Spring temperature shapes West Nile Virus (WNV) transmission in Europe
CC Attribution 3.0 Germany:
You are free to use, adapt and copy, distribute and transmit the work or content in adapted or unchanged form for any legal purpose as long as the work is attributed to the author in the manner specified by the author or licensor.
Manica, Mattia
Mattia Manicai is a researcher at Fondazione Edmund Mach. His presentation will be centered around one of the most recent publications he has contributed to, titled “Spring temperature shapes West Nile Virus (WNV) transmission in Europe”. Mattia explains how spatio-temporal conditions will shape WNV transmissions, why WNV circulation tends to be higher in warmer regions, how the impact of change of temperatures due to approaching spring time on WNV transmissions can be predicted, and why new results in conjunction with previous findings can serve as indicators for the reliability of surveillance systems and WNV’s overwintering capacity.
 So I will talk about how spring temperatures shape the Western light values transmission in Europe. This work was led by Giovanni Marini, who unfortunately cannot attend today's presentation. So I hope it will be as interesting as it was him presenting. Next slide, please. The outline of the presentation is a little brief background on the West Nile virus, and then I'm going to talk about the previous work on West Nile transmission in Emilia Romagna, which is a region of Northern Italy, and then how we try to extend it to the whole of Europe and try to investigate how spring temperature may affect humans below. Next slide, please. So West Nile is a virus which is mainly circulating among mosquito and birds, where mosquito act as a vector, and given that the mosquito species, which is the primary vector also bites other animals like horses and human. It can happen to have an incidental infection to again horses and humans, which are dead at host. So an infected human is not able to infect the mosquito vector. And so to is not able to further transmission. So this kind of disease is a symptomatic, but in rare cases, people infected can develop severe neuroinvasive implications. So most of the time, the disease goes undetected by the public health system. And when the system starts to see cases, the circulation is already widespread or happening for a quite long amount of time. So next slide, please. So obviously, given that there is a mosquito vector involved, temperature plays a key role, because it affects mosquito dynamics. Several works have shown how warmer conditions can speed up the development and the life cycle of the mosquito and also increased a biting rate to the host, which can be bird, in some cases also human or horses rather mammals. And similarly, also temperature plays a role by affecting the pathogen transmission as warmer condition may decrease the incubation period in both the host or in the vector and increase the transmission probability to other hosts. Next slide, please. So as I said, there has been a previous work led by Giovanni, which was in which he analyzed the West Nile virus transmission in northern Italy, in particular in the region of familiar Romania in the northern Italy, which is characterized to be the region where the low land of the river is located. And as you can see the points are the traps deployed by the surveillance systems. And in this work is a top of entomological model coupled with an epidemiological model. So in the first entomological. So we analyze the life cycle of the mosquito and the model was calibrated to mosquito captures and as that input data was the temperature by this model one is able to estimate the expected mosquito bundas. The second part, coupled with the epidemiological model which models the transmission between birds and mosquitoes. In this way, one can compute the prevalence on the on the board on the mosquito which has the vector and from that compute the human risk given the mosquito prevalence. And the model was computed. The model was calibrated on the data in the past year, and it's very interesting to note how the 2018 was a very exceptional year. And also, later in the season in about 100 narrow evasives human cases, which are a lot more than the total number recorded in the previous year. This is not specific to the Emilia Romania region but it's also happened in 2019 in almost all Europe. So, here's the next slide. These are some results, as you can see spring was exceptionally warm in 2019 and this seems to have amplified that the was not virus transmission at the beginning of the season. So, having a higher transmission at the beginning of the season resulted in more circulation during the season and therefore in a lot of more cases than observed by the public as system. So, next slide please. So, we observed that pattern in Emilia Romania region without trying to explore if this pattern is also observable interest of Europe. Again, there's a problem of data, which are not available as in Emilia Romania for the rest of the Europe. So we collated data from the CDC, which report cases from all Europe for the period from 2011 to 2019 at the nuts three level. As you can see in the upper right graph 2018 is a very exceptional year for all the Europe in terms of West Nile virus transmission. So we taking consideration this data and we applied a statistical analysis to quantify how any spring temperature can influence you must be lower at an European scale. So, please switch to the next slide. The model we use was to consider the laboratory number of laboratory confirmed cases for an area that's three level and year specific here. We assume that given the CDC record only report positive cases when an area has no cases so is not present in the database, but it has already as add cases in previous year, we assume that there is a zero reported cases so we inflate the database with zero. And also we take into input the land surface temperature as an independent variable inter statistical model. Can you please go ahead. Yeah, thank you. The model was a zero inflated negative binomial models. It's a two part model. The first part is a binomial part which models the present of absence of cases. We use this, we use this framework because we inflate the database with zero. And now we think we it was important to model also this part. And the second part of the model is modeling the number of positive cases recorded. So as you can see by the formula and equation here to dependent variable was the number of laboratory confirmed cases, while the independent variable where the average spring temperature in the period 2003 2010. And these act as a gradient differentiating south to northern Europe. And the second dependent variable was the temperature anomaly so we normalize the temperature as a difference between the average temperature so this variable indicates characterized each year if it is a warmer or colder year with respect to the mean. And then we also consider the temperature of the area. And we also consider if the West Nile was already circulating in that area for that year, in the previous year. So we can see simple and explorative statistical model to just see if we can find some relationship between temperature and the number of cases recorded by the CDC at European level. So next slide please. Indeed, there is a correlation as expected. And as you can see in the upper panel. So the average number of cases expected conditional to the temperature in the average temperature in the upper left panel, or with respect to the temperature anomaly in the upper right panel. As you can see, there is an increasing pattern. So the warmer, the on the average spring temperature more cases and the warmer specific springs. So the higher number of cases. On the other hand, the lower panel models the probability of serving as zero or a positive number of cases. As you can see, again, the role of temperature. And that the more warmer the temperature, the less likely to serve as your record. So next slide please. So to conclude, the previous work on the middle of Romania region and the following work that is now published in the tropical. European level shows that there is an association between previous was not detection and larger number of cases in the following years. It's more likely to have an higher circulation in usually warm region. That is further in as if the specific spring temperature or a fire is warmer than the average temperature. And that considering a warmer temperature spring in particular warmer temperature as a nearly warning signal signal for West Nile virus humans below. I think that thank you for your attention. And if there is any question. I would be glad to answer it. Thank you, my dear, my apologies for some technical problems but we're still on time. So please questions. Maybe I will start off somebody has a really, really question. I think that's me. Nice talk. Thank you. Your first point there previous West Nile detection is associated with larger outbreaks. That implies that, I mean, if you've had a large outbreak, let's say in 2018 that conclusion would suggest you're likely to have another one in 2019 is that right now surely not. A large outbreaks is probably catch phrase for the presentation. You just say that. This is the next year. Obviously, quite. Were there also far more places, as opposed to cases. The Nile is spreading across Europe and teams that most of most area. It's when it's a pair then it's bound to stay. And I think that's the problem going on with the detection. They think because given that is not really symptomatic for most cases is difficult to detect but if you look for it in the mosquito vector when you are an area that results positive for what's not by circulation. And in time, you will pile up cases and when again, 2018 was an exceptional year but probably condition align warmer temperature previous circulation, a lot of vertical transmission from the previous you have this outbreak happening. At the moment was 2018 was the highest level, the peak level. Observed. Okay, thank you. We have one more question from Veeam, I think. Yeah, thank you. I thank you for your nice talk on West now I see for your easy DC data you use data from 2011 to 2019. And there's no specific reason not to have 2010 to the database this was also the start of the West now to the large West now to circulation in Europe a large outbreak in Greece. And we also very specific climatic conditions. 2010 was also a very strong winter. I think I don't know what the spring temperature did. Was there any specific reasons, not having 2010 in your analysis. So, you know, the temperature data from 2003 2010 as a reference. And then we started the database from the independent for the dependent variable from the 2011. As you say, I think the 2010 is the first year of the appearance of us nine in Europe for the DCC so we, so we discarded the first year, I think. Because it was also a very large outbreak. Of course, very, I think at a time many focusing on Greece so so it was included in your reference point. No, at the moment, no. Okay. Okay, thanks. Any other questions. I have a question. I was wondering, do you plan to include other variables except a temperature like rainfall. Yeah, we would like water areas. The model. Yeah, it would be nice. I think it's a future direction for research to include more variable. Again, this was to explore an association we find the founder on Emilia, Romania region in the previous work. But yeah, with my virus circulation is a very complex system and mechanism and cannot be explained only by temperature. And yes, indeed, we should include more variables also relating to the climate change and future temperatures. Have you have you tried doing that, like just extrapolating the model you fitted with the future like projected climate scenarios. It would be nice to do that but we, we were not so much confident about the reliability of the result. Because again, it's an indication that with temperature things in terms of West Nile are going worse and worse. So, if we expected to be an increasing temperature. Then we have to expect an increase in West Nile virus transmission and circulation across all Europe. Temperature most likely affect the transmission of us now because it acts as a boost the mosquito population, which act as a vector because warmer condition can speed up development of mosquito and also can speed up the incubation period within the vector of the viruses. So, if you have a warmer condition, most likely, you can have more aggressive or more abundant vector population. And if the viruses is introduced in the system or in the area, then it can be circulating with much more velocity. So, yeah, that's the main reason I think for temperature being very important in the modeling and in understanding of the West Nile virus circulation. There's a lot of different factor because we know that birds are the primary host but there's not so much information which kind type species of birds is really maintaining and sustaining the viruses in the environment. So, we're able to winter through the mosquito population, which entering the oppose and then by vertical transmission and start the cycle in the next season. So, we have an observation of the opposing mosquito vector, which are infected with West Nile, founded during winter time, and at least in the USA, there are also horizontal transmission between birds, but there are no evidence of that because there's a lack of lack of studio in Europe about the role of birds in the maintaining or even wintering of West Nile. So, yeah, I think we're wintering at the moment is probably due to the mosquito vector, which carry on the virus to the next season. I don't think there is much of relation between the two viruses, but for sure, the spread and the spread and the present situation with COVID is worsening a possible West Nile virus outbreak because the restriction measurement at the moment for COVID, again, limit the movement of people, render more difficult, maybe do some entomological control or intervention, and also I've stressed the public health care system so maybe symptomatic West Nile virus cases could have less access to cure or be confused with the other. I mean, if you develop a fever now, there's likely a fear that you have COVID rather than West Nile, so you may have one diagnosis at the moment. So, yeah, surely COVID have impacted the health care system and is reflected to West Nile, but it's too early in the season to predict or have an idea how much the West Nile will circulate or well. But yeah, if you're going to see another outbreak like 2018, it will be worsened by the COVID situation.
 So I will talk about how spring temperatures shape  the Western light values transmission in Europe.  This work was led by Giovanni Marini, who unfortunately cannot attend today's presentation.  So I hope it will be as interesting as it was him presenting.  Next slide, please.  The outline of the presentation is a little brief background on the West Nile virus, and then I'm going to talk about the previous work on West Nile transmission in Emilia Romagna, which is a region of Northern Italy,  and then how we try to extend it to the whole of Europe and try to investigate how spring temperature may affect humans below.  Next slide, please.  So West Nile is a virus which is mainly circulating among mosquito and birds, where mosquito act as a vector, and given that the mosquito species, which is the primary vector also bites other animals like horses and human.  It can happen to have an incidental infection to again horses and humans, which are dead at host. So an infected human is not able to infect the mosquito vector. And so to is not able to further transmission.  So this kind of disease is a symptomatic, but in rare cases, people infected can develop severe neuroinvasive implications.  So most of the time, the disease goes undetected by the public health system. And when the system starts to see cases, the circulation is already widespread or happening for a quite long amount of time.  So next slide, please.  So obviously, given that there is a mosquito vector involved, temperature plays a key role, because it affects mosquito dynamics.  Several works have shown how warmer conditions can speed up the development and the life cycle of the mosquito and also increased a biting rate to the host, which can be bird, in some cases also human or horses rather mammals.  And similarly, also temperature plays a role by affecting the pathogen transmission as warmer condition may decrease the incubation period in both the host or in the vector and increase the transmission probability to other hosts.  Next slide, please.  So as I said, there has been a previous work led by Giovanni, which was in which he analyzed the West Nile virus transmission in northern Italy, in particular in the region of familiar Romania in the northern Italy, which is characterized to be the region where the low land of the  river is located. And as you can see the points are the traps deployed by the surveillance systems.  And in this work is a top of entomological model coupled with an epidemiological model. So in the first entomological.  So we analyze the life cycle of the mosquito and the model was calibrated to mosquito captures and as that input data was the temperature by this model one is able to estimate the expected mosquito bundas.  The second part, coupled with the epidemiological model which models the transmission between birds and mosquitoes. In this way, one can compute the prevalence on the on the board on the mosquito which has the vector and from that compute the human risk given the mosquito  prevalence.  And the model was computed.  The model was calibrated on the data in the past year, and it's very interesting to note how the 2018 was a very exceptional year.  And also, later in the season in about 100 narrow evasives human cases, which are a lot more than the total number recorded in the previous year.  This is not specific to the Emilia Romania region but it's also happened in 2019 in almost all Europe.  So, here's the next slide.  These are some results, as you can see spring was exceptionally warm in 2019 and this seems to have amplified that the was not virus transmission at the beginning of the season.  