Experimental Infection Of Cattle Research Articles

Emergence of bluetongue virus serotype 6 in Europe—German field data and experimental infection of cattle

In late 2008, bluetongue virus (BTV) serotype 6 (BTV-6), which had never occurred in Europe before, was first detected in the Netherlands and Germany. While the origin of the virus remains unknown, the prevalence of infections in cattle was investigated in a virological ( N = 28,658) and serological ( N = 2075) field survey in Lower Saxony, where 45 cases confined to the district Grafschaft Bentheim were found. Blood from affected animals was used for the experimental infection of three cattle with different BTV antibody status, leading to sustained viraemia in one animal naïve for BTV. Of two animals that had detectable antibodies against BTV serotype 8, one became transiently infected and seroconverted for BTV-6 while the other did not react. In conclusion, while only a very limited spread of BTV-6 could be observed in the field, experimental infection of cattle did not show substantial differences of the course of infection in comparison to other BTV serotypes.

Distinct Ecologically Relevant Strains ofAnaplasma phagocytophilum

Distinct Ecologically Relevant Strains of<i>Anaplasma phagocytophilum</i>

Development and evaluation of a polymerase chain reaction assay to detect Bovine herpesvirus 1

A polymerase chain reaction (PCR) assay to detect Bovine herpesvirus 1 (BoHV-1) has been developed and evaluated. This assay was based on the amplification of a viral thymidine kinase (tk) gene fragment, for which a novel pair of primers and different PCR conditions were tested. Under optimal conditions, no amplification was observed from heterologous herpesviruses or other bovine viruses. However, a 202 bp band from BoHV-1 was obtained confirming the specificity of the assay. An analytical sensitivity of 0.15 TCID50 per reaction led to the detection of BoHV-1 in nasal swab supernatants up to 11 days after experimental infection of cattle. There was a 100% match on the results until six days post-infection when PCR was compared with virus isolation and fluorescent antibody test (FAT). Starting the seven day post-infection and until the end of the experiment most of the positive samples were obtained by PCR. This simple PCR strategy do not use co-solvents in order to eliminate non-specific products or increase the efficiency of the amplification, provides a rapid method for diagnosis of BoHV-1 diseases in contrast with semi-nested or nested protocols with more time-consuming and a higher contamination rate, moreover, can be easily used in all diagnosis laboratories.

Experimental infection of cattle with highly pathogenic avian influenza virus (H5N1).

Four calves were experimentally inoculated with highly pathogenic avian influenza virus A/cat/Germany/R606/2006 (H5N1) isolated from a cat in 2006. All calves remained healthy, but several animals shed low amounts of virus, detected by inoculation of nasal swab fluid into embryonated chicken eggs and onto MDCK cells. All calves seroconverted.

Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

BackgroundOur laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.ResultsSera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease.ConclusionCollectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.

The extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following Neospora caninum infection in early and late gestation correlates with foetal death

The protozoan parasite Neospora caninum is the most frequently diagnosed abortifacient in the UK and a leading cause of abortion worldwide but the mechanisms leading to abortion are not fully understood. The distribution of parasites and the histopathological changes in the placenta and foetus were compared in 12 cows following experimental infection of cattle with N. caninum in early ( n = 6) and late ( n = 6) gestation, by PCR, immunohistology, light microscopy and transmission electron microscopy. Twelve uninfected pregnant cattle were used as controls. Infection in early gestation led to foetal death. In the placentae of cattle immediately following foetal death, N. caninum DNA was detected and there was evidence of widespread parasite dissemination. This was associated with extensive focal epithelial necrosis, serum leakage and moderate maternal interstitial mononuclear cell infiltration. In the foetuses, parasites were evident in all tissues examined and were associated with necrosis. In the placenta of cattle infected in late gestation, N. caninum DNA was detected sporadically but parasites were not evident immunohistologically. Small foci of necrosis were seen associated with mild interstitial mononuclear cell infiltration. Detection of N. caninum DNA in the foetuses was sporadic and parasites were demonstrated immunohistologically in brain and spinal cord only, with an associated mononuclear cell infiltration. This data is consistent with uncontrolled parasite spread in an immunologically immature foetus and could, via multiparenchymal necrosis of foetal tissues or the widespread necrosis and inflammation observed in the placenta, be the cause of Neospora-associated abortions.

