Transmission of classical swine fever virus by artificial insemination.

Classical swine fever (CSF) virus was introduced into an artificial insemination centre during the CSF epizootic of 1997-1998 in the Netherlands. The risk of further spread of CSF virus via contaminated semen was recognised, but could not be assessed because scientific data on this issue were not available. An animal experiment was performed to determine whether CSF virus could be transmitted via artificial insemination with contaminated semen. Three boars were inoculated with a CSF virus field isolate and from Day 5 till Day 18 thereafter, ejaculates were collected and prepared for insemination. Ruttish sows were inseminated with the extended semen from Day 5 till Day 18 after inoculation of the boars. All the inoculated boars remained healthy throughout the experiment and developed CSF neutralising antibodies between 14 and 21 days after inoculation. Virus was isolated from several semen samples collected from 5 till 11 days after inoculation. Two out of six sows inseminated with CSF contaminated semen seroconverted after insemination. All the other sows remained seronegative. In the foetuses of both the seropositive sows, CSF virus was detected at approximately 35 days post insemination. These results demonstrate that adult boars infected with CSF virus can excrete virus with semen and can, subsequently, transmit the virus to sows and their foetuses via artificial insemination.     

Akabane virus infection in the pregnant ewe. 1. Growth of virus in the foetus and the development of the foetal immune response

Three different pools of the CSIRO 16 strain of Akabane virus differing in their laboratory passage histories were used to inoculate 39 ewes between 32 and 36 days pregnant; 22 pregnant ewes received inocula containing no virus. There was no difference in the development, duration and titre of the viraemia and neutralising antibody response between the three infected groups of ewes. Both infected and control ewes had 141% foetuses when autopsied at 69 to 105 days gestation. Of the 55 foetuses from infected ewes 44 (80%) had gross developmental abnormalities.At autopsy of the dams Akabane virus was isolated only from the uterine caruncle. From foetal samples virus was isolated from a wide range of tissues, from one foetus at 69 days and from the blood of four foetuses at 95 to 106 days gestation. Virus was also isolated from 24 of the choriolllantoic fluid samples and from 37 placentomes of the 44 foetuses with developmental defects, in concentrations ranging from 10² to 10^5.5 TCID₅₀/ml or/g. No virus was isolated from the tissues of the control ewes or their foetuses.Neutralising antibody to Akabane virus was detected in 78% of the foetal sera from the infected group, titres ranging from 2 to 64. IgM and IgG₁ and neutralising antibody were detected in sera of 40 foetuses with developmental abnormalities including three that were of 76 to 78 days gestation. Neutralising antibody was detected only in serum that contained IgG₁ but may also have been associated with IgM in infected foetuses. IgM was detected in the serum of most foetuses including the non-infected controls, but sera from the control foetuses did not contain IgG₁ or neutralising antibody to Akabane virus. No IgG₂ or IgA were detected in any foetal serum.     

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.     

The effects of bovine viral diarrhoea-mucosal disease (BVD) virus on the ovine foetus

Bovine viral diarrhoea-mucosal disease (BVD) virus has been incriminated as a cause of abortion, hairy birth coat and unthriftiness in sheep. Intravenous inoculation of 40 ewes 34 to 45 days pregnant with the V/TOB strain of virus produced death in two of four foetuses 9 days after inoculation and in all but one of 31 foetuses between 11 and 56 days. The highest levels of virus in placentomes and foetal tissues occurred between 9 and 15 days after inoculation and in foetal fluids between 11 and 18 days. Virus was not detected in any foetus later than 21 days after inoculation. Groups of 10 ewes infected between 59 and 62 days (Group B) and 70 and 76 days (Group C) of gestation had 73% and 62%, respectively, of abortions or perinatal foetal deaths. Birth weights of lambs born to infected ewes in groups B and C were significantly lower than those born to uninfected control ewes. Virus was recovered consistently from the cotyledons of the foetal membranes of live lambs, and irregularly from the tissues of full term foetuses that were dead at birth but on no occasion from mummified foetuses. There were no specific gross or microscopic lesions in tissues selected from aborted foetuses and the results highlight the difficulties associated with the diagnosis of BVD abortions and perinatal death of foetuses under field conditions.     

