Study on Correlation of Maedi-Visna Virus (MVV) with Ovine Subclinical Mastitis in Iran

Maedi-Visna is an important slow viral disease of sheep leading to progressive pneumonia, encephalitis and mastitis. Udder is one of the organs affected by MVV. Despite the fact that in Iran Maedi-Visna is known since 2000, to the authors’ knowledge correlation of subclinical mastitis and infection with MVV has not been assayed. In this study 50 milk samples from 10 flocks in East Azerbaijan Province of Iran were tested. None of the animals exhibited any clinical signs of the disease. Forty samples were collected from CMT positive ewes and ten were taken from CMT negative ewes. Milk samples were analyzed using PCR targeting gag sequence. Presence of provirus DNA was detected in one sample from CMT negative and seven samples from CMT positive ewes. These data demonstrate that 16.5 % of sheep with subclinical mastitis were infected to MVV. Thus this virus can be considered one of the main pathogenic agents of mastitis and can be dramatically transmitted to lambs by milk.     

Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep

The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.     

Association of Maedi Visna virus with Brucella ovis infection in rams

Maedi Visna Virus (MVV) is the etiological agent of a systemic disease of sheep, which causes lesions in lungs, the central nervous system, joints, and mammary glands. It has been speculated that the association with Brucella ovis may lead to the venereal shedding of the virus. In this work, samples of epididymis from ten rams positive for MVV and infected experimentally with Brucella ovis, were subjected to liquid-phase PCR, immunohistochemistry (IHC) and in situ PCR tests, aimed at identifying the pathogens in a tissue context. IHC was carried out using a monoclonal antibody raised against p28 MVV protein and a polyclonal antibody to B. ovis. Liquid phase- and in situ PCR were designed to amplify a portion of MVV proviral DNA Pol sequence. In the animals showing B. ovis-related histopathological changes, IHC clearly demonstrated a positivity for B. ovis and MVV in interstitial and epithelial ductal cells. In situ PCR assessed the presence of MVV proviral DNA in macrophages and elements inside the epithelium. The unaffected and reagent control samples constantly gave negative results. Taken together, these data demonstrate that MVV may affect ovine epididymis, apparently taking advantage of the concurrent infection by B. ovis. The tropism of MVV for the epididymal epithelial cells, may be responsible for its excretion with the semen.     

Evolution of B cell lineage lymphomas in mice with a retrovirus-induced immunodeficiency syndrome, MAIDS

Mice infected with LP-BM5 murine leukemia virus develop lymphadenopathy, splenomegaly, hypergammaglobulinemia, and profound immunosuppression associated with enhanced susceptibility to infection. In this study, molecular genetic analyses of spleen and lymph node cells from infected mice showed the early course of disease was associated with polyclonal proliferations of both B and T cells but that by 12 wk oligoclonal expansions of B or T cells could be detected. When near death, the mice were killed and almost all exhibited clonally restricted populations of B cells, and continuous cultures of B lineage cells were established from three of 19 mice. Histologically, lymph nodes with polyclonal lymphoproliferative lesions were indistinguishable from nodes with clonally restricted populations of cells. However, aggressive immunoblastic lymphomas of characteristic morphology were seen in nonlymphoid organs, particularly in the brain. The demonstration of terminal B cell lymphomas in murine AIDS extends the similarities between this syndrome and AIDS in humans.     

The maedi-visna virus Tat protein induces multiorgan lymphoid hyperplasia in transgenic mice.

Sheep infected with maedi-visna virus experience immunological disorders leading to progressive chronic diseases involving the brain, lung, spleen, and lymph nodes. To study the biological activity of the viral transactivating Tat protein, we generated transgenic mice carrying the tat gene. Analysis of the transgenic mouse tissues for tat mRNA revealed that while low at the messenger level, the expression of the transgene correlated with dramatic follicular lymphoproliferative disorders involving the lung, spleen, lymph nodes, and skin. This finding suggests that the viral protein possesses a high pathological potency. Our findings show that the maedi-visna virus tat gene product contributes to the pathogenesis of multiorgan proliferative disorders associated with maedi-visna virus infection.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Immune response to individual maedi-visna virus gag antigens

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individual gag antigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+ T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all three gag antigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinant gag antigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+ T lymphocytes. All three gag antigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.     

