Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds.

METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination.

RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58–0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive.

CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.     

The effect of Johne's vaccination on tuberculin testing in farmed red deer (Cervus elaphus)

AIM: To assess the degree of interference with bovine tuberculin testing in farmed red deer that vaccination of young deer with an oil-adjuvanted vs aqueous formulation of live attenuated Mycobacterium paratuberculosis Strain 316F vaccines would be likely to cause, and to compare immunological responses between vaccine formulations.

METHODS: Five-month-old red deer (n=45) were randomly allocated to three treatment groups of 15 animals, which received either no vaccine, a single 2-ml dose of an oil-adjuvanted formulation or two 2-ml doses, 6 weeks apart, of an aqueous formulation of live attenuated M. paratuberculosis Strain 316F vaccine injected subcutaneously (S/C) in the neck (Control, Oil-adjuvant Ptb, and Aqueous Ptb groups, respectively). Injection-site reactions were described and measured on Weeks 3, 6 and 9. Animals were weighed and lymphocyte transformation tests (LTT) and antibody enzyme-linked immunosorbent assays (ELISA) using avian, bovine and Johnin tuberculin were conducted on blood samples collected at Weeks 0, 6, 12, 15, 24, 27, 36 and 39. A bovine mid-cervical skin test (MCT) was applied at Week 12, and comparative cervical skin tests (CCTs) at Weeks 24 and 36. At Week 42, the animals were slaughtered at a commercial deer slaughter premises and subjected to rigorous meat inspection.

RESULTS: Two animals were eliminated at the start of the trial due to a positive cross-reaction with bovine tuberculin in the initial LTT. Almost all animals reacted to the MCT at Week 12, with mean skin thicknesses of 3.9, 2.9 and 1.0 mm for the Oil- adjuvant Ptb, Aqueous Ptb and Control groups, respectively. When the CCT was conducted at Week 24, 2/15 Oil-adjuvant Ptb, 2/14 Aqueous Ptb and 1/14 Control animals were classified as CCT-positive to bovine tuberculin. By Week 36, all animals were CCT-negative. The Oil-adjuvant Ptb vaccination resulted in high persistent levels of antibody that reacted with bovine tuberculin, compared with negligible levels in the Aqueous Ptb group.

Overall, a single dose of the Oil-adjuvant Ptb vaccine in deer stimulated a vigorous, cross-reactive immune response, evidenced by high LTT, skin-test and antibody reactions to bovine tuberculin, with both cell-mediated and humoral characteristics. By comparison, two doses of the Aqueous Ptb vaccine produced less cross-reactivity and a bias towards a cell-mediated response. The Oil-adjuvant Ptb vaccine resulted in moderate injection- site lesions that were quite persistent, whereas the Aqueous Ptb vaccine resulted in smaller nodules that regressed more quickly.

CONCLUSIONS: Vaccination of farmed deer with an oil-adjuvanted Johne's vaccine has the potential to cause significant interference with routine tuberculin skin testing. The cross-reactivity should decline with time and the CCT should be able to clear MCT-positives, but there is a risk of false-positives to the blood test for tuberculosis (BTB), due to high persistent levels of antibody. The CCT could be used as a primary skin test in vaccinated deer on some farms.

The Aqueous Ptb caused fewer problems with skin testing and produced significantly less bovine antibody than the Oil-adjuvant Ptb, but stimulated persistent cell-mediated immune responses that may provide some protection against Johne's disease.     

Idiopathic eosinophilic meningoencephalitis in Rottweiler dogs: three cases (1992–1997)

We report three cases of eosinophilic meningitis in young male Rottweiler type dogs in New Zealand. No cause for the disease was identified. There were variable clinical signs referable to central nervous system dysfunction, and a variable response to treatment.     

In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self‐renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21‐day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Etiology and real-time polymerase chain reaction-based detection of gremmeniella- and phomopsis-associated disease in norway spruce seedlings

ABSTRACT In spring 2002, an unusual disease outburst was recorded on Norway spruce seedlings in southeast Norway. Extensive damage was recorded on 1- and 2-year-old Norway spruce seedlings that either had wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterized by leader shoot dieback and stem necroses on the upper or lower part of the shoot from 2001. Gremmeniella abietina and a Phomopsis sp. frequently were isolated from the diseased seedlings. Internal transcribed spacer (ITS) ribosomal (r)DNA sequence analysis and random amplified microsatellites profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to the large-tree type (LTT) ecotype of the European race of G. abietina var. abietina, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on ITS rDNA sequence analysis, the Phomopsis strains associated with diseased seedlings did not represent any characterized Phomopsis spp. associated with conifers. The Phomopsis sp. was not pathogenic in inoculation tests, indicating that it may be a secondary colonizer. ITS-based real-time polymerase chain reaction assays were developed in order to detect and quantify G. abietina and Phomopsis in the nursery stock. We describe here the G. abietina-associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst likely was related to the G. abietina var. abietina epidemic caused by the LTT on large Scots pines in 2001.     

Evaluation of action thresholds for chronic rice insect pests in the Philippines. I. Less frequently occurring pests and overall assessment

Action thresholds as decision tools for insecticide application were developed and tested against the major insect pests of rice at four sites in the Philippines over a 13-year period. Action threshold treatments were compared to the farmers' practice, prophylactic insecticide usage, and an untreated check. Yield loss data using the insecticide check method partitioned yield losses over three crop growth stages in the same test fields. Chronic pests that exceeded action thresholds in 79% of fields were whorl maggot Hydrellia philippina Ferino (Diptera: Ephydridae), defoliators Naranga aenescens Moore and Rivula atimeta (Swinhoe) (Lepidoptera: Noctuidae), leaffolders Cnaphalocrocis medinalis (Guenée) and Marasmia patnalis Bradley (Lepidoptera: Pyralidae), and stemborers Scirpophaga incertulas (Walker) and S. innotata (Walker) (Lepidoptera: Pyralidae). Minor chronic pests reached threshold levels in only one site each: rice bug Leptocorisa oratorius (F.) (Koronadal), whitebacked planthopper Sogatella furcifera (Horvath) (Zaragoza) and green leafhopper Nephotettix virescens (Distant) (Guimba); brown planthopper Nilaparvata lugens (Stål) did not exceed a threshold in any field. Stemborers were the most important pest group in terms of yield loss. Despite the insecticide check method underestimating losses, a mean crop loss of 0.62 t/ha was measured which showed ample scope for corrective action. But loss was evenly distributed across crop growth stages (0.15?–?0.24 t/ha) reducing the impact of insecticides. Action threshold treatments overall outyielded the untreated check, more so in the two sites with highest pest density. The benefit of thresholds was to reduce insecticide usage, as a cost saving. However all the practices showed poor economic returns including the farmers' practice. Farmers' practice employed low insecticide dosages and timing was not consistent with pest damage, but yields were often similar to threshold treatments. Farmers appear to use insecticide more for risk aversion than for profit. The best threshold characters when evaluated against resulting pest density and yield loss criteria showed accuracies >?90% correct decisions. Future work is needed to improve the insecticide response rather than monitoring tools. Thresholds need to be incorporated into improved crop management, which was often found suboptimal by farmers, to take advantage of the high levels of tolerance in modern high tillering cultivars. Crop husbandry practices which improve yield potential such as selection of longer maturing varieties and nitrogen fertilizer may be a more effective pest management strategy than insecticides.     

Dermatoses caused by infestations of immature Ixodes spp. on dogs and cats in Sydney, Australia

Infestations of larval and nymphal Ixodes spp. were identified in 16 dogs and 16 cats from several small animal clinics in Sydney. Cases occurred in late summer or autumn, peaking in February, and were seasonally recurrent in some individuals. Clinical signs of infestation included a papular dermatitis and irritation or pruritus that ranged from severe to mild or absent. The distribution of tick attachment tended to be cranial and ventral, with the face, legs, axillae and ventrum the most commonly affected sites. The estimated number of ticks in each infestation varied from less than 10 to more than 100. Basic morphological examination of ticks collected from affected animals was performed by attending veterinarians using light microscopy, and larvae and nymphs belonging to the Ixodes genus were identified. Ticks collected from 17 animals and submitted to the Department of Medical Entomology, Westmead Hospital were putatively identified as I. trichosuri (57%) and I. holocyclus (25%) larvae. Histopathological samples of attachment sites collected from three dogs and one cat were characterised by ticks attached in well-demarcated invaginations of the skin ('tick craters') associated with variable epidermal and/or dermal necrosis, focal eosinophilic intraspinous pustules, mild to marked eosinophilic and neutrophilic, superficial to deep, dermal perivascular to interstitial inflammation, and moderate to marked superficial dermal oedema and red cell extravasation. A range of topical acaricidal preparations, including fipronil and synthetic pyrethroids, were used for treatment.