Experimental in utero infection of pig foetuses with porcine parvovirus (PPV).

Pig foetuses of various gestational ages were exposed to experimental infection with porcine parvovirus (PPV) in utero. Inoculation of 40-, 50- and 60-day-old foetuses with PPV caused foetal death and mummification and spread of the infection to non-inoculated foetuses. Inoculation at 80 and 100 days gestation caused pathological lesions of various degrees whereas spread of infection occurred only sporadically. Serological examinations of foetuses of different ages suggest that immunocompetence for PPV develops before 70 days gestation. The present results strongly indicate that intrauterine spread of PPV is a route of transmission of this virus between pig foetuses.     

Bluetongue virus serotype 20: experimental infection of pregnant heifers

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.     

A morphometric study of foetal and newborn cardiac growth in the horse

A morphometric study of hearts in 81 equine foetuses, ranging in age from 190 to 330 days of foetal age, and in 26 newborn foals is reported. The mean weight, external dimensions, ventricular wall thickness and circumference of the atrio-ventricular orifice were measured. Features of the main associated vessels of the heart were also recorded. All cardiac measurements of foetuses increased linearly throughout the latter half of pregnancy and were highly correlated with foetal age. This linear growth pattern was also found in the parameters of the associated arterial trunks. The ratio of the right ventricular weight to the total ventricular weight in the equine foetuses increased gradually from 0.27 (Day 190) to 0.34 (full term), but in newborn foals, this ratio began to decrease soon after birth, declining from 0.32 to 0.28 by 11 days of age. The interventricular septum was thickest followed by the right ventricular wall, and the left ventricular wall was thinnest during the period of gestation studied. The right ventricle was 3.2 per cent to 31.5 per cent thicker than the left ventricle.     

Detection of PI-3 viruses, BVD-MD viruses and parvoviruses in bovine fetuses after experimental intrauterine infections

Growing organ cultures prepared from foetuses experimentally infected in utero by the viruses PI-3 and BVD-MD or bovine parvovirus proved to be suitable in tests with the re-isolation of these viruses 7, 14, 21, 35, 42 and 70 days after infection. The attempts at demonstrating them by the traditional method of the inoculation of the primary cell culture of foetal kidneys were successful only at the re-isolation of the PI-3 virus seven days after infection. The PI-3 virus without cytopathic effect was demonstrated in the primary cell culture prepared from the spleen, lungs, kidneys and testes of foetuses delivered seven days from infection. The cytopathic effect manifested itself after cell transplantation in the first passage when further multiplication of the virus occurred.     

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.     

Transplacental transmission of Bluetongue virus serotype 8 in ewes in early and mid gestation

The ability of Bluetongue virus serotype 8 (BTV-8) originating from the 2006 European outbreak to cross the ovine placenta during early and mid gestation was investigated in two separate experiments. In the first experiment, 16 ewes were infected with BTV-8 at 70-75 days gestation. The foetuses were collected at 18-19 days after infection (dpi). BTV-8 could be isolated from at least two organs of 19 out of 40 lambs and from 11 out of 16 infected ewes. In the second experiment, 20 BTV-8 infected ewes in early gestation (day 40-45) were euthanized at 10 days (10 ewes) or 30 days (10 ewes) after infection. The presence of BTV could be demonstrated in two foetuses from two ewes at 10 dpi and in 4 foetuses from four ewes at 30 dpi. The main pathological findings in the foetuses in mid gestation were meningo-encephalitis and vacuolation of the cerebrum. In the foetuses early at gestation, haemorrhages in various foetal tissues and necrosis and haemorrhages in the placentomes were found. These experiments demonstrate for the first time the presence of infectious BTV in lamb foetuses at different stages of gestation, combined with a difference in transmission rate depending on the gestation stage. The high transmission rate found at mid term gestation (69%) makes our model very suitable for further research into the mechanisms of transplacental transmission and for research into the reduction of this route of transmission through vaccination.     

