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Supplementary Table 2 from Contribution of Abcc10 (Mrp7) to In Vivo Paclitaxel Resistance as Assessed in Abcc10−/− Mice

Supplementary Figure Legend from Contribution of Abcc10 (Mrp7) to In Vivo Paclitaxel Resistance as Assessed in Abcc10−/− Mice

Supplementary Figure 1 from Contribution of Abcc10 (Mrp7) to In Vivo Paclitaxel Resistance as Assessed in Abcc10−/− Mice

Supplementary Table 1 from Contribution of Abcc10 (Mrp7) to In Vivo Paclitaxel Resistance as Assessed in Abcc10−/− Mice

The objectives of this study were to identify the prevalence, incidence and range of udder defects in non-dairy breed ewes in New Zealand and to investigate the range of bacteria present in milk sa...

Case summary An 11-year-old female spayed Maine Coon cat was referred for assessment of hyporexia, weight loss, vomiting and diarrhoea. An abdominal ultrasound revealed an enlarged and hypoechoic pancreas containing two large fluid-filled structures. Fine-needle aspiration of the cyst-like structures was performed, and cytology revealed moderate-to-marked predominantly suppurative inflammation with yeast cells. Candida glabrata was cultured from the fluid. The patient was treated with oral itraconazole and the clinical signs resolved, although repeat abdominal ultrasound and cytology revealed persistence of the infected cyst-like structures. The patient remained asymptomatic for 8 months after the discontinuation of antifungal medications, despite the persistence of the pancreatic infection with C glabrata. Relevance and novel information To our knowledge, this is the first report of pancreatic infection with Candida species in a cat, followed by a chronic subclinical infection persisting for at least 8 months after treatment discontinuation.

We performed a survey aimed at analyzing milk samples collected from cows with mastitis for the presence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. Single-quarter mastitic milk samples obtained from 400 cows in 23 Greek dairy herds with a history of E. coli mastitis were processed for the selective isolation of ESBL-producing E. coli. The antimicrobial susceptibility of the ESBL-producing isolates was analyzed using agar disk diffusion, and minimum inhibitory concentrations of colistin were determined by broth microdilution. We used PCR followed by DNA sequencing to characterize the β-lactamases and mcr-1 (colistin resistance) genes, and for phylotyping and multilocus sequence typing. We found a total of 89/400 (22.25%) E. coli isolates from 12/23 (52%) farms. Six isolates originating from 6 cows on a single farm were ESBL producers and were resistant to cefquinome, amoxicillin-clavulanic acid, aztreonam, ampicillin, and colistin. Five of these isolates were resistant to trimethoprim-sulfamethoxazole, and 5 to streptomycin. The 6 ESBL producers were mcr-1-positive and carried blaTEM-1 genes; 3 also carried blaCTX-M genes, and 3 carried blaSHV genes. All of the ESBL producers belonged to phylogroup A, multilocus sequence type ST666 (n = 5), or a single locus variant of ST666 (n = 1). To our knowledge, this is the first report of endemic bovine mastitis caused by mcr-1-positive, ESBL-producing E. coli. These results highlight the value of active surveillance of antimicrobial resistance not commonly tested by diagnostic laboratories for the early detection of novel resistant strains.

Simple Summary Invasive rats and field mice were captured under the project RESTO CON LIFE “Island conservation in Tuscany, restoring habitat not only for birds”, aiming to improve habitats preservation on the Italian islands of Pianosa, Elba, Montecristo, and Giannutri. Bartonella henselae DNA was detected in one captured animal. This is the first report of the presence of B. henselae DNA in rodents in Italy. B. henselae is the causative agent of cat scratch disease, and the detection of this bacterium in rodents might have public health implications. Abstract Wild rodents are reservoirs of several Bartonella species that cause human bartonellosis. The aim of this study was to assess the presence of Bartonella spp. DNA in wild rodents in Pianosa island, Italy. Rats (Rattus spp.; n = 15) and field mice (Apodemus spp.; n = 16) were captured and spleen DNA tested for the presence of Bartonella spp. by means of an initial screening using a qPCR amplifying a short segment of the 16S-23S rRNA gene intergenic transcribed spacer region (ITS, ~200 bp) followed by conventional PCR amplification of a longer ITS fragment (~600 bp) and of a citrate synthase (gltA, ~340 bp) gene segment. A total of 25 spleen DNA samples obtained from 31 rodent carcasses (81%) yielded positive qPCR results. Bartonella genus was confirmed by amplicon sequencing. By conventional PCR, eight out of 25 samples (32%) yielded bands on gels consistent with ITS segment, and 6/25 (24%) yielded bands consistent with the gltA locus. Amplicon sequencing identified B. henselae and B. coopersplainsensis in 1/25 (4%), and 4/25 (16%) samples, respectively. Moreover, 5/25 (20%) of Bartonella spp. positive samples showed gltA sequences with about 97% identity to B. grahamii. These results provide support to recently published observations suggesting that B. henselae circulates in wild rodent populations.

