Essential role of Rnd1 in innate immunity during viral and bacterial infections.

Intracellular and cell surface pattern-recognition receptors (PRRs) are an essential part of innate immune recognition and host defense. Here, we have compared the innate immune responses between humans and bats to identify a novel membrane-associated protein, Rnd1, which defends against viral and bacterial infection in an interferon-independent manner. Rnd1 belongs to the Rho GTPase family, but unlike other small GTPase members, it is constitutively active. We show that Rnd1 is induced by pro-inflammatory cytokines during viral and bacterial infections and provides protection against these pathogens through two distinct mechanisms. Rnd1 counteracts intracellular calcium fluctuations by inhibiting RhoA activation, thereby inhibiting virus internalisation. On the other hand, Rnd1 also facilitates pro-inflammatory cytokines IL-6 and TNF-α through Plxnb1, which are highly effective against intracellular bacterial infections. These data provide a novel Rnd1-mediated innate defense against viral and bacterial infections.

Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens.

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.

Pharmacogenomics and Personalized Medicine in Type 2 Diabetes Mellitus: Potential Implications for Clinical Practice.

Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, and is rising in incidence with widespread prevalence. Multiple gene variants are associated with glucose homeostasis, complex T2DM pathogenesis, and its complications. Exploring more effective therapeutic strategies for patients with diabetes is crucial. Pharmacogenomics has made precision medicine possible by allowing for individualized drug therapy based on a patient's genetic and genomic information. T2DM is treated with various classes of oral hypoglycemic agents, such as biguanides, sulfonylureas, thiazolidinediones, meglitinides, DPP4 inhibitors, SGLT2 inhibitors, α-glucosidase inhibitors, and GLP1 analogues, which exhibit various pharmacogenetic variants. Although genomic interventions in monogenic diabetes have been implemented in clinical practice, they are still in the early stages for complex polygenic disorders, such as T2DM. Precision DM medicine has the potential to be effective in personalized therapy for those suffering from various forms of DM, such as T2DM. With recent developments in genetic techniques, the application of candidate-gene studies, large-scale genotyping investigations, genome-wide association studies, and "multiomics" studies has begun to produce results that may lead to changes in clinical practice. Enhanced knowledge of the genetic architecture of T2DM presents a bigger translational potential. This review summarizes the genetics and pathophysiology of T2DM, candidate-gene approaches, genome-wide association studies, personalized medicine, clinical relevance of pharmacogenetic variants associated with oral hypoglycemic agents, and paths toward personalized diabetology.

SARS-CoV-2 Delta Variant among Asiatic Lions, India.

In May 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in Asiatic lions in a zoological park in India. Sequence and phylogenetic analyses showed the SARS-CoV-2 strains were the B.1.617.2 (Delta) variant. To reduce transmission of variants of concern, surveillance of SARS-CoV-2 in wild animal populations should be increased.

Particulate matter (PM10) enhances RNA virus infection through modulation of innate immune responses.

Sensing of pathogens by specialized receptors is the hallmark of the innate immunity. Innate immune response also mounts a defense response against various allergens and pollutants including particulate matter present in the atmosphere. Air pollution has been included as the top threat to global health declared by WHO which aims to cover more than three billion people against health emergencies from 2019 to 2023. Particulate matter (PM), one of the major components of air pollution, is a significant risk factor for many human diseases and its adverse effects include morbidity and premature deaths throughout the world. Several clinical and epidemiological studies have identified a key link between the PM existence and the prevalence of respiratory and inflammatory disorders. However, the underlying molecular mechanism is not well understood. Here, we investigated the influence of air pollutant, PM10 (particles with aerodynamic diameter less than 10 µm) during RNA virus infections using Highly Pathogenic Avian Influenza (HPAI) - H5N1 virus. We thus characterized the transcriptomic profile of lung epithelial cell line, A549 treated with PM10 prior to H5N1infection, which is known to cause severe lung damage and respiratory disease. We found that PM10 enhances vulnerability (by cellular damage) and regulates virus infectivity to enhance overall pathogenic burden in the lung cells. Additionally, the transcriptomic profile highlights the connection of host factors related to various metabolic pathways and immune responses which were dysregulated during virus infection. Collectively, our findings suggest a strong link between the prevalence of respiratory illness and its association with the air quality.