Effect of Rift Valley fever virus on pregnant and non-pregnant local Barki ewes after artificial infection

Studies were conducted into effects of Rift Valley fever virus on oestrous and vaginal cytology in non-pregnant ewes. 6 pregnant and 8 non-pregnant local Barki ewes were subcutaneously inoculated with different doses of virus isolated from Sharkia Province. Observed were irregularities in the oestrous cycle (20-28 days inoculation) and anoestrum. The anoestrum smears included large non-cornified epithelial cells with large well stains centrally located nuclei together with a large number of bacteria. Abortion and retention of placenta occurred in pregnant ewes with prolonged puerperal heat up to 70 days. Inoculation of virus was followed by thermal response. Re-isolation of virus in BHK cells was possible from aborted foetuses and placenta.     

口蹄疫病毒受体整联蛋白在绵羊不同发育阶段组织中mRNA转录水平的分析

口蹄疫病毒野毒株利用整联蛋白（integrin）αvβ1、αvβ3、αvβ6和αvβ8作为受体侵入细胞。本研究采用实时逆转录聚合酶链反应（qRT-PCR）技术,定量检测绵羊胎儿（45、75和110 d的绵羊胎儿）、新生羔羊和成年绵羊组织中整联蛋白亚单位αv、β3、β6和β8转录水平。比较分析发现,在蹄冠上皮组织中,成年绵羊和新生羔羊的β6 mRNA水平低于75和110 d胎儿的;在心组织中,成年绵羊、新生羔羊和110 d胎儿的β6 mRNA高于45和75 d胎儿的;在肌肉组织中,胎儿和新生羔羊的β6以及β8 mRNA水平高于成年绵羊的;在舌上皮组织中,成年绵羊的β3 mRNA水平低于其他发育时期的。在软腭组织、扁桃体中整联蛋白mRNA水平在胎儿、新生儿和成年绵羊之间变化不大。本研究首次获得相关整联蛋白mRNA在绵羊不同发育阶段组织中分布的定量数据,为了解整联蛋白在口蹄疫病毒感染组织嗜性中的作用机制提供新的科学依据。     

Studies of foetuses from cows clinically affected with bovine leucosis

Five foetuses varying stages of gestation were recovered from cows showing clinical signs of infection with bovine leukaemia virus (BLV). No pathological changes were found in the foetuses although BLV was isolated from 2 of these foetuses. No antibody to BLV could be detected by virus-neutralisation or immunodiffusion (ID) techniques in these two foetuses, although neutralising antibody alone was detected in one of the other 3 foetuses which were all negative for virus.     

Wesselsbron disease: a cause of congenital porencephaly and cerebellar hypoplasia in calves

Fifteen pregnant cows were inoculated subcutaneously and intravenously between 101-147 days of gestation with the wild-type Wesselsbron disease virus. In addition, 2 foetuses were injected directly through the uterine wall after surgical exposure of the pregnant horn. The clinical symptoms, viraemia and serology in the cows are reported, as also the gross- and histopathology and the virological and serological results of the calves and foetuses. Abortion was not an important manifestation of experimental Wesselsbron disease in cows, as it occurred in 3 animals only. Apart from a short temperature reaction in some cows no other clinical symptoms were recorded. A viraemia was not always present in these cows and, when detected, was of low magnitude and short duration. One cow, in which the foetus was ioculated at 115 days of gestation, aborted at 231 days. The foetus showed marked porencephaly and cerebellar hypoplasia.     

Akabane virus infection in the pregnant ewe. 2. Pathology of the foetus

A strain of Akabane virus (CSIRO 16) isolated from Culicoides brevitarsis was given three different passage treatments in the laboratory and then inoculated into ewes that were 32 to 36 days pregnant. The foetuses from these ewes were examined between the 69th and 106th days of gestation. The 39 infected ewes produced 55 foetuses of which 44 (80%) had severe developmental defects. Arthrogryposis and agenesis of the brain or hydranencephaly, were present in 43 of the foetuses. Other gross defects affecting variable proportions of foetuses were porencephaly, brachygnathism, scoliosis, hypoplasia of the lungs and agenesis or hypoplasia of the spinal cord. Histopathological findings covered a wide spectrum of defects that have previously been considered to occur over an extended range of foetal ages. These defects included skeletal muscle atrophy and degeneration, and in the brain, particularly in the cerebrum, cystic areas and malacia, general oedema, subependymal gliosis, perivascular cuffing and mineralised plaques. Similar lesions were seen in the pons and cerebellum. Extensive lesions, with and without inflammation were seen in the spinal cord.     

