Experimental infection of pregnant ewes with Listeria monocytogenes

Eight 18-month-old ewes were infected orally with Listeria monocytogenes between 77 and 91 days of pregnancy. Only one ewe aborted, 10 days after the first infecting dose, at 94 days of gestation; L monocytogenes was isolated from several sites in both its aborted fetuses. Two days after the first infecting dose all the ewes exhibited mild illness and pyrexia lasting for two to three days but the ewe which aborted was seriously ill until nine to 10 days after aborting. Agglutination tests carried out on 2-mercaptoethanol reduced sera revealed a strong immunological response in all the infected ewes but in the ewe which aborted this response was delayed. Four uninfected ewes which were kept as controls remained healthy throughout the experiment and showed no evidence of 2-mercaptoethanol resistant antibodies to L monocytogenes. Growth retardation lines, occurring at the time of and after experimental infection, were found in the bones of 14 of 17 newborn lambs in both the infected and control groups; in the aborted lambs these lines occurred before the infection.     

The production and survival of lambs persistently infected with border disease virus.

From 1985 to 1989 lambs persistently infected with border disease virus (BDV) were produced for comparative immunological studies by infecting 57 susceptible pregnant ewes between 50 and 60 days' gestation with Moredun or Oban strains of BDV. Ewes were infected either by injection with virus grown in cell culture or by housing with lambs excreting BDV. There was no significant difference in the outcomes of these different methods of infection. There was a significant difference in the number of viable lambs born to ewes receiving the two viruses. Of 41 ewes infected with Moredun virus 21 produced 32 live lambs of which 17 were reared to 1 month old (53% viability). Of 16 ewes receiving Oban virus 10 gave birth to 17 live lambs of which 15 were reared to 1 month old (88% viability). All the lambs born to ewes infected with Moredun BDV had varying signs of tremor and increased hairiness ("hairy-shakers") while those born to ewes infected with the Oban virus had no obvious clinical signs. Survival of the lambs was poor. Up until February 1991, 14 Moredun and 10 Oban sheep between the ages of 4 months and 5.5 yr had died from a variety of causes. The two commonest causes were a chronic wasting syndrome and a mucosal disease-like syndrome which was associated with the recovery of cytopathic BDV. Mating of unrelated persistently infected sheep was largely unproductive although 2 lambs were reared.     

Experimentally induced ovine border disease: extensive hypomyelination with minimal viral antigen in neonatal spinal cord

Border disease (BD) was experimentally induced in 9 lambs by inoculation of their dams with the BD-31 strain of border disease virus (BDV) at 50 days of gestation. These ewes developed serum-neutralizing antibody titers to BDV. The diagnosis of BD in their lambs was confirmed by hairy birthcoats, intrauterine growth retardation, tremors, and hypomyelination. Three clinically healthy age-matched control lambs, whose dams had been given an inoculum containing only BDV-free cell culture supernatant, were studied in parallel. There were significant differences in birth weights and in the lengths of the right tibiae and radii between the affected and the control lambs. There was a gradient in severity of clinical neurologic signs among the affected lambs, which directly correlated with the severity of hypomyelination in their spinal cords. However, the difference in severity of the neurologic deficits did not correlate with differences either in the precolostral serum-neutralizing antibody titers to BDV in these lambs or in the number of BDV antigen-containing cells in their spinal cords. Only approximately 0.01% to 0.3% of spinal cord cells, both in gray matter and white matter, were BDV antigen positive in the affected lambs. These results indicate that extensive infection of CNS cells with their subsequent destruction or functional alteration may not be a critical part of the pathogenesis of the hypomyelination in BD.     

