<div style="text-align:justify">Rapid industrialization and successful green revolution have introduced a wide array of chemicals into our environment; some of these chemicals entered in ecosystem; gets accumulated and exert serious health and ecological problems. These toxic substances can enter the food chain and emphasize pathological changes which damage either cell, organ or system (circulatory, immune, respiratory, digestive, nervous, reproductive and musculo-skeletal etc.) by altering structure and/or function of biological components; DNA, RNA, proteins, lipids and carbohydrates. This review article provides certain molecular diagnostic techniques used for their robust and accurate detection at molecular level. Investigations conducted during 2020-2021 where various review and research articles were surveyed and then extracted to enlist congestive datum for rapid detection of toxicological changes in animals. In our investigations we concluded that toxic substances present in our environment affects health of animals by altering structure and functioning of biomolecules and their concerned system. These cytological and systemic changes can be detected with the help of molecular diagnostic techniques including dideoxysequencing, pyrosequencing, allele specific RT-PCR, CRISPER/Cas, genotyping and microarrays etc. present collection of data will provide congestive information for rapid toxicological detection at molecular level.</div>

Molecular Diagnostic Strategies for the Detection of Toxicological Changes in Animals: A Review

<div style="text-align:justify">Background: Pulicat Lake is one of India&rsquo;s most valuable ecosystems. Correct species identification is essential for the conservation and management of finite resources. Molecular signatures and character-based keys could be used for faster identification. This will be a powerful tool to obtain a significant amount of accurate information quickly and conveniently and could be used for forensic applications and the conservation of fish in Indian waters. 
 
Methods: In addition to morphological identification, the species were identified using various approaches, such as Neighbour-joining (NJ), Maximum Likelihood (ML) trees and the Character-based method. For each species, the LOGic (BLOG 2.0) DNA Barcode analysis method was used to identify different positions of key diagnostic nucleotides in a given set. 
 
Result: DNA barcodes were generated from twenty-nine species of fish representing seven orders, 26 genera and 21 families in this study. The current study&rsquo;s findings show that the 16S rRNA barcodes facilitated successful fish species identification and also provided phylogenetic information to differentiate the fishes. The character-based molecular diagnostic keys for fishes were also developed in-silico from the data set comprising 393 mitochondrial 16S rRNA sequences including the sequences generated in the present study and other published resources.</div>

Character-based Diagnostic Keys, Molecular Identification and Phylogenetic Relationships of Fishes based on Mitochondrial Gene from Pulicat Lake, India: A Tool for Conservation and Fishery Management Purposes

Background: Bean common mosaic disease (BCMD) caused by Bean common mosaic virus (BCMV) has been regarded as one of the most important seed-borne and aphid vector transmitted destructive disease of cowpea not only affecting its yield but also reducing its grain quality globally. Since the dawn of crop improvement, germplasm served as the source of resistance for various biotic stresses. Currently, host plant resistance is the best, reliable, economic and environmental friendly practice of virus disease management. The objective of this study was to assess the response of diverse cowpea germplasm against BCMV under natural and controlled conditions followed by serological and molecular detection. 
 
Methods: In this study, 85 cowpea germplasm accessions and one known BCMV susceptible (C152) and one resistant (CP55) varieties are evaluated for their response to the BCMD in augmented block design (ABD). The reaction of cowpea accessions were assessed using percent disease incidence (PDI). Transmission Electron microscopy (TEM), Direct Antigen Coating-Enzyme-linked Immunosorbent Assay (DAC-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were used for confirming the BCMV. 
 
Result: Natural and controlled condition screening data analysis revealed a significant difference among diverse cowpea germplasm for BCMV diseases incidence. Based on field screening at Ranchi and New Delhi (2019 and 2020) and artificial confirmation. 24 out of 85 accessions were found to be immune and confirmed through DAC-ELISA and RT-PCR. As a consequence these accessions could be a potential new source of disease resistance for BCMV in breeding programs.Further, DAC ELISA of seed coat and Embryo revealed the presence of virus in 5% and 15% of samples tested. Accession IC418505 recorded highest disease incidence (40.0%).

Germplasm Screening for Identification of BCMV Resistance Sources from Diverse Cowpea [Vigna unguiculata (L.) Walp.] Germplasm using Serological and Molecular Diagnostics

This review analyses the use of molecular techniques in rumen microbial identification. The use of regular methods like roll tube and most probable number resulted in under estimation of rumen microbial growth. So molecular biology acts as an advanced tool for rumen microbial culture and identification of various new species. These techniques give complete descriptions of individual ruminal populations. Use of molecular techniques like PCR, DDGE and FISH, which also pave pathway for genetic manipulation of rumen microbes in the field of rumen manipulation. So the combination of traditional and molecular assays gives accurate and satisfactory results.

Molecular techniques in rumen biotechnology: A review

Background: In India, canine Babesiosis has been emerged as a serious canine haemoprotozoan disease caused by protozoa namely Babesia vogeli and Babesia gibsoni which owe their sustainment and transmission to the tick vectors. Reliable, rapid and efficient molecular detection technique is necessary for choosing the effective treatment protocol and planning of control strategies against this emerging canine health problem. 
 
Methods: Babesia gibsoni and Babesia vogeli were detected by molecular means and characterized from extracted DNA of canine blood samples from 8 selected districts of Odisha, India, by an internally controlled multiplex PCR targeting the 18S rRNA gene.&nbsp; The assay was standardized and in-house validated using positive controls and random field samples (n=61) with history of tick infestation. The kappa values were analysed for diagnostic specificity and sensitivity of multiplex PCR assay for detection of naplasma. platys, B. gibsoni, Erhlichia canis, Hepatozoon canis and B. vogeli considering singleplex PCR counterparts as standard test. The PCR products found positive, were subjected to sequencing and phylogenetic analysis. The epidemiological survey (n=280) was performed using both microscopy and the standardized mPCR and risk factors were analyzed.&nbsp; 
 
Result: The mPCR assay generated amplicons of 602 bp and 489 bp corresponding to B. vogeli and B. gibsoni along with amplicon of 218 bp targeting canine &beta;-actin gene as IAC to avoid false negative result. The kappa values for diagnostic specificity and sensitivity of multiplex PCR assay in detection of B. gibsoni and B. vogeli revealed very good agreement with their singleplex PCR counterparts (n=61). The epidemiological survey (n=280) revealed 17.14% prevalence of Babesiosis with high prevalence of B. gibsoni in Khordha districts and incidence of infection were high in summer season. Presence of ticks and limited outdoor activity were found to be significantly associated with rise in incidence of Babesiosis. The Odisha isolates aligned well within the established genetic boundaries for these species, solidifying the utility of gene markers for epidemiological tracking and comparative studies.&nbsp;

Development of Multiplex PCR for Detection of Babesiosis in Dogs along with Molecular Characterization of Pathogens

Molecular markers are effective tools used to &lsquo;flag&rsquo; the location of a specific gene or the inheritance of a definite trait. Markers are unique DNA fragments that can be identified inside the entire genome. The development of molecular markers combined with high throughput technologies have paved the way for achieving the desirable traits as well as induced biotic and abiotic stress tolerance in plant, which enhanced the crop breeding. Highly polymorphic molecular markers are developed for gene mapping, estimation of genetic diversity, finding out the evolution and phylogeny of crop, analysis of heterosis, assessment of diploid/haploid crops and genotyping of cultivars along with Marker Assisted Breeding (MAB)/Marker Assisted Selection (MAS). These are the most significant objectives for crop breeding. This review reveals about the role of various recently developed molecular markers in the improvement of crop. Molecular markers act as a &ldquo;milestone&rdquo; for the researchers who aim to enhance crop breeding.

