Shambhu Dayal Singh

Tuberculosis in Birds: Insights into the Mycobacterium avium Infections

Tuberculosis, a List B disease of World Organization for Animal Health, caused by M. avium or M. genavense predominantly affects poultry and pet or captive birds. Clinical manifestations in birds include emaciation, depression and diarrhea along with marked atrophy of breast muscle. Unlike tuberculosis in animals and man, lesions in lungs are rare. Tubercular nodules can be seen in liver, spleen, intestine and bone marrow. Granulomatous lesion without calcification is a prominent feature. The disease is a rarity in organized poultry sector due to improved farm practices, but occurs in zoo aviaries. Molecular techniques like polymerase chain reaction combined with restriction fragment length polymorphism and gene probes aid in rapid identification and characterization of mycobacteria subspecies, and overcome disadvantages of conventional methods which are slow, labour intensive and may at times fail to produce precise results. M. avium subsp. avium with genotype IS901+ and IS1245+ causes infections in animals and human beings too. The bacterium causes sensitivity in cattle to the tuberculin test. The paper discusses in brief the M. avium infection in birds, its importance in a zoonotic perspective, and outlines conventional and novel strategies for its diagnosis, prevention and eradication in domestic/pet birds and humans alike.

Diclofenac is toxic to the Steppe Eagle Aquila nipalensis: widening the diversity of raptors threatened by NSAID misuse in South Asia

Three Critically Endangered Gyps vultures endemic to South Asia continue to decline due to the use of diclofenac to treat livestock. High nephrotoxicity of diclofenac to Gyps vultures, leading to death, has been established by experiment and observation, in four out of five Gyps vulture species which occur in South Asia. Declines have also been observed in South Asia’s four other non- Gyps vulture species, but to date there has been no evidence about the importance of diclofenac as a potential cause. Neither is there any evidence on the toxicity of diclofenac to the Accipitridae other than vultures. In this study, gross and microscopic lesions and diclofenac tissue levels in Steppe Eagles Aquila nipalensis found at a cattle carcass dump in Rajasthan, India, show evidence of the toxicity of diclofenac for this species. These findings suggest the possibility that diclofenac is toxic to other accipitrid raptors and is therefore a potential threat to much wider range of scavenging species in South Asia.

Isolation and molecular characterization of infectious bronchitis virus from recent outbreaks in broiler flocks reveals emergence of novel strain in India

In this study, two isolates of infectious bronchitis virus (IBV) from field outbreaks in 2008 (India/LKW/56/IVRI/08) and 2010 (India/NMK/72/IVRI/10) from broiler chickens in India were isolated and characterized. Reverse transcription polymerase chain reaction–restriction fragment length polymorphism of the entire S1 gene revealed that these isolates belong to two different genotypes, India/LKW/56/IVRI/08 as Mass strain whereas India/NMK/72/IVRI/10 as of different genotype. Nucleotide sequencing analysis showed that India/LKW/56/IVRI/08 shared 99xa0% homology with THA280252 (Thailand) and India/NMK/72/IVRI/10 shared greater than 99xa0% homology with 4/91 pathogenic strain (UK), JP/Wakayama/2/2004 (Japan) and TA03 (China), while the two Indian IBV isolates shared 73xa0% identity between them. Phylogenetic data allowed classification of two Indian isolates, India/LKW/56/IVRI/08 as having unique lineage within Mass genotype and India/NMK/72/IVRI/10 as of 4/91 genotype. Our study confirmed the presence of 4/91 (793/B) IBV nephropathogenic strain for the first time in India by virus isolation and sequencing.

Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses.

