Distribution of Mycobacterium avium subsp. paratuberculosis in a Subclinical Naturally Infected German Fleckvieh Bull.

Although it has been known for years that Mycobacterium avium subsp. paratuberculosis (MAP) is detectable in the reproductive organs and semen of infected bulls, only few studies have been conducted on this topic worldwide. This study surveyed the MAP status of a bull, naturally infected due to close contact with its subclinically infected parents over a period of 4 years. From the age of 7 weeks to necropsy, faecal, blood and, after sexual maturity, semen samples were drawn repeatedly. Already at the first sampling day, MAP-DNA was detected in faeces by semi-nested PCR. True infection was confirmed by the detection of MAP-DNA in blood at the age of 40 weeks. In total, MAP-DNA was present in 25% faecal (34/139), 16% blood (23/140) and 5% semen (4/89) samples, including MAP-free intervals of up to 9 weeks. MAP genome equivalents (MAP-GE) of up to 6.3 × 106 /g faeces and 1.8 × 105 /ml blood were determined. Cultivation of MAP occurred only in three of 137 faecal and two of 109 blood, but never in semen samples. Over the whole period, the bull was a serological negative MAP shedder. During necropsy, 42 tissue samples were collected. Neither macroscopic nor histological lesions characteristic of a MAP infection were observed. Cultivation of MAP in tissue sections failed. However, MAP-DNA was spread widely in the host, including in tissues of the lymphatic system (7/15), digestive tract (5/14) and the urogenital tract (5/9) with concentrations of up to 3.9 × 106 MAP-GE/g tissue. The study highlighted the detection of MAP in male reproductive organs and semen. It supports the hypothesis that bulls may probably transmit MAP, at least under natural mating conditions. In artificial insemination, this might not be relevant, due to antibiotics included currently in semen extenders. However, the survivability of MAP in this microenvironment should be investigated in detail.

Evaluation of two commercial, rapid, ELISA kits testing for scrapie in retro-pharyngeal lymph nodes in sheep.

AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes. METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID). RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3-75.3)% for ELISA-BR and 91.6 (95% CI=88.2-94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID. CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity. CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.

A longitudinal study to characterize the distribution patterns of Mycobacterium avium ssp. paratuberculosis in semen, blood and faeces of a naturally infected bull by IS 900 semi-nested and quantitative real-time PCR.

Johne's disease is caused by Mycobacterium avium ssp. paratuberculosis (MAP) and has been recognized as an important bacterial infection in ruminants. Although MAP has been detected in semen and within the reproductive organs of bulls, the bacterial distribution and shedding patterns are currently not well characterized. Our investigation was performed to detect and quantify MAP in faeces, semen and blood samples repeatedly drawn from a naturally infected but asymptomatic 18-month-old German Simmental breeding bull candidate over a period of 3 years (June 2007-November 2010). Qualitative and quantitative polymerase chain reaction (PCR) techniques were used to correlate the presence and matrix-specific amounts of MAP. In total, 65 sampling dates were selected. Mycobacterium avium ssp. paratuberculosis was detected intermittently in all matrices with MAP-free intervals of up to 18 weeks by an IS900 semi-nested PCR. The number of MAP-positive results from semen and blood samples was higher than from faecal samples. A quantitative polymerase chain reaction detected the highest MAP contents in faeces (10(3) -10(6) MAP/g), while lower amounts were found in semen and blood samples (10(2) -10(5) MAP/ml). Although no significant agreement was calculated between the presence of MAP in faeces and blood, a statistically significant positive correlation between its occurrence in semen and blood was determined (r = 0.38, P < 0.05, n = 29). The present study contributes to a more detailed understanding of MAP distribution patterns in faeces, semen and blood of a subclinically infected breeding bull candidate. It highlights the possible role of breeding bulls as a source of MAP transmission and indicates the need for further monitoring and hygienic measures to prevent the spread of the infection via semen.

Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.

The paraffin-embedded tissue (PET) blot method was used to investigate sections of the central nervous system and lymphatic tissues from 24 cases of classical scrapie and 25 cases of atypical/Nor98 scrapie in sheep and four healthy control sheep. The PET blot detected deposits of PrP(Sc) in the brain tissue of all 49 sheep with scrapie but no PrP(Sc) labelling could be detected in the control sheep. By contrast, not all the atypical/Nor98 scrapie cases were detectable by immunohistochemistry. The high sensitivity of the PET blot method made it possible to observe that in some atypical/Nor98 cases, deposits of PrP(Sc) may be restricted to supratentorial brain structures and that the diagnosis may be missed when only testing the obex area, where deposits are common in classical scrapie, and the cerebellar structures, where deposits are considered to be common in atypical/Nor98 cases.

[Case-report. Malignant nerve sheath tumor in a cow with symptoms of suspected BSE]. / Fallbericht: Maligner Nervenscheidentumor bei einer Kuh mit BSE-verdächtiger Symptomatik.

We report here on a 3 1/2-year-old mother cow with a malignant perineural tumour near the pontine angle of the cerebellum, but which first drew attention because of clinical signs of BSE. Neurological symptoms that manifested during the course of the disease included disturbances in behaviour, movement and aesthesia, as described by BRAUN et al. (2001) in cases of BSE. Inconsistent with a diagnosis of BSE were focal neurological disturbances (head held aslant to the right, tendency to fall to the right, right-sided facial weakness, left-sided nystagmus and ventral strabismus). Following euthanasia, magnetic resonance imaging (MRI) revealed a tumour in the cerebellopontine angle. Histological findings describe a malignant peripheral nerve tumour of the vagal nerve with rhabdoid differentiation (a so-called Triton tumour) with an intracranial and an extracranial part.