In New Zealand, the major risk factor for campylobacteriosis has been identified as poultry consumption. New Zealanders consume different types of chicken meat which undergo different processing before entering the retail chain. The manipulations and jointing of chicken carcasses into pieces and the subsequent processing and packaging have the potential to cross-contaminate and reshuffle bacterial pathogens among the different products sold. The aim of this study was to analyse: (a) the differences in the viable count and population genetic structure between Campylobacter isolated from chicken drumsticks and whole carcass meat for retail sale over a 1-year period; and (b) the genetic relatedness of human and chicken isolates collected concurrently. Enumeration of Campylobacter was performed using a spiral plater combined with manual spread plating. Campylobacter isolates were identified by polymerase chain reaction and typed by multilocus sequence typing (MLST). C. jejuni was the dominant species among both whole carcasses (63.5%) and drumsticks samples (73.8%), followed by C. coli (27% and 23.1%, respectively). After sample weight adjustment, whole carcasses showed significantly higher Campylobacter counts than drumsticks, with a significant difference in the counts between the commercial suppliers in both types of retail meat. MLST revealed 28 different sequence types among the two types of meat. Using permutational multivariate analysis of variance, statistically significant differences in the population genetic structures were observed between different suppliers but were not observed between the two types of chicken retail meat. In conclusion, we found differences in Campylobacter viable counts, suggesting consumption of whole carcasses may determine an exposure to a higher number of Campylobacter bacteria than consumption of chicken drumsticks. The Campylobacter population genetic structure did not differ between the two types of chicken retail meat. Therefore, source attribution studies based on MLST are unlikely to be biased by the selection of these types of retail meat during sampling.

AIM To identify Bartonella spp. in rats from New Zealand using molecular methods. METHODS DNA was extracted from the spleens of 143 black rats (Rattus rattus) captured in the Tongariro National Park, New Zealand. PCR was performed using Bartonella genus-specific primers amplifying segments of the 16S-23S rRNA internal transcribed spacer and citrate synthase (gltA) and beta subunit of the RNA polymerase (rpoB) genes. PCR products were sequenced and compared online with sequences stored in the database of the National Center for Biotechnology Information of the United States of America. RESULTS DNA sequences matching Bartonella coopersplainsensis and B. henselae were detected in samples from 22/143 (15.4%) and 3/143 (2.1%) rats, respectively. Co-occurrence of B. coopersplainsensis and B. henselae sequences was observed in the sample from one rat. CONCLUSIONS AND CLINICAL RELEVANCE Gram-negative fastidious bacteria belonging to the genus Bartonella are associated with a range of human diseases. Rodents play an important role as reservoirs of a broad range of Bartonella species. To our knowledge, this is the first report of a molecular detection of Bartonella spp. DNA in rodents from New Zealand, and the first identification of B. henselae DNA in rats, worldwide. Whereas the public health significance of B. coopersplainsensis remains undefined, B. henselae is the agent of cat scratch disease, and the presence of this bacterium in rats may have public health implications. Our results are preliminary and additional analyses of larger samples, preferably by bacterial culture, would provide more information on the prevalence and diversity of Bartonella spp., in particular B. henselae, in rats.

Background: Lower urinary tract infections are common in dogs, and Escherichia coli is the most common bacterial pathogen isolated. The literature has conflicting evidence regarding the inhibitory effects of urine concentration and pH on E. coli growth. Hypothesis/Objectives: To determine the effect of different pH and urine concentrations on E. coli growth in vitro. Animals: Voided urine samples from 10 apparently healthy spayed female dogs were used. Methods: A matrix of 9 urine specific gravity (USG; 1.010, 1.020, and 1.030) and pH (5.5, 7.0, and 8.5) combinations was prepared by diluting and titrating filtered voided urine samples. Three E. coli isolates were obtained from urine of female dogs with signs of lower urinary tract infection and cultured at different urine pH and USG combinations in wells of a microtiter plate. The number of E. coli colony‐forming units (CFU) per mL of urine was calculated after aerobic incubation of the urine at 37°C for 18 hours, and statistically compared. Results: Significant differences were identified in the mean log CFU/mL among different combinations of pH and USG. The lowest log CFU/mL were observed in alkaline concentrated urine (pH 8.5 and USG 1.030). Conclusions and Clinical Importance: Escherichia coli in vitro growth was higher in neutral to acidic and diluted urine compared to alkaline and concentrated urine. The impact of non‐alkalizing diluting diets on the incidence of E. coli lower urinary tract infections should be further explored.

