Prevalence and risk factor for H9N2 avian influenza virus in poultry retail shops of Madhya Pradesh.

H9N2 avian Influenza virus subtype is highly neglected but have the potential to emerge as a next pandemic influenza virus, by either itself evolution or through the donation of genes to other subtype. So to understand the extent of H9N2 virus prevalence and associated risk factors in poultry of retail shops and their surrounding environment a cross sectional study was carried out. A total of 500 poultry tissue and 700 environmental samples were collected from 20 district of Madhya Pradesh. Virus isolation was carried out in egg inoculation and harvested allantoic fluid was tested for HA and further molecular confirmation of subtypes by RT-PCR using H9 specific primers. Prevalence was calculated and positive samples were statistically associated with observed risk factors using univariate and multivariate logistic regression analysis. A total of 9.4% and 9.7% prevalence in tissue samples and environmental samples has been reported respectively and out of 20 districts 10 (50%) were found positive for the virus. Out of 21 studied risk factors only two risk factors named as "keeping total number birds slaughtered per day" and "procuring birds from wholesaler" were found significantly associated with the H9N2 positivity in multivariate logistic regression analysis. This high level of H9N2 positivity in birds with no clinical manifestations providing a great opportunity for avian influenza virus for amplification, co-infection in other animals like dogs, cats, pigs and in human through genetic re-assortment that may lead to emergence of a novel influenza virus with high zoonotic potential. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00865-y.

Identification and molecular characterization of H9N2 viruses carrying multiple mammalian adaptation markers in resident birds in central-western wetlands in India.

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.

Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

Survivability of low pathogenic (H9N2) avian influenza virus in water in the presence of Atyopsis moluccensis (Bamboo shrimp).

Low pathogenic avian influenza virus (LPAIV) exhibits an ecological climax with the aquatic ecosystem. The most widely prevalent subtype of LPAIV is H9N2. Wild aquatic birds being the natural reservoirs and ducks, the "Trojan horses" for Avian Influenza Virus (AIV), can contaminate the natural water bodies inhabited by them. The virus can persist in the contaminated water from days to years depending upon the environmental conditions. Various aquatic species other than ducks can promote the persistence and transmission of AIV; however, studies on the role of aquatic fauna in persistence and transmission of avian influenza virus are scarce. This experiment was designed to evaluate the survivability of H9N2 LPAIV in water with and without Atyopsis moluccensis (bamboo shrimp) for a period of 12 days. The infectivity and amount of virus in water were calculated and were found to be significantly higher in water with A. moluccensis than in water without A. moluccensis. The study also showed that A. moluccensis can accumulate the virus mechanically which can infect chicken eggs up to 11 days. The virus transmission potential of A. moluccensis requires further studies.

Survivability of Highly Pathogenic Avian Influenza H5N1 Virus in Poultry Faeces at Different Temperatures.

Highly pathogenic avian Influenza (HPAI) is an important zoonotic disease and is becoming a great threat to poultry industry. India has experienced continual outbreaks of H5N1 HPAI virus since February, 2006 especially in Eastern India. Survivability in poultry faeces is an important determinant in evaluating the persistence of the virus in the poultry sheds and their vicinity. In this paper, survivability of Indian H5N1 HPAI virus in dry and wet poultry faeces at 42, 37, 24 and 4 °C, respectively is reported. The effect of different temperatures was determined by linear regression model and defined in terms of linear equation. The virus survived up to 18 h at 42 °C, 24 h at 37 °C, 5 days at 24 °C and 8 weeks at 4 °C in dry and wet faeces, respectively. The coefficients of determination (R(2)) values for dry and wet faeces revealed that the difference in viral persistence in dry and wet faeces at all temperatures was not very marked. Results of the present study indicated that H5N1 HPAI virus may remain viable for extended periods of time in faeces at low temperatures and may act as a long term source of influenza virus in the environment.

Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.

A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 µl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.

Isolation and characterization of influenza A virus (subtype H5N1) that caused the first highly pathogenic avian influenza outbreak in chicken in Bhutan.

We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other "Qinghai-like" H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids ((222)Q and (224)G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1-100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds.

Phylogenetic evidence of multiple introduction of H5N1 virus in Malda district of West Bengal, India in 2008.

Outbreaks of H5N1 avian influenza virus were reported in 15 districts of West Bengal State in India in early 2008 and subsequent re-occurrence in 5 districts in December, 2008 to May, 2009. We have sequenced complete genome of 12 viruses isolated from early 2008 outbreak and from recurrent outbreak and determined the phylogenetic relationship between the viruses isolated from the two outbreaks. One of the virus isolated in early 2008 from Malda district (A/chicken/West Bengal/81760/2008) clustered with Korean and Russian isolates of 2006 in European-Middle Eastern-African (EMA) 3 sub-lineage of sub-clade 2.2, whereas other viruses showed close genetic relationship with 2007-2009 isolates of Bangladesh. Nucleotide sequence analysis revealed that the PB1-F2 protein expression might be completely abolished due to mutated start codon ((95)ATG(97)→(95)ACG(97)) in this isolate but in all other isolates it was completely expressed. Hence, we conclude that there were two separate introductions of H5N1 viruses in Malda district and this H5N1 virus was not epidemiologically dominant as the viruses isolated subsequently from the same district and region did not share close relationship with this virus. The failure of this virus to spread to adjoining areas suggests that the culling and disposal operations initiated by Government of India were effective.

Emergence of amantadine-resistant avian influenza H5N1 virus in India.

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008-2009) and avian and human isolates from Bangladesh (2007-2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.

Influence of dose of inocula on outcome of clinical disease in highly pathogenic avian influenza (H5N1) infections--an experimental study.