[The effect of vitamin A and beta-carotene on the vitamin E status, ejaculation parameters and health of boar used for insemination]. / Einfluss von Vitamin A und beta-Carotin auf Vitamin E-Haushalt, Ejakulatparameter und Gesundheit beim Besamungseber.

In this study consequences of vitamin A-supplementation to the vitamin E-status was investigated in the boar. Three groups of boars, each with 9 animals were fed over a period of seven month with 30000 I.E. Vit. A/kg concentrate (group A), 90 mg b-carotene + 1000 I.E. Vit. A/kg (group B) and 1000 I.E. Vit. A/kg (group C). Every boar was given 100 mg Vit. E/kg plus 50 ml soybean oil/kg to induce oxidative stress. After four month group C showed a higher amount of tocopherol in serum (p < 0.05). The amount of tocopherol in serum of the group B were exactly between group A and C. The amount of retinol in serum of the group C began to decrease after three month due to the high reserve capacity of the liver (p < 0.01). The retinyl ester in serum reflected the state of supply. 90 mg b-carotene led to an efficiency of 15000 I.E. Vit. A. The vitamin antagonism between Vit. A and Vit. E is not based on an antagonism of the intestinal resorption. There was no influence on the daily sperm production caused by different supplementations. The sperm quality was lowered in group C; the number of defective sperm increased (p < 0.001). The supplementation of soybean oil lead to an increase of the saturated fatty acids in the fatty acid pattern of the sperm cells. The increase of saturated fatty acids was the lowest in group C that showed the highest amount of tocopherol in serum.

[Follow-up studies of the detection of IgG1 antibodies to Coxiella burnetii in blood and milk from cattle in relation to the reproductive cycle and milk production]. / Verlaufsuntersuchungen zum Nachweis von IgG1-Antikörpern gegen Coxiella burnetii in Blut und Milch beim Rind unter Berücksichtigung von Reproduktionszyklus und Milchleistung.

During a 14 month period, 36 dairy cattle were selected from a herd (n = 103), after IgG1 antibodies to Coxiella burnetii had repeatedly been detected in blood and milk, as measured by ELISA. ELISA results were analyzed with reference to reproductive cycle, daily milk yield, as well as daily concentration, and output of milk protein. The rate of positive blood samples slightly grew from 47.6% in the non-pregnant period, and 46.8% in the first half of pregnancy to 52.0% in the second half of pregnancy. In contrast, results of milk ELISA changed significantly from non-pregnant period to first, and second half of pregnancy, with increases from 23.8 to 38.5, and 64.6% of positive milk samples. Moreover, milk samples showed negative correlations between ELISA OD405 values and daily milk yield, as well as daily output of milk protein.

[Evaluation of blood parameters from dairy cow stock with fertility disorders]. / Auswertung von Blutparametern aus fruchtbarkeitsgestörten Milchviehbeständen.

The evaluation is based on 1329 blood samples from 199 farms using the offer of a fertility and feeding advisory programme. Total bilirubin, AST (GOT), GLDH, glucose, urea, inorganic phosphorus, and beta-carotene served as parameters. Considerable variations of all the parameters depending on the lactation level could be demonstrated. The problem of standardisation is discussed. Increased total bilirubin values (acute metabolic stress) often occurred one or two weeks post partum (26.9% of the cows). During lactation, the marginal value was exceeded by 24.6% of the cows. AST (GOT) values beyond the standard were seen in 19.4-30.1% of the cows, depending on the lactation level. These liver diseases were verified by a 25.5% increase in GLDH-values. Hypoglycaemic cows ranged between 18.5 and 28.5%. Oversupply of proteins shows a decreasing tendency (25.7%). During the winter months the beta-carotene supply is still a problem, with 40% of the cows not reaching the standard values.

On the interaction of bull and boar acrosins with the zona pellucida of different mammalian species in vitro.

Acrosin was prepared from boar and bull spermatozoa and its lytic effect in vitro on the zona pellucida of mouse, golden hamster, rabbit, pig and cow was investigated. Depending upon the species studied, ovarian oocytes, ovulated oocytes and preimplantation embryos were obtained for the experiments. While in golden hamster and rabbit the zona pellucida was removed by both acrosins, this effect was absent for bull acrosin in cow and mouse eggs and for boar acrosin in pig and mouse eggs. In those species in which the zona pellucida was not removed by the acrosins after an incubation period of 2 hours even a prolongation up to 24 hours with higher amounts of acrosin, the addition of acrosomal extracts to the incubation buffer (Tyrode solution pH 7.2) or an increase of the pH value up to 8.6 of the acrosin solution had no effect upon the zona pellucida. Our results indicate that at least in vitro, acrosin does not possess the capacity to lyse the zona pellucida in a species specific fashion. Since the lytic effect of boar and bull acrosin on the zona pellucida of ovarian oocytes and preimplantation embryos is not different from that on ovulated oocytes it can be assumed that neither the maturation of the zona pellucida during oogenesis nor its modification after fertilization, change the susceptibility of the zona pellucida to acrosin digestion.