Acquisition of haemoglobin-bound iron by Histophilus somni

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2,336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an approximately 120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.     

Loss of reserves of Cu in liver when Cu supplements are withdrawn from dairy herds in the Waikato region

Abstract

AIM: To monitor the consequences of withdrawing mineral Cu supplements from two dairy herds with initially high concentrations of Cu in liver.

METHODS: Two herds were selected from dairy farms in the Waikato region of New Zealand that participated in an earlier survey of Cu supplementation practices and Cu status of dairy cows. The herds were fed pasture, grass and maize silage, plus palm kernel expeller (PKE) containing 25–30 mg Cu/kg dry matter (DM) fed at 2–4 kg/cow/day. No mineral Cu supplements were supplied from January 2009. Pasture samples were collected for mineral analysis in September 2008 and April 2009. Concentration of Cu in liver biopsies from the same 9–10 cows per herd was measured on three occasions between April 2009 and May 2010.

RESULTS: Pastures on both farms contained 10 mg Cu/kg DM, 0.1–0.5 mg Mo/kg DM and 3.5–4.0 g S/kg DM. The initial herd mean concentrations of Cu in liver were 1,500 (SD 590) and 1,250 (SD 640) μmol Cu/kg fresh tissue. In the absence of mineral Cu supplements, those mean concentrations decreased over 12 months to 705 (SD 370) and 1,120 (SD 560) μmol Cu/kg fresh tissue, respectively. For cows in the first herd, the rate of depletion of liver Cu reserves was influenced by initial concentration of Cu, such that high concentration led to faster loss according to first-order kinetics.

CONCLUSIONS: Mineral Cu supplementation was not necessary over 12 months for two dairy herds with mean concentrations of Cu in liver >1,250 μmol Cu/kg fresh tissue, grazing pastures containing 10 mg Cu/kg DM and concentrations of Mo <1 mg/kg DM. The quantity and particularly the duration of feeding PKE appeared to be a factor in whether or not the herd lost substantial reserves of Cu in liver during the year. However, the Cu status of both herds in this study was more than adequate to support late pregnancy and mating.

CLINICAL REVELANCE: Copper status of the herd should be monitored and on-farm management of Cu nutrition should take into account all sources contributing to daily intake of Cu. Where Cu supplementation has been excessive and there is risk of chronic Cu toxicity, mineral Cu supplements may be withdrawn for a period commensurate with the expected rate of liver Cu depletion.     

Evaluation of age-dependent susceptibility in calves infected with two doses of Mycobacterium avium subspecies paratuberculosis using pathology and tissue culture

The longstanding assumption that calves of more than 6 months of age are more resistant to Mycobacterium avium subspecies paratuberculosis (MAP) infection has recently been challenged. In order to elucidate this, a challenge experiment was performed to evaluate age- and dose-dependent susceptibility to MAP infection in dairy calves. Fifty-six calves from MAP-negative dams were randomly allocated to 10 MAP challenge groups (5 animals per group) and a negative control group (6 calves). Calves were inoculated orally on 2 consecutive days at 5 ages: 2 weeks and 3, 6, 9 or 12 months. Within each age group 5 calves received either a high – or low – dose of 5 × 10⁹ CFU or 5 × 10⁷ CFU, respectively. All calves were euthanized at 17 months of age. Macroscopic and histological lesions were assessed and bacterial culture was done on numerous tissue samples. Within all 5 age groups, calves were successfully infected with either dose of MAP. Calves inoculated at < 6 months usually had more culture-positive tissue locations and higher histological lesion scores. Furthermore, those infected with a high dose had more severe scores for histologic and macroscopic lesions as well as more culture-positive tissue locations compared to calves infected with a low dose. In conclusion, calves to 1 year of age were susceptible to MAP infection and a high infection dose produced more severe lesions than a low dose.     

A non-invasive technique for standing surgical repair of urinary bladder rupture in a post-partum mare: a case report

An 11-year-old mare presented 36 hours after foaling with a ruptured bladder. Uroperitoneum was diagnosed on ultrasound and from the creatinine concentration of the peritoneal fluid. Bladder endoscopy demonstrated tissue necrosis and a rent in the dorsocranial aspect of the bladder. Following stabilisation, including abdominal drainage and lavage, the mare was taken to standing surgery. Under continuous sedation and epidural anaesthesia, and after surgical preparation, a Balfour retractor was placed in the vagina. Using sterile lubricant and moderate force, it was possible to insert a hand into the bladder. The tear was easily palpable on the dorsal portion of the bladder. Two fingers were inserted through the tear and used to provide traction to evert the bladder completely into the vagina where it could grasped with the surgeons other hand to prevent further trauma. A second surgeon could then visualise the entire tear and repaired this using a single layer of size zero PDS suture in a single continuous pattern. As soon as the bladder was repaired, it was replaced via the urethra. The mare did well after surgery and was discharged after 48 hours, apparently normal.This report is the first describing repair of the bladder without an abdominal incision or incision into the urethral sphincter. This greatly reduces the chance of possible complications such as urine pooling after surgery with the previously described standing technique or bladder trauma due to traction with abdominal surgery.     