So, having a higher transmission at the beginning of the season resulted in more circulation during the season and therefore in a lot of more cases than observed by the public as system.  So, next slide please.  So, we observed that pattern in Emilia Romania region without trying to explore if this pattern is also observable interest of Europe.  Again, there's a problem of data, which are not available as in Emilia Romania for the rest of the Europe.  So we collated data from the CDC, which report cases from all Europe for the period from 2011 to 2019 at the nuts three level.  As you can see in the upper right graph 2018 is a very exceptional year for all the Europe in terms of West Nile virus transmission.  So we taking consideration this data and we applied a statistical analysis to quantify how any spring temperature can influence you must be lower at an European scale.  So, please switch to the next slide.  The model we use was to consider the laboratory number of laboratory confirmed cases for an area that's three level and year specific here.  We assume that given the CDC record only report positive cases when an area has no cases so is not present in the database, but it has already as add cases in previous year, we assume that there is a zero reported cases so we inflate the database with zero.  And also we take into input the land surface temperature as an independent variable inter statistical model.  Can you please go ahead. Yeah, thank you.  The model was a zero inflated negative binomial models. It's a two part model. The first part is a binomial part which models the present of absence of cases.  We use this, we use this framework because we inflate the database with zero.  And now we think we it was important to model also this part. And the second part of the model is modeling the number of positive cases recorded.  So as you can see by the formula and equation here to dependent variable was the number of laboratory confirmed cases, while the independent variable where the average spring temperature in the period 2003 2010.  And these act as a gradient differentiating south to northern Europe.  And the second dependent variable was the temperature anomaly so we normalize the temperature as a difference between the average temperature so this variable indicates characterized each year if it is a warmer or colder year with respect to the mean.  And then we also consider the temperature of the area.  And we also consider if the West Nile was already circulating in that area for that year, in the previous year.  So we can see simple and explorative statistical model to just see if we can find some relationship between temperature and the number of cases recorded by the CDC at European level.  So next slide please.  Indeed, there is a correlation as expected.  And as you can see in the upper panel.  So the average number of cases expected conditional to the temperature in the average temperature in the upper left panel, or with respect to the temperature anomaly in the upper right panel.  As you can see, there is an increasing pattern.  So the warmer, the on the average spring temperature more cases and the warmer specific springs.  So the higher number of cases.  On the other hand, the lower panel models the probability of serving as zero or a positive number of cases. As you can see, again, the role of temperature.  And that the more warmer the temperature, the less likely to serve as your record.  So next slide please.  So to conclude, the previous work on the middle of Romania region and the following work that is now published in the tropical.  European level shows that there is an association between previous was not detection and larger number of cases in the following years.  It's more likely to have an higher circulation in usually warm region.  That is further in as if the specific spring temperature or a fire is warmer than the average temperature.  And that considering a warmer temperature spring in particular warmer temperature as a nearly warning signal signal for West Nile virus humans below.  I think that thank you for your attention.  And if there is any question.  I would be glad to answer it.  Thank you, my dear, my apologies for some technical problems but we're still on time. So please questions.  Maybe I will start off somebody has a really, really question.  I think that's me.  Nice talk. Thank you.  Your first point there previous West Nile detection is associated with larger outbreaks.  That implies that, I mean, if you've had a large outbreak, let's say in 2018 that conclusion would suggest you're likely to have another one in 2019 is that right now surely not.  A large outbreaks is probably catch phrase for the presentation. You just say that.  This is the next year. Obviously, quite.  Were there also far more places, as opposed to cases.  The Nile is spreading across Europe and teams that most of most area.  It's when it's a pair then it's bound to stay.  And I think that's the problem going on with the detection. They think because given that is not really symptomatic for most cases is difficult to detect but if you look for it in the mosquito vector when you are an area that results positive for what's not by circulation.  And in time, you will pile up cases and when again, 2018 was an exceptional year but probably condition align warmer temperature previous circulation, a lot of vertical transmission from the previous you have this outbreak happening.  At the moment was 2018 was the highest level, the peak level.  Observed.  Okay, thank you. We have one more question from Veeam, I think.  Yeah, thank you. I thank you for your nice talk on West now I see for your easy DC data you use data from 2011 to 2019.  And there's no specific reason not to have 2010 to the database this was also the start of the West now to the large West now to circulation in Europe a large outbreak in Greece.  And we also very specific climatic conditions. 2010 was also a very strong winter. I think I don't know what the spring temperature did. Was there any specific reasons, not having 2010 in your analysis.  