Summary Objectives Staphylococcus aureus skin and soft tissue infection (Sa-SSTI) places a significant burden on healthcare systems. New Zealand has a high incidence of Sa-SSTI, and here most morbidity is caused by a polyclonal methicillin-susceptible (MSSA) bacterial population. However, MSSA also colonise asymptomatically the cornified epithelia of approximately 20% of the population, and their divide between commensalism and pathogenicity is poorly understood. We aimed to see whether MSSA are genetically differentiated across colonisation and SSTI; and given the close interactions between people and pets, whether strains isolated from pets differ from human strains. Methods We compared the genomes of contemporaneous colonisation and clinical MSSA isolates obtained in New Zealand from humans and pets. Results Core and accessory genome comparisons revealed a homogeneous bacterial population across colonisation, disease, humans, and pets. The rate of MSSA colonisation in dogs was comparatively low (5.4%). Conclusions In New Zealand, most Sa-SSTI morbidity is caused by a random sample of the colonising MSSA population, consistent with the opportunistic infection model rather than the paradigm distinguishing strains according to their pathogenicity. Thus, studies of the factors determining colonisation and immune-escape may be more beneficial than comparative virulence studies. Contact with house-hold pets may pose low zoonotic risk.

In 2006, New Zealand had the highest notification rate of campylobacteriosis in the world, and poultry was considered the leading source of campylobacteriosis. Implementation of food safety interventions by the poultry industry led to a decrease in the campylobacteriosis notification rate. The aim is to examine the impact of targeted food safety interventions implemented by the New Zealand poultry industry on the source attribution of Campylobacter jejuni infections in a sentinel region. Campylobacter jejuni isolates collected from the Manawatu region of New Zealand between 2005 and 2007 (“before intervention”) and 2008 and 2015 (“after intervention”) from human clinical cases, chicken meat, ruminant feces, environmental water, and wild bird sources were subtyped by multilocus sequence typing. Viable counts of Campylobacter spp. from carcasses were analyzed using a zero-inflated Poisson regression model. In the period before intervention, sequence type 474 (ST-474) was the most common sequence type (ST) recovered from human cases, accounting for 28.2% of the isolates. After intervention, the proportion of human cases positive for ST-474 reduced to 9.3%. Modeling indicated that chicken meat, primarily from one supplier, was the main source of C. jejuni infection in the Manawatu region before intervention. However, after intervention poultry collectively had a similar attribution to ruminants, but more human cases were attributed to ruminants than any single chicken supplier. Viable counts on carcasses were lower in all poultry suppliers after intervention. This study provides evidence of changes in the source attribution of campylobacteriosis following targeted food safety interventions in one sector of the food supply chain. IMPORTANCE This study provides a unique insight into the effects of food safety interventions implemented in one sector of the food industry on the transmission routes of a major foodborne agent. Following the implementation of food safety interventions by the poultry industry, shifts in the molecular epidemiology of Campylobacter jejuni infections in a sentinel region of New Zealand were observed. Targeted interventions to reduce disease incidence are effective but require continued surveillance and analysis to indicate where further interventions may be beneficial.

Background Bacterial urinary tract infections are uncommon in cats in general but the prevalence increases to 29% in older cats with comorbidities (Veterinary Clinical Pathology 2008, 37, 317; Journal of Feline Medicine & Surgery 2007, 9, 124; Veterinary Microbiology 2009, 136, 130). Frequently, the infections are subclinical. The clinical relevance of subclinical bacteriuria (SB) is uncertain, and the optimal treatment requires clarification. Objective Prospective, observational study to: (i) identify the prevalence and incidence count of SB in older (≥7 years), nonazotemic cats, (ii) evaluate specific risk factors for SB, and (iii) investigate the potential relationship between untreated SB and survival. Animals Sixty-seven, nonazotemic cats were tested on 5 occasions over 3 years. Methods Urine samples were obtained by cystocentesis for quantitative urine culture and blood samples for measurement of serum creatinine concentration. Episodes of SB were not treated. Serum creatinine concentration, body weight, urine specific gravity, sex, and age were evaluated as potential risk factors for a positive urine culture. The association between urine culture results and survival was evaluated with Cox's proportional hazard model. Results A total of 256 urine samples was obtained. The prevalence of SB varied between 10 and 13%, and incident infections were uncommon. Female cats were 21 times more likely to have a positive urine culture than were male cats (odds ratio [OR], 21.2; confidence interval [CI], 4.1–110; P = .00028). Subclinical bacteriuria was not significantly associated with survival. Conclusion and clinical importance Subclinical bacteriuria is common in nonazotemic, older cats. Although antimicrobial treatment was withheld, the presence of SB was not adversely associated with survival.

Knowledge of the within-host genetic diversity of a pathogen often has broad implications for disease management. Cryptosporidium protozoan parasites are among the most common causative agents of infectious diarrhoea. Current limitations of in vitro culture impose the use of uncultured isolates obtained directly from the hosts as operational units of Cryptosporidium genotyping. The validity of this practice is centred on the assumption of genetic homogeneity of the parasite within the host, and genetic studies often take little account of the within-host genetic diversity of Cryptosporidium. Yet, theory and experimental evidence contemplate genetic diversity of Cryptosporidium at the within-host scale, but this diversity is not easily identified by genotyping methods ill-suited for the resolution of DNA mixtures. We performed a systematic bibliographical search of the occurrence of within-host genetic diversity of Cryptosporidium parasites in epidemiological samples, between 2005 and 2015. Our results indicate that genetic diversity at the within-host scale, in the form of mixed species or intra-species diversity, has been identified in a large number (n=55) of epidemiological surveys of cryptosporidiosis in variable proportions, but has often been treated as a secondary finding and not analysed. As in malaria, there are indications that the scale of this diversity varies between geographical regions, perhaps depending on the prevailing transmission pathways. These results provide a significant knowledge base from which to draw alternative population genetic structure models, some of which are discussed in this paper.

