Non-invasive genotyping with a massively parallel sequencing panel for the detection of SNPs in HPA-axis genes.

We designed a genotyping panel for the investigation of the genetic underpinnings of inter-individual differences in aggression and the physiological stress response. The panel builds on single nucleotide polymorphisms (SNPs) in genes involved in the three subsystems of the hypothalamic-pituitary-adrenal (HPA)-axis: the catecholamine, serotonin and corticoid metabolism. To promote the pipeline for use with wild animal populations, we used non-invasively collected faecal samples from a wild population of Assamese macaques (Macaca assamensis). We targeted loci of 46 previously reported SNPs in 21 candidate genes coding for elements of the HPA-axis and amplified and sequenced them using next-generation Illumina sequencing technology. We compared multiple bioinformatics pipelines for variant calling and variant effect prediction. Based on this strategy and the application of different quality thresholds, we identified up to 159 SNPs with different types of predicted functional effects among our natural study population. This study provides a massively parallel sequencing panel that will facilitate integrating large-scale SNP data into behavioural and physiological studies. Such a multi-faceted approach will promote understanding of flexibility and constraints of animal behaviour and hormone physiology.

Phylogeographic analysis of African swine fever virus based on the p72 gene sequence.

African swine fever virus (ASFV) outbreak has been considered as an emerging and re-emerging disease for almost a century. Diagnostically, simple polymerase chain reaction and sequencing-based molecular detection could be employed for both viral identification and genotyping. This study established a novel phylogenetic analysis and epidemiology comparison based on 205 bp of p72 gene sequences. Based on this partial p72 fragment, an updated list of 44 different genotypes from a total of 516 ASFV sequences compiled from GenBank was generated. Nucleotide diversity was 0.04325 ± 0.00231. The analysis of spatial genetic variation divided the ASFV populations of the African continent into four clades (clade A: central and upper eastern Africa; clade B: eastern Africa; clade C: eastern and southern Africa; and clade D: southern Africa). These results and the developed protocol could serve as useful molecular tools for ASFV diagnosis from degraded DNA or putrefied samples, and also provide the phylogeographic perspective to identify the origin of viral outbreaks, facilitating the decision planning to limit their spread.

The elephant interferon gamma assay: a contribution to diagnosis of tuberculosis in elephants.

Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-γ in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require its application in large populations of non-infected and infected elephants.

Y-chromosomal variation confirms independent domestications of swamp and river buffalo.

Y-chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.

Expression of mouse mammary tumor virus superantigen accelerates tumorigenicity of myeloma cells.

To investigate whether superantigen (SAG) from endogenous mouse mammary tumor virus functions as an immunogenic or a tumorigenic factor in tumor development, the BALB/c myeloma cell line FO was transfected with the SAG gene from the 3' Mtv-50 long terminal repeat (LTR) open reading frame (ORF), the product of which was specific for Vbeta6. All five transfectants expressing Mtv-50 LTR ORF mRNA showed stimulatory activity for Vbeta6 T-cell hybridomas in vitro; this activity was inhibited by the addition of anti-Mtv-7 monoclonal antibody (MAb) or anti-major histocompatibility complex class II I-A(d) and I-E(d) MAb. All transfectants with the SAG gene grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation, whereas all clones, including mock transfectants, grew equally well in athymic nude mice. A significant fraction of Vbeta6 T cells selectively expressed activation markers, including CD44(high), CD62L(low), and CD69(high), and produced large amounts of interleukin 5 (IL-5) and IL-6 in BALB/c mice inoculated with transfectants. These results suggested that the expression of viral SAG enhances the tumorigenicity of a myeloma cell line through the stimulation of SAG-reactive T cells.

T cells bearing Vbeta8 are preferentially infected with exogenous mouse mammary tumor virus.