Role of Molecular Markers in Crop Breeding: A Review

Background: The research work was undertaken to investigate the efficacy of various diagnostic tests by using conventional microscopic examination, rapid Ab detection test and molecular technique, haemato-biochemical changes, Abdominal organs changes using ultrasonography, and therapeutic efficacy of different treatment protocols on Canine Monocytic Ehrlichiosis (CME) infected dogs from in and around Navsari, Gujarat, India.

Methods: 69 dogs suspected of ehrlichiosis based on clinical signs and hematological values and then subjected to blood and buffy coat smear examination, rapid Ab detection test&nbsp;and PCR based assay targeting 16S rRNA gene. The pre and post-treatment haematology and biochemical analysis of treatment groups along with all the positive dogs were performed. Further, all the treatment group dogs were subjected to ultrasound for evaluating organ abnormalities. Modified Clinical Sign Grading (MCSG) score for CME-infected dogs was carried out to evaluate therapeutic responses based on minimizing the clinical score. Descriptive statistics along with Student t-test and One-way analysis of variance (ANOVA) followed by DMRT was performed to compare differences between means wherever applicable.

Result: CME incidence among the 69 suspected cases was 49.28%. Out of 69 suspected dogs, Ehrlichial inclusions or morulae in the monocytes could be identified in only 4.35% of blood smears and 10.14% of buffy coat smears examined by Giemsa and or Field-stained and 15.94% of dogs were found positive for Ehrlichia genus by PCR. 72.5 per cent of dogs were found positive for antibodies to E. canis, out of 40 dogs. The sensitivity of blood smear examination, buffy coat smear examination, and thrombocytopenia was 10.34%, 24.14%&nbsp;and 89.66%, respectively. The specificity of all the diagnostic tests was 100%. The anemia along with leucopenia, were the most significant hematological alteration recorded while ALP, creatinine, and BUN were elevated and albumin was significantly decreased in CME-infected dogs. Significant improvement of haemato-biochemical parameters towards normalcy in both treatment groups. However, a higher percentage of reduction was observed in the clinical score of imidocarb treated CME affected dogs than in the oxy-doxycycline therapy after 14 days.

Clinico-diagnostic and Therapeutic Management of Ehrlichiosis in Dog

Background: Aspergillus aflatoxiformans, Fusarium humuli and Curvularia geniculata are fungal pathogens of agricultural and clinical significance, associated with crop diseases, mycotoxin production and opportunistic human infections. Accurate identification is essential for understanding their taxonomy, pathogenic potential and ecological roles. 
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Methods: Infected wheat grains and leaves were collected from the Meerut region, India. Fungi were isolated using surface sterilization and cultured on potato dextrose agar. Morphological identification was performed via colony characterization and microscopic examination. Molecular identification was conducted through internal transcribed spacer region amplification, sequencing and BLAST analysis. Phylogenetic relationships and genetic diversity were assessed using Neighbor-Joining and Maximum Composite Likelihood methods. 
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Result: Fusarium spp. exhibited rapid growth with sickle-shaped macroconidia; Aspergillus spp. formed yellow-green, powdery colonies with globose conidia; Curvularia spp. produced dark brown, velvety colonies with geniculate conidia. Internal transcribed spacer sequencing yielded amplicons of 553 bp (Fusarium humuli), 588 bp (Aspergillus aflatoxiformans) and 618 bp (Curvularia geniculata), showing high homology to reference strains (98&ndash;100%). Phylogenetic analysis clustered isolates with their respective species, revealing clear genetic distinctiveness and potential regional adaptations. Integration of morphological and internal transcribed spacer-based molecular approaches effectively identified Fusarium humuli, Aspergillus aflatoxiformans and Curvularia geniculata highlighting their pathogenic potential and genetic distinctiveness. These findings underscore the importance of combined diagnostic methods for accurate fungal identification, which is crucial for crop protection, food safety and public health.