MicroRNA hsa-miR-324-5p Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2.

MicroRNAs (miRNAs) are small noncoding RNAs that are crucial posttranscriptional regulators for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA virus infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we showed that expression of cellular miRNA miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses H5N1, A/PR8/H1N1, and Newcastle disease virus (NDV) and transfection with poly(I·C). We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of type I interferon, type III interferon, and interferon-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of the JAK1-STAT3 pathway. Together, these findings highlight that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003 to 2017, H5N1 HPAIV caused 453 human deaths, giving it a high mortality rate (52.74%). This work shows that miR-324-5p suppresses H5N1 HPAIV replication by directly targeting the viral genome (thereby inhibiting viral gene expression) and cellular CUEDC2 gene, the negative regulator of the interferon pathway (thereby enhancing antiviral genes). Our study enhances the knowledge of the role of microRNAs in the cellular response to viral infection. Also, the study provides help in understanding how the host cells utilize small RNAs in controlling the viral burden.

Emerging avian influenza infections: Current understanding of innate immune response and molecular pathogenesis.

The highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in gallinaceous poultry species, domestic ducks, various aquatic and terrestrial wild bird species as well as humans. The outcome of the disease is determined by complex interactions of multiple components of the host, the virus, and the environment. While the host-innate immune response plays an important role for clearance of infection, excessive inflammatory immune response (cytokine storm) may contribute to morbidity and mortality of the host. Therefore, innate immunity response in avian influenza infection has two distinct roles. However, the viral pathogenic mechanism varies widely in different avian species, which are not completely understood. In this review, we summarized the current understanding and gaps in host-pathogen interaction of avian influenza infection in birds. In first part of this article, we summarized influenza viral pathogenesis of gallinaceous and non-gallinaceous avian species. Then we discussed innate immune response against influenza infection, cytokine storm, differential host immune responses against different pathotypes, and response in different avian species. Finally, we reviewed the systems biology approach to study host-pathogen interaction in avian species for better characterization of molecular pathogenesis of the disease. Wild aquatic birds act as natural reservoir of AIVs. Better understanding of host-pathogen interaction in natural reservoir is fundamental to understand the properties of AIV infection and development of improved vaccine and therapeutic strategies against influenza.

Emergence of a deviating genotype VI pigeon paramyxovirus type-1 isolated from India.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1 (APMV-1), which infects pigeons. The virus causes high morbidity and mortality, creating an alarming state for the poultry industry. The present work describes the molecular and pathogenic characterization of a PPMV-1 strain isolated from pigeon in Bhopal, India. Complete genome sequence analysis revealed a genome of 15,192 nucleotides encoding six genes organized in the order 3'-N-P-M-F-HN-L-5'. The fusion gene sequence analysis showed the presence of multiple basic amino acids 112R-R-Q-K-R-F117 at the cleavage site corresponding to pathogenic strains. The mean death time and intracerebral pathogenicity index values indicated a mesogenic nature for the PPMV-1 isolate. On phylogenetic analysis, the strain clustered with genotype VI viruses, including isolates from pigeon and dove. The Bhopal strain showed significant intra and inter-genotype evolutionary distance, suggesting the emergence of a new sub-genotype, VIj.

Duck gut viral metagenome analysis captures snapshot of viral diversity.

BACKGROUND: Ducks (Anas platyrhynchos) an economically important waterfowl for meat, eggs and feathers; is also a natural reservoir for influenza A viruses. The emergence of novel viruses is attributed to the status of co-existence of multiple types and subtypes of viruses in the reservoir hosts. For effective prediction of future viral epidemic or pandemic an in-depth understanding of the virome status in the key reservoir species is highly essential. METHODS: To obtain an unbiased measure of viral diversity in the enteric tract of ducks by viral metagenomic approach, we deep sequenced the viral nucleic acid extracted from cloacal swabs collected from the flock of 23 ducks which shared the water bodies with wild migratory birds. RESULT: In total 7,455,180 reads with average length of 146 bases were generated of which 7,354,300 reads were de novo assembled into 24,945 contigs with an average length of 220 bases and the remaining 100,880 reads were singletons. The duck virome were identified by sequence similarity comparisons of contigs and singletons (BLASTx E score, <10(-3)) against viral reference database. Numerous duck virome sequences were homologous to the animal virus of the Papillomaviridae family; and phages of the Caudovirales, Inoviridae, Tectiviridae, Microviridae families and unclassified phages. Further, several duck virome sequences had homologous with the insect viruses of the Poxviridae, Alphatetraviridae, Baculoviridae, Densovirinae, Iflaviridae and Dicistroviridae families; and plant viruses of the Secoviridae, Virgaviridae, Tombusviridae and Partitiviridae families, which reflects the diet and habitation of ducks. CONCLUSION: This study increases our understanding of the viral diversity and expands the knowledge about the spectrum of viruses harboured in the enteric tract of ducks.

