Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

Infectivity and persistence of an outbreak strain of Salmonella enterica serotype Typhimurium DT160 for house sparrows (Passer domesticus) in New Zealand

AIM: To examine the infective dose, incubation period and disease progression of an isolate of Salmonella enterica serotype Typhimurium definitive type 160 (DT160) originating from a naturally-infected house sparrow (Passer domesticus) during an outbreak of the disease in New Zealand.

METHODS: Thirty-six house sparrows captured from the wild and free of Salmonella spp were divided into six groups of six birds, housed individually, and inoculated orally with phosphate buffered saline (PBS) or 10¹, 10², 10³, 10⁵, 2 × 10⁸ colony forming units (cfu) of the outbreak strain of S. Typhimurium DT160. The birds were observed for 10 days for clinical signs and/or mortality, and faecal samples were collected to determine excretion of S. Typhimurium. The birds were eutha- nised 11 days post-inoculation (p.i.) and a wide range of tissue samples were collected for histopathological examination, and culture and typing of Salmonella spp. Macro-restriction profiling by pulsed-field gel electrophoresis (PFGE) using XbaI was performed for the epidemiological typing of S. Typhimurium DT160 isolates.

RESULTS: Mortality in house sparrows inoculated with S. Typhimurium DT160 was dose-dependent, and 2/6 birds inoculated with ¹⁰⁵ cfu and all six birds inoculated with 2 × 10⁸ cfu died during the study. Infected sparrows displayed few clinical signs, apart from diarrhoea and/or polyuria, fluffed plumage, and sitting on the floor of the cage. Faecal excretion of DT160 occurred briefly in two birds inoculated with 10² cfu and four birds inoculated with 10³ cfu, on most days in five birds inoculated with 10⁵ cfu, and continuously in six birds inoculated with 2 × 10⁸ cfu. DT160 was isolated from the livers of three birds which received 10³ cfu, five birds dosed with 10⁵ cfu, and all six birds given 2 × 10⁸ cfu. Following necropsy, histopathological lesions similar to those seen in the natural disease were observed in the liver or spleen of three birds which received 10³ cfu, and all birds dosed with ≥10⁵ cfu.

CONCLUSION: The results indicate that an isolate of S. Typhimurium DT60 originating from house sparrows in New Zealand is pathogenic to these birds and that the response is dose- dependent. The persistence and excretion of the pathogen may last for at least 10 days. This confirms that sparrows infected with DT160 could be a source of infection to humans and other in-contact animals.     

Study of Sperm Concentration,Seminal Plasma Composition and their Physiological Correlation in the Persian Sturgeon,Acipenser persicus

The objectives of the present study were to determine ionic and organic composition of seminal plasma, sperm concentration and their relationships in the Persian sturgeon (Acipenser persicus). In this regard, ionic content (Na⁺, K⁺, Cl^?, Ca²⁺ and Mg²⁺) and organic content (total protein, glucose, cholesterol and triglyceride) along with sperm concentration were measured in 17 specimens of the Persian sturgeon. The seminal plasma contained 59.53 ± 2.56 mm /l sodium, 9.1 ± 1.42 mm chloride, 4.72 ± 0.3 mm potassium, 1.45 ± 0.075 mm calcium and 0.7 ± 0.072 mm magnesium. The following organic contents were found: total protein 0.11 ± 0.02 g/dl, glucose 22.18 ± 4.16 mg/dl, cholesterol 6.67 ± 1.04 mg/dl and triglyceride 15.2 ± 0.65 mg/dl. The mean sperm concentration was estimated to be 1.6 ± 0.12 (×10⁹ sperm/ml). A significant relationship was found between sperm concentration and K⁺ of seminal plasma (r = 0.533, p < 0.05). Significant correlations were observed between ionic contents: Na⁺ vs Cl^? (r = ?0.854, p < 0.01) and Mg²⁺ vs K⁺ (?0.583, p < 0.05). Also, level of triglyceride was negatively correlated with Mg²⁺ (r = ?0.503, p < 0.05). Presented data could be considered as a complementary study for developing special extenders and protectant solutions for improving artificial fertilization in this valuable species.     

Optimization Protocol for Storage of Goldfish (Carassius auratus) Embryos in Chilled State

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0°C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat‐stage goldfish embryos were chilled at 0, 4 or 8°C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8°C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8°C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non‐significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat‐stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 μg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

Magnetic microstructure of magnetotactic bacteria by electron holography

Off-axis electron holography in the transmission electron microscope was used to correlate the physical and magnetic microstructure of magnetite nanocrystals in magnetotactic bacteria. The magnetite crystals were all single magnetic domains, and the magnetization directions of small superparamagnetic crystals were constrained by magnetic interactions with larger crystals in the chains. Shape anisotropy was found to dominate magnetocrystalline anisotropy in elongated crystals. A coercive field between 300 and 450 oersted was determined for one chain.     

