Pathogenicity of the bacterium New Zealand rickettsia-like organism (NZ-RLO2) in Chinook salmon Oncorhynchus tshawytscha smolt.

Farmed New Zealand Chinook salmon Oncorhynchus tshawytscha Walbaum have been found to be infected by rickettsia-like organisms (NZ-RLO). While these Gram-negative intra-cellular bacteria are closely related to Piscirickettsia salmonis, a significant pathogen for farmed salmon globally, the pathogenicity of NZ-RLO is unknown. The aim of the present study was to determine if one strain, NZ-RLO2, causes disease in Chinook salmon. Post-smolt salmon were inoculated with NZ-RLO2 by intraperitoneal injection at high, medium and low doses and observed for 30 d. All fish in the high and medium dosed groups died by the end of the study and 63% of the low dose group died within 30 d of inoculation. Necropsy revealed the fish inoculated with NZ-RLO2 had internal multifocal haemorrhages. The most consistent histological finding in fish inoculated with NZ-RLO2 was neutrophilic and necrotizing pancreatitis and steatitis with intra-cytoplasmic organisms often visible within areas of inflammation. Other histological lesions included multifocal hepatic necrosis, haematopoietic cell necrosis and splenic and renal lymphoid depletion. The presence of NZ-RLO2 within the inoculated fish was confirmed by replication in cell culture and qPCR. The results suggest NZ-RLO2 can cause disease in Chinook salmon and therefore could be a significant pathogen in farmed Chinook salmon.

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand.

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons. LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D752 that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak. DIAGNOSIS: Equine herpesvirus myeloencephalopathy. CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.

New Zealand juvenile oyster mortality associated with ostreid herpesvirus 1-an opportunistic longitudinal study.

During the 2010-11 summer outbreak of ostreid herpesvirus 1 (OsHV-1) in New Zealand, an opportunistic longitudinal field study was conducted. OsHV-1 PCR-negative oyster spat (Crassostrea gigas) were relocated to an OsHV-1 PCR-positive area of the North Island of New Zealand that was experiencing juvenile oyster mortalities. Over a period of 13 d, spat were monitored for mortality, sampled for histopathology, and tested for the presence of OsHV-1 using real time PCR and Vibrio culture. Histopathology showed some evidence of tissue pathology; however, no consistent progressive pathology was apparent. Field mortalities were evident from Day 6 on. After 5 and 7 d of exposure, 83 and 100% of spat, respectively, tested positive for the virus by real time PCR. Vibrio species recovered during the longitudinal study included V. splendidus and V. aestuarianus. This study offers insight into the rapidity of onset and virulence of the virus in naïve oyster spat in New Zealand waters.

Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

Development and validation of real-time PCR for the detection of Yersinia ruckeri.

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish.

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

Survey of bulk tank milk in New Zealand for Mycoplasma bovis , using species-specific nested PCR and culture.

AIM: To survey the dairy cattle population in New Zealand for the presence or absence of Mycoplasma bovis. METHODS: A random cross-sectional survey of bulk tank milk from dairy herds in New Zealand based on regionally proportioned sampling, weighted towards herds with a high bulk tank milk somatic cell count (SCC) was used to detect M. bovis at a between-herd prevalence of 2%, with 99% confidence. Bulk tank milk samples collected on-farm were tested using a nested M. bovis PCR, and bacteriological culture employing enrichment in mycoplasma broth and direct plating onto mycoplasma agar. RESULTS: Mycoplasma bovis was not detected in any of the 244 bulk tank milk samples by either PCR or culture. CONCLUSIONS: This survey provides further evidence that M. bovis is not present in the dairy cattle population in New Zealand.

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae.

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster.

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.

Failure to detect Salmonella species in a population of wild tuatara (Sphenodon punctatus).

AIM: To assess the prevalence of faecal excretion of Salmonella serovars by wild tuatara (Sphenodon punctatus) on Stephens Island, New Zealand. METHODS: One hundred cloacal swabs obtained as part of health-screening for the translocation of adult tuatara from Stephens Island were subjected to general aerobic culture and enrichment, and cultured specifically for Salmonella spp. RESULTS: No Salmonella spp were cultured from any of the cloacal samples, which suggests that, at the 95% confidence interval, the maximum prevalence of tuatara in the island population that were shedding Salmonella spp not detected by our sample size was 1.5%. Mixed bacteria were grown from the 70 cloacal swabs cultured aerobically. A predominant organism was evident in 30 cultures, and these were identified as Hafnia alvei type 1 (n=16) and type 2 (n=7), Corynebacterium spp (n=4), Klebsiella oxytoca (n=2), and Moraxella spp (n=1). CONCLUSIONS: The absence of intestinal carriage of Salmonella spp by the tuatara sampled in this study may indicate either lack of exposure, or an innate resistance to intestinal colonisation in tuatara. The significance of the other bacteria cultured as potential pathogens to the tuatara and as zoonotic risks is also uncertain. Wildlife managers should screen translocated reptiles for Salmonella spp, and thereby avoid exposing wild and managed populations to infection.

Characterization of a Brucella sp. strain as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in New Zealand.

Naturally acquired infection of humans with a marine mammal-associated Brucella sp. has only been reported once previously in a study describing infections of two patients from Peru. We report the isolation and characterization of a strain of Brucella from a New Zealand patient that appears most closely related to strains previously identified from marine mammals. The isolate was preliminarily identified as Brucella suis using conventional bacteriological tests in our laboratory. However, the results profile was not an exact match, and the isolate was forwarded to four international reference laboratories for further identification. The reference laboratories identified the isolate as either B. suis or B. melitensis by traditional bacteriological methods in three laboratories and by a molecular test in the fourth laboratory. Molecular characterization by PCR, PCR-restriction fragment length polymorphism, and DNA sequencing of the bp26 gene; IS711; the omp genes omp25, omp31, omp2a, and omp2b; IRS-PCR fragments I, III, and IV; and five housekeeping gene fragments was conducted to resolve the discrepant identification of the isolate. The isolate was identified to be closely related to a Brucella sp. originating from a United States bottlenose dolphin (Tursiops truncatus) and common seals (Phoca vitulina).

Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA.

Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.

Evaluation of diagnostic tests for Johne's disease in young cattle.

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.

Characteristics of intrafamilial and extrafamilial child sexual abuse.

OBJECTIVE: The precise nature of the differences between intrafamilial and extrafamilial child sexual abuse is not clear. The purpose of the present study is to provide clarification of these differences. METHOD: Archival data containing 1,037 cases of child sexual abuse were obtained from police files in two western Canadian cities with populations of about 180,000. Two trained research assistants coded and transcribed the data. RESULTS: Results showed: (1) earlier onset, longer duration, higher level of intrusion, and greater physical and emotional injury for intrafamilial victims; (2) less use of physical/verbal force, or enticements, and greater use of instructions "not to tell" by intrafamilial offenders; (3) more convictions and longer jail sentences for intrafamilial offenders; and (4) no intra-extrafamilial differences in victim sex preference. CONCLUSIONS: Boys are younger than girls at the time of first abuse in samples of criminal justice and hospital referrals, although only for older aged victims, for example, 8 to 17 years. Although statistically significant, there is little difference in level of intrusion perpetrated by intrafamilial and extrafamilial offenders, both are highly intrusive. Both intrafamilial and extrafamilial offenders use physical/verbal force, with older victims: extrafamilial offenders more often choose older victims, and therefore more often use force. Intrafamilial victims suffer greater physical and emotional injury, resulting from greater intrusion not duration of abuse. It's not clear that victim sex preferences of intra- and extrafamilial offenders exist.