Morphological and Molecular Characterization of the Fungi Isolated from Infected Wheat Plant

<div>Background: Pathomicrobial and molecular investigations of respiratory system diseases was undertaken on twelve adult buffalo carcasses received for necropsy examination at post mortem facility of Department of Veterinary Pathology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana during the period from August, 2018 to February, 2019.&nbsp; 
 
Methods: After collection of the samples, laboratory work was undertaken in the laboratories of Department of Veterinary Pathology, Department of Animal Biotechnology and Central Laboratory of the College, LUVAS, Hisar, Haryana (India) in the year 2018-2019 regarding the examination of the clinical history, pathological, microbiological and molecular investigations. 
 
Result: The gross pathological changes observed in lungs were variable degree of vascular changes, mild to severe consolidation, fibrin deposition along with adhesions to the thoracic wall. Histopathological findings revealed abnormalities of inflation such as pulmonary emphysema, atelectasis, pulmonary congestion and haemorrhages which was associated with different types of pneumonia viz. fibrinous bronchopneumonia, suppurative giant cell pneumonia, interstitial pneumonia and serous pneumonia. Lung, heart blood and tracheal swab samples collected from the buffalo carcasses revealed eighteen bacterial isolates which were identified by Vitek 2 system. These include E. coli (11 isolates), Salmonella enteric enterica (2 isolates), Acinetobacter ursingii (1 isolate), Staphylococcus haemolyticus (1 isolate), Staphylococcus sciuri (1 isolate), Staphylococcus warneri (1 isolate) and Staphylococcus hominis (1 isolate) mostly belonging to opportunistic pathogen category. E. coli serotypes confirmed from these cases were O83, O149 and O8. The results of in vitro drug sensitivity testing revealed that most of bacterial strains were found sensitive to cefoperazone/sulbactum and co-trimoxazole. Molecular studies confirmed Bovine Herpes Virus-1 (BHV-1) infection in one case with Fibrinous bronchopneumonia through real-time quantitative PCR indicating the prevalence of the infection in the state.</div>

Pathomicrobial and Molecular Investigations of Respiratory System Diseases Affecting Buffaloes

<div>Background: Riemerella anatipestifer (R. anatipestifer) is a gram negative, microaerophilic, non-motile, bipolar bacteria. High genetic diversity and molecular differentiation were reported among field isolates. Although the bacterium causes one of the most economically important duck diseases in the north-eastern region of India, little work has been done on isolation, identification and molecular characterization of the bacteria. Hence, the present investigation was undertaken with a view to characterize the R. anatipestifer isolates from ducks of Assam.</div>

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Methods: Phenotypic and molecular identification of R. anatipestifer isolates from domesticated ducks of Assam, India were carried out during the period from February, 2019 to January 2020. A total of 624 samples (Ocular swab, throat swab, liver, spleen, kidney, brain, heart, lung) from ducks comprising of apparently healthy, ailing and dead ducks were collected from five districts of Assam, India were processed to isolate and identify the bacteria. The tentative identification of the bacteria was done based on phenotypic characteristics viz., colony morphology, growth characteristics and biochemical reactions. All the phenotypically positive isolates were further subjected to molecular identification based on PCR assay targeting 16S rRNA gene and ERIC sequence. 
 
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<div>Result: The bacteria could be isolated from different field samples. The highest percentage of the samples that yielded the bacteria are from brain (76%) followed by spleen (74%) of dead ducks and less number of ocular swab (33%) from apparently healthy ducks were found positive. Sequencing of the amplified product of the selected R. anatipestifer isolates targeting 16S rRNA gene revealed homology percentage of 96.5-100%. Further, sequences representing five geographical locations were submitted to NCBI gene bank. Phylogenetic studies of the isolates indicated that there is prevalence of at least two genetically different strains of R. anatipestifer in the study area. The study suggested that the R. anatipestifer infection is endemic in Assam causing varying rate of morbidity (39%) and mortality (53%) and molecular based confirmation is necessary besides phenotypic identification.</div>