Border Disease Virus Transmitted to Sheep and Cattle by a Persistently Infected Ewe: Epidemiology and Control

In a Swedish sheep flock comprising 202 ewes and 13 rams, a pair of twin lambs born in the spring of 1990 demonstrated signs of border disease (BD) and were persistently infected (PI) with border disease virus (BDV). Investigation showed that BDV had been introduced in the preceding tupping period 5-6 months earlier by a bought-in ewe which, on the basis of immunoperoxidase- and polymerase chain reaction techniques, was shown to be PI with BDV. Only 7 of the ewes, all of which had been in close contact with the PI ewe, seroconverted during the subsequent gestation. Apart from the PI twin lambs the losses caused by BDV were restricted to 2 barren ewes. The twin lambs, the PI ewe and lambs from the other 4 ewes that seroconverted were removed from the flock. The flock was thereafter free from an ongoing infection with BDV as shown by the absence of seroconversion.In addition, 5 heifers in late pregnancy most probably seroconverted to bovine virus diarrhoea virus (B VDV) when kept in close contact with the same PI ewe during the winter of 1989-90. When these heifers were reintroduced to the BVDV-free dairy herd from which they originated, their serum antibody titres ranged between 1:250 and 1:1250. Neither these heifers - nor their calves–caused any spread of the infection in the herd, as indicated by the absence of seroconversion in 70 cows. The present investigation shows that in the control of both BDV in sheep and BVDV in cattle, it is important to ensure that the risk of transmission of pestivirus between the 2 species is minimized.     

Repetitive abortion in Neospora-infected ewes.

Pregnant ewes inoculated with cultured Neospora caninum tachyzoites in 1995, 1996, or 1995 and 1996 aborted or delivered weak or clinically normal lambs in 1996. Nine of 11 ewes in the study had previously produced infected lambs or fetuses after being experimentally infected in 1995. Fetuses and lambs produced in 1996 showed histologic lesions and zoites indicative of Neospora. Serologic responses and production of infected fetuses/lambs indicated systemic neosporosis in the ewes during gestation, although tachyzoites could not be cultured from maternal tissues. The repetitive infection of fetuses, and resulting clinical and histopathologic findings in the present study are similar to those reported in naturally infected cattle, adding to the already established similarities of neosporosis between sheep and cattle.     

Observations on transplacental infection with bluetongue virus in sheep

Twenty-four ewes were inoculated with 1 of 2 strains of bluetongue virus type 4 at 40, 60, or 80 days of gestation. Two ewes aborted, 2 ewes died, and 1 was killed during the experiment, but their fetuses were recovered. At term, 2 mummified fetuses, 4 dead lambs, and 17 clinically healthy lambs were produced by 12 sheep, and the remaining 7 sheep were barren. Porencephaly and cerebellar dysgenesis were found in term lambs born to sheep inoculated at 40 and 60 days of gestation. Radiographic examination of 12 fetuses showed developmental ages far less than their chronologic age; 8 fetuses had skeletal growth-retardation lines, which were also observed in the dead lambs. A systemic lymphoreticular hyperplasia was observed in the dead lambs and in all lambs at 12 weeks of age; in 4 of the latter, granulomatous reactions were present in the liver and kidney. Lungs of the full-term lambs were reduced in weight and showed poor alveolar development and mononuclear cell infiltration, which persisted in the 12-week-old lambs. It was concluded that bluetongue virus is capable of causing not only gross abnormalities of the CNS, but also generalized growth retardation and fetal lymphoreticular hyperplasia.     

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

Studies on the response of ewes to live chlamydiae adapted to chicken embryos or tissue culture.

Ewes infected before gestation with chicken embryo or tissue culture adapted chlamydial strain B-577 were challenge inoculated with the homologous strain at four to 18 weeks of gestation. The ewes responsed with group specific complement fixing antibody titers of 1:8 to 1:256 by the second week after initial infection. A secondary antibody response in the surviving challenge inoculated ewes occurred at the time of lambing and reached titers of 1:32 to 1:256 by the second week after parturition. Group specific complement fixing antibodies did not appear to play a significant role in resistance to chlamydial infection. Ewes infected with the chicken embryo adapted strain B-577 excreted chlamydiae in their feces 60 days after inoculation. However, chlamydiae were not recovered from feces of ewes infected with the tissue culture adapted strain B-577. Placentas of ewes challenge inoculated by the intravenous route were consistently infected. Chlamydiae were recovered from placentas, some fetuses and lambs. In two instances when challenge inoculation was given by the intramuscular route, infection was detected only by the direct fluorescent antibody method.     

