A Review of Methods Used in the Laboratory Diagnosis of Fireblight1)

Most well–known laboratory methods which can be used in the diagnosis of fireblight, as well as more recent developments in identification, have been discussed. They vary from the use of simple culture media to the use of immunofluorescent microscopy. The use of any one method alone is not advised, but by combining methods, accurate and rapid diagnosis is possible. In order to minimize diagnostic errors, especially among less experienced workers, the use of 5 % sucrose–nutrient agar for bacterial isolation followed by serological control is recommended. For further information reference is given particularly to the procedures described by Lelliott at the first EPPO Conference on Fireblight held in 1967 at Canterbury.     

Seroprevalence of Canine Herpesvirus‐1 in the Belgian Dog Population in 2000

Canine herpesvirus‐1 (CHV‐1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme‐linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV‐1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV‐1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non‐breeding dogs.     

Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus)

Abstract

CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summer of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds.

CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birdsbelonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater).

DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined.

CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.     

Identification of the DDAH2 Protein in Pig Reproductive Tract Mucus: A Putative Oestrus Detection Marker

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus‐specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous‐ and oestrous‐stage mucus proteins were purified and analysed with proteomic tools such as two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization–time‐of‐flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6‐fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti‐DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types.

LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR.

A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%).

DIAGNOSIS: Bovine haemoplasmosis.

CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.     

Effect of Dimethyl Sulfoxide on Cell Cycle Synchronization of Goldfish Caudal Fin Derived Fibroblasts Cells

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin‐derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.     

Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.     

Captive Breeding of Cheetahs in South Africa – 30 Years of Data from the de Wildt Cheetah and Wildlife Centre

The de Wildt Cheetah and Wildlife Centre was established in 1971 and the first cheetah cubs were born in 1975. During the period 1975–2005, 242 litters were born with a total of 785 cubs. Mean cub survival from 1 to 12 months and greater than 12 months of age was 71.3 and 66.2%, respectively. The majority of losses (84.9%) occurred during the first month postpartum whereas only 15.1% deaths took place between 1 and 12 months of age. Females were first bred at an age of approximately 3 years, reached maximum reproductive age at 6–8 years, where after fertility declined. Males reached peak reproduction at 6 and maintained this for up to 12 years of age. Male fertility was best correlated with sperm morphology. During recent years, for practical purposes, males were allocated to 'good' (≥70% normal), 'fair' (40–70% normal) and 'poor' (<40% normal) categories according to sperm morphology count. The breeding males were selected from the good (preferably) and fair categories but poor category males were also used at times. Average litter sizes for 'good', 'fair' and 'poor' males were 3.44 (n = 21), 3.14 (n = 18) and 2.28 (n = 18), respectively. In females the heritability for litter size was high at 0.5848 (532 progeny, 1975–2007) and the maternal heritability for cub mortality was estimated to be 0.596. The data from the de Wildt Cheetah and Wildlife Centre and two other centres in the world (Kapama and Wassenaar) demonstrate that cheetah can be bred successfully in captivity.