Persistence of Granulocytic Ehrlichia Infection During Wintertime in Two Sheep Flocks in Norway

Granulocytic Ehrlichia infection in sheep is common in Norway in areas with Ixodes ricinus. In this study, 2 sheep flocks that had been grazing on I. ricinus infested pastures the previous season, were blood sampled after being housed indoors for nearly 6 months during wintertime. Thirty animals from each flock were examined for granulocytic Ehrlichia infection in the peripheral blood by blood inoculation studies, stained blood smear evaluation, polymerase chain reaction (PCR) analysis and serology (IFAantibodies). The animals were sampled twice within a three-week period, the first time before and the second time after lambing. Two sheep in one flock were found Ehrlichia positive by both blood smear evaluation and PCR before lambing, and 3 sheep were found positive after lambing; 2 by blood smear examination and 3 by PCR. In the other flock, no sheep was found infected before lambing, but 2 ewes were found positive after lambing by both blood smear evaluation and PCR. In the first flock, 87% of the animals were found seropositive before lambing, and the mean antibody titre (log 10 ± SD) to E. equi was 2.45 ± 0.401. In the second flock, 40% were found seropositive before lambing, and the mean antibody titre was 1.93 ± 0.260. Seroprevalence and mean antibody titre in these 2 flocks were significantly different (p < 0.001). The present study indicates that sheep may be a reservoir host for granulocytic Ehrlichia infection from one grazing season to the next under natural conditions in Norway.     

Phenotypic and genetic relationships between udder morphology and udder health in dairy ewes

The aim of this paper was studying the phenotypic and genetic relationships between udder morphology traits and udder health in dairy sheep. From 2000 to 2008 udder traits, somatic cell count (SCC) and clinical mastitis cases were recorded in an experimental flock consisting each year of around 900 ewes. A logistic regression was performed to evaluate the risk for a ewe of showing either a mastitis or at least 2 daily SCC records greater than 1 × 10⁶ cells/ml in one of its lactations as function of udder traits scored in 1st lactation. 1587 individual udder scores and 39,950 SCC daily records were used for the analysis. Secondly, genetic correlation between lactation mean of somatic cell score and udder traits were estimated by a REML method applied to four bi-trait animal models, using data from 2251 primiparous ewes. Logistic regression results indicated that the risk of mastitis or high SCC values during the productive life increased as the cistern height increased and the degree of udder suspension and udder depth decreased. This suggests that the appraisal of the udder is a useful tool for culling decision aimed at increasing the sanitary status of the flock. The genetic correlation between lactation SCS and udder traits were favourable for udder depth (−0.50 ± 0.12), teat placement (0.39 ± 0.011) and degree of udder suspension (−0.42 ± 0.011) and closed to zero for the degree of separation of the two halves. Thus selection for udder morphology, already implemented in some dairy sheep breeds with the aim of improving machine milkability, will lead to a favourable correlated genetic response on SCC.     

Lack of risk of transmission of caprine arthritis-encephalitis virus (CAEV) after an appropriate embryo transfer procedure

The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.     

Lymph node cell responsiveness in BALB/c mice infected with Leishmania mexicana

In the present study we measured the blastogenic response of lymph node cells from BALB/c mice infected with Leishmania mexicana throughout the course of infection. Results showed that infected mice displayed normal blastogenic responses in the lymph nodes until twenty weeks of infection. Thereafter, there was a gradual suppression. Comparison of the immunoresponsiveness in the spleen and lymph nodes, revealed normal responses in the lymph nodes several weeks after suppression in the spleen had occurred. Suppression of blastogenic responses in the lymph nodes was related to an adherent macrophage-like cell which actively suppressed normal proliferative responses to mitogens.     

New mastitis phenotypes suitable for genomic selection in meat sheep and their genetic relationships with udder conformation and lamb live weights

Mastitis can prove expensive in sheep reared for meat production due to costs associated with treatment methods, poor lamb growth and premature culling of ewes. The most commonly used method to detect mastitis, in dairy systems, is somatic cell counts. However, in many meat-producing sheep flocks ewes are not routinely handled, thus regular milk sampling is not always possible. It is, therefore, worthwhile to investigate alternative phenotypes, such as those associated with udder conformation and methods of evaluating somatic cell counts in the milk, such as the California Mastitis Test. The main objectives of this study were therefore: (a) to estimate genetic parameters of traits relating to mastitis and udder conformation in a meat sheep breed; (b) estimate the level of association between somatic cell counts and the California Mastitis Test and (c) assess the relationships between mastitis and both udder conformation and lamb live weights. Data were collected from Texel ewes based on 29 flocks, throughout the UK, during 2015 and 2016. The ewes were scored twice each year, at mid- and late-lactation. Eight different conformation traits, relating to udder and teat characteristics, and milk samples were recorded. The data set comprised of data available for 2957 ewes. The pedigree file used contained sire and dam information for 31 775 individuals. The animal models used fitted relevant fixed and random effects. Heritability estimates for traits relating to mastitis (somatic cell score and the California Mastitis Test), ranged from 0.08 to 0.11 and 0.07 to 0.11, respectively. High genetic correlations were observed between somatic cell score and the California Mastitis Test (0.76 to 0.98), indicating the California Mastitis Test to be worthwhile for assessing infection levels, particularly at mid-lactation. The strongest correlations observed between the mastitis traits and the udder conformation traits were associated with udder depth (0.61 to 0.75) also at mid-lactation. Negative phenotypic correlations were estimated between mastitis and the weight of lamb reared by the ewe (−0.15 to −0.23), suggesting that lamb weights fell as infection levels rose. Genetic correlations were not significantly different from zero. Reducing mastitis will lead to improvements in flock productivity and the health and welfare of the animals. It will also improve the efficiency of production and the resilience to disease challenge. The economic benefits, therefore, of these results combined could be substantial not only in this breed but also in the overall meat sheep industry.     

Exploring of Primate Models of Tick-Borne Flaviviruses Infection for Evaluation of Vaccines and Drugs Efficacy

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.     

Protective immune response to experimental infection with Sarcocystis neurona in 57BL/6 mice

Immunocompetent C57BL/6 mice were infected with Sarcocystis neurona merozoites to assess the protective immune response to active infection. Using a direct agglutination test, all infected mice seroconverted to S. neurona merozoite antigens by day 14 postinfection (PI). Further, mice developed splenomegaly and bilateral symmetrical lymphadenopathy by day 14 PI, which appeared to be resolving by day 28 PI. Histologic analysis revealed a marked increase in germinal center formation in the spleen and lymph nodes by day 14 PI. Corresponding to gross and histopathological changes, the percentage of B-cells decreased significantly by day 14 PI but then increased significantly and persisted at day 28 PI in the blood, spleen, and multiple lymph nodes. There was a sharp nonspecific significant decrease in CD4 percentages by day 14 PI in the blood, spleen, and lymph nodes. Early-activation CD8 lymphocytes (CD62/CD8) were significantly down-regulated coinciding with a significant compensatory up-regulation of memory (CD44/CD8) lymphocytes in multiple organs. We propose that the protective cell-mediated immune response to S. neurona involves both CD4 and CD8 cells, with CD8 lymphocytes appearing to play a more critical role.     

Pen size and parity effects on maternal behaviour of Small-Tail Han sheep

The aim of this experiment was to study the effects of pen size and parity on maternal behaviour of twin-bearing Small-Tail Han ewes. A total of 24 ewes were allocated to a 2×2 design (six per pen), with parity (primiparous or multiparous) and pen size (large: 6.0×3.0 m; small: 6.0×1.5 m) as main effects at Linyi University, Shandong Province, China. Behaviour was observed from after parturition until weaning. All ewes were observed for 6 h every 5 days from 0700 to1000 h and from 1400 to 1700 h. Continuous focal animal sampling was used to quantify the duration of maternal behaviours: sucking, grooming and following as well as the frequency of udder accepting, udder refusing and low-pitched bleating. Oestradiol and cortisol concentrations in the faeces (collected in the morning every 5 days) were detected using EIA kits. All lambs were weighed 24 h after parturition and again at weaning at 35 days of age. The small pen size significantly reduced following (P<0.005), grooming (P<0.001) and suckling durations (P<0.05), as well as the frequency of udder refusals (P<0.001). However, there was a significant interaction with ewe parity, with decreased grooming and suckling in the small pen largely seen in the multiparous ewes (P<0.001). Independent of pen size, multiparous ewes accepted more sucking attempts by their lambs (P<0.05) and made more low-pitched bleats than primiparous ewes (P<0.001). Multiparous ewes had higher faecal oestradiol concentrations than primiparous ewes (P<0.001), and ewes in small pens had higher faecal cortisol levels compared with ewes in larger pens (P<0.001). As lambs increased in age, the duration of maternal grooming, following and suckling as well as frequency of udder acceptance and low-pitched bleating all declined, and the frequency of udder refusing increased (P<0.001 for all). Ewe parity, but not pen size, affected lamb weight gain during the period of observation (P<0.001). This is the first study to show that pen size, interacting with parity, can affect the expression of maternal behaviour in sheep during lactation. The study is also the first to report on the maternal behaviour of Chinese native sheep breeds (Small-Tail Han sheep), with implications for the production of sheep in China.     

Increase of canine leishmaniasis in a previously low-endemicity area in Tunisia

An epidemiological study of canine leishmaniasis (CanL) was carried out in nine districts of Sfax, in the southern central part of Tunisia. Sera from 250 dogs were tested by two serological methods: the indirect immunofluorescence antibody test and the counter-immunoelectrophoresis. Seven to eight months later, before the next season of transmission, seropositive dogs from the first test were re-examined and a second sampling was performed. Infection status was assessed by serology and by other methods. PCR, in vitro culture and direct examination were applied on blood and other samples (bone marrow, liver, lymph node, spleen and cutaneous biopsies). The seroprevalence of the infection in dogs was 6%. Infection was then confirmed by at least one other method. The PCR is the method which agreed most with serology, all seropositive dogs were found PCR-positive. The sensitivity of the direct examination and the culture was only 33% and 55% respectively as compared with serology. A similar value of seroprevalence has been observed previously in Sousse, in the northern central part of Tunisia. The present report suggests a significant increase of CanL in the Sfax area and confirms that the disease is continuing to move southwards in Tunisia.     

Trypanosoma congolense. II. Histopathologic findings in experimentally infected cattle

Heart, kidney, lung, liver, brain, spleen, lymph nodes, tongue, and diaphragm of 9 cattle experimentally infected with the Trans Mara I strain of Trypanosoma congolense, were examined histologically. A haemosiderosis, infiltrations in the kidney, changes in the vascular wall mainly of the arteries of the lung, scattered local perivascular, and meningeal infiltrations, and small juxtavascular glial nodules in the CNS were found.     

Morphopathological findings in dog's acute toxoplasmosis

Toxoplasmosis was clinically diagnosed in two dogs of 4 and respectively 18 months. A seven-day treatment remained inefficient and the animals died. Autopsy revealed a global inflammation of the lung, with necrotic lesions of bronchial lymph nodes and acute hyperplastic reaction of the spleen. Histologically, there were identified a diffuse, serofibrinous inflammation of the lung with necrotic foci, fibrinoleucocytic and necrotic exudate of bronchial and portal lymph nodes, small necrotic or fibrinonecrotic foci of the liver and spleen, lymphoglial foci and edema at the base of the cerebellum. In myocardium, as well as in the above mentioned organs, toxoplamas are free or accumulated in cysts. Inoculation of infected lung material into mice and newborn dogs gave positive results.     

Enzootic toxoplasmosis in sheep in north-central United States

Of 1,564 serum samples from adult ewes from 33 farms in Iowa, Minnesota, South Dakota, Kansas, and Nebraska where toxoplasmosis-induced ovine abortions had been diagnosed, 65.5% were found positive for Toxoplasma gondii antibodies using the modified agglutination test. Toxoplasma gondii antibody titers of ewes were: less than 64 (34.5%), 64 (14.9%), 256 (22.0%), 1,024 (14.5%), and greater than 4,096 (13.8%). Thus, 28.3% of sheep had high titers (greater than 1,024) indicating recently acquired T. gondii infection. On certain farms, up to 95% of ewes were seropositive. Prevalence of T. gondii antibodies increased with age of the ewe. Of 665 ewes, 53.6% of 1-yr-old ewes were seropositive (titers greater than 64) versus 75% of 5-yr-old ewes. Results indicate that T. gondii infection in sheep in the United States is widespread.