<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>Background:&nbsp;Ticks are of great importance in transmission of various canine tick borne diseases. Several characteristics of ticks make them outstanding vectors of pathogenic agents, the wide host range and slow feeding habit along with tendency to feed on several hosts during life cycle ensures ample opportunity to acquire and transmit pathogens. 
 
Methods: This study focuses on status of canine tick vectors and molecular detection of haemoparasites in these ticks and their host, in and around Kolkata. The blood and tick samples were collected from Dog Ward, Department of Teaching Veterinary and Clinical Complex; Faculty of Veterinary and Animal Sciences, WBUAFS at Belgachia; Veterinary Clinic of Kolkata Police Dog Squad at Alipore; Veterinary Clinic of Barrackpore Police dog Squad and samples from stray dogs were also collected from inside the University campus through a period of one year (August, 2016 to July, 2017).&nbsp; 
 
Result: The tick infestation was recorded at 41% with Rhipicephalus sanguineus, being the only tick. Nine primer sets were used for detection of Babesia spp, Ehrlichia canis, Ehrlichia chaffensis, Babesia gibsoni, Hepatozoon canis, Mycoplasma haemocanis, Anaplasma platys and Theileria annae from the respective tick samples and blood sample of hosts. Tick samples were found positive for Babesia spp, Ehrlichia spp. and H. canis where as the corresponding blood samples were found positive for Babesia spp, Ehrlichia spp., Mycoplasma spp. and H. canis. This study conclusively provides evidence of high rates of incidence of haemoparasitic infection or canine tick borne diseases infection and tick infestation, with at least four haemoparasites infecting the dog population and at least one tick species (Rhipicephalus sanguineus) infesting the dogs in and around Kolkata.</div>

Incidence of Canine Tick Vectors and Molecular Detection of Haemoparasites in Vectors and Hosts

The present study was undertaken with the objectives of molecular characterization and detection of genetic polymorphisms in the HSPB6 gene of Sahiwal cattle. A total of 100 Sahiwal cattle maintained at LRC, NDRI, Karnal (Haryana) were included in the study. HSPB6 gene has been mapped on Bos taurus autosome 18 (BTA 18) and spans nearly 2620 bp comprising 3 exons and 2 introns. Four sets of forward and reverse gene-specific oligonucleotide primers of HSPB6 gene were designed using Primer3 software and amplicon size of 529 bp, 460 bp, 670 bp and 502 bp were ascertained. On the basis of comparative sequence analysis, total three polymorphic loci (SNPs) were detected at locus G436A, A2152G and A2417T in HSPB6 gene as compare to Bos taurus (NCBI GenBank AC_000175.1). Out of these three SNPs, two (A2152G &amp; A2417T) were found in 3&acute;-UTR region and third one (G436A) was detected in intronic region 1. However, monomorphic pattern was found in exon 2 of HSPB6 gene in Sahiwal cattle. Further research need be directed to find out the association of allelic variants in major heat shock protein genes to establish markers for the future genetic improvement of livestock through marker assisted selection.

Molecular characterization and polymorphism detection in HSPB6 gene in Sahiwal cattle

<div>Background: Poultry enteritis is an important multifactorial disease. Chicken Astrovirus (CAstV) usually associated with enteritis. The aim of this study was to investigate the occurrence of CAstV in poultry enteritis cases, its molecular characterization, phylogenetic analysis and gross and microscopic examination of intestine and liver specimen affected with CAstV. 
 
Methods: Total 604 dead poultry birds from commercial poultry farms affected with enteritis were examined for presence of CAstV. Intestinal samples of four birds were pooled to make one biological sample. CAstV was detected by reverse transcriptase PCR (RT-PCR) using ORF-1b gene specific primers. Molecular characterization was carried out by partial gene sequencing. 
 
Result: CAstV was detected in 20.52% (31/151) of samples. Highest prevalence (49.29%) was observed in 0-1 week old chicks. The partial molecular characterization revealed high similarity of the nucleotide sequence from India (97% to 93%) and from USA, Brazil, Poland and Korea (94 to 92%). Further similarity of amino acid sequences of CAstV from India (100% to 98%) and from USA, Brazil, Poland and Korea (98 to 97%) was observed. Histopathological examination revealed villous atrophy, congestion and atrophic cystic glands in sub-mucosa of intestine. Further severe congestion and hemorrhages along with infiltration of inflammatory cells in liver parenchyma was observed.</div>

Molecular Detection and Phylogenetic Analysis of Chicken Astrovirus Associated with Poultry Enteritis

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

<div>Background: To investigate seroprevalence, clinical manifestations, molecular diagnosis of Lumpy Skin Disease in cattle in an around Parbhani during outbreak of LSD. 
 