Genome Wide Host Gene Expression Analysis in Chicken Lungs Infected with Avian Influenza Viruses.

The molecular pathogenesis of avian influenza infection varies greatly with individual bird species and virus strain. The molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV) or the low pathogenic avian influenza virus (LPAIV) infection in avian species remains poorly understood. Thus, global immune response of chickens infected with HPAI H5N1 (A/duck/India/02CA10/2011) and LPAI H9N2 (A/duck/India/249800/2010) viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAI H5N1 virus induced excessive expression of type I IFNs (IFNA and IFNG), cytokines (IL1B, IL18, IL22, IL13, and IL12B), chemokines (CCL4, CCL19, CCL10, and CX3CL1) and IFN stimulated genes (OASL, MX1, RSAD2, IFITM5, IFIT5, GBP 1, and EIF2AK) in lung tissues. This dysregulation of host innate immune genes may be the critical determinant of the severity and the outcome of the influenza infection in chickens. In contrast, the expression levels of most of these genes was not induced in the lungs of LPAI H9N2 virus infected chickens. This study indicated the relationship between host immune genes and their roles in pathogenesis of HPAIV infection in chickens.

The microRNA miR-485 targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication.

MicroRNAs (miRNAs) are small noncoding RNAs that are responsible for dynamic changes in gene expression, and some regulate innate antiviral responses. Retinoic acid-inducible gene I (RIG-I) is a cytosolic sensor of viral RNA; RIG-I activation induces an antiviral immune response. We found that miR-485 of the host was produced in response to viral infection and targeted RIG-I mRNA for degradation, which led to suppression of the antiviral response and enhanced viral replication. Thus, inhibition of the expression of mir-485 markedly reduced the replication of Newcastle disease virus (NDV) and the H5N1 strain of influenza virus in mammalian cells. Unexpectedly, miR-485 also bound to the H5N1 gene PB1 (which encodes an RNA polymerase required for viral replication) in a sequence-specific manner, thereby inhibiting replication of the H5N1 virus. Furthermore, miR-485 exhibited bispecificity, targeting RIG-I in cells with a low abundance of H5N1 virus and targeting PB1 in cells with increased amounts of the H5N1 virus. These findings highlight the dual role of miR-485 in preventing spurious activation of antiviral signaling and restricting influenza virus infection.

Low prevalence of CCR5-Δ32, CCR2-64I and SDF1-3'A alleles in the Baiga and Gond tribes of Central India.

Human immunodeficiency virus-1 (HIV-1) which causes acquired immune deficiency syndrome (AIDS), by infecting CD4(+) immune cells and hence weakening the host defense mechanism till death, is one of the major factor responsible for human demises worldwide. Both innate (monocytes and macrophages) and adaptive (T cells) immune cells expresses chemokines receptors (2 and 5) and stromal cell derived factor-1 (SDF-1) which play crucial role in HIV-1 virus entry and progression. Allele variants of genes CCR5 (CCR5-Δ32), CCR2 (CCR2-64I) and SDF1 (SDFA-3'A; the ligand of CXCR4) are known to slow down the HIV-1 progression in infected individual. In the present study, the frequency of CCR5-Δ32, CCR2-64I and SDF1-3'A alleles in primitive tribe (Baiga) and a non-primitive tribe (Gond) of central India were investigated. A total 200 seronegative samples for HIV from healthy individuals of tribes were analyzed and observed allele frequencies of CCR5-Δ32, CCR2-64I and SDF1-3'A were (0, 0.035, 0.080) and (0, 0.110, 0.100) in Baiga and Gond respectively. Minor allele frequency of these alleles of Gond and Baiga tribes were compared with different populations of the world for relative hazard (RH), which indicate the risk of progression after infection of HIV1. The RH values were calculated based on genotypic frequency, showed the high RH value (RH1-AIDS1993-0.98, RH2-AIDS1987-0.98 and death/RH3-0.97) in Baiga tribe, indicates the low level of resistance against HIV-1 progression after infection.