So, you know, the temperature data from 2003 2010 as a reference. And then we started the database from the independent for the dependent variable from the 2011.  As you say, I think the 2010 is the first year of the appearance of us nine in Europe for the DCC so we, so we discarded the first year, I think.  Because it was also a very large outbreak. Of course, very, I think at a time many focusing on Greece so so it was included in your reference point.  No, at the moment, no.  Okay.  Okay, thanks.  Any other questions.  I have a question.  I was wondering, do you plan to include other variables except a temperature like rainfall.  Yeah, we would like water areas.  The model.  Yeah, it would be nice. I think it's a future direction for research to include more variable. Again, this was to explore an association we find the founder on Emilia, Romania region in the previous work.  But yeah, with my virus circulation is a very complex system and mechanism and cannot be explained only by temperature.  And yes, indeed, we should include more variables also relating to the  climate change and future temperatures. Have you have you tried doing that, like just extrapolating the model you fitted with the future like projected climate scenarios.  It would be nice to do that but we, we were not so much confident about the reliability of the result.  Because again, it's an indication that with temperature things in terms of West Nile are going worse and worse. So, if we expected to be an increasing temperature.  Then we have to expect an increase in West Nile virus transmission and circulation across all Europe.  Temperature most likely affect the transmission of us now because it acts as a boost the mosquito population, which act as a vector because warmer condition can speed up development of mosquito and also can speed up the incubation period within the vector of the viruses.  So, if you have a warmer condition, most likely, you can have more aggressive or more abundant vector population.  And if the viruses is introduced in the system or in the area, then it can be circulating with much more velocity.  So, yeah, that's the main reason I think for temperature being very important in the modeling and in understanding of the West Nile virus circulation.  There's a lot of different factor because we know that birds are the primary host but there's not so much information which kind type species of birds is really maintaining and sustaining the viruses in the environment.  So, we're able to winter through the mosquito population, which entering the oppose and then by vertical transmission and start the cycle in the next season.  So, we have an observation of the opposing mosquito vector, which are infected with West Nile, founded during winter time, and at least in the USA, there are also horizontal transmission between birds, but there are no evidence of that because there's a lack of lack of studio  in Europe about the role of birds in the maintaining or even wintering of West Nile. So, yeah, I think we're wintering at the moment is probably due to the mosquito vector, which carry on the virus to the next season.  I don't think there is much of relation between the two viruses, but for sure, the spread and the spread and the present situation with COVID is worsening a possible West Nile virus outbreak because the restriction measurement  at the moment for COVID, again, limit the movement of people, render more difficult, maybe do some entomological control or intervention, and also I've stressed the public health care system so maybe symptomatic West Nile virus cases could have less access to cure or be confused with the other.  I mean, if you develop a fever now, there's likely a fear that you have COVID rather than West Nile, so you may have one diagnosis at the moment.  So, yeah, surely COVID have impacted the health care system and is reflected to West Nile, but it's too early in the season to predict or have an idea how much the West Nile will circulate or well.  But yeah, if you're going to see another outbreak like 2018, it will be worsened by the COVID situation.
Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_0_83.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_84_215.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_216_731.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_732_1499.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_1500_2219.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_2220_3275.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_3276_4619.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_4620_7943.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_7944_8819.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_8820_9143.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_9144_10547.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_10548_10919.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_10920_11723.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_11724_14483.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_14484_16271.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_16272_18659.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_18660_21683.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_21684_21743.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_21744_21803.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_21804_21863.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_21864_27623.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_27624_27719.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_27720_27851.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_27852_27947.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_27948_29747.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_29748_29855.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_29856_31079.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_31080_31187.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_31188_33275.png Mattia_Manica_Feb2021_Spring_Temperatures_Shape_West_Nile_Virus_Transmission_In_Europe_webinar_33276_33279.png