Besides the vaccine strains, the Malaysian variant (MV) and QX-like are the predominant IBVs detected on commercial poultry farms. These two virus strains are distinct based on genomic and pathogenicity studies. In this study, we determined the sequence of the S1 gene and compared the pathogenicity of serial passage 70 (P70) of Malaysian QX-like (QX/P70) and MV (MV/P70) strains with that of their respective wild-type viruses. The nucleotide and amino acid sequences of the complete S1 genes of QX/P70 and MV/P70 showed 1.4 to 1.6% and 3.0 to 3.3% variation, respectively, when compared to the wild-type virus. Most of the mutations were insertions and substitutions in the hypervariable regions (HVRs), primarily in HVR 3. Furthermore, selection pressure analysis showed that both viruses are under purifying selection. A pathogenicity study in specific-pathogen-free (SPF) chickens showed a reduction in respiratory and kidney lesions in chickens inoculated with MV/P70, but not with QX/P70, when compared to the respective wild-type viruses. However, MV/P70 is still pathogenic and can cause ciliary damage. In conclusion, the MV IBV strain is more responsive than the QX-like IBV strain following the attenuation process used for the development of a live attenuated IBV vaccine.
Biochar has proven to be a feasible additive for mitigating nitrogen loss during the composting process. This study aims to evaluate the influence of biochar addition on bacterial community and physicochemical properties changes, including ammonium (NH4+), nitrite (NO2-) and nitrate (NO3-) contents during the composting of poultry manure. The composting was carried out by adding 20% (w/w) of biochar into the mixture of poultry manure and rice straw with a ratio of 2:1, and the same treatment without biochar was prepared as a control. The finished product of control compost recorded the high contents of NO2- and NO3- (366 mg/kg and 600 mg/kg) with reduced the total NH4+ content to 10 mg/kg. Meanwhile, biochar compost recorded a higher amount of total NH4+ content (110 mg/kg) with low NO2- and NO3- (161 mg/kg and 137 mg/kg) content in the final composting material. The principal component analysis showed that the dynamics of dominant genera related to Halomonas, Pusillimonas, and Pseudofulvimonas, all of which were known as nitrifying and denitrifying bacteria, was significantly correlated with the dynamic of NO2- and NO3- content throughout the composting process. The genera related to Pusillimonas, and Pseudofulvimonas appeared as the dominant communities as the NO2- and NO3- increased. In contrast, as the NO2- and NO3- concentration decreased, the Halomonas genus were notably enriched in biochar compost. This study revealed the bacterial community shifts corresponded with the change of physicochemical properties, which provides essential information for a better understanding of monitoring and improving the composting process.
Storage temperature and duration plays an important role in meat processing. Observations in poultry processing plants have shown a serious deviation in storage condition compared to the recommended procedures. Furthermore, there is still a paucity of evidence on the effects of storage temperature and duration on meat quality and microbial population. The aim of this study was to determine the effects of different temperature and duration during storage on physico-chemical properties and microbiological quality of broiler chicken Pectoralis major muscle. Eighty birds were slaughtered and processed, following which the packed boneless breast (PBB) (each bird was to provide two breast muscle samples; left breast and right breast) was divided into four groups, each consisted of 40 PBB. Each group was subsequently assigned to storage either at 4 °C, -10 °C, -18 °C or -40 °C, for 24 h before 20 PBB samples from each group were transported to the respective laboratory for meat quality and microbiological analysis. The remaining 20 PBB from each storage temperature were stored for 72 h before being transported for analysis. Results have shown significant increases in drip loss and cooking loss as the storage temperature decreases. Similarly, storage duration significantly affected cooking loss, of which, samples stored for 72 h exhibited higher cooking loss compared to those stored for 24 h. For color, significant differences were only observed in lightness (L*) and redness (a*) values. Longer duration of storage had significantly improved tenderness whereby, samples that have been stored for a shorter duration presented higher pH values. Populations of coliform and Salmonella decreased significantly with decreasing temperature and increasing storage duration.
Inappropriate use of antibiotics in animal production system is one of the major factors leading to the antibiotic resistance (ABR) development. In Vietnam, the ABR situation is crucial as antibiotics have been used indiscriminately for disease prevention and as growth promoters in animals. Thus, a thorough understanding on the ABR in veterinary settings would be beneficial to the Vietnam public health authority in formulating timely interventions. This review aimed to provide information on the current status of antibiotic usage in animal husbandry in Vietnam, identified gaps in research, and suggested possible solutions to tackle ABR. To this end, data on ABR in animals were extracted from 3 major electronic databases (PubMed, Web of Science, and ScienceDirect) in the period of January 2013-December 2020. The review findings were reported according to PRISMA, which highlighted the emergence and persistence of ABR in bacterial isolates, including Escherichia coli, Enterococcus spp., and Salmonella species, obtained from pigs and poultry. The lack of awareness of Vietnamese farmers on the antibiotic utilization guidelines was one of the main causes driving the animal ABR. Hence, this paper calls for interventions to restrict antibiotics use in food-producing animals by national action plan and antibiotics control programs. Additionally, studies to evaluate knowledge, attitude, and practice (KAP) of the community are required to promote rational use of antibiotics in all sectors.
