Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Comparison of Serum Amylase and Lipase Levels between Predialysis and Maintenance Haemodialysis CKD Patients.

This cross sectional study was done to compare serum levels of amylase and lipase between predialysis and maintenance haemodialysis chronic kidney disease (CKD) patients and also to find out their relationship between degrees of renal impairment in Mymensingh Medical College Hospital and National Institute of Kidney Diseases and Urology, Dhaka, Bangladesh from May 2016 to April 2017. A total of 80 patients were included purposively as study subjects and made into two groups namely predialysis CKD group comprising 50 patients and other as maintenance haemodialysis group comprising of 30 patients. Among the predialysis group majority of the CKD was caused by glomerulonephritis (48%) followed by diabetes (26%), HTN (2%) and large portion undiagnosed (24%) whereas in the haemodialysis group ESRD was caused by diabetes (46%) followed by glomerulonephritis (16%), HTN (13%) and undiagnosed (23%). This study showed that mean serum amylase (158±718U/L vs. 111±41U/L) did not significantly differ between study groups except being above reference level but serum lipase (739±888U/L vs. 434±214U/L) was significantly higher in the predialysis group. There was a correlation between rising serum creatinine with serum amylase and lipase.

Response of Proliferative Lupus Nephritis with Pulse Cyclophosphamide Therapy during Induction Period: A Single Centre Study in Bangladesh.

This prospective study was conducted to assess the response of proliferative lupus nephritis with pulse cyclophosphamide therapy during induction period in the department of Nephrology of Dhaka Medical College, Dhaka, Bangladesh from December 2012 to November 2013. A total of 35 clinically diagnosed SLE patients of class III/IV lupus nephritis were included. But 3 patients were dropped out during follow-up, therefore finally 32 patients (class III = 4, class IV = 28) were studied. The patients were evaluated for response on the basis of proteinuria, serum creatinine & active sediment in urine after 6th cycle of cyclophosphamide and 62.5% patients achieved complete response, 25% patients achieved partial response & 12.5% patients achieved no response. The factors favored complete response was early clinical presentation (7 months duration), proteinuria ≤3gm/day& normal renal function during their initial presentation. And higher anti ds DNA titre was an independent predictor for partial response/no response.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Radiation hybrid map assignments of 11 ESTs obtained from a 28-day-old swine embryo cDNA library to the IMpRH map.

In order to improve the map resolution and to locate more genes on the porcine radiation hybrid map, expressed sequence tags (ESTs) were isolated from a 28-day-old normal pig embryo cDNA library. The ESTs were sequenced from the 5'-end and similarities were checked with sequences registered in the NCBI DNA database (http://www.ncbi.nlm.nih.gov/blast/). The ESTs sequences which have high identity scores (>80%) against human genes or ESTs were further sequenced from the 3' untranslated region. The ESTs which were sequenced successfully were used to design primers for PCR analysis of the radiation hybrid panel. Eleven ESTs were physically mapped to porcine chromosomes 2, 4, 8, 10, 13, 14 and X. The localizations are in agreement with the comparative mapping data between human and pig. The results will provide unique information to the comparative map of human and pig.

Directed isolation and mapping of microsatellites from swine Chromosome 1q telomeric region through microdissection and RH mapping.

Several quantitative trait loci (QTLs) (vertebrate number, birth weight, age at puberty, growth rate, gestation length, and backfat depth) have been independently mapped to the distal region of swine Chromosome (SSC) 1q in several resource populations. In order to improve the map resolution and refine these QTLs more precisely on SSC1q, we have isolated and mapped additional microsatellites (ms), using chromosome microdissection and radiation hybrid (RH) mapping. Five copies of the telomeric region of SSC1q were microdissected from metaphase spreads and pooled. The chromosomal fragment DNA was randomly amplified by using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), enriched for ms, and subcloned into a PCR vector. Screening of subsequent clones with ms probes identified 23 unique ms sequences. Fifteen of these (65%) were subjected to radiation hybrid (RH) mapping by using the INRA-University of Minnesota porcine RH panel (IMpRH); and the remaining eight were not suited for the RH mapping. Twelve microsatellites were assigned to SSC1q telomeric region of IMpRH map (LOD >6), and three remain unlinked (LOD <6). Out of the 15 microsatellite markers, 9 were polymorphic in NIAI reference population based on the Meishan and Göttingen miniature pig. In summary, we have used microdissection and radiation hybrid mapping to clone and map 12 new microsatellites to the swine gene map to increase the resolution of SSC1q in the region of known QTLs.