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8(+) cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed ‘pigeon PMV-1’ (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Downregulation in cytokines profiles and immunopathological changes in chicks infected with chicken infectious anaemia virus

The present study was designed to study cytokine profile and lymphocytic proliferation activity in chicken infectious anaemia virus (CIAV) infected chicks at various intervals of clinically susceptible age. Briefly, ninety specific pathogen free (SPF) chicks were divided into five experimental groups (A to E). Groups A to D were inoculatedxa0with 0.5 ml of 104.5TCID50xa0of CIAV intramuscularly at 1, 14, 21 and 28-day-old, respectively, whilexa0group E served as uninfected control. Comparative cytokine gene expression levels of interleukin (IL)-1β, IL-10, IL-12β and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured at 4 and 7 days post infection (dpi) by qRT-PCR. Also, lymphocytic proliferation activity was measured at 1, 2 and 3 week post infection (wpi) by lymphocyte transformation test (LTT).xa0The down regulatory changes in cytokines and depressedxa0cell mediated immune (CMI) responses were observed in chicks of all infected groups (A to D), which were significantlyxa0(p<0.05)xa0low as compared to control group. Higher intensities and more pronounced effects on cytokine downregulation and blastogenic responses of lymphocytes were seen at younger ages. Apoptosis in thymic DNA as detected by nucleosomal fragmentation was highest on 7 and 10 dpi and lowest on 14 dpi, which correlated with down regulated cytokine profile in all infected groups.xa0The development of clinical signs and lesions along with PCR testing confirmed the establishment of CIAV infection in experimental chicks.xa0Increased apoptosis, decreased PCV, depletion of lymphocytes, down regulated cytokine profile and reduced lymphocyte activity explain involvement of thymic and hematopoietic precursor cells leading to CMI suppression caused by CIAV. Altogether, immunopathological changes were more severe in chicks infected at younger ages as compared to later ages of the clinically susceptible period.However, further studies are suggested regarding the impact of viral load on the various cytokine profiles and revealing more about immunopathogenesis of CIAV both in young and adult birds. n n xa0 n n Key words:xa0Chicken infectious anemia virus, cell mediated immune response, polymerase chain reaction (PCR), qRT-PCR, cytokine, apoptosis.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India

ABSTRACT Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

A sensitive haemadsorption technique based RT-PCR for concentration and detection of Newcastle disease virus from clinical samples and allantoic fluid

The present study describes the exploitation of haemadsorption (HAd) property of the Newcastle disease virus (NDV) for the development of a novel sensitive HAd technique based RT-PCR for detection of NDV from clinical samples of virus infected experimental birds. The NDV propagated allantoic fluid from the infected embryonated chicken eggs or supernatant of the processed clinical samples (tissue triturate, cloaca and tracheal swabs) from the experimentally infected birds were added with chicken red blood cells (RBC) to adsorb the virus on RBC’s surface. The virus adsorbed RBCs were subjected to trizol method of RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) for detection of NDV. The HAd based RNA extraction showed better yield of 700–900xa0ng RNA and when subjected to RT-PCR detection revealed a 100 times higher sensitivity than the conventional RNA extraction and RT-PCR detection system. This could be an alternate technique which can be exploited in low NDV load situations in clinical samples.

Baruscapillaria obsignata: a serious cause of enteropathy and high mortality in turkeys (meleagris gallopavo)

ABSTRACT Background: Capillariasis, an important parasitic disease of birds is caused at least by seven different genera of trichurid nematodes with clinical outcome ranging from mild enteritis to high mortality. Objective: This study was aimed to investigate the causative agent involved in high mortality associated with severe enteric illness among turkey flocks in an organized commercial poultry farm at Bareilly, India. Materials and Methods: Turkey carcasses (n = 119) and fecal samples from the affected deep litter pen constituted as the study materials. The disease was investigated by systematic necropsy, direct microscopy and histopathology. Representative samples were screened for other enteric pathogens. Results: Microscopic examination of mucosal scraping revealed capillarid worms and their eggs in all the samples. The morphological features of adult worms were typically consistent to Baruscapillaria obsignata. Histopathology exhibited thickened muscular and mucosal layers, mononuclear and heterophilic infiltration in the lamina propria, blunting and clubbing of villi, epithelial denudation and sections of capillarid worms. Administration of levamisole at 80 ppm in drinking water reduced the mortality, clinical illness and worm load after three days of therapy. Conclusions: The capillarid worms in different avian hosts can cause different clinical manifestations and outcomes. From India, this is the first report describing intestinal pathology caused by B. obsignata in turkeys. We conclude that the B. obsignata infection is capable of causing life threatening enteropathy in turkeys and, hence, routine screening, scheduled deworming and good litter management are crucial to control the infection and its associated loss.