Twelve-week-old Vanaraja (an Indian native dual purpose breed) chickens were inoculated intranasally with different doses (100, 1000, and 10,000 mean embryo infective dose [EID50]) of H5N1 virus, and the clinical disease and pathologic changes were compared. Although the overall severity of clinical signs was more severe in the 100 EID50 group, the progression of the clinical disease was slower with delayed onset of mortality when compared with the other two groups. The mean death time of the 100 EID50 group (4.57 days) differed significantly from that of the 10,000 EID50 group (3.60 days) and from that of the 1000 EID50 group (3.33 days). Similarly, overall severity of gross lesions was expressed more in the 100 EID50 group. The histopathologic lesions were of a more hemorrhagic and necrotic nature in the 100 EID50 group, histopathologic lesions were of an inflammatory/proliferative nature in the 1000 EID50 group, and a tendency for intravascular coagulopathy was observed in the 10,000 EID50 group. These differences may be assigned to the influence of dose in the outcome of disease.

Isolation and molecular characterization of a H5N1 virus isolated from a Jungle crow (Corvus macrohynchos) in India.

In 2008, India experienced widespread outbreaks of H5N1 virus in West Bengal, Tripura, and Assam. The virus was detected in Kamrup district of Assam in November 2008 and subsequently spread to eight more districts. Two Jungle or Large billed crows (Corvus macrohynchos) were found dead in a hospital campus at about 8 km from the foci of initial detection of the virus in the same district. One of the crows was positive for H5N1 avian influenza virus by virus isolation, real time RT-PCR, and RT-PCR tests. Full length sequencing of all the eight segments of the virus was carried out. The phylogenetic analysis indicated that all the eight genes grouped with clade 2.2 viruses and were closely related to the human isolate of Bangladesh and avian isolates from India, Bangladesh, Kuwait, Germany, and Saudi Arabia. The molecular analysis indicated avian receptor (alpha 2,3 sialic acid) specificity, susceptibility to oseltamivir and amantadine group of antivirals and lower pathogenicity to mice.

Seroprevalence of toxoplasmosis in a district of Assam, India.

Toxoplasmosis is one of the important zoonoses of man and has been known to cause serious problems particularly in females. A study on seroprevalence of toxoplasmosis was undertaken amongst the human population in Assam to determine the level of exposure of the population to the infection by using commercial ELISA kits. Of the 241 sera belonging to different age groups, sex and religion and having varying levels of exposure to the animals examined, 23 (9.54%) were positive for toxoplasmosis. No significant difference in the prevalence amongst males and females was observed. Some occupational groups like veterinarians, pet keepers and farmers were found to infect more frequently. Although the overall prevalence rate of toxoplasmosis was relatively low, higher prevalence rate of toxoplasmosis amongst the exposed groups warrants due care by these groups when they are handling the animals.

Genetic analysis of H9N2 avian influenza viruses isolated from India.

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R-S-N-R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.

Isolation, phage typing and antibiogram of Salmonella from man and animals in northeastern India.

BACKGROUND AND OBJECTIVE: Salmonella is an important zoonotic pathogen and its prevalence in the animals acts as a continuous threat to man. The present study was carried out to report the isolation along with the serotypes, phage types and antibiogram pattern of Salmonella among man, livestock and poultry in the northeastern India. METHODS: A total of 654 samples from diarrhoeic livestock and humans were processed for the isolation of Salmonella. All the isolates were subjected to antibiogram studies against 15 antimicrobials. Representative isolates of S. Typhimurium and S. Enteritidis were phage typed. RESULTS: Ninety five isolates of Salmonella enterica belonging to 5 serotypes- S. Typhimurium, S. Enteritidis, S. Gallinarum, S. Paratyphi B and S. Bareilly were obtained with an overall prevalence rate of 14.40 per cent. S. Typhimurium isolates were distributed among four phages- DT003, DT004, DT096 and DT193 and all the S. Enteritidis isolates belonged to a single phage type, PT13a/7. Interspecies sharing of the phages was observed. Norfloxacin, enrofloxacin, gentamycin and ciprofloxacin were most effective, whereas, doxycycline, ampicillin, amoxycillin and tetracycline were relatively less effective. INTERPRETATION AND CONCLUSION: Our findings showed that three of the five serovars as well as some of the phage types of these serovars were shared by animals and humans indicating the zoonotic potential of the organism. Thus, it is imperative that salmonellosis control measures adopted for humans should give adequate importance to its control in the animals particularly their products.

Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals.

Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed.

Distribution of virulence genes in Salmonella serovars isolated from man & animals.

BACKGROUND & OBJECTIVES: Pathogenesis of Salmonellosis depends upon a large number of factors controlled by an array of genes that synergise into the actual virulence of Salmonella. A study was undertaken to observe the distribution of three such genes, namely, Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef and plasmid encoded fimbrial (pef genes, among different serovars of Salmonella enterica isolated from man and animals. METHODS: A total of 95 isolates belonging to S. Typhimurium (51), S. Enteritidis (36), S. Bareilly (3), and S. Paratyphi B (5) serovars were subjected to polymerase chain reaction (PCR) assay for the detection of stnl ssf and pef genes using their specific primers and the PCR products were analysed by 1 per cent agarose gel electrophoresis for the presence of the respective genes. RESULTS: Varying distribution pattern of these genes was observed amongst the isolates. While, stn was found in all the 95 strains, sef was found only among the S. Enteritidis isolates. The pef gene was found to be absent in 10 isolates including the three S. Bareilly isolates. INTERPRETATION & CONCLUSION: Findings indicated that the stn gene is widely distributed among Salmonella irrespective of the serovars and source of isolation. However, the sef gene appears to be serovar specific. Since the stn gene is found in all the isolates, it can be a viable target gene to explore the possibility of direct detection of Salmonella from samples from biological sources.