The incidence of infections with extended spectrum β-lactamase producing E. coli (ESBL-E) is increasing both in humans and animals. There is a paucity of data about the rate of faecal carriage of ESBL-E in pets. In this study, we analysed the ESBL-E faecal carriage rate in cats and dogs in Auckland, New Zealand, and the factors associated with carriage. Faecal swabs collected from 586 pets (225 cats; 361 dogs) were analysed for the presence of ESBL-E by culture, and a questionnaire was delivered to the owners. The ESBL-E were characterised, and data elicited by the questionnaires were used to investigate the factors associated with carriage. The prevalence of ESBL-E in faecal swabs was 6.4%. The β-lactamase genes detected in the ESBL-E were the blaCTX-M-14 (n = 2) and blaCMY-2 (n = 34). Several isolates displayed multilocus-sequence-types (MLST) associated with human and animal infections. Multiple isolates sharing the same MLST displayed different antibiograms and β-lactamase genes, reflecting horizontal gene transfer between and within MLSTs. Factors associated with increased odds of ESBL-E carriage were: animal received systemic antimicrobial treatment in the six months before the sampling; the presence of household members working in veterinary clinics; the presence of household members travelling overseas in the six months before the sampling. We conclude that pets are colonised by ESBL-E which are genotypically similar to the bacteria found to infect humans and animals. The statistical analysis shed light on the eco-epidemiological factors associated with ESBL-E carriage. In particular, they suggest veterinary clinics may represent hot-spots of antimicrobial resistance.

To compare the antimicrobial susceptibility patterns of three common mastitis pathogens (Staphylococcus aureus, Streptococcus uberis and Str. dysgalactiae) isolated from milk samples from New Zealand and the USA.A total of 182 S. aureus, 126 Str. uberis and 89 Str. dysgalactiae isolates from New Zealand (107, 106 and 41, respectively) and the USA (75, 20 and 48, respectively) were assessed using the disk diffusion test.Susceptibility varied among the bacterial species. All isolates were susceptible to the amoxicillin-clavulanic acid combination. Resistance to lincomycin was most frequent (susceptibility of 8.6%) across all species. Non-susceptible (i.e. resistant or intermediate) isolates of S. aureus were identified for the three non-isoxazolyl penicillins (amoxicillin, ampicillin and penicillin: 20.6% and 36.0%) and lincomycin (99.9% and 94.6%) for NZ and the USA, respectively. Resistance to erythromycin (5.3%) and tetracyclines (6.7%) was detected only in isolates from the USA. There were differences in susceptibility between Str. uberis and Str. dysgalactiae; all streptococcal isolates demonstrated resistance to aminoglycosides (neomycin 52.4% and streptomycin 27.9%) and enrofloxacin (28%). Resistance of Str. dysgalactiae to tetracycline was almost 100.0% and to oxytetracycline 89.9%.Most of the isolates tested were susceptible to most of the antimicrobials commonly used for treatment of bovine mastitis, with the exception of the lincosamides. Susceptibility to a selected class-representative antimicrobial and at the genus level should be interpreted with caution. Differences between NZ and the USA confirm the value of national surveys to determine the susceptibility patterns of mastitis pathogens.

Performance efficiency is always a top concern for DBAs and developers. Indexes are a front runner to improve data delivery processes. However, the logical tree structure of XML data cannot be indexed in the same way as plain old relational data. Therefore, with the introduction of SQL Server 2005, Microsoft added an indexing mechanism for XML data type columns. XML data is not the same as scalar data in a table’s column, therefore the XML indexing mechanism provides for specialized primary and secondary XML indexes. In SQL Server 2012, the XML indexes are enhanced with a Selective XML Index, which provides an improvement in search efficiency for big XML data.

In Chapter 2, we discussed how to build XML from an SQL query result set. Before we begin reviewing the options on how to shred XML data, we first need to know how to store the XML result on the storage (disk, SSD drive, SAN, etc.) and how to upload an XML file into a table. This chapter will demonstrate the various options to manipulate the XML files.