We previously reported that a mouse mammary tumor virus (MMTV(II-TES14)), encoding a superantigen specific for TCR Vbeta14, can infect lymph node (LN) cells of mice in an I-E-independent manner. Here we examined the kinetics of cell types infected with exogenous MMTV in the draining LN after s.c. injection of II-TES milk containing MMTV(II-TES14). The infectivity was assessed in LN cells sorted into each cell subset by a semiquantitative analysis of MMTV provirus using PCR with a primer specific for MMTV(II-TES14). Only B cells in the LN were infected by the MMTV on day 6 after injection, but CD8+ T cells and, to a lesser extent, CD4+ T cells were also found to be detectably infected on day 14 after the injection of II-TES milk. Among the T cells we examined, Vbeta8 T cells were most preferentially infected with MMTV, but no Vbeta14 T cells specific for MMTV(II-TES14) superantigen were infected on day 14 after infection. The transfer of Vbeta8 T cells sorted from mice injected with II-TES milk 14 days previously resulted in the deletion of CD4+ Vbeta14 T cells and in the MMTV infection of normal B6 mice. No MMTV infection of T cells occurred in IgM knockout mice, which lack a mature B cell compartment. These results suggest that MMTV surviving in B cells is transferred to Vbeta8 T cells, which may play an important role in MMTV longevity.

CD4 expression is important but not essential for infection with exogenous mouse mammary tumor virus.

We studied local events in the popliteal lymph nodes of CD4-deficient mice following foot pad injection with an MMTV strain which carries the gene for a V beta 14-specific superantigen. Injection of the V beta 14-specific MMTV induced vigorous expansion of V beta 14+ CD4+ T cells and B cells in their lymph nodes of CD4+/- heterozygous control mice. On the other hand, CD4-/- mice injected with the MMTV showed a proliferation of V beta 14+ T cells among the population of TCR alpha beta + CD4-CD8- T cells, although to a lesser extent. This phenomenon was not accompanied by vigorous B cell expansion. A PCR assay revelated that the MMTV definitely infected the lymph nodes cells of the CD4-/- mouse. However, the infectivity of the MMTV in CD4-/- mice was approximately 20 times lower than that in CD4+/- mice. These findings indicate that, in MMTV infection of CD4-deficient mice, the superantigen-reactive T cells among the population of TCR alpha beta +CD4-CD8- T cells substitute for the superantigen-reactive CD4- T cells of normal mice, and that the absence of CD4 molecules decreased the infectivity of MMTV because of insufficient expansion of the superantigen-reactive T cells.

A novel V beta 2-specific endogenous mouse mammary tumor virus which is capable of producing a milk-borne exogenous virus.

We have previously reported new Mtv loci, Mtv-48 and -51, in the Japanese laboratory mouse strains CS and NC. Here we show by backcross analysis that both Mtv-48 and -51 cosegregate with very slow deletion of T cells bearing V beta 2. The nucleotide sequences of the open reading frames in the 3' long terminal repeats of Mtv-48 and -51 were very similar to those of Mtv-DDO, mouse mammary tumor virus C4 [MMTV(C4)], and MMTV(BALB/cV), which encode V beta 2-specific superantigens. Furthermore, backcross female mice carrying Mtv-48 but not Mtv-51 were found to be able to produce milk-borne MMTV(CS), which can vigorously stimulate V beta 2-expressing T cells after local injection in vivo in an I-E-dependent manner. On the other hand, mice carrying Mtv-51 but not Mtv-48 could not produce such an MMTV in milk. The nucleotide sequences of MMTV(CS) open reading frame were completely matched with those of Mtv-48. These results indicate that the provirus Mtv-48 but not Mtv-51 is capable of producing a milk-borne virus of which the superantigen stimulates V beta 2-expressing T cells.

Concomitant infection with exogenous mouse mammary tumor virus encoding I-E-dependent superantigen in I-E-negative mouse strain.