Peripheral immune responses in pregnant cattle following Neospora caninum infection

Experimental infection of cattle with Neospora caninum in early gestation causes foetal death, but the foetus survives infection in late gestation. An immunological mechanism of abortion has been suggested; therefore changes in the maternal immune response during pregnancy could account for these differences. We have investigated the peripheral immune responses of pregnant cattle following an intravenous inoculation with 10(7) N. caninum tachyzoites in early and late gestation. Percentages of CD2+ and CD4+ T-cells in peripheral blood mononuclear cells (PBMC) increased 1-2 weeks after infection in both early (day 70) and late (day 210) gestation, and percentages of CD8+ T-cells increased 1-2 weeks after infection at day 70. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression in PBMC increased 1-2 weeks after infection at day 210 and IL-4 increased 1-2 weeks after infection at day 70. Immunomagnetic isolation of CD4+ cells from PBMC showed that they were a major source of IL-4 and IFN-gamma, and expression of both cytokines increased in CD4+ cells after infection in early and late gestation. These results suggest that CD4+ cells proliferate and express IL-4 and IFN-gamma in response to N. caninum irrespective of the stage of gestation when infection occurs.

Cytotoxic T-cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis.

Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+gammadelta and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies.

Maximal predicted duration of viremia in bluetongue virus-infected cattle.

Central to the development of rational trade policies pertaining to bluetongue virus (BTV) infection is determination of the risk posed by ruminants previously exposed to the virus. Precise determination of the maximal duration of infectious viremia is essential to the development of an appropriate quarantine period prior to movement of animals from BTV-endemic to BTV-free regions. The objective of this study was to predict the duration of detectable viremia in BTV-infected cattle using a probabilistic modeling analysis of existing data. Data on the duration of detectable viremia in cattle were obtained from previously published studies. Data sets were created from a large field study of naturally infected cattle in Australia and from experimental infections of cattle with Australian and US serotypes of BTV. Probability distributions were fitted to the pooled empirical data, and the 3 probability distributions that provided the best fit to the data were the gamma, Weibull, and lognormal probability distributions. These asymmetric probability distributions are often well suited for decay processes, such as the time to termination of detectable viremia. The analyses indicated a > 99% probability of detectable BTV viremia ceasing after < or = 9 weeks of infection in adult cattle and after a slightly longer interval in BTV-infected, colostrum-deprived newborn calves.

Antigenic and genetic stability of bovine immunodeficiency virus during long-term persistence in cattle experimentally infected with the BIV(R29) isolate.

Experimental infection of cattle with bovine immunodeficiency virus (BIV) is characterized by persistent, low levels of virus replication in the absence of clinical disease. A virus neutralization (VN) assay was developed to examine the role of VN antibodies in controlling virus replication in cattle experimentally infected with the BIV(R29) isolate of BIV. All animals developed VN antibody, but there was no correlation between VN titres and restriction of virus replication in vivo. BIV infection did not induce high-titred, cross-neutralizing antibody and there was no evidence for antigenic variation through more than 4 years in vivo. Genetic comparisons among the BIV(R29) inoculum virus and viruses isolated from infected animals identified only limited genetic variation during 4 years in vivo. Moreover, there was no evidence that the observed variation was due to selection. Analyses of genetic diversity in the virus stock used for inoculation indicated a fairly homogeneous population. In the absence of high levels of virus replication and overt clinical disease, there appeared to be little selection of virus variants, resulting in antigenic and genetic stability of BIV(R29) during long-term, persistent infection.

Experimental infection of cattle with several Borrelia burgdorferi sensu lato strains; immunological heterogeneity of strains as revealed in serological tests

Twenty-three experimental cattle, mainly calves, were each inoculated 1–3 times with one of ten Finnish Borrelia burgdorferi sensu lato strains. All three genospecies were represented. Borreliae were administered mainly by both intravenous (about 10 6 to 10 9 spirochaetes) and intradermal (10 4) routes, and on six occasions subcutaneously (10 3) only. For infectivity control and comparison purposes mice and rabbits were inoculated simultaneously. Immune responses in cattle were monitored both with whole-cell sonicate enzyme-linked immunosorbent assay (IgG-ELISA) and indirect immunofluorescent assay (IgM–IgG-IFA). Five Finnish strains and the American strain B31 were used as antigens. No clinical signs of borreliosis were observed. Of the strains, 7/10 were interpreted by the immune responses to have caused relatively short-term subclinical infections of varying intensity. Borreliae could not be isolated from blood or other organ specimens of cattle. A rough estimate of the mean infectious dose in the conditions of experiments is 10 6 to 10 7 organisms. In conclusion, the overall result appears to argue a low susceptibility of cattle to clinical borreliosis, at least when infected by Finnish strains of the agent. Significant antigen-specific differences were observed both by ELISA and IFA in detection and quantification of immune responses. As a rule, the homologous antigen was found to be the most sensitive. Genospecies differences were mostly distinct. Antigens of two Borrelia garinii isolates proved practically equal in sensitivity, whereas major differences were displayed between two Borrelia afzelii antigens. In an IFA study, an American (B31) and a Finnish B. burgdorferi sensu stricto strain proved equally sensitive as antigens. In two relatively strong primary immune responses the antigen-specific measurement differences were such that diagnostically in a cross-sectional study only the homologous antigen or an antigen of the same genospecies would have been sufficiently sensitive to show a positive result.

Histamine and Eicosanoid Levels in Plasma and Peripheral Blood Leucocyte Cultures During Experimental Infection of Cattle with Bluetongue Virus Serotype 11

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.

Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie

Cattle are susceptible to experimental infection with the Stetsonville isolate of the transmissible mink encephalopathy (TME) agent. To determine if they are susceptible to other TME isolates, two groups of calves were inoculated intracerebrally with homogenate of mink brain containing the Hayward isolate or the Blackfoot isolate. For comparison, a third group was inoculated with a brain homogenate from a steer infected with the Stetsonville isolate in its primary cattle passage and a fourth group was inoculated with a pool of brain homogenate from three cattle experimentally infected with a sheep and goat scrapie agent in its primary cattle passage. Clinical signs of neurological disease appeared in each steer of every group between 15 and 25 months after inoculation. An encephalopathy characterized by severe spongiform change and pronounced astrocytosis occurred in the three groups inoculated with the TME agent. In contrast, the neurohistological changes in the steers inoculated with the cattle-passaged scrapie agent were slight and subtle. Analysis of the octapeptide repeat region of the bovine protease-resistant protein (PrP) gene showed that variations in incubation period, clinical signs, and neurohistological changes were unrelated to the homozygous or heterozygous condition of six or six/five octapeptide repeats.

Rapid detection of bovine herpesvirus 1 (BHV 1) using the polymerase chain reaction

A polymerase chain reaction (PCR) assay based on primers from the viral gl glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1 TCID 50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size was amplified using DNA from herpesvirus isolated from reindeer, red deer and goats. The PCR assay was able to detect virus in nasal swabs up to 14 days after experimental infection of cattle and there was a good correlation when PCR was compared with virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful.

The effect of the mode of sampling on BHV-1 detection in infected cattle by dot-blot hybridization

The BHV-1 genome in nasal swabs and washings was detected by dot-blot hybridization using the 32P-pUR-1 probe (1.8 kb EcoRI- HindIII random fragment of BHV-1 DNA ligated into the pUC-9 plasmid) as early as on day 1 after the experimental infection of cattle. In dependence on the sampling method, differences were observed in the maximum of hybridization signals. During nasal swab analyses maximum amounts of BHV-1 differed in the individual samples (day 1–3). Hybridization signals obtained at the analysis of BHV-1 DNA nasal washings did not vary but showed a continuous maximum on day 2 after infection. Nasal washings proved to be more advantageous for detection of the BHV-1 genome by the hybridization technique.

