A multi-screening Fast qPCR approach to the identification of abortive agents in ruminants.

Abortion in ruminants represents an important economic concern for farmers. Microbial agents, such as Brucella spp., Chlamydia spp., Coxiella burnetii, Leptospira spp., Neospora caninum, Salmonella spp. and Toxoplasma gondii, are among the main infectious causes of abortion and require rapid and reliable diagnosis. This study describes the development of a multi-screening assay using Fast Real-Time PCR (Fast qPCR) that allows, in a single test, the simultaneous identification of the above-mentioned abortive agents. This multi-screening approach is characterized by a mean diagnostic sensitivity and specificity of 100% and 97%, respectively; it has a limit of detection (LOD) ranging from 5 × 103 to 4 × 104 genomic copies/g of tissue and a very good concordance with traditional end-point PCR assays used in routine diagnostic activity. The proposed method represents a rapid approach to the simultaneous detection of the main abortive agents in ruminants that allows to make an accurate diagnosis and to set up appropriate control measures in a short period of time.

Caracterização morfológica e imuno-histoquímica de lesões em casos de aborto bovino bacteriano e viral no sul de Minas Gerais / Morphological and immunohistochemical characterization of the lesions in cases of bacterial and viral bovine abortion in southern Minas Gerais, Brazil

O presente estudo avaliou a participação de agentes bacterianos e virais em abortos em bovinos de propriedades rurais do sul de Minas Gerais. Foi realizada análise histopatológica e imuno-histoquímica dos casos de aborto recebidos pelo Setor de Patologia Veterinária da Universidade Federal de Lavras no período de 1999 a 2013. De 60 fetos analisados, em 30 (50%) foram observadas lesões microscópicas. Destes, oito apresentavam lesões compatíveis com infecção por agentes bacterianos e três apresentaram lesões sugestivas de agentes virais. Dos abortos bacterianos, um feto tinha lesões compatíveis com leptospirose, caracterizadas por icterícia e colestase, nefrite intersticial linfoplasmocítica e nefrose tubular. Sete fetos apresentaram pneumonia ou broncopneumonia purulenta; num deles havia também pleurite e peritonite fibrinosas; e em dois desses fetos houve imunomarcação para Brucella abortus. Dos três fetos com lesões sugestivas de aborto viral ocorreu imunomarcação anti-Herpesvírus bovino em um. Os resultados demonstram a ocorrência de abortos de origem bacteriana e viral na Região do estudo e que medidas profiláticas devem ser adotadas nas propriedades. O trabalho demonstra também que a imuno-histoquímica (IHQ); associada à histopatologia; é uma ferramenta útil e viável para o diagnóstico, especialmente quando provas microbiológicas e/ou sorológicas não estão disponíveis.

The paper evaluates the participation of bacterial and viral agents in bovine abortions in farms of southern Minas Gerais state, Brazil. Histopathological and immunohistochemical analysis was performed in aborted fetuses of cattle received by the Setor de Patologia Veterinária, Universidade Federal de Lavras, from 1999 to 2013. From 60 fetuses studied, 30 (50%) had microscopic lesions. From these, eight had lesions consistent with bacterial agents and three had lesions suggestive of viral agents. In the bacterial abortions, one fetus presented lesions compatible with leptospirosis, characterized by jaundice, cholestasis, lymphoplasmacytic intersticial nephritis, and tubular nephrosis. Seven fetuses had purulent pneumonia or bronchopneumonia and one of them had also fibrinous pleuritis and peritonitis; two of them presented positive immunostaining for Brucella abortus. One of the three fetuses with lesions of viral infection revealed positive imunostaining for bovine herpesvirus. The results showed that abortions of bacterial and viral origin occur in the Region of this study and prophylactic measures should be adopted on the farms. This study also demonstrates that immunohistochemistry associated with histopathology is a useful and viable tool for the diagnosis, especially when microbiological and/or serological tests are not available.

Experimental infection of pregnant cows with noncytopathogenic bovine viral diarrhoea virus between days 26 and 50 postbreeding.

The effect of bovine viral diarrhoea virus (BVDV) infection on early pregnant cows between 10 and 24 days after virus inoculation at day 26 of pregnancy was determined. Four cows were inoculated intravenously with either BVDV (treated, n=3) or growth medium (control, n=1). The treated cows were euthanized on either day 10, 17 or 24 post-infection and the control cow was euthanized on day 24 post-infection. The level of serum 2-5A synthetase increased in all of the three treated cows. Progesterone levels decreased to below 1.0 ng/ml between 10 and 22 days after inoculation in two of the three treated cows and the embryos/foetuses of two cows died. Therefore, BVDV may be a cause of early embryonic or feotal loss in early pregnant cows and serum 2-5A synthetase may be useful as an indicator of viral infection in cows.

Effects of Gram-positive bacterial pathogens in ewes: peptidoglycan as a potential mediator of interruption of early pregnancy.