We found that milk from II TES mice contained two species of exogenous mouse mammary tumor viruses (MMTV). Sequence analysis of the open reading frame (ORF) in the MMTV 3' long terminal repeat indicated that the two MMTV, MMTV (II TES2) and MMTV (II TES14), encode superantigens specific for V beta 2+ T cells and V beta 14+ T cells, respectively. In an experiment of subcutaneous injection of II TES milk, both T cells bearing TCR V beta 2 and V beta 14 proliferated vigorously in the draining lymph node from BALB/c mice (H-2d I-E+), whereas only V beta 14+ T cells showed significant proliferation in C57BL/6 mouse (B6 H-2b I-E-) lymph nodes. These findings indicated that the superantigen encoded by MMTV (II TES2) required MHC class II I-E molecules exclusively for Ag presentation, but MMTV (II TES14) stimulated V beta 14+ T cells even in the absence of I-E molecules. Semiquantitative analysis of MMTV proviruses using PCR revealed that B6 mice were not infected with MMTV (II TES2) by injection of this MMTV alone. However, injection of II TES milk containing both MMTV (II TES14) and MMTV (II TES2) induced infection of B6 mice with MMTV (II TES2) besides MMTV (II TES14), in spite of no expansion of V beta 2+ T cells in this mouse strain. These results suggested that I-E-negative mice were concomitantly infected with MMTV (II TES2) with the help of I-E independent T cell activation mediated by MMTV (II TES14).

Delay in expression of a mammary tumor provirus is responsible for defective clonal deletion during postnatal period.

A gene-encoding ligand for deletion of T cells bearing TcRV beta 6 and V beta 8.1 cosegregates a new mammary tumor provirus locus, Mtv-50 in NC mice. The sequence of the open reading frame (ORF) in the 3' long terminal repeat (LTR) of Mtv-50 was strikingly similar to those of Mtv-7, Mtv-43 and exogenous mouse mammary tumor virus (SW) with properties of minor lymphocyte stimulating antigen 1a. Consistent with previous reports, clonal deletion of mature thymocytes bearing TcRV beta 6 was defective during the early postnatal period of mice carrying Mtv-50. Appreciable levels of mRNA corresponding to common Mtv ORF and Mtv-6 ORF were expressed in the neonatal thymus, while little, if any, mRNA corresponding to Mtv-50 ORF was detected in the thymus at the early postnatal stage. Delay in expression of Mtv-50 ORF during the postnatal period may be responsible for the failure of clonal deletion of V beta 6-T cells in the early postnatal life of mice carrying Mtv-50.

Neuroblastoma in a transgenic mouse carrying a metallothionein/ret fusion gene.

We have recently succeeded in producing transgenic mice carrying a hybrid gene consisting of mouse metallothionein promoter-enhancer and the ret oncogene (MT/ret). (Iwamoto et al., 1991b). A retroperitoneal tumour developed in one of 17 MT/ret transgenic founder mice. Histological analysis revealed that the tumour consisted of undifferentiated neuroblasts and differentiated ganglion cells, the latter of which were strongly positive for neuron specific enolase. Expression of the ret transgene was observed at high levels in RNA from the tumour, but not in those of other normal tissues. In addition, a 100kDa ret protein was detected in the cell lysate of the tumour. Taken together with our previous data, these results suggest a possible role for the ret oncogene in the proliferation of neural crest cells.

Expression of proto-oncogenes and tumor suppressor genes in in vitro cell lines derived from a thymus, thymoma, and malignant thymoma of rats.

