence of degeneration or vesicle formation. From this preliminary study, it appears that, in the guinea pig, the virus is widely disseminated to foot pads and tongue, with epidermal lesions resulting only in selected areas.     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs: 4. Contact Infection from Orally Inoculated Pigs*

In 3 experiments, 13 pigs were inoculated orally with 0.5 mg Mycobacterium avium daily for 5 days (1 mg = 32–68×10⁶ viable units). Five to 8 days after inoculation, 16 non-inoculated pigs were added to the inoculated pigs.Cultures from faeces showed excretion of M. avium from all the inoculated pigs for some time within the period 16 to 65 days after the last inoculation; the greatest numbers of organisms (62000 per 100 g faeces) were found at about the middle of the excretion period (Table 2). Two of the contact pigs were found to excrete M. avium in small numbers, 1 at 15, the other at 37 and 44 days after contact.All the inoculated pigs and 13 of the contact pigs showed positive intradermal tuberculin reactions. Post-mortem examination showed tuberculous lesions in all the inoculated pigs (Table 3). M. avium was transmitted to 15 of 16 pigs by contact. Five of these pigs showed gross lesions, 10 of them microscopic lesions only; in 9 of them the infection was proved by culture.     

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.