Longitudinal studies of naturally acquired Brucella abortus infection in sheep.

Naturally acquired Brucella abortus infections were studied during consecutive pregnancies in eight sheep and in their lambs over a period of 40 months to evaluate epizootiologic aspects of natural infection in sheep. Brucella abortus was isolated from the ewes following 16 of 26 natural terminations of pregnancy: from 5 of 6 ewes in the first year, from six of eight ewes in the second year, from two of six ewes in the third year, and from three of six ewes in the fourth year. Vaginal swab samples and milk samples were the most consistent source of the brucella organisms. Brucella abortus was isolated from three ewes when standard tube test seroagglutination titers were less than 1:100. In contrast, results of supplemental tests (card, 2-mercaptoethanol, complement-fixation, and Rivanol) remained positive during the study. During the 40 months, B abortus was isolated from 4 of 4 aborted fetuses, 2 of 5 stillborn lambs, 10 of 37 living lambs, and as an indicator of continuing infection, from 6 of 12 lambs born during the fourth year. Although B abortus has a definite host preference for cattle, this study demonstrated that under appropriate management conditions, sheep may be naturally infected and may remain infected for more than 40 months. Epizootiologic evaluation of all factors, including husbandry practices and exposure potential, should be utilized in determining the need to test other species that may have been exposed to cattle infected with B abortus.     

Colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes

Maedi visna virus (MVV) vertical transmission in sheep via infected colostrums is a very important route of infection in lambs. To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes.     

Effects on ovine fetuses of exposure to ovine progressive pneumonia virus

Fetuses of 20 pregnant ewes at 4 gestational periods (45, 55, 85, and 100 days) were inoculated with ovine progressive pneumonia virus. Fourteen of 16 fetuses exposed to virus before gestational day 80 were either resorbed or expelled, whereas 10 of 15 fetuses exposed to virus after day 80 were normal at birth. Three of the 9 expelled fetuses and 1 of 2 newborn lambs had accumulations of lymphoid cells in the lungs. Virus was readily isolated from the tissues of expelled fetuses and newborn lambs. Lambs did not have precipitating antibody to the virus at birth, but 3 to 5 lambs had specific antiviral antibody at 18 months of age.     

Experimental infection of pregnant ewes with bovine viral diarrhea virus type-2 (BVDV-2): effects on the pregnancy and fetus

The reproduction effects of bovine viral diarrhea virus type-2 (BVDV-2) infection were investigated in ewes inoculated with a non-cytopathic BVDV-2 isolate at three stages of gestation. Virus inoculation was followed by a transient viremia, accompanied by a transient and mild hyperthermia and nasal discharge in a few animals. Some ewes were sacrificed at different time-points after virus inoculation to study the kinetics of fetal infection. Infectivity and viral antigens were detected in placentomes from day 7 to 36 post-inoculation (pi) and in fetal fluids and tissues between days 10 and 28 pi. Cardiac petechial hemorrhages and hemoperitoneum accompanied by a severe fibrinous ulcerative placentitis were observed in fetuses examined at days 21, 28 and 36 pi. Inoculation of ewes at days 55-60 of gestation resulted in a prolonged virus replication in placentomes and fetal tissues; ewes that were allowed to proceed with pregnancy had 77% of abortions or fetal and perinatal deaths. Seven stillbirths, unviable and viable lambs born to these ewes were virus-positive at birth. Infectious virus was repeatedly isolated from leukocytes of two lambs up to 2 and 6 months of age, indicating they were persistently infected. Ewes inoculated at days 65-70 of gestation had 66.6% of fetal and perinatal losses. Three viable lambs born to these ewes were healthy, BVDV antibody-positive and virus-negative. A transient viral replication in placentomes and in a few fetal tissues, followed by the rise of fetal neutralizing antibodies and virus clearance was the result of inoculating ewes at days 120-125 of gestation. Lambs born to these ewes were healthy, antibody-positive and virus-negative. These results demonstrate that the biology of BVDV-2 infection in pregnant sheep is essentially similar to that of BVDV-1 in pregnant cattle and sheep. These features make this species an attractive animal model for studying the pathogenesis of congenital BVDV-2 infection.     