Crypt abscesses in the caeca of feral rabbits in a rabbit haemorrhagic disease endemic region of New Zealand

AIM: To describe the pathology of crypt abscesses in the caeca of feral rabbits in the Manawatu region of New Zealand, and to examine the possible relationship between their prevalence and rabbit haemorrhagic disease (RHD) virus (RHDV) infection.

METHODS: During the course of a 3-year study of RHD, 173 wild rabbits (Oryctolagus cuniculus) were shot on three pastoral livestock farms in the Manawatu region of New Zealand. All rabbits were necropsied, and tissue samples of the caeca were examined histologically. The age of each animal was determined, and blood samples collected for the detection of RHDV antibodies. Logistic regression was used to model the odds of rabbits having crypt abscesses.

RESULTS: At necropsy, 63/173 (36.4%) rabbits were found to have small circular black nodules on the mucosal surface of their appendix caecum and/or sacculus rotundus. Microscopically, these were identified as small crypt abscesses composed of dilated sacs at the base of the mucosa that were often lined by a thin layer of attenuated epithelial cells. They usually contained large amounts of concentrically-laminated mucopolysaccharide material that was sometimes pigmented, inflammatory debris, and were often the site of a moderate multifocal appendicitis. Although RHDV was active in the study area, no association was found between RHDV antibodies in serum and the presence of lesions. The lesions were more common in older individuals and those born in summer or autumn.

CONCLUSIONS: This is the first report of crypt abscesses with inflammation and necrotic debris in the caeca of rabbits. No association between the occurrence of crypt abscesses and RHDV infection was identified. Wild rabbits born in a particular season were presumably exposed very early in life to conditions that caused the crypt abscesses to develop. Alternatively, association with season of birth may represent rabbits that were of similar age in a later stage of their lives, when they became exposed to the cause of the lesions, which remains unidentified.     

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll^®, Puresperm^® and Bovipure^?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure^? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll^® and Puresperm^® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll^® selected less live and more apoptotic spermatozoa than Puresperm^® and Bovipure^? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.     

Growth Hormone Receptors in the Porcine Testis During Prepuberty

Supplementation of exogenous growth hormone (GH) during prepuberty advances onset of spermatogenesis in boars, but the mechanism of action is unknown. The present study is an investigation of the presence and characteristics of testicular growth hormone receptors (GHR). A total of 36 boars were castrated, three boars every 10 days, between the ages of 10 and 120 days. Testicular membrane preparations of 10, 20, 30, 50, 70, 100 and 120‐day‐old boars were used to determine ¹²⁵I‐bGH binding and Scatchard analysis. Liver from a 60‐kg barrow was used for comparison. Specific ¹²⁵I‐bGH binding to testicular membrane preparations occurred in all age groups with the exception of 20‐day‐old boars at levels of 30–40% of liver binding. At 30 days of age the unlabelled bGH at 1.1 ng/tube achieved half maximal inhibition (ID₅₀). Results of Scatchard analysis indicated a single class of binding sites. Binding affinity was 2.89 × 10⁹m with a binding capacity of 12 fmole/mg membrane protein. The results from this study suggest that GH may act directly on the cells of the prepubertal boar testis.     

Fatal levamisole toxicosis of captive kiwi (Apteryx mantelli)

CASE HISTORY: Nine of 24 captive kiwi treated with oral levamisole at a dose between 25–43 mg/kg showed signs of respiratory distress. Six died within 4 h of treatment and the remaining three made a full recovery within 24 h.

CLINICAL AND PATHOLOGICAL FINDINGS: Within 3–4 h of treatment, the affected birds had an elevated respiratory rate, mucoid nasal discharge and rapidly became comatose. Post mortem examination revealed accumulation of thick mucus in the oral cavity and trachea. There was severe pulmonary congestion and oedema and early bronchopneumonia in the lungs of five of the birds. In two birds, there was acute hepatic degeneration and necrosis and one bird had acute pancreatic degeneration and necrosis.

DIAGNOSIS: Acute levamisole toxicity.

CLINICAL RELEVANCE: Kiwi were acutely sensitive to levamisole toxicity at doses that are well within the safe range for domestic poultry. Levamisole should not be used as an anthelmintic in kiwi.