Biofilm formation is of growing concern in human and animal health. However, it is still unclear how biofilms are related to mastitis infections in dairy cattle. In this study, a comparison between two tests for biofilm formation and the association between biofilm and the presence of genes associated with biofilm formation were investigated for 92 Staphylococcus aureus isolates from clinical mastitis cases. Congo red agar (CRA) and microtitre test assay (MTA) in vitro phenotypic tests were used to evaluate biofilm formation. The presence of icaA, icaD, and bap genes associated with biofilm formation was confirmed using the polymerase chain reaction. Results show that most of the S. aureus isolates, though not possessing one of the biofilm-forming genes, were able to produce biofilms. MTA was more frequently positive in identifying biofilm-forming isolates than CRA.

Several Cryptosporidium species are known to infect cattle. However, the occurrence of mixed infections with more than one species and the impact of this phenomenon on animal and human health are poorly understood. Therefore, to detect the presence of mixed Cryptosporidium infections, 15 immunofluorescence-positive specimens obtained from 6-week-old calves' faeces (n=60) on one dairy farm were subjected to PCR-sequencing at multiple loci. DNA sequences of three Cryptosporidium species: C. parvum (15/15), C. bovis (3/15) and C. andersoni (1/15), and two new genetic variants were identified. There was evidence of mixed infections in five specimens. C. parvum, C. bovis and C. andersoni sequences were detected together in one specimen, C. parvum and C. bovis in two specimens, and C. parvum and C. parvum-like variants in the remaining two specimens. Sequencing of gp60 amplicons identified the IIaA19G4R1 (8/15) and IIaA18G3R1 (4/15) C. parvum subgenotypes. This study provides evidence of endemic mixed infections with the three main Cryptosporidium species of cattle and new genetic variants, in calves at the transition age of six weeks. The results add to the body of evidence describing Cryptosporidium isolates as genetically heterogeneous populations, and highlight the need for iterative genotyping to explore their genetic makeup.

Halofuginone lactate (HL) is registered in several countries for the prevention of calf cryptosporidiosis, but the compound's utility in the presence of co-infection with other enteropathogens is not well understood. We performed a randomized controlled field trial of the efficacy of HL for the prevention of natural calf cryptosporidiosis, in the presence of co-infection with rotavirus and Salmonella Typhimurium. Newborn calves on one farm were sequentially enrolled and allocated to a full dose (n = 15), half dose (n = 15), or a placebo control group (n = 15), using a randomized block design. The Cryptosporidium oocysts in fecal specimens collected on Days 6, 8, 10, 14 and 20 were counted and the severity of the diarrhea was assessed using fecal consistency scores (solid, semisolid, or liquid). The oocyst numbers and fecal consistency scores were statistically compared between the groups. Ninety one percent of the calves shed Cryptosporidium parvum oocysts during the trial. The full dose group had a longer prepatent period than the control group, but no statistical difference in the number of oocysts was identified between the groups after controlling for the effects of sex and breed. The fecal consistency scores and mortality rates did not differ between the groups. These results indicated that the anti-Cryptosporidium activity and clinical benefit of HL were limited. It is concluded that in order to maximize the clinical efficacy of HL in the field, diagnostic efforts should aim to rule out the presence of other enteropathogens.

Abrupt dietary transitions and feeding of rapidly fermentable diets are common practices in the horse industry and have been associated with digestive and metabolic disorders that can impair the performance of horses. The present study investigated the effect of dietary transition from pasture grazing to confinement with concentrate feeding, and back, on fecal pH and bacterial populations of Streptococcus spp and Lactobacillus spp. Six Thoroughbred fillies, previously grazing perennial ryegrass and white clover-based pasture, were housed in individual stalls and fed an increasing ratio of concentrate to conserved forages for 13 days (days 1-13), followed by an abrupt transition back to only pasture-grazing for 3 days (days 14-16). The concentrate was initially offered at 0.83 kg dry matter (DM)/d and increased to 5 kg DM/d, whereas ensiled alfalfa was initially offered at 0.61 kg DM/d, increasing to 1.22 kg DM/d. Meadow hay was initially offered at 6.73 kg DM/d, decreasing to 1.6 kg DM/d. Fecal specimens were collected daily for determination of pH, and every 2 days for quantitative analysis of Streptococcus spp and Lactobacillus spp. Mean fecal pH increased significantly from pasture baseline values (pH 6.18) during the initial confinement and supplementation on day 1 (6.37), day 2 (6.52), day 3 (6.58), and day 4 (6.43) (standard error of mean [SEM]: 0.056; P < .001). By day 5, mean fecal pH values had decreased to, and remained at, baseline values until the horses returned to pasture, when another increase occurred at day 15 (6.45). Fecal colony forming units (cfu) of Streptococcus spp and Lactobacillus spp increased linearly (r = 0.94; P < .001) from 6.0 and 6.1 log10 cfu/g on day -4, to 7.8 log10 cfu/g on day 14 (SEM: 0.2 P < .001), respectively. Fecal cfu decreased on return to a pasture-only diet (P < .001). In this study, the increment of bacterial populations was associated with a relatively stable fecal pH and highlights the difficulty in identifying the effects of dietary transition on the equine hindgut health, without microbial culture.

AIMS: To assess the occurrence of, and characterise, extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC)-producing Enterobacteriaceae isolated by veterinary diagnostic laboratories from infection sites in companion animals in New Zealand. METHODS: Selected Enterobacteriaceae isolates were submitted by seven New Zealand veterinary diagnostic laboratories. They were isolated from infection sites in companion animals between June 2012 and June 2013, and were resistant to amoxicillin-clavulanic acid, fluoroquinolones, or any combination of two or more antimicrobials. Based on disk diffusion test results, the isolates were phenotypically categorised according to production of ESBL and AmpC. Genes for ESBL and AmpC production were amplified by PCR and sequenced. Escherichia coli isolates were also typed by multilocus sequence typing. RESULTS: A total of 115 isolates matching the inclusion criteria were obtained from the participating laboratories, of which 74 (64%) originated from dogs and 29 (25%) from cats. Seven bacterial species were identified, of which E. coli was the most common (87/115, 76%). Of the 115 isolates, 10 (9%) expressed the ESBL phenotype, 43 (37%) the AmpC phenotype, and seven (6%) both ESBL and AmpC phenotypes. Of the 60 ESBL and AmpC-producing isolates, 36 (60%) were E. coli. Amongst these isolates, 27/60 (45%) were classified as multidrug resistant, compared with 15/55 (27%) non-ESBL or AmpC-producing isolates (pblaCTX-M-14 and blaCTX-M-15, and the dominant plasmid-encoded AmpC gene was blaCMY-2. Thirty-eight E. coli multilocus sequence types (ST) were identified, and the most prevalent were ST12 (12/89, 13%), ST131 (6/89, 7%) and ST648 (6/89, 7%). ESBL and AmpC-producing isolates accounted for 35/1,082 (3.2%) of the Enterobacteriaceae isolated by one laboratory network over the study period. CONCLUSIONS AND CLINICAL RELEVANCE: ESBL and AmpC-producing Enterobacteriaceae were associated with clinical infections in companion animals in New Zealand, and were often multidrug resistant. In this study, these organisms accounted for

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRβ locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRβ locus leading to a dramatically altered usage of Vβ segments for Vβ-to-DβJβ recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer–promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRβ locus supports the emerging role of CTCF in governing VDJ recombination.

Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as ‘coreceptor gene imprinting’. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4+ cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus.

The incidence of clinical mastitis and infection status at calving was assessed in quarters treated with 1 of 2 internal teat sealants at the time of dry off. Two contralateral quarters per cow (n=63 cows) were treated with a sealant that contained 0.5% chlorhexidine; the other quarters were treated with a commercial teat sealant. Ten cows were untreated (controls). On d 2, 4, and 16 after dry off, cows were challenged with Streptococcus uberis S210 strain. Cows were examined daily for 34 d after drying off and cases of clinical mastitis were recorded. Milk samples were collected for culture from any quarters that developed clinical mastitis during the first 34 d after drying-off and from all quarters on d −5 and 0 relative to treatment and at the first and twentieth milking after calving. The incidence of clinical mastitis during the examination period was lower in treated quarters (n=7/252; 1.5%; lower incidence for those treated with chlorhexidine-containing teat sealant n=3/126; 1.2%) than in untreated quarters (n=13/40; 26.8%). The protection against intramammary infection after calving, adjusted for the effect of cow, was higher in quarters treated with the novel teat sealant (89/105; 15.2%; 95% CI=9.6–23.4) than in those treated with the commercial teat sealant (71/104; 31.7%; 95% CI=23.5–41.3) and untreated controls (6/28; 78.6%; 95% CI=59.8–90.0), respectively. Quarters treated with teat sealants were less likely to have an intramammary infection after calving and had a lower incidence of clinical mastitis during the early dry period than did untreated controls in this challenge study.

Recently, we reported that the ATP-binding cassette transporter 10 (ABCC10), also known as multidrug resistance protein 7 (MRP7), is able to confer resistance to a variety of anticancer agents, including taxanes. However, the in vivo functions of the pump have not been determined to any extent. In this study, we generated and analyzed Abcc10−/− mice to investigate the ability of Abcc10 to function as an endogenous resistance factor. Mouse embryo fibroblasts derived from Abcc10−/− mice were hypersensitive to docetaxel, paclitaxel, vincristine, and cytarabine (Ara-C) and exhibited increased cellular drug accumulation, relative to wild-type controls. Abcc10−/− null mice treated with paclitaxel exhibited increased lethality associated with neutropenia and marked bone marrow toxicity. In addition, toxicity in spleen and thymus was evident. These findings indicate that Abcc10 is dispensable for health and viability and that it is an endogenous resistance factor for taxanes, other natural product agents, and nucleoside analogues. This is the first demonstration that an ATP-binding cassette transporter other than P-glycoprotein can affect in vivo tissue sensitivity toward taxanes. Cancer Res; 71(10); 3649–57. ©2011 AACR.

SUMMARYThe occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations.Cryptosporidium parvumwas the only enteropathogen identified in 4/7 faecal specimens analysed. TheC. parvumisolates carried a rareIIaGP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. A total of 25/80 respondents to a questionnaire were defined as cases using a clinical case definition (31% attack rate). The inferred median incubation period was 5 days (range 0–11 days), and the median illness duration was 5–6 days (range 2–23 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a significant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis.

A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.

To describe clinically overt infections with methicillin resistant Staphylococcus aureus (MRSA) in animals in New Zealand, characterise clinical isolates, and track their sources.MRSA isolates identified in 2005 and 2006 by a veterinary diagnostic laboratory were referred to Massey University for confirmation and characterisation. Clinical information was extracted from the laboratory records or obtained from referring clinicians.Seven MRSA isolates from animals and contact persons were characterised. All the isolates belonged to the British epidemic MRSA 15 strain (EMRSA-15). Three EMRSA-15 were isolated from post-operative infections in two dogs. An EMRSA-15 indistinguishable from the isolate recovered from one dog was isolated from the anterior nares of a healthy hospital staff member involved in the care of the animal, suggesting nosocomial transmission. Other EMRSA-15 isolates of uncertain clinical significance were isolated from the femoral head of a cat, and from a sample of cow's milk. AlleMRSA-15 isolates were resistant to ciprofloxacin, and four were resistant to erythromycin; the latter four isolates also exhibited inducible resistance to clindamycin.MRSA can cause clinically overt and difficult-to-treat infections in animals in New Zealand. The rapid emergence of EMRSA-15 as the dominant MRSA strain in humans has resulted in infection spill over to animals.Little is known about the incidence of clinically overt infections with MRSA in animals. The cases described here illustrate the complexities involved in the pharmacological management of EMRSA-15 infections, which is compounded by the universal resistance to beta-lactams, and by the strain's fluoroquinolone resistance and frequent inducible resistance to clindamycin. Such complexities indicate there is a need to develop specific empirical antimicrobial treatment strategies and antibiotic susceptibility testing protocols in countries where EMRSA-15 is dominant.

Campylobacteriosis is one of the most important foodborne diseases worldwide and a significant health burden in New Zealand. Campylobacter jejuni is the predominant species worldwide, accounting for approximately 90% of human cases, followed by Campylobacter coli . Most studies in New Zealand have focused on C. jejuni ; hence, the impact of C. coli strains on human health is not well understood. The aim of this study was to genotype C. coli isolates collected in the Manawatu region of New Zealand from clinical cases, fresh poultry meat, ruminant feces, and environmental water sources, between 2005 and 2014, to study their population structure and estimate the contribution of each source to the burden of human disease. Campylobacter isolates were identified by PCR and typed by multilocus sequence typing. C. coli accounted for 2.9% ( n = 47/1,601) of Campylobacter isolates from human clinical cases, 9.6% ( n = 108/1,123) from poultry, 13.4% ( n = 49/364) from ruminants, and 6.4% ( n = 11/171) from water. Molecular subtyping revealed 27 different sequence types (STs), of which 18 belonged to clonal complex ST-828. ST-1581 was the most prevalent C. coli sequence type isolated from both human cases ( n = 12/47) and poultry ( n = 44/110). When classified using cladistics, all sequence types belonged to clade 1 except ST-7774, which belonged to clade 2. ST-854, ST-1590, and ST-4009 were isolated only from human cases and fresh poultry, while ST-3232 was isolated only from human cases and ruminant sources. Modeling indicated ruminants and poultry as the main sources of C. coli human infection. IMPORTANCE We performed a molecular epidemiological study of Campylobacter coli infection in New Zealand, one of few such studies globally. This study analyzed the population genetic structure of the bacterium and included a probabilistic source attribution model covering different animal and water sources. The results are discussed in a global context.

To compare the ability of four strains of Streptococcus uberis at two doses to induce clinical mastitis in lactating dairy cows after intramammary inoculation in order to evaluate their usefulness for future experimental infection models.Four field strains of S. uberis (26LB, S418, and S523 and SR115) were obtained from cows with clinical mastitis in the Wairarapa and Waikato regions of New Zealand. Twenty-four crossbred lactating cows, with no history of mastitis and absence of major pathogens following culture of milk samples, were randomly allocated to four groups (one per strain) of six cows. Each cow was infused (Day 0) in one quarter with approximately 10(4) cfu and in the contralateral quarter with approximately 10(6) cfu of the same strain. The other two quarters remained unchallenged. All four quarters were then inspected for signs of clinical mastitis, by palpation and observation of the foremilk, twice daily from Days 0-9, and composite milk samples were collected from Days 0-8 for analysis of somatic cell counts (SCC). Quarters were treated with penicillin when clinical mastitis was observed. Duplicate milk samples were collected and cultured on presentation of each clinical case and on Day 4 from challenged quarters with no clinical signs.Clinical mastitis was diagnosed in 26/48 (54%) challenged quarters. Challenge with strain S418 resulted in more cases of mastitis (12/12 quarters) than strains SR115 (7/12), 26LB (6/12) or S523 (1/12), and the mean interval from challenge to first diagnosis of mastitis was shorter for S418 than the other strains (p0.001). The proportion of quarters from which S. uberis could be isolated after challenge was less for strain 26LB (1/6) than SR115 (6/7) (p0.05), and SCC following challenge was lower for strain S523 than the other strains (p0.05).There were significant differences between the strains in the proportion of quarters developing clinical mastitis, the interval to mastitis onset, SCC following challenge and the proportion of clinical cases from which S. uberis could be isolated. These results illustrate the difference in the ability of S. uberis strains to cause mastitis and the severity of the infections caused.Experimental challenge models can be used to compare infectivity and pathogenicity of different strains of mastitis-causing bacteria, the efficacy of pharmaceutical products and host-responses in a cost-effective manner.