The aims of this communication were to study characterization of serogroups among Salmonella isolates and the relationship of antimicrobial resistance to serogroups. Multiple antimicrobial resistance (MAR) was performed on 189 Salmonella enterica isolates associated with 38 different serovars that were recovered from poultry and four types of indigenous vegetables.
Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.
Village chicken or Ayam Kampung, common to Southeast Asian countries, has always been regarded as superior in comparison to commercial broiler chicken in terms of wholesomeness and health benefits. The current study investigates the prevalence and risk factors of Salmonella among village chicken flocks from the central and southern states of Peninsular Malaysia. A total of 35 village flocks were sampled from Selangor (n = 19), Melaka (n = 10), Johor (n = 4), and Negeri Sembilan (n = 2). In total, 1,042 samples were collected; these included cloacal swabs (n = 675), eggs (n = 62), pooled drinking water (n = 175), pooled feeds (n = 70), and pooled flies (n = 60). Isolation of Salmonella from cloacal swabs, poultry drinking water, and feeds was carried out according to the protocols and recommendations of the World Organization for Animal Health (OIE) terrestrial manual. The prevalence of Salmonella at an individual bird-level was 2.5% (17/675, 95% CI: 1.6 to 4.0). All eggs screened were negative; in the case of environmental samples, however, Salmonella was detected in 5.14% (9/175), 7.14% (5/70), and 5.0% (3/60) for water, feed, and flies, respectively. A total of 34 isolates and 8 Salmonella serotypes were identified. Weltevreden (20.6%) was the most common, followed by Typhimurium and Agona (17.6%), Albany and Enteritidis (8.8%), Molade (5.9%), Corvallis and Schleissheim (2.9%), and others grouped as Salmonella spp. (11.8%). Multivariable logistic regression models revealed that Salmonella positivity among flocks could be strongly predicted by storage of feeds (uncovered feeds; OR = 10.38; 95% CI: 1.25 to 86.39; p = 0.030) and uncovered water tanks (uncovered tank; OR = 6.43; 95% CI: 1.02 to 40.60; p = 0.048). The presence of Salmonella in village chickens in the study area was lower than that of commercial chickens in Malaysia.
Campylobacter is reported as a major cause of foodborne illness worldwide. Consumption of contaminated chicken meat is considered a significant risk factor of Campylobacter infection in humans. This study investigated the occurrence of non-Campylobacter jejuni-Campylobacter coli, in broiler chickens (n = 210) and chicken meat (n = 109). The samples were collected from seven broiler chicken farms (n = 210 cloacal swabs), 11 markets (n = 84 chicken meat), and 5 supermarkets (n = 25 chicken meat) located in different districts of Selangor State. Campylobacter were isolated from cloacal swabs using the Cape Town Protocol and from meat samples using the method of Duffy et al. (2007) with some modifications for Campylobacter isolations which were reported effective in the isolation of non-C. jejuni-C. coli Campylobacter species. The isolates were identified by Gram staining for cellular morphology, wet mount for motility and biochemical tests. Confirmation of presumed Campylobacter isolates was carried out using multiplex PCR (mPCR). One hundred seven (107/210) or 50.9% and twenty-nine (29/109) or 26.6% of chickens and chicken meat samples respectively were positive for Campylobacter species. Among the Campylobacter isolates from chickens, C. jejuni was the most predominantly isolated species (69.5%), followed by C. coli (16.2%). Campylobacter fetus and C. upsaliensis were the non-C. jejuni-C. coli Campylobacter species isolated in this study, at 9.3% and 2.5% respectively. Overall, the findings indicated broiler chickens were colonized not only by the common Campylobacter species but also by other Campylobacter species. We found the Cape Town Protocol useful to detect the occurrence of non-C. jejuni-C. coli isolates in chickens.
Despite the availability of Newcastle disease (ND) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. The aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated Newcastle disease virus (NDV) strain IBS025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype VII isolate. Firstly, high titre of IBS025/13 was exposed to various concentrations of binary ethylenimine (BEI) to determine the optimal conditions for complete inactivation of the virus. The inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (BSO) or Freund's incomplete adjuvant (FIA) and used as vaccines in specific pathogen-free chicken. Efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\VII NDV isolate. The results indicate that exposure of NDV IBS025/13 to 10 mM of BEI for 21 h at 37 °C could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. More so, the inactivated vaccines adjuvanted with either BSO- or FIA-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. Thus, genotype VII-based NDV-inactivated vaccines formulated in BSO could substantially improve poultry disease control particularly when combined with live attenuated vaccines.