Development and persistence of Cowdria ruminantium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test

Different breeds of cattle were experimentally infected with Palm River, a Zimbabwean isolate, or Ball-3, a South African isolate of Cowdria ruminantium, derived from tissue culture or tick or blood stabilates. C. ruminantium specific antibody responses were detected by an indirect fluorescent antibody test (IFAT) using C. ruminantium-infected bovine aortic endothelial (BAE) cell cultures as antigen. The first detection of antibodies to C. ruminantium generally coincided with the peak of the febrile reaction and the antibodies remained detectable for a period of 8–30 weeks in the Palm River infected group and 18–30 weeks in the Ball-3 infected group. Peak reciprocal antibody titres in both groups ranged from 64 to 2048 between 3 and 6 weeks post-infection. No apparent serological differences were observed among the various C. ruminantium isolates when tested in homologous and heterologous IFATs. Post-infection sera to Anaplasma marginale, Theileria parva parva, Babesia bigemina and Rickettsia conorii did not exhibit reactivity with the C. ruminantium antigen. These results indicate the possible use of C. ruminantium-infected cultures as antigen in IFATs to detect similar C. ruminantium-specific antibody responses in the field in clinically sick, recovered and carrier animals.

Experimental infection of cattle in early pregnancy with a cytopathic strain of bovine virus diarrhoea virus

Nine pregnant heifers, in early gestation (63 to 107 days), were infected intranasally or in utero with cytopathic bovine virus diarrhoea virus (BVDV) and each dam seroconverted. All nine calves developed to full term; four were stillborn, of which one had seroconverted but virus was not recovered from their tissues. One of the five liveborn calves appeared to have seroconverted in utero to an adventitious BVDV infection in late pregnancy but the remaining four were not viraemic and showed a normal secondary antibody response to BVDV infection at about six months old. Thus, in contrast to results with noncytopathic virus there was no evidence that infection in utero with cytopathic virus could result in a persistent viraemia or immunotolerance. It is suggested that cells able to support a persistent viraemia with cytopathic virus may not be developed in the young fetus.

Herpesvirus infection in Cervidae

Summary: The earliest evidence of herpesvirus infections of Cervidae came from testing free-living deer for antibody to bovine herpesviruses pathogenic to cattle. Antibody to the cattle alphaherpesvirus bovine herpesvirus type 1 (BHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), has been demonstrated in mule deer, white-tailed deer and caribou in North America and in red deer, roe deer, reindeer and fallow deer in Europe. Alphaherpesviruses serologically related to BHV-1 have been isolated from red deer and reindeer. The red deer virus, herpesvirus of Cervidae type 1 (HVC-1), was recovered from farmed red deer calves with ocular disease. The reindeer virus was isolated from an animal which had been treated with dexamethasone and no disease attributable to a herpesvirus has been seen in this species. Biochemical and serological studies have demonstrated that these two viruses are distinct from each other and from BHV-1. Experimental infections of cattle with the two viruses have induced no or mild disease. The one serious disease recognised in Cervidae due to herpesvirus infection is malignant catarrhal fever (MCF). The wildebeest-associated gammaherpesvirus, alcelaphine herpesvirus-1 (AHV-1), has caused deaths of white-tailed deer, sika deer, barasingha deer and reindeer among captive populations in zoological parks, while MCF due to the sheep-associated agent, believed to be a gammaherpesvirus, is common among several species of farmed deer and represents a serious threat to these new domestic livestock. Deer do not shed infectivity, thus the disease does not spread from affected animals. The disease has not been reported to affect free-living animals.

Besnoitia besnoiti: studies on the definitive host and experimental infections in cattle.

Domestic cats, 11 other species of carnivorous mammals, 6 species of snakes, and white-backed vultures were tested for their possible role as definitive hosts of Benoitia besnoiti by feeding with cystic material from chronically infected bovines. None of the species tested is a definitive host; hence, the life cycle of this parasite remains obscure. In attempts to produce clinical cases of besnoitiosis by experimental infection, bovines were inoculated IV, SC, and IP with cystozoites or tachyzoites. Immunosuppression of the animals was essential for the development of severe cases and skin lesions; cystozoites proved to be more pathogenic than tachyzoites.

Development of specific antibodies mediating antibody-dependent cell-mediated cytotoxicity following experimental infection of cattle with bovine herpesvirus 1 and subsequent viral reactivation

Evolution of sensitizing antibodies involved in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-complement cell lysis and complement-facilitated ADCC was followed in bulls after primary infection by a wild strain of infectious bovine rhinotracheitis virus (bovine herpesvirus 1; BHV-1) and after experimental reactivation of the virus. These antibodies were detected between the 4th and the 7th day after primary infection, reached a maximum level after 2 weeks and rose slightly after reactivation of the virus following dexamethasone treatment. The presence of endogenous complement slightly enhanced the ADCC reaction.