To analyze the processes of the development of thymoma and malignant thymoma from normal thymic epithelial cells, we have examined the expression of 15 proto-oncogenes and tumor suppressor genes in seven in vitro epithelial cell lines established from a normal thymus (TuD1-1, TuD1-3, and TuD1-5), thymoma (TaD1-3 and TaD1-8), and malignant thymoma (MTHC-1 and MTHC-3) of rats. Northern blot analysis indicated that most of these genes examined were transcribed at similar levels. However, higher levels of transcription of epidermal growth factor receptor (EGFR) were observed in TuD1-1, TuD1-3, TuD1-5, TaD1-3, and TaD1-8 cells than in MTHC-1 and MTHC-3 cells. Conversely, four of the former five cell lines showed no TGF-beta transcription while the latter two cell lines had high levels of its expression. In addition, c-fos proto-oncogene was highly expressed in TuD1-5 cells, which showed the fastest growth rate among the seven cell lines. These results denote that some molecular changes in the regulation of gene expression occurred in the processes of malignant transformation of thymic epithelial cells.

Low frequency of rearrangements of the ret and trk proto-oncogenes in Japanese thyroid papillary carcinomas.

We investigated the frequency of rearrangements of the ret and trk proto-oncogenes in Japanese thyroid tumors. DNAs from 38 thyroid papillary carcinomas and 14 follicular adenomas were analyzed by Southern blotting. Rearrangements of the ret and trk proto-oncogenes were detected in one and two papillary carcinomas, respectively, but not in follicular adenomas. Analysis by a reverse transcriptase-polymerase chain reaction method showed that the ret rearrangement-positive tumor contained the PTC/retTPC chimeric transcript, which was reported to be found specifically in thyroid tumors and adenomatous goiter. We also found that rearranged mRNA of the trk proto-oncogene was expressed at high levels in one of two trk rearrangement-positive tumors. Our results indicated that the frequency of rearrangements of these proto-oncogenes in Japanese papillary carcinomas was much lower than that in Italian patients.

Tissue distribution of mouse mammary tumor virus (MMTV) antigens and new endogenous MMTV loci in Japanese laboratory mouse strains.

The distribution of mouse mammary tumor virus (MMTV) antigens was studied by the immunoperoxidase method in the II-TES and I-TES mouse strains as well as their progenitors, CS and DBA/2 strains. In the II-TES, I-TES and CS strains, and BALB/c mice foster-nursed with these strains, MMTV antigens were found not only in epithelial cells of the mammary glands but also in those of other tissues including the seminal vesicle, vas deferens, epididymis, prostate, parotid, submandibular, lacrimal, sebaceous, and urethral glands. In DBA/2 and BALB/cfDBA/2 mice, however, the MMTV antigens were found only in the mammary glands. Electron microscopic examination showed MMTV particles in these organs. When we examined the presence of Mtv-1 and 2 proviruses, which are known to be responsible for MMTV expression, in the genomes of the II-TES, I-TES, CS and DBA/2 strains by Southern blotting, Mtv-2 was not found in any of the mice and Mtv-1 was found in the II-TES and DBA/2 mice but not in the I-TES and CS mice. Instead, four new endogenous MMTV loci, which have never previously been reported in laboratory mouse strains, were detected in the genomes of the II-TES, I-TES and CS strains. One (designated Mtv-42) was common in the three strains and the other three (designated Mtv-43, 44 and 45) were common in the II-TEX and I-TES strains or the II-TES and CS strains. These results thus suggest that new endogenous MMTV loci may be responsible for MMTV expression in a variety of tissues of these three strains.

Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/ret fusion gene.

We generated four independent transgenic mouse lines that showed severe melanosis of the whole body by introducing the ret oncogene fused to the mouse metallothionein (MT)-I promoter-enhancer (MT/ret). Whereas melanogenesis was accelerated without distinct proliferative disorders in one line, melanocytic tumours frequently developed in the other three lines. Northern hybridization and in situ hybridization analyses showed that tumour cells and non-tumorous melanin-producing cells expressed the transgene at high levels. The aberrant melanogenesis and tumour development were influenced by genetic and environmental factors. Furthermore, crossbreeding experiments between the transgenic mice and Wv mice suggested that the ret gene product can partially compensate for the defect of melanocyte development in Wv mice. This is a novel mammalian model in which melanosis and melanocytic tumours develop stepwise, triggered by a single transgene.