In-silico search of virus-specific host microRNAs regulating avian influenza virus NS1 expression.

Avian influenza is a highly contagious viral infection caused by avian influenza virus type A of the family Orthomyxoviridae primarily affecting the avian species. The non-structural protein 1 (NS1) encoded by the NS1 gene of the virus is critical in establishing the infection. NS1 protein acts to suppress the virus-induced host interferon response and also inhibit Protein kinase R activation thereby helping the virus to establish the infection. MicroRNAs (miRNA) are small regulatory endogenous non-coding RNAs of ~22 nucleotides in length located within introns of coding and non-coding genes, exons of non-coding genes or inter-genic regions. miRNAs can target the gene at various sites and effectively reduce or shut down its expression. In this study, set of differentially expressed chicken miRNA identified by deep sequencing H5N1 infected and SPF chicken lung were computationally analyzed, to identify targets in the NS1 gene. 300 differentially expressed miRNAs were then analyzed individually for target sites in gi|147667147|gb|EF362422.1| influenza A virus (A/chicken/India/NIV33487/06(H5N1)) segment 8, complete sequence using RNAhybrid 2.2. The analysis yielded gga-miR-1658* as the potential miRNA which is targeting the NS1 gene of H5N1 genome.

Analysis of the crow lung transcriptome in response to infection with highly pathogenic H5N1 avian influenza virus.

The highly pathogenic avian influenza (HPAI) H5N1 virus, currently circulating in Asia, causes severe disease in domestic poultry as well as wild birds like crow. However, the molecular pathogenesis of HPAIV infection in crows and other wild birds is not well known. Thus, as a step to explore it, a comprehensive global gene expression analysis was performed on crow lungs, infected with HPAI H5N1 crow isolate (A/Crow/India/11TI11/2011) using high throughput next generation sequencing (NGS) (GS FLX Titanium XLR70). The reference genome of crow is not available, so RNA seq analysis was performed on the basis of a de novo assembled transcriptome. The RNA seq result shows, 4052 genes were expressed uniquely in noninfected, 6277 genes were expressed uniquely in HPAIV infected sample and of the 6814 genes expressed in both samples, 2279 genes were significantly differentially expressed. Our transcriptome profile data allows for the ability to understand the molecular mechanism behind the recent lethal HPAIV outbreak in crows which was, until recently, thought to cause lethal infections only in gallinaceous birds such as chickens, but not in wild birds. The pattern of differentially expressed genes suggest that this isolate of H5N1 virus evades the host innate immune response by attenuating interferon (IFN)-inducible signalling possibly by down regulating the signalling from type I IFN (IFNAR1 and IFNAR2) and type II IFN receptors, upregulation of the signalling inhibitors suppressor of cytokine signalling 1 (SOCS1) and SOCS3 and altering the expression of toll-like receptors (TLRs). This may be the reason for disease and mortality in crows.

The role of TLR9 polymorphism in susceptibility to pulmonary tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis (PTB), a major health problem that leads to 1.5 million deaths annually. Host genetic factors play a significant role in disease resistance/susceptibility by altering immunity against MTB. Toll-like receptor (TLR) sensors such as TLR2, TLR4, TLR8, and TLR9 are known to play a pivotal role in PTB via modulating sensor expression and/or effector responses. Single-nucleotide polymorphism (SNP) rs187084 (T-1486C) of the TLR9 promoter is associated with various autoimmune disorders and cancers. A recent bioinformatic analysis predicted that the T-1486C SNP is involved in PTB, although its potential role is unclear. To investigate the role of T-1486C in PTB, we stimulated PBMCs with the H37Rv whole cell lysate. We found that the presence of the "C" allele increases the transcriptional activity of the TLR9, which in turn induces high levels of Interferon gamma-induced protein 10 (IP-10), a biomarker for PTB. However, the expression of protective cytokines such as IFNγ and TNFα was observed significantly less with "C" allele in comparison to "T" allele. We further selected three different tribe populations showing differential susceptibility to PTB and performed genotypic analyses for the TLR9 promoter. We found a significantly lower minor allele frequency (MAF) of T-1486C in the Baiga tribe, wherein fewer PTB cases were reported, than that in the Gond and Korku tribes. Collectively, these data suggest that the minor "C" allele at rs187084 locus may be associated with susceptibility to PTB, which may explain the relatively lower PTB rates observed in Baiga tribe members.