Cell-mediated and humoral immunity and phagocytic ability in chicken Lines divergently selected for serum immunoglobulin M and G levels.

Humoral and cell-mediated immunities and phagocytic ability were examined at the third and fourth generations of selection in two pairs of chicken lines (at 10 wk of age) that were divergently selected for levels of high serum Ig M (HIM), low serum Ig M (LIM), high serum Ig G (HIG), and low serum Ig G (LIG). Cell-mediated immunity was examined by splenomegaly assay at 12 wk of age. At 20 and 23 wk of age, 20 birds from the respective lines were injected Brucella abortus (BA), and blood samples were collected at 7 and 14 d postprimary immunization (PPI) and postsecondary immunization (PSI). Phagocytic ability was measured by carbon clearance assay at 25 and 30 wk of age. The results showed that the LIG line had higher degree of splenomegaly indices than the HIG line in both generations. The HIM and HIG lines had significantly (P < 0.05) higher total antibody titers to BA than their low counterparts. Similarly, mercaptoethanol-resistant (MER) antibody titers to BA, as measured only in the fourth generation, were significantly (P < 0.05) higher in the HIM and HIG lines than their low counterparts. In both generations, the HIM and HIG lines had significantly (P < 0.01) faster carbon clearance ability than the LIM and LIG lines. The results suggest that both pairs of selected lines exhibited divergence in immunocompetence, although they had been selected for serum Ig isotypes.

Direct and correlated response to divergent selection for serum immunoglobulin M and G levels in chickens.

Two pairs of chicken lines were divergently selected for serum immunoglobulin M and G levels at 10 wk of age to examine the effect of selection on direct and correlated traits. After three generations of selection, the lines were tested for their ability of antibody production against SRBC, a T cell dependent antigen. At 14 and 17 wk of age, the lines were immunized intravenously with 1 mL of 5% SRBC solution. Blood samples were collected at 7 and 14 d of post primary immunization (PPI) and post secondary immunization (PSI). Selection was effective (P < 0.01) in changing the serum immunoglobulin M levels. The G levels were changed in a downward direction initially in the high line. The differences between the high immunoglobulin M (HIM) and low immunoglobulin M (LIM) lines as well as high immunoglobulin G (HIG) and low immunoglobulin G (LIG) lines became larger during the course of selection. Selection differentials varied considerably during the selection, but the selection response was almost constant. Realized heritability was relatively high, ranging from 0.58 to 0.66 and from 0.40 to 0.60 for the IgM and IgG selected lines, respectively. Total antibody titers to SRBC differed significantly (P < 0.01) between the HIM and LIM lines at 7 and 14 d PPI, and 7 d PSI. The HIM line also produced significantly higher mercaptoethanol resistant antibody titers to SRBC than the LIM line at 7 and 14 d PPI and PSI. The LIG line had significantly higher total antibody titers to SRBC than the HIG line at both 7 and 14 d of PPI and PSI. From these results, it is suggested that selection of chickens on the basis of serum immunoglobulin isotypes may change antibody producing cells as well as other immunocompetent cells that modulate the immune response of the selected lines.

Chronic diarrhoeal diseases in adults (a preliminary report).

The relationship of chronic diarrhoeal diseases in adults on socio-economic condition, parasitic infestation and degree of anaemia have been investigated. The disease was more prevalent in poor socio-economic group of people. Most of the patients had mixed parasitic infestations. Anaemia was present in all the cases. Difference in the degree of anaemia was not significant amongst the patients having either Ascaris or hookworm infestation.