Morphogenesis of Fusarium sp-induced tibial dyschondroplasia in chickens

Tibial dyschondroplasia was induced in female broiler chicks by the incorporation of 2% Fusarium roseum "Alaska" culture into their starter ration. Chicks were placed on this diet at one day of age and maintained until they were killed at four days or one week. Proximal tibial physes were grossly thickened into cone-shaped masses of cartilage by one week of age. Microscopically, lesions were in both ages of treated chickens and were characterized by thickening of the transitional zone which was especially prominent in the center of the growth plate. This zone was unmineralized, avascular, and contained chondrocytes which were crenated and densely eosinophilic. The cartilage matrix was pale and contained some patchy eosinophilic foci. Four growth plates with tibial dyschondroplasia and four normal growth plates from each of the four-day and one-week-old age groups were evaluated based on the following parameters: number of metaphyseal vascular sprouts, distance between the proliferative/transitional junction and the tip of the metaphyseal vascular sprouts, width of the tips of the metaphyseal sprouts, distance between tips of adjacent metaphyseal vascular sprouts, and number of perforating vessels in the proliferative zone. The distance between the proliferative/transitional junction and the metaphyseal sprout tips was greatly increased (p less than 0.01) in the affected four-day and one-week-old chickens compared to age-matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype)

To evaluate the pathogenicity of local isolates of ovine pestiviruses (BDV-4 genotype), 13 virus- and antibody-negative, artificially inseminated pregnant ewes were challenged on days 108 (5 ewes), 76 (5 ewes) and 55 of pregnancy (3 ewes) with 2 ml of ovine pestivirus containing 10⁶ TCID₅₀. Viraemia was detected by RT-PCR from 2 to 15 days pi in most ewes. No abortion due to the infection was observed but the number of stillbirths was high (32%), and bodyweight at lambing was significantly reduced compared to the experimental flock of origin used as control. Clinical symptoms in live lambs consisted on tremors, gait anomalies and inability to stand unaided. Skeletal abnormalities (brachygnathia, prognathia, arthrogryposis) were present in 44% of the lambs. Only 20% of the lambs were clinically normal. RT-PCR was a very sensitive technique compared to antigen ELISA in detecting viral presence in experimentally infected ewes and their progeny.     

Vertical transmission of Toxoplasma gondii in naturally infected ewes in the semiarid region of Brazil

To evaluate transplacental transmission of Toxoplasma gondii in naturally infected ewes, blood samples were collected from 55 pregnant ewes and their offspring, before ingestion of colostrum. From 16 offspring of positive ewes and nine offspring from negative ewes, blood samples were obtained after 48 h and 7, 14, 21, 28, 35, 42, 49 and 56 days after birth. T. gondii antibodies were detected in serum samples using the indirect fluorescence antibody test (IFAT ≥ 64). Four of the 30 positive ewes (13.3 %) had offspring positive for T. gondii before ingesting colostrum (vertical transmission). The colostrum antibody titers decreased every week, and only 20 % (2/10) of the lambs in continued to present detectable antibody titers until day 56 after birth. Therefore, vertical transmission of T. gondii in lambs was indication of occur and is an important route for transferring and maintaining the agent in sheep herds in the Brazilian semiarid region.     

Kinetics of infection and effects on the placenta of Chlamydophila abortus in experimentally infected pregnant ewes

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.     

Linear hoof defects in sheep infected with foot-and-mouth disease

During the epidemic of foot-and-mouth disease (FMD) in The Netherlands in 2001, a sheep farm was identified that had been subclinically infected with the disease. The FMD virus genome was detected in 12 of 16 probang samples collected from the sheep and the virus was isolated from four of these samples. Linear defects were observed, 1 to 3 cm from the coronary band, in the hooves of several of the sheep. The defects were thought to have been caused by the FMD infection. It was thought that the distance of the defects from the coronary band might be an indication of the time since the animals had been infected. To determine the growth rate of the claws of sheep, the growth of the hoof horn of uninfected lambs and ewes was measured; in the lambs the growth rate was 0.44 mm per day and in the ewes it was 0.29 mm per day.     

Immunization protocols against Cryptosporidium parvum in ovines: protection in suckling lambs

Ovine colostrum and milk from immunized ewes were tested for their ability to prevent cryptosporidiosis in the lambs experimentally infected with 10(6) oocysts of Cryptosporidium parvum at 36-48 h of age (day 0 post-infection). All lambs became infected and developed clinical cryptosporidiosis. However, lambs fed by immunized dams have shown shedding involved, significantly, fewer oocysts and lasted for a shorter period than in control lambs. In addition, diarrhoea was less severe. The best results emerged in lambs of ewes immunized by intramuscular injection of an emulsion of 2 ml of Freund's complete adjuvant and 2 ml of C. parvum antigen in sterile phosphate buffered saline solution, administrated four weeks before parturition, together with an intramammary infusion of 25 microg of antigen in 2 ml of sterile PBS emulsified in 2 ml of Freund's incomplete adjuvant, which showed the highest anti-C. parvum titres in lacteal secretions. In their case, the onset of output of oocysts was delayed by two days, the patent period was shortened by three days, their diarrhoea continued for only three days, and the quantity of oocysts shed decreased by 77%. The outcome was that at the end of the study they had a live weight gain of 2 kg more than the lambs in the control group. These results indicate that lactogenic immunoprophylaxis should help mitigate the financial losses caused by cryptosporidiosis in small ruminants, as well as reducing the risk of infection of humans through the decreased contamination of the environment with oocysts.     

Analysis of the kinetics, isotype and specificity of serum and coproantibody in lambs infected with Cryptosporidium parvum

Enteric cryptosporidiosis was studied in colostrum-deprived lambs each infected at five days old with 10(6) oocysts. The prepatent period was three to five days and faecal oocyst concentration fell below detectable levels by day 16 after infection. Specific IgA, the only isotype detected by immunofluorescent assay in faecal extracts from infected lambs, was first evident on day 10 and titres continued to rise until day 16 of infection in association with declining oocyst output. Specific IgM and IgG antibodies were first detected in serum seven days after infection. No specific antibody was detected in uninfected control lambs. Immunoblotting methods showed that serum antibody and faecal IgA had similar profiles of antigen recognition. Antigens with approximate molecular weights of 180,000, 23,000 and 15,000 were consistent features on immunoblots performed with convalescent sera and faecal extracts. The results suggest that specific IgA in intestinal secretions has an important role in immunity to cryptosporidiosis.     

IgG1 antibody in milk protects lambs against rotavirus diarrhoea

Newborn gnotobiotic lambs were fed a diet of diluted evaporated milk supplemented either with normal ewes' milk or with milk obtained from ewes injected parenterally during gestation with rotavirus. Lambs fed 150 ml per day of milk collected 5 days after lambing from normal ewes were susceptible to rotavirus infection and diarrhoea, while lambs fed milk from vaccinated ewes collected either 5 or 12 days after lambing were protected. Analysis of the milk by column chromatography showed the anti-rotavirus activity to be in the fractions containing IgG1.