Superinfection with porcine cytomegalovirus initiating transplacental infection

Four sows with circulating antibody were exposed to porcine cytomegalovirus. Virus was detected in 8 of 24 foetuses by immunofluorescence and/or virus isolation from 2 sows with low levels of antibody. In 6 of the infected foetuses, the virus was in capillary endothelium and macrophages of the lung, and was associated with interlobular oedema in 2 of these. Virus was also detected in the nasal mucosa, spleen and brain. The majority of the virus-positive foetuses were grossly normal.     

Evaluation of the effects of long-term storage of bovine ear notch samples on the ability of 2 diagnostic assays to identify calves persistently infected with bovine viral diarrhoea virus

Research aimed at optimising diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses, resulting in reproductive losses due to foetal death or persistently infected calves that die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of infection. This poses important challenges to overall animal/herd health and can cause major losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected with BVDV may adversely affect the ability of diagnostic assays to detect the virus efficiently. In order to test this hypothesis, ear notch samples from 7 animals were divided into 2 groups. One set was subjected to prompt formalin fixation and the other set stored either as fresh samples without preservatives at -2 degrees C, or soaked overnight in phosphate buffered saline followed by freezing of the supernatant fluid at -2 degrees C. Frozen ear notches and ear notch supernatant yielded positive results with an antigen-capture, enzyme linked immunosorbent assay (AC-ELISA) for the duration of the study (6 months) and optical density (OD) values remained significantly within range. There was no significant difference between storing fresh ear notch samples or PBS at -2 degrees C. However, positive immunohistochemistry (IHC) staining on formalin fixed ear notches started to fade between Day 17 and Day 29 when stored at room temperature. It was concluded that fresh ear notches could safely be stored at -2 degrees C for a period of 6 months prior to testing for BVD viral antigens.     

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG‐1 are used for pregnancy diagnosis and as a marker of placental/foetal well‐being, while those of PAG‐2 may be an indicator of abortion risk in Neospora caninum‐infected cows. Studies have shown that N. caninum infection modifies PAG‐1 and PAG‐2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG‐1, PAG‐2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG‐2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non‐infected foetuses. In infected foetuses, significantly higher concentrations of PAG‐1 (p < 0.001) and PAG‐2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non‐infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG‐1 and PAG‐2 levels and pH values than fluids from non‐infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.     

Multiplication of the IBR, PI-3 and BVD-MD viruses in cell and organ cultures of bovine fetuses after experimental intrauterine infections

The intrauterine infection of four- to nine-month-old bovine foetuses with the PI-3, BVD-MD viruses, performed 7 to 72 days prior to their delivery, did not exert any significant influence upon the susceptibility of primary cell cultures from foetal organs and tissues to further viral infection in vitro. The BVD-MD and IBR viruses multiplied in the primary cell cultures from the organs of a foetus infected with the PI-3 virus seven days before delivery even in the presence of endogenous PI-3 virus. Persisting infection with the PI-3 virus also failed to influence the susceptibility of foetal organ cultures to infection with the IBR and PI-3 viruses in vitro. The IBR virus and endogenous PI-3 virus multiplied simultaneously to high titres in the organ cultures of thymus and lungs whereas in the organ cultures of kidneys, spleen and testes the multiplication of endogenous PI-3 virus was suppressed.     

An association between encephalomyocarditis virus infection and reproductive failure in pigs

An outbreak of reproductive failure, characterised by mummified foetuses and stillbirths, was investigated in an intensive piggery. Six foetuses that died towards the end of gestation had multifocal myocardial necrosis and encephalomyocarditis virus was recovered from 4 of these foetuses but not from 6 mummified foetuses. There was also a significant increase in failure of conception or early embryonic deaths in sows mated at the same time as sows which produced affected litters.