Bacterial cell walls contain peptidoglycan (PTG), which, among other actions, induces fever. The present experiment evaluated the effects of PTG treatment on early pregnancy and blood plasma concentrations of reproductive hormones. Ewes were injected i.v. with saline or 15, 30 or 60 microg kg(-1) sonicated PTG (Streptococcus pyogenes) on day 5 after mating. Each dose of PTG induced fever. Pregnancy rate at day 25 was not related to incidence of fever but tended to differ among treatments (control, 100%; low, 100%; medium, 67%; high, 60%; P < 0.08). Combined pregnancy rate in ewes from control and low dose groups (100%) was greater than that in ewes from medium and high dose groups (64%, P < 0.01). Ewes with high 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) concentrations had lower pregnancy rates (6 of 10) than those with low concentrations of PGFM (11 of 11; P < 0.05). Mean cortisol concentrations were higher in treated (2.8 +/- 0.28 microg dl(-1)) than in control (1.1 +/- 0.03 microg dl(-1)) ewes (P < 0.01); the pattern of secretion was biphasic and increased in all treated ewes (P < 0.01). Neither means nor profiles of oestradiol differed with treatment. Mean concentrations and the pattern of concentrations of progesterone were reduced in all treated ewes, as indicated by the time by treatment and linear interaction with treatment (1.2 +/- 0.1 versus 1.6 +/- 0.1 ng ml(-1), P < 0.01). Patterns of LH pulses did not differ from 0 to 4 h or 24 to 28 h after treatment; mean plasma LH concentration was lower in ewes treated with 0, 15 or 30 microg PTG kg(-1) than with 60 microg PTG kg(-1) (P < 0.01). Pregnancy status was not related to plasma concentrations or patterns of LH, oestradiol, progesterone or cortisol. Inflammatory mediators, such as PGF(2alpha), may act directly on the embryo or uterus in ewes treated with PTG.

Immune regulation during pregnancy and host-pathogen interactions in infectious abortion.

The immunological mechanisms that govern the success of pregnancy in outbred mammals are complex. During placental formation the invasion of fetal cells into maternal tissue must be controlled to prevent damage to the mother. Equally, maternal recognition of pregnancy must be such that allorejection of the fetus does not occur. Despite the complexity of this phenomenon, it is clear that cytokines play a crucial role at the maternofetal interface and in the periphery to ensure that pregnancy proceeds successfully. Inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) can exert detrimental effects in the placenta and tend to be present at low concentrations, whereas the regulatory cytokines interleukin (IL)-10 and tranforming growth factor-beta (TGF-beta) are beneficial and tend to predominate. This means that infection with pathogens that target the placenta and that elicit inflammatory responses may cause abortion by giving rise to a detrimental combination of cytokines that causes damage but does not control the disease. Infectious abortion is discussed in the context of the modulation of host immune responses during pregnancy, taking into account the different placental structures present in human beings, rodents and ruminants.

A study of the pathogenesis of equid herpesvirus-1 (EHV-1) abortion by DNA in-situ hybridization.

The polymerase chain reaction and DNA in-situ hybridization were used to study sections of uterine tissue collected from mares near the time of abortion due to equid herpesvirus-1 (EHV-1) infection. These techniques revealed viral nucleic acids in endothelial cells of endometrial arterioles, in accordance with previously published immunohistological data. In addition, however, they revealed nucleic acids in cellular debris within endometrial glands and diffusing across the placenta at sites of microcotyledonary infarction. Perivascular leucocytes were generally negative for viral DNA, despite marked perivascular cuffing. These data provided further support for the central role of the vascular endothelial cell in the pathogenesis of EHV-1 abortion and demonstrated direct transplacental spread of nucleic acids at sites of microcotyledonary infarction and across the endometrial glands in the vicinity of vascular lesions.

The role of endothelial cell infection in the endometrium, placenta and foetus of equid herpesvirus 1 (EHV-1) abortions.

One of three mares in the last trimester of pregnancy became paraplegic 7 days after experimental infection with EHV-1 and was killed 10 days after infection (d.p.i.). The other two mares aborted foetuses at 12 and 14 d.p.i. In the first mare, virus was detected by immunofluorescence (IIF) and immunoperoxidase (IP) staining in endothelial cells of the endometrium, placenta and umbilical vein, but not in any other foetal tissues. In the experimentally aborted foetuses, and in two other independent field cases of abortions, endothelial cell infection was also detected in the foetuses, both in major blood vessels and in capillaries or sinusoidal cells associated with parenchymal lesions. In these four cases there were also positive endothelial placental lesions detected by IIF or IP, although it was not always possible to isolate virus from these tissues, as it was from the foetuses. The evidence suggests that infection of maternal endothelial cells has a major role in the pathogenesis of abortion, and that endothelial cells are also involved in dissemination of virus within the foetus.