Mycoplasma synoviae (n = 26) and M. gallisepticum (n = 11) isolates were gained from 164 clinical samples collected from China, India, Indonesia, Malaysia, Philippines, Republic of Korea and Thailand. Most isolates were from commercial chicken production systems. A method of filtering (0.45 μm) samples immediately after collection was convenient allowing over a week for transit to the laboratory. Minimum inhibitory concentrations (MICs) were characterized by a broth microdilution method to enrofloxacin, difloxacin, oxytetracycline, chlortetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tiamulin, florfenicol, lincomycin, spectinomycin and lincomycin and spectinomycin combination (1:2). Increased MICs to various antimicrobials were seen in different isolates but appeared largely unrelated to the antimicrobial treatment histories. Overall, the results were similar to other MIC surveys around the world. Generally, low MICs to tetracyclines, tiamulin and tylvalosin were observed. Increased tilmicosin MICs were observed in both M. synoviae and M. gallisepticum isolates (≥64 μg/ml MIC90 values) and this was seen in all isolates with high tylosin MICs. Increases in lincomycin MICs were mostly associated with increases in tilmicosin MICs. The results also suggested that antimicrobial use after mycoplasma vaccination may interfere with vaccine strain persistence and efficacy (field strains were more commonly observed in flocks that had treatments after vaccination) and this area warrants more investigation. The study shows that isolation and MIC determination can be done from remote locations and suggests that this may provide information that will allow more effective use of antimicrobials or other methods of control of avian mycoplasma in chickens (e.g. live vaccines) and therefore more responsible use of antimicrobials from a one health perspective.
This paper reports the experimental transmission of a bird parasite into jirds. Infective larvae of Cardiofilaria nilesi obtained from laboratory colonized Coquillettidia crassipes mosquitoes which had fed on an infected chicken were inoculated subcutaneously into jirds. The number of larvae per jird varied from 10 to 228. Microfilaraemia appeared 22 to 89 days after inoculation of the infective larvae. Experiments were carried out with 24 jirds through six generations extending over a period of 22 months and 17 produced patent infections. At present 8 infected jirds are being maintained in the laboratory; their patent periods ranging from 6 to 13 months. However, the longest patent period observed was about thirteen months. The percentage of adults recovered in autopsied jirds ranged from 0 to 40 with an average of 16. The chicken showed a microfilarial periodicity with the peak microfilarial density around 2200 hours. However, in jirds there was a change in sub-periodicity. This model in the jird may be very useful for the screening of filaricides and in immunological work.
Eight-five clinical and 15 poultry isolates of Campylobacter species were characterised by biotyping, serotyping and by using a radiolabelled DNA probe. A total of 80% of the isolates from both sources were identified as C. jejuni. Also amongst the clinical strains were 5 c. jejuni subsp. doylei, 7 C. coli, 3 C. lari and 8 were untypable. The similarity in the distribution of C. jejuni in the clinical and poultry isolates adds credibility to published reports of chickens being the most common source of Campylobacter infections. Although the gold standard for identification of C. jejuni is the DNA probe, serotyping is more discriminating while biotyping is the most feasible method in most laboratories.
Colibacillosis is one of the main causes of economic loss in the poultry industry worldwide. Although antibiotics have been used to control this infection, the emergence of antibiotic-resistant bacteria poses a threat to animal and human health. Phage therapy has been reported as one of the potential alternative methods to control bacterial infections. However, efficient phage therapy is highly dependent on the characteristics of the phage isolated. In the present study the characteristics of a lytic phage, ØEC1, which was found to be effective against the causative agent of colibacillosis in chickens in a previous in vivo study, are reported.
Duck Tembusu virus (TMUV) is a recently identified pathogenic flavivirus that causes severe egg drop and encephalitis in Chinese ducks and geese. It has been found to be most closely related to the mosquito-origin Tembusu virus and chicken Sitiawan virus reported in Malaysia. However, the ecological characteristics and the pathogenesis of duck TMUV are largely unknown. We report the construction of full-length cDNA clone of duck TMUV strain JXSP. The virus genome was reverse transcribed, amplified as seven overlapping fragments and successively ligated into the low copy number vector pWSK29 under the control of a T7 promoter. Transfection of BHK-21 cells with the transcribed RNA from the full-length cDNA clone resulted in production of highly infectious progeny virus. In vitro growth characteristics in BHK-21 cells and virulence in ducklings and BALB/c mice were similar for the rescued and parental viruses. This stable infectious cDNA clone will be a valuable tool for studying the genetic determinants of duck TMUV.
Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain.
Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection.
Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.
Duck Tembusu virus (DTMUV), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT-PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV, which was the most closely related DTMUV. It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUVs, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUVs; however, it was genetically distinctive to any of the currently circulating DTMUVs. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUVs in Asia.
Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.
Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 10⁵ 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain.