A longitudinal study of the dynamics of Mycoplasma bovis antibody status in primiparous cows and bulk tank milk in Swedish dairy herds.

Mycoplasma (M.) bovis is an important pathogen causing pneumonia, mastitis, and arthritis in cattle all over the world entailing reduced animal welfare and economic losses. In this longitudinal study, we investigated the presence of M. bovis antibodies in bulk tank milk (BTM) and in milk from primiparous (PP) cows at 4 sampling occasions over 2 years. Herd characteristics associated with a positive antibody test result in PP cows were investigated. The participating dairy herds (n = 149) were situated in southern Sweden, samples were collected and analyzed with ID Screen antibody ELISA. Information on herd characteristics was retrieved from the national Dairy Herd Improvement database. To identify herd characteristics associated with the presence of antibodies in PP cows, mixed linear regression with herd and sample as random factors were used. The apparent herd-level prevalence of M. bovis infection based on antibodies in BTM was 17% but with the addition of PP cows the prevalence increased to 28%. The results showed that larger herds and introduction of cattle was associated with higher antibody levels in PP cows. In conclusion, this study showed a clear difference in the apparent prevalence of M. bovis infection based on antibodies in BTM or in PP cows, the no. of positive herds were almost doubled when including PP cows. This motivates repeated sampling of a few PP cows to find newly infected herds in an early stage. Finally, the results showed that introduction of cattle influences the level of M. bovis antibodies. This is important in the control and prevention of further spread of the infection. It is essential for free herds to know their M. bovis status and antibody testing is highly recommended if introducing cattle.

Herd-level prevalence of Mycoplasma bovis in Swedish dairy herds determined by antibody ELISA and PCR on bulk tank milk and herd characteristics associated with seropositivity.

Mycoplasma bovis is an important pathogen causing pneumonia, mastitis, and arthritis in cattle, leading to reduced animal welfare and economic losses worldwide. In this cross-sectional study, we investigated the prevalence of M. bovis in bulk tank milk (BTM) and herd characteristics associated with a positive antibody test result in Swedish dairy herds. Bulk tank milk samples from all Swedish dairy herds (n = 3,144) were collected and analyzed with ID Screen antibody ELISA and PCR. Information on herd characteristics was collected from the national Dairy Herd Improvement database. To identify herd characteristics associated with the presence of antibodies in BTM, logistic regression was used in 4 different models. The apparent herd-level prevalence of M. bovis infection based on antibodies in BTM was 4.8%, with large regional differences ranging from 0 to 20%. None of the BTM samples was positive by PCR. All the antibody-positive herds were situated in the south of Sweden. The logistic regression model showed that larger herds had higher odds of detectable antibodies in BTM (herd size >120 cows, odds ratio = 8.8). An association was also found between antibodies in BTM and both a higher late calf mortality (2-6 mo) and a higher young stock mortality (6-15 mo). This study showed a clear regional difference in the apparent prevalence of M. bovis infection based on antibodies. The relatively low prevalence of M. bovis in Sweden is a strong motivator for the cattle industry to take steps to prevent further spread of the infection. It is essential that the M. bovis status of free herds be known, and the regional differences shown in this study suggest that testing is highly recommended when live cattle from high-prevalence areas are being introduced into herds. We do not recommend using PCR on BTM to detect infected herds, owing to the low detection frequency in this study.

Blood profiles in dairy cows with displaced abomasum.

An observational study was carried out in Swedish dairy herds to investigate differences between cows with and without displaced abomasum (DA), in concentrations of glucose, insulin, fructosamine, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, cholesterol, haptoglobin, increased enzyme activity of aspartate aminotransferase and glutamate dehydrogenase, and the revised Quantitative Insulin Sensitivity Check Index (RQUICKI). A secondary aim was to study how blood profiles for DA cows vary with time in relation to calving. Sixty-nine DA cows within 2 to 56 d postpartum, in 60 herds, were clinically examined and blood samples were drawn at the time DA was diagnosed. At the same time, 104 healthy control cows matched by herd and stage of lactation were also sampled. The blood parameters were studied using mixed linear models, including herd as a random effect, and DA (case or control), parity, breed, sampling time in relation to calving, other diseases, and the interaction between DA and time as fixed effects. Concentrations were higher in DA cows than in control cows for NEFA (least squares means 1.36 vs. 0.34 mmol/L), ß-hydroxybutyrate (1.56 vs. 0.90 mmol/L), aspartate aminotransferase (1.96 vs. 0.97 µkat/L), glutamate dehydrogenase (197 vs. 78 µkat/L), and haptoglobin (0.76 vs. 0.17 g/L), whereas concentrations were lower in DA cows than in control cows for insulin (3.61 vs. 8.48 mU/L) and cholesterol (3.04 vs. 3.75 mmol/L). Glucose (2.83 vs. 2.79 mmol/L) and fructosamine (266 vs. 252 µmol/L) concentrations were similar in both groups; however, a tendency toward lower RQUICKI values (0.42 vs. 0.46) in the DA cows was found, indicating reduced insulin sensitivity. For most blood parameters, differences between DA cows and controls remained constant over time. Seventy-two percent of the DA cows had at least one other disease in the period from 1 wk antepartum to 1 wk after the DA was diagnosed. Haptoglobin could potentially be used to detect treatable infectious or inflammatory conditions in the early postpartum period, possibly reducing the incidence of DA. Consequently, there were major changes in blood profiles in cows with DA compared with healthy control cows, indicating a negative energy balance, liver cell damage, and an inflammatory response. The results contribute to an understanding of the metabolic changes in DA cows.

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum.

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature. The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves.

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks. In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.

Serum concentrations of thyroid hormones in calves with a transient or persistent infection with bovine viral diarrhoea virus.

Calves persistently infected with bovine viral diarrhoea virus (BVDV) often appear small for their age and it is possible that the virus interferes with their body metabolism by affecting the production of hormones. In this study, the serum concentrations of thyroxine (T4) and triiodothyronine (T3) were measured in calves with transient or persistent BVDV infections. The mean (SD) concentrations of T3 and T4 were lower (P < 0.001) in the persistently infected calves (1.79 [0.67] and 69.2 [23.8] nmol litre-1, respectively) from three dairy herds than in age-matched control calves (2.39 [0.72] and 93.4 [22.1] nmol litre-1, respectively). In each herd, the thyroid hormone levels were also lower in the persistently infected calves than in the controls, the differences being significant (P < 0.05) with the exception of the T3 level in one herd. The girth of the infected calves over the heart was less (P < 0.001) than that of the controls, and, among the infected calves but not among the controls, there was a significant correlation between heart girth and the levels of T3 (r = 0.72, P < 0.001) and T4 (r = 0.60, P < 0.01). Six calves not previously exposed to BVDV were infected through nose-to-nose contact with a persistently infected calf. Their T3 and T4 levels were decreased seven days (P < 0.05 and not significant, respectively) and nine days (P < 0.001 and P < 0.01, respectively) after the infection, when the clinical signs of the disease appeared.

Failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus.

Previous reports on the spread of bovine virus diarrhoea virus (BVDV) from animals primarily infected with the agent are contradictory. In this study, the possibility of transmission of BVDV from calves simultaneously subjected to acute BVDV and bovine coronavirus (BCV) infection was investigated. Ten calves were inoculated intranasally with BVDV Type 1. Each of the 10 calves was then randomly allocated to one of two groups. In each group there were four additional calves, resulting in five infected and four susceptible calves per group. Virulent BCV was actively introduced in one of the groups by means of a transmitter calf. Two calves, susceptible to both BVDV and BCV, were kept in a separate group, as controls. All ten calves actively inoculated with BVDV became infected as shown by seroconversions, and six of them also shed the virus in nasal secretions. However, none of the other eight calves in the two groups (four in each) seroconverted to this agent. In contrast, it proved impossible to prevent the spread of BCV infection between the experimental groups and consequently all 20 study calves became infected with the virus. Following infection, BCV was detected in nasal secretions and in faeces of the calves and, after three weeks in the study, all had seroconverted to this virus. All calves, including the controls, showed at least one of the following clinical signs during days 3-15 after the trial started: fever (> or =40 degrees C), depressed general condition, diarrhoea, and cough. The study showed that BVDV primarily infected cattle, even when co-infected with an enteric and respiratory pathogen, are inefficient transmitters of BVDV. This finding supports the principle of the Scandinavian BVDV control programmes that elimination of BVDV infection from cattle populations can be achieved by identifying and removing persistently infected (PI) animals, i.e. that long-term circulation of the virus without the presence of PI animals is highly unlikely.