Identification of Chicken Pulmonary miRNAs Targeting PB1, PB1-F2, and N40 Genes of Highly Pathogenic Avian Influenza Virus H5N1 In Silico.

Highly pathogenic Avian influenza (HPAI) is a notifiable viral disease caused by avian influenza type A viruses of the Orthomyxoviridae family. Type A influenza genome consists of eight segments of negative-sense RNA. RNA segment 2 encodes three proteins, PB1, PB1-F2, and N40, which are translated from the same mRNA by ribosomal leaky scanning and reinitiation. Since these proteins are critical for viral replication and pathogenesis, targeting their expression can be one of the approaches to control and resist HPAI. MicroRNAs are short noncoding RNAs that regulate a variety of biological processes such as cell growth, tissue differentiation, apoptosis, and viral infection. In this study, a set of 300 miRNAs expressed in chicken lungs were screened against the HPAI virus (H5N1) segment 2 with different screening parameter like thermodynamic stability of heteroduplex, seed sequence complementarity, conserved target sequence, and target-site accessibility for identifying miRNAs that can potentially target the transcript of segment 2 of H5N1. Chicken miRNAs gga-mir-133c, gga-mir-1710, and gga-mir-146c* are predicted to target the expression of PB1, PB1-F2, and N40 proteins. This indicates that chicken has genetic potential to resist/tolerate H5N1 infection and these can be suitably exploited in designing strategies for control of avian influenza in chicken.

Novel molecular beacon probe-based real-time RT-PCR assay for diagnosis of Crimean-Congo hemorrhagic fever encountered in India.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

De novo assembly and analysis of crow lungs transcriptome.

The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1,510,303 high-quality sequence reads with 581,198,230 bases was de novo assembled into 22,169 isotigs (isotig represents an individual transcript) and 784,009 singletons. Using these isotigs and 581,681 length-filtered (greater than 300 bp) singletons, 20,010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46,604 (70.29%) and 51,642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Transcripts putatively involved in the immune response were identified. Furthermore, 20,599 single nucleotide polymorphisms (SNPs) and 7525 simple sequence repeats (SSRs) were retrieved from the assembled transcript database. This resource should lay an important base for future ecological, evolutionary, and conservation genetic studies on this species and in other related species.

Molecular differentiation of Peroxysome proliferator activated receptor coactivator-1 among different breeds of Bubalus bubalis.

Peroxysome proliferator activated receptor coactivator-1 gene (PPARGC1A) is a positional and functional candidate gene for milk fat yield. It has key role in energy, fat and glucose metabolism. Single nucleotide polymorphisms (SNPs) in Exon-8 of PPARGC1A are reported to be associated with milk fat yield in dairy cattle. In the present investigation PPARGC1A was partially amplified (around 767bp) by designing gene specific primer and confirm by sequencing the amplicon and its comparison with the PPARGC1A gene of bovine. Comparative study of PPARGC1A among different breeds of buffaloes reveals different level of mutations with respect to its gene sequence 0.013-1.69% and protein sequence 0.42% to 2.99%, Similarly the protein structures modeled from their sequences were compared by structural superposition that shows variations (RMSD) from 0.736 to 1.507. Furthermore, the sequences were used to generate a dendrogram. It reveals that Murrah and reference are very close to each other, similarly Toda, Bhadawari and Surti are closely related, whereas Pandharpuri is separated from both the cluster. Especially the variations are more at the binding site of this protein that may be the cause that different breeds have different percentage of milk fat. Further study is underway to detect polymorphism and associate them with milk fat related traits in buffalo.