Methods: The cattle irrespective of their age, breed and gender were screened for detection of LSDV. Duration of study period was from December, 2020 to August, 2021. The cattle showed high fever, presence of nodules on body, nasal discharge, ocular discharge, brisket oedema and lameness were screened for Lumpy Skin Disease and were further confirmed using molecular tests. Total 478 cattle were screened for the seroprevalence of Lumpy Skin Disease by ELISA and PCR tests. The cattle were categorized into different age groups as calves (less than 0-2 years), young (between 2-4 years) and adults (4 years and above). The various breeds such as Khillar, Red Kandhari, Gir, Holstein-Friesen, cross-bred and non-descript cattle were screened. The seroprevalence of Lumpy Skin Disease in female and male was also recorded. 
 
Result: The molecular assay of LSD revealed that, out of 478 cattle screened, 267 (55.85%) serum samples were found positive by ELISA test. Whereas, among 267 blood samples and 8 scab samples tested by PCR, 82 blood samples (30.7%) and 5 scab samples (62.5%) were found positive for LSDV in cattle. LSD is an emerging, transboundary and economically important viral disease of cattle. The overall seroprevalence of LSD in cattle was 55.85%. Prominent clinical manifestations in LSD affected cattle was Prominent clinical manifestations in LSD affected cattle were fever followed by generalized appearance of skin nodules on body. In addition, LSD affected cattle showed oculo-nasal discharge, enlarged lymph nodes, brisket oedema, lameness, corneal opacity and anorexia. The scab samples were more reliable material for LSDV confirmation by PCR.</div>

Molecular Detection and Seroprevalence of Lumpy Skin Disease in Cattle

Background: Picobirnavirus (PBV) is an enteric virus with potential for interspecies transmission, affecting various animal species and possibly humans. This study aimed to investigate the prevalence and molecular characterization of PBV in faecal samples from various animal species in Jabalpur, Madhya Pradesh, India. Diarrhoeic and non-diarrhoeic samples from calves (cattle and buffalo), goats, pigs, dogs and poultry were collected for analysis. 
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Methods: A total of 150 faecal samples, including diarrhoeic and non-diarrhoeic samples, were collected from calves (cattle and buffalo), goats, pigs, dogs and poultry. Faecal suspensions were prepared and RNA extraction was performed. Both RNA-PAGE (Polyacrylamide Gel Electrophoresis) and PCR techniques were employed to detect PBV, with PCR also used for Rotavirus detection. Various primer sets targeting the RdRp gene of segment 2 were utilized for PBV detection, while specific primers were applied for Rotavirus detection. Sequencing and phylogenetic analysis were conducted on selected samples to characterize viral genetic similarity. 
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Result: Among the 150 faecal samples, 4.0% (6/150) were positive for PBV by RNA-PAGE, with three buffalo and three dog samples displaying a three-band pattern. RT-PCR using different primer sets revealed a 7.33% (11/150) prevalence of PBV, with buffalo and goats showing the highest rates. Co-infection of PBV and Rotavirus was observed in some samples, suggesting possible interactions between these viruses. Nucleotide identities of the goat isolate S14 (GenBank OP866965) with other PBV sequences in GenBank ranged from 93.40% to 95.73%. 
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Detection and Molecular Analysis of Picobirnavirus and Rotavirus from Animal Faecal Samples

<div>Background: Abscessed wounds in cattle represent a significant veterinary concern, often leading considerable economic losses due to decreased productivity, treatment cost, potential culling and even death. This study aims to investigate the incidence rate of Trueperella pyogenes in abscessed wounds of cattle using molecular technique with phylogenetic analysis of study isolates and estimation the relationship of infection to animal risk factors. 
 
Methods: A total of 271 cattle of different ages and sexes admitted to private veterinarian clinics or being visited to their farms in Wasit province (Iraq) were diagnosed clinically with abscessed wounds. Then, samples of abscess swab were collected and examined molecularly by the conventional PCR. Finally, the positive T. pyogenes isolates were sequenced, submitted in the NCBI database and analysed phylogenetically.&nbsp; 
 
Result: Findings of clinical examination revealed a significant increase in abscessed wounds at abdomen but decreased in neck and back when compared to other body regions; head, forelimbs, hindlimbs and pelvis. Targeting of 16S rRNA gene, 7.38% from abscessed wounds of study cattle were positively infected with T. pyogenes. Phylogenetic analysis of the study T. pyogenes isolates revealed its identity to NCBI-BLAST USA T. pyogenes isolate (KX592206.1) at rates of similarity and mutation/changes ranged 98.47-99.86% and 0.0003-0.001%, respectively. Relation to risk factors, incidence and risk of positivity in body parts were elevated significantly in abdomen but decreased in neck and back, head, forelimbs and hindlimbs when compared to pelvis. Concerning age, although insignificant variation in incidence of positivity was seen among age groups of study animals, cattle aged 1-4 years and &gt;4 years were appeared at higher risk of infection than those of &lt;1 year. For sex, also insignificant differences in incidence rate of positivity was detected between females and males; however, females were detected at higher risk of infection than males.</div>

Clinical and Molecular Phylogenetic Detection of Trueperella pyogenes in Abscessed-wounds of Cattle

<div>Background: Infectious coryza is an acute, highly contagious respiratory disease of chickens caused by Avibacterium paragallinarum causing heavy economical loss to the poultry farmers. The laboratory diagnosis of Av. paragallinarum infection is based mainly on conventional methods such as isolation and identification of the organisms. In modern poultry industry, the rapid diagnosis of Av. paragallinarum is essential along with detection of variation among the serotypes.&nbsp;&nbsp; 
 
Methods: A total of 128 samples (viz., heart blood swabs, tracheal, eye and infra orbital swabs) suspected for infectious coryza were screened for the presence of Av. paragallinarum by isolation and molecular characterization. Information regarding farm/flock strength, age of birds, method of rearing, production performance, clinical symptoms manifested and mortality patterns from various organized poultry farms were recorded. A total of five isolates of Av. paragallinarum have been recovered from suspected materials processed for isolation. Two isolates were sequenced and analysed.&nbsp; 
 
Result: All the isolates showed same biochemical characters but exhibited variability in the fermentation of mannose, mannitol and sorbitol. The nucleotide variation of Av. paragallinarum present in Indian subcontinent differs in their virulence and pathogenicity. The study was also focused for the molecular characterisation of Avibacterium species from different isolates. Though the clinical signs and gross pathological signs were similar, the nucleotide variation between isolates was observed. Sequencing of &lsquo;hagA&rsquo; gene showed that the two isolates were closely related to the strains of China, Australia, Mexico and India which shared 94-100% homology.</div>

Detection and Molecular Characterization of Avibacterium paragallinarum from Poultry

<div>Background: Newcastle disease is one of the major highly contagious disease of birds only next to the highly pathogenic avian influenza virus infection. Ducks are found to be natural reservoirs transmitting the virus to other birds-in-contact. The present study was conducted with the aim to isolate the NDV virus from free ranging ducks and detection of the genotype and pathotype based on HN and F genes. 
 
Methods: The virus was isolated in chicken embryo fibroblast (CEF) primary cell culture and confirmed by cytopathic effects as well as by hemagglutination and hemagglutination inhibition tests. Molecular detection was performed using primers specific for HN and F genes. Amplified partial HN and F genes were cloned and sequenced. Phylogenetic tree was prepared based on both partial HN and F genes. Deduced amino acid alignment of protease cleavage site of F protein was performed to determine the strain. 
 
Result: The isolated virus could be confirmed by CPE as well as molecular detection by PCR. The target genes, partial HN and F genes exhibited an amplicon of 652 bp and 534 bp, respectively by PCR. On phylogenetic analysis, the present isolate was found to be genotype Xlll and on cleavage site analysis of fusion protein, which was found to be similar in pattern with velogenic pathotype of NDV.</div>

Molecular Detection and Characterization of Hemagglutinin-neuraminidase (HN) and Fusion (F) Genes of Newcastle Disease Virus (NDV) from Duck in NER India