Nationwide survey of antibodies to bovine coronavirus in bulk milk from Swedish dairy herds.

Bulk milk samples from 2236 dairy herds randomly selected throughout Sweden in proportion to region and herd size were analysed for antibodies to bovine coronavirus (BCV) in an ELISA. The results were expressed as optical density (OD) values and an OD > 0.04 was considered positive. Eighty-nine per cent of the samples were antibody-positive and 52 per cent had high levels of antibodies to BCV (an OD > 0.70). There were significantly higher OD values (P < 0.001) and fewer antibody-negative samples (P < 0.001) from larger herds than from smaller herds. There were also significantly higher OD values and fewer antibody-negative samples from herds in southern Sweden than from herds in northern Sweden (P < 0.001 and P < 0.001, respectively). These results indicate a higher frequency of BCV infections in larger herds and in herds in southern Sweden.

Winter dysentery diagnosed by farmers in dairy herds in central Sweden: incidence, clinical signs and protective immunity.

The occurrence of winter dysentery, diagnosed by farmers and defined as an outbreak of diarrhoea among at least 30 per cent of adult cattle in a herd, was monitored in 256 dairy herds in an area of central Sweden. The cumulative incidence of winter dysentery between April 1988 and March 1989 was 28.5 per cent. A typical outbreak lasted for one to two weeks and 74 per cent of the outbreaks occurred between November and January. A decrease in milk yield was reported in 90 per cent of the affected herds and the cows showed respiratory signs in 57 per cent of them. There was a significant (P < 0.05) association between the occurrence of fever and coughing. In 31 per cent of the outbreaks the farmer also noticed diarrhoea among the calves. One-third of the affected herds had experienced an outbreak within the previous four years and 18 per cent had at least one further outbreak during the following two years. There was a significantly (P < 0.05) lower disease score in herds that had had an outbreak within the previous four years than in herds which had had a less recent or no previous outbreak, indicating the development of temporary immunity to the causative agent.

Serum antibodies to bovine coronavirus in Swedish sheep.

Altogether 218 sheep sera from 40 flocks in different parts of Sweden were screened for antibodies to bovine coronavirus (BCV). Nineteen per cent of the sera were positive and there was a significantly higher frequency (p < 0.05) of at least one positive sample in flocks with more than 100 adult sheep than in smaller flocks. There was also a significantly higher frequency (p < 0.001) of positive samples from sheep older than 4 years than from younger ones. Only a weak relationship between BCV positivity (2 or more positive samples, p < 0.05) and cattle contact was demonstrated in this study. Possible transmission routes and other factors that could have affected the result are discussed. In light of our finding that all 5 sheep experimentally exposed to BCV through contact with infectious cow faeces seroconverted, we conclude that the antibodies found in Swedish sheep are probably the result of BCV infections directly or indirectly transmitted from cattle.

A longitudinal study of the dynamics of bovine corona virus and respiratory syncytial virus infections in dairy herds.

The objective of this study was to examine the dynamics of bovine corona virus (BCV) and bovine respiratory syncytial virus (BRSV) infections in dairy herds over a 3-year period. The status of 79 dairy herds located in two Northern and two Southern Regions of Sweden were surveyed by measuring antibody concentrations to BCV and BRSV in pooled milk samples from primiparous cows, and in bulk-tank milk twice annually. In the Southern Regions the percentage of antibody-positive herds remained persistently high (75-100%), whereas in herds based in the Northern Region, the percentage of positive herds for BCV and BRSV was 38-80% and 0-80%, respectively, with antibody levels to BRSV decreasing over time. Pooled milk samples of 'home-bred' primiparous animals were found to be most useful in terms of monitoring herd status but could gradually be replaced by bulk-tank sampling once freedom from infection was established.

High- and low-purity glycerine supplementation to dairy cows in early lactation: effects on silage intake, milk production and metabolism.