Effects of soil compaction and arbuscular mycorrhiza on corn (Zea mays L.) nutrient uptake

Soil compaction is of great importance, due to its adverse effects on plant growth and the environment. Mechanical methods to control soil compaction may not be economically and environmentally friendly. Hence, we designed experiments to test the hypothesis that use of plant symbiotic fungi, arbuscular mycorrhiza (AM) may alleviate the stressful effects of soil compaction on corn (Zea mays L.) growth through enhancing nutrient uptake. AM continuously interact with other soil microorganisms and its original diversity may also be important in determining the ability of the fungi to cope with the stresses. Hence, the objectives were: (1) to determine the effects of soil compaction on corn nutrient uptake in unsterilized (S1) and sterilized (S2) soils, and (2) to determine if inoculation of corn with different species of AM with different origins can enhance corn nutrient uptake in a compacted soil. Using 2 kg weights, soils (from the field topsoil) of 10 kg pots were compacted at three and four levels (C1, C2, C3 and C4) (C1 = non-compacted control) in the first and second experiment, respectively. Corn (cv. 704) seeds were planted in each pot and were inoculated with different AM treatments including control (M1), Iranian Glomus mosseae (M2), Iranian G. etunicatum (M3), and Canadian G. mosseae, received from GINCO (Glomales In Vitro Collection), Canada (M4). Corn leaf nutrient uptake of N, P, K, Fe, Mn, Zn and Cu were determined. Higher levels of compaction reduced corn nutrient uptake, however different species of AM and soil sterilization significantly increased it. The highest increase in nutrient uptake was related to P (60%) and Fe (58%) due to treatment M4S2C3. Although it seems that M3 and M4 may be the most effective species on corn nutrient uptake in a compacted soil, M2 increased nutrient uptake under conditions (C3 and C4 in unsterilized soil) where the other species did not. Through increasing nutrient uptake AM can alleviate the stressful effects of soil compaction on corn growth.     

African Horse-Sickness Killed-Virus Tissue Culture Vaccine

Formalized African horse-sickness (AHS) type 9 virus cultivated in monkey kidney stable (MS) cell cultures was experimentally used for immunizing horses. Inactivated vaccines prepared either from viscerotropic or neurotropic type 9 AHS virus produced antibodies in vaccinated horses. Immunity developed in all horses vaccinated with various amounts of the vaccine, and protected them from infection, when challenged 5 weeks after vaccination.     

Evaluation of Fertilizing Potential of Frozen-thawed dog Spermatozoa Diluted in ACP-106® using an In Vitro Sperm–Oocyte Interaction Assay

The aim of present study was to evaluate frozen canine semen with ACP-106 (Powder Coconut Water) using an in vitro sperm--oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1% and 94.3 +/- 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 was efficient for maintain the in vitro fertility potential of dog spermatozoa.     

Polymerase chain reaction and other laboratory techniques in the diagnosis of long incubation rabies in Australia

SUMMARY Blood and post-mortem tissues from a 10-years-old girl were submitted to the Australian Animal Health Laboratory. Clinical signs and histopathological lesions had suggested a diagnosis of rabies, but, an unusually long incubation period of at least 5 years did not encourage such a diagnosis. Serological examinations by the rapid fluorescent focus inhibition test revealed a dramatic increase in rabies virus-neutralising antibody during the 10-day period of hospitalisation. The results of a fluorescent antibody test on brain smears, and an immunoperoxidase test on formalin-fixed sections of brain were also consistent with a diagnosis of rabies. Attempts to isolate virus were unsuccessful. Polymerase chain reactions (PCRs) were conducted on a 10% suspension of a post-mortem sample from the patient's brain, using primers based on the published sequence of the Pasteur virus strain of rabies virus. 413 and 513 bp fragments from the nucleoprotein gene and a 403 bp fragment from the glycoprotein gene were amplified. Subsequent sequencing of these fragments, and comparison with equivalent regions of known rabies viruses, confirmed that the fragments originated from a virus belonging to the rabies virus serotype. This case demonstrated the advantage of using a range of laboratory techniques to obtain a definitive diagnosis. In particular, a PCR-based test may allow a diagnosis, even in the face of conditions that preclude virus isolation such as apparently occurred in this case. Finally, this case demonstrated that an unusually long incubation period should not discourage a tentative clinical diagnosis of rabies.     

Factors affecting the breeding success of the African Black Oystercatcher (Haematopus moquini): a perspective on protection and food availability

Breeding success (fledglings pair^?1 y^?1) of the Red-listed African Black Oystercatcher (Haematopus moquini) is highly variable, both spatially and temporally. Despite a diversity of natural factors causing this variability, there is evidence that two anthropogenic factors, i.e. disturbance and an introduced mussel (Mytilus galloprovincialis), are having an impact on the local breeding success of this species. Using a data set comprising 87 site-years of nest-monitoring data across most of the species’ breeding range, we analysed the extent and causes of variability in breeding success. Breeding success differed across three population categories defined by varying levels of human disturbance: island populations, protected mainland populations, and unprotected mainland populations. Differences in breeding success between island populations and protected mainland populations were likely due to differing exposure to predators; however, differences between protected and unprotected mainland populations were unlikely caused by this as both experience equivalent predation levels (although from different predators). Protection only improved the breeding success of oystercatchers in very high-quality habitats (with a high biomass of alien mussels), and where populations were ‘released’ from high levels of human disturbance. In unprotected mainland areas, human activity impacted on the breeding success of local populations primarily through predation of small chicks by uncontrolled dogs, and by rising tides drowning chicks that were hiding from human disturbance. The findings of this study note the potential conservation dilemma resulting from an invasive species improving the conservation status of a Red-listed species, and encourage the implementation of restricted sites in high-quality habitats with high breeding pair densities.