AIMS: To characterise methicillin-resistant Staphylococcus aureus (MRSA) isolates from infection sites in animals in New Zealand and assess the prevalence of subclinical MRSA colonisation in dogs and cats attending veterinary clinics in Auckland. METHODS: MRSA isolates from clinical specimens obtained by the main New Zealand veterinary diagnostic laboratories between June 2012 and June 2013, were genotypically characterised by DNA microarray hybridisation analysis and spa typing. In addition, nasal or perineal skin swabs collected from a cross-sectional sample of dogs (n=361) and cats (n=225) attending 29 veterinary clinics in Auckland during the same period were analysed for MRSA by culture. RESULTS: Eight MRSA clinical isolates were submitted for characterisation by the participating laboratories. The isolates originated from five dogs, including two isolates from the same dog, one foal, and one isolate had no identification of the source. The strain-types identified were AK3 (ST-5 SCCmecIV t045; n=1), USA500 (ST8 SCCmecIV t064; n=1), WSPP (ST30 SCCmecIV t019; n=1), Rhine Hesse (ST5 SCCmecII t002; n=2), and EMRSA-15 (ST22 SCCmecIV t032; n=3). No MRSA were isolated from 586 cultured swabs. Methicillin-susceptible S. aureus were detected in 9/257 (3.5%) swabs and non-aureus staphylococci in 22/257 (8.5%) swabs. The estimated true MRSA subclinical colonisation prevalence was 0%, with an upper 95% CI boundary of 1.9% for cats and 1.4% for dogs. CONCLUSIONS: The modest number of MRSA isolates submitted for this study by the participating laboratories suggests clinical MRSA infection in animals in New Zealand continues to be sporadic. The wide variety of strain-types found mirrored the evolving strain-type diversity observed in humans. We cannot rule out bias due to the non-random sampling of dogs and cats, but the apparent colonisation prevalence of 0% was consistent with the low prevalence of subclinical colonisation in humans in New Zealand. These similarities indicate the epidemiology of animal and human MRSA infections are linked. CLINICAL RELEVANCE: In the last decade, the prevalence of human MRSA infections in New Zealand has steadily increased. This is the second published study of MRSA in animals in New Zealand. The results indicate clinical MRSA infection in animals remains sporadic, but the diversification of the strain-types may pose new therapeutic challenges to veterinarians, due to their diverse resistome.

The 2001/2002 and 2002/2003 breeding seasons of New Zealand sea lions (NZSLs) on the Auckland Islands were marked by a high pup mortality caused by acute bacterial infections. As part of a health survey from 1998/1999 to 2004/2005, tissues and swabs of lesions had been collected at necropsy to identify the bacteria associated with pup mortality. Klebsiella pneumoniae was grown in pure culture from 83% of various organs and lesions in 2001/2002 and 76% in 2002/2003, and less frequently in the following seasons (56% in 2003/2004 and 49% in 2004/2005). Pup isolates of K. pneumoniae showed identical minimal inhibitory concentrations (MIC) of cefuroxime, neomycin, cephalotin, cephalexin and dihydrostreptomycin, suggesting clonal aetiology of the pathogen. Isolates also tested negative for production of extended-spectrum beta-lactamases (ESBLs), which was not in favour of an anthropogenetic origin of the epidemic strain. Pulsed-field gel electrophoresis (PFGE) of XbaI DNA macrorestriction fragments was performed on isolates of K. pneumoniae and Klebsiella oxytoca from 35 pups, thee NZSL adult females, and from three human patients for comparison. PFGE showed that pup isolates of K. pneumoniae were genetically indistinguishable but were neither related to K. pneumoniae from humans and from NZSL adults, nor to K. oxytoca from NZSLs. It is concluded that the 2001/2002 and 2002/2003 epidemics at Sandy Bay rookery were caused by a single K. pneumoniae clonal lineage, genetically different from the strain carried by adult NZSLs. An anthropogenic origin of the K. pneumoniae clone could not be confirmed, but further investigations are required to rule-out such occurrence.