This study evaluated the effects of supplemental low- and high-purity glycerine on silage intake, milk yield and composition, plasma metabolites and body condition score (BCS) in dairy cows. A total of 42 cows of the Swedish Red Breed, housed in individual tie stalls, were fed 0.25 kg of low- or high-purity glycerine on top of concentrate, twice daily, during the first 4 weeks of lactation. One-third of the cows acted as controls, receiving no glycerine. Silage was fed for ad libitum intake and concentrate was fed at restricted level of intake, about 6 kg/day for primiparous cows and 7 kg/day for multiparous cows. Feed refusals were weighed daily. Cows were milked twice daily, milk yield was recorded on four occasions per week and milk samples were collected simultaneously. Blood samples were drawn from the coccygeal vessel once a week. Low- and high-purity glycerine had no effect on silage or total dry matter intake (P = 0.38 and P = 0.75, respectively) or on BCS (P = 0.45). Cows fed high-purity glycerine tended to have higher milk yield than control cows (P = 0.06). Milk composition tended to differ among treatments. No main effects of treatment on concentration of glycerine (P = 0.44), glucose (P = 0.78), insulin (P = 0.33), non-esterified fatty acids (P = 0.33) and ß-hydroxybutyrate (P = 0.15) in plasma. These data indicate that high-purity glycerine has the potential to increase milk yield, as well as enhance the milk protein concentration and milk fat + protein yield.

Relationship between incidence of milk fever and feeding of minerals during the last 3 weeks of gestation.

This study evaluated whether differences in milk fever incidence among Swedish dairy herds could be explained partly by differences in mineral feeding during the last weeks of gestation. A case-control study was performed on dietary risk factors for a high incidence of milk fever at the herd level using information regarding feeding and management of the dry cows collected in a written questionnaire distributed by post in spring 2008. The study was conducted from September 2004 to August 2007. Data on the diets fed to dry cows, with an emphasis on the amounts of minerals (Ca, P, Mg and K) fed 3 weeks before calving and at calving, were obtained from 30 herds belonging to the 100 Swedish herds with the highest recorded incidence of veterinary treatment for milk fever (>8.8%) and from 22 herds with no recorded milk fever treatments. Multivariable logistic regression analysis showed that a linear increase in the total amount of K and less than 26 g of Mg/day fed to dry cows 3 weeks before calving was associated with an increased risk of high milk fever incidence. A large increase in the amount of dry matter (DM) fed (>3.1 kg DM extra per day at calving compared with 3 weeks before calving) was associated with a higher incidence of milk fever, but no differences were found for Ca or P intake. Breed composition, herd average milk yield and age composition of the herd did not explain any of the observed differences between the case and the control herds. The results indicate that differences in the frequency of milk fever among herds can be associated with differences in mineral feeding of the dry cows. A high amount of K in the diet may increase the risk of milk fever linearly, whereas Mg should probably be fed at a higher level than the current Nordic recommendation to prevent milk fever.

Risk factors for displaced abomasum or ketosis in Swedish dairy herds.

Risk factors associated with high or low long-term incidence of displaced abomasum (DA) or clinical ketosis were studied in 60 Swedish dairy herds, using multivariable logistic regression modelling. Forty high-incidence herds were included as cases and 20 low-incidence herds as controls. Incidence rates were calculated based on veterinary records of clinical diagnoses. During the 3-year period preceding the herd classification, herds with a high incidence had a disease incidence of DA or clinical ketosis above the 3rd quartile in a national database for disease recordings. Control herds had no cows with DA or clinical ketosis. All herds were visited during the housing period and herdsmen were interviewed about management routines, housing, feeding, milk yield, and herd health. Target groups were heifers in late gestation, dry cows, and cows in early lactation. Univariable logistic regression was used to screen for factors associated with being a high-incidence herd. A multivariable logistic regression model was built using stepwise regression. A higher maximum daily milk yield in multiparous cows and a large herd size (p=0.054 and p=0.066, respectively) tended to be associated with being a high-incidence herd. Not cleaning the heifer feeding platform daily increased the odds of having a high-incidence herd twelvefold (p<0.01). Keeping cows in only one group in the dry period increased the odds of having a high incidence herd eightfold (p=0.03). Herd size was confounded with housing system. Housing system was therefore added to the final logistic regression model. In conclusion, a large herd size, a high maximum daily milk yield, keeping dry cows in one group, and not cleaning the feeding platform daily appear to be important risk factors for a high incidence of DA or clinical ketosis in Swedish dairy herds. These results confirm the importance of housing, management and feeding in the prevention of metabolic disorders in dairy cows around parturition and in early lactation.

Risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds.

A cross-sectional study was conducted to evaluate the association between herd-level characteristics, management routines and farm-level antibody status of bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) in 257 Swedish dairy herds. The possible spatial clustering of positive herds compared with negative herds was also investigated. For each herd, a pooled milk sample from five primiparous cows was analysed for the presence of antibodies to BCV and BRSV. Herd-level information was obtained by a questionnaire. Logistic regression was used to evaluate the association between predictors and antibody status to BCV and BRSV. Large herd size, being located in southern Sweden, and not providing boots for visitors were found to be associated with being antibody-positive to BCV and BRSV. A short distance to the nearest cattle herd was an additional risk factor for BCV. One of the studied areas was suitable for spatial analysis. Positive herds were not spatially autocorrelated when compared with negative herds as estimated by the K-function regarding both BCV and BRSV. This indicates that local factors such as daily visiting milk trucks and wild animals were unlikely to be important sources of infection in this area. Moran's I statistics and semi-variogram showed no evidence of spatial autocorrelation in the residuals, indicating that remaining unidentified factors are not spatially dependent in the areas under study.

Mononuclear cell subsets in bronchoalveolar lavage fluid during Dictyocaulus viviparus infection of calves: a potential role for gamma/delta TCR-expressing cells in airway immune responses?

Mononuclear cell populations in the lungs of calves infected with Dictyocaulus viviparus were studied during primary infection and reinfection in order to identify cells involved in development of protective immunity to parasitic bronchitis. Three groups of calves were either inoculated with 500 third-stage larvae at both weeks 0 and 10 (n = 6), inoculated only at week 10 (n = 6), or remained uninfected (n = 3). The animals were monitored weekly by collection of bronchoalveolar lavage fluid (BALF), blood and faeces. Among mononuclear BALF-cell populations, the gamma/delta TCR-expressing cells showed a pronounced transient increase in proportion as well as in relative cell size 2 weeks post primary infection, whereas CD4-, CD8-, Ig- and CD14-expressing cells showed no significant differences related to the infection. The increase in gamma/delta TCR-expressing cells coincided with significantly increased proportions of eosinophils and recovery of adult worms in BALF. After reinfection, gamma/delta TCR-expressing cells increased again, but not until week 3 post inoculation, whereas eosinophils were increased by week 2 and reached higher levels than after primary infection. After reinfection, establishment of D. viviparus was less successful than after primary infection. In conclusion, these results indicate a role for gamma/delta TCR-expressing lymphocytes in the pathogenesis of D. viviparus infection.

Impaired glucose tolerance and heterogeneity of insulin responses in cows with abomasal displacement.

Injections of glucagon in dairy cows resulted in a rapid and marked increase in serum insulin and also in plasma glucose. Glucagon injection appears to be a simple and useful method for testing insulin secretion and glucose release. There was a somewhat reduced insulin response 2 weeks after calving in normal cows. Cows given high intensity feeding before calving showed higher plasma glucose levels in the glucagon test 3 days post partum and showed signs of reduced sensitivity to insulin action. Cows with abomasal displacement showed impaired glucose tolerance and heterogeneity of insulin responses indicating insulin resistance and signs of Type 2 (non-insulin-dependent) diabetes mellitus.

Primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf.

Six calves, aged 24 to 58 days and not previously exposed to bovine viral diarrhoea virus (BVDV), were infected with this agent by nose-to-nose contact with a persistently BVDV viraemic calf. The study was conducted in two trials, using 3 calves in each. All 6 calves showed a peak interferon level in serum at 4 days post infection (dpi), and they seroconverted to BVDV at 16-21 dpi. The calves in trial 1 had diarrhoea for 2 or 3 days between 2 and 6 dpi and one calf again from 9 to 11 dpi. During the periods of fever, the calves were slightly depressed. Those in trial 2 were more depressed and their oral and nasal mucous membranes were reddened but they never had diarrhoea. In both trials, fever (up to 41.3 degrees C) was a prominent symptom at 8 to 9 dpi and 2 calves showed a diphasic fever course. Respiratory affection was mild and no medical treatment was required. Haematological assessment demonstrated a transient but significant leukopenia and lymphopenia at 4 dpi (P less than 0.01 and P less than 0.05 respectively) and 11 dpi (P less than 0.05 and P less than 0.01 respectively). A significant decrease in thrombocyte count was seen at 4 dpi (P less than 0.05, n = 3). This study has demonstrated that nose-to-nose contact is an effective way of transmitting BVDV from persistently infected to susceptible cattle.

Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses.

Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3'-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.