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 Academic Journal of Science, Engineering and Technology 

Vol.6, Issue 1; January - Febuary 2021; 

1252 Columbia Rd NW, Washington DC, United States 

https://topjournals.org/index.php/AJSET/index; mail: topacademicjournals@gmail.com 

  

 

 

83 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

THE NEED FOR FURTHER RESEARCH INTO THE TRANSMISSION OF 

INFLUENZA A VIRUSES BETWEEN MIGRATORY WATERFOWL AND DOMESTIC 

DUCKS 

 

Janet Marie Williams and Robert Michael Brown 

University of Texas at San Antonio  

  

Abstract: Migratory waterfowl are the natural reservoir and source point for influenza viruses. Artificial water 

ponds in rural and urban communities are potential sites of the human-animal interface for influenza A virus 

(IAV). This paper reviews the literature on the transmission of IAV between migratory waterfowl and domestic 

ducks in community ponds, and the potential for this to lead to human infection. 

Keywords: Influenza A virus (IAV), Migratory waterfowl, Domestic ducks, Community ponds, Human infection, 

Transmission 

 

Introduction and Background   

Numerous research studies have indicated that migratory waterfowl are the natural reservoir and a source point 

for influenza viruses. Artificial water ponds in rural and urban communities are potential sites of the human-

animal interface for  

IAV.   

Webster, Bean, Gorman, Chambers, and Kawaoka (1992) described this human-animal interface with the example 

of domestic ducks in community ponds attracting migratory waterfowl. The migratory waterfowl introduce 

influenza virus to that community’s water pond from fecal contamination. The contaminated community water 

pond now becomes a potential source of influenza virus to both humans and animals.  Influenza disease emergence 

data are collected year-round, but economic strain on global public health to prevent and treat human influenza 

outbreaks has been enormous. Therefore, it is imperative to identify potential sources of the virus to help minimize 

outbreak occurrence.   

Statement of the Problem   

There are gaps in knowledge about the association between molecular structure, epidemiologic and clinical 

characteristics, and the impact of ecological and other contextual aspects of IAV. Gaps in understanding the role 

of the physical and biogeochemical environment as an integral part of the IAV transmission also exist (Lang et 

al., 2008).More importantly, gaps in knowledge about the burden of IAV in rural and urban community settings 

remain present. The problem was that public health science professionals have been battling emerging human 

influenza diseases with tactile and reactionary methods because there was a lack of knowledge and data at the 

human-animal interface. The purpose of this baseline study of the proportion of IAV in urban and rural community 

settings was to provide knowledge and biological data of significant interest at the human-animal interface.   

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84 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

Emerging influenza viruses have continued to challenge public health officials. Influenza viral infections appear 

with such regular annual frequency that it has been common to refer to the phenomenon as flu season or seasonal 

flu. However, pandemic influenza events have not been regular or predictable.   

Taubenberger and Morens (2006) identified and provided a brief synopsis of 13 pandemic events between 1510 

and 1978.Wali and Music (2011) provided a summary of public health outcomes of nine U.S. influenza epidemics 

that occurred between the period 1972 to 1973 through 1991 to 1992.Keeler (2011) provided a brief history of 

influenza pandemics from the time of discovery of the influenza virus through the last decade. Further, Keeler 

notably identified the sharp increase in public awareness of influenza and the potential human health threat of the 

virus. 

The outcomes of this study and detection of IAV along the eastern Pacific flyway were in alignment with the 

World Health Organization (2011) description of the human-animal interface as a complex juncture at which new 

paradigms are emerging. Austin and Hinshaw (1984) suggested that surveillance of healthy ducks and the aquatic 

environment they frequent may be of significant interest to monitoring and controlling IAV. This research study 

is a model that can be modified to monitor IAV in the aquatic environment.   

Purpose of the Study  

In this study, the proportion and probability of the presence of IAV were investigated in recirculating artificial 

ponds in rural and urban geographical locations. Rural ponds were viewed as one population and urban ponds as 

another population. The dependent agent variables were IAV. The research question was as follows: Is there a 

difference in the burden of IAV in rural ponds compared to urban ponds?   

Definition of Terms  

Several not commonly used terms-outside of the topic of influenza-are necessary for this research study. The 

manuscript by Reid, Taubenberger, and Fanning (2004) includes definition of these terms.   

Antigenic drift: Minor changes in viral antigens due to gradual accumulation of mutations over time.    

Antigenic shift: Sudden change in viral antigens due to acquisition of one or more novel surface-protein-encoding 

genes by the process of reassortment.  

M gene sequence: The membrane protein (M) gene of IAV is 1,027 nucleotides long and encodes two proteins, 

M1 and M2. M1 protein is the most abundant protein in the influenza a virus viron (Webster et al., 1992).  

Reassortment: Due to the segmented nature of the IAV genome (eight individual RNA segments), influenza 

viruses can undergo a process of genetic reassortment to produce new variant strains of virus.   

In a cell infected with two different IAV strains, gene segments from each can be packaged into viable hybrid 

virus strains.  

Subtype: A designation for IAV describing the antigenic group to which the two dominant surface glycoprotein’s 

— haemagglutinin (HA) and neuraminidase (NA) — belong, written in the form HXNX, wherein one of the 16 

possible HAs and one of the nine possible NAs is listed, for example, H1N1 or H3N2. These terms are necessary 

and used throughout this study for clarity. 

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 Academic Journal of Science, Engineering and Technology 

Vol.6, Issue 1; January - Febuary 2021; 

1252 Columbia Rd NW, Washington DC, United States 

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85 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

Assumptions and Limitations 

There are several assumptions about IAV that are believed, but cannot be demonstrated to be true. Firstly, the 

unique and problematic property of IAV to evade hosts immune responses. Secondly, the phenomena of human-

animal transmission are not well understood.   

It is possible the ability to evade host immune responses and the humananimal transmission are the evolutionary 

traits that give IAV a “natural fluid” presence resulting in seasonal outbreaks. Keeler (2011) and others asserted 

the emergence of novel IAV through antigenic drift or antigenic shift can result in significant increases in 

morbidity and mortality during any given influenza season. The outcome of antigenic drift or antigenic shift are 

routinely identified, however the mechanism of the processes are insofar only theorized.  

A pandemic is the outbreak of infection, arising in a specific geographical area and spreads throughout the world 

resulting in a high percentage of infected individuals and resulting in increased mortality rates (Potter, 2001).It 

has been assumed that pandemics may be caused by a new IAV subtypes. The new IAV subtype may have an HA 

of which is not related to that of influenza viruses circulating immediately before the outbreak, and could not have 

arisen from those viruses by mutation (Potter, 2001).The 1918 Spanish flu is assumed to be caused by a “new 

IAV” subtype. Investigation into the influenza virus responsible for the 1918 Spanish flu has not yet been fully 

discovered. Investigation into the molecular mechanisms of IAV is prominent in current literature. This may be 

due to recent advances in molecular and genetic sciences.  

Artificial water ponds in rural and urban communities are potential sites of the human-animal interface. The 

investigation of the burden of IAV in artificial rural and urban ponds may be of significant interest to the scientific 

community. The intent of this study was to investigate the burden of IAV in artificial reticulating water ponds in 

the geographic locations of rural and urban Californian communities. The geographical sampling region for rural 

and urban ponds was the eastern Pacific flyway for migratory birds in California.   

For this study, it was necessary to make the assumption that artificial water ponds in rural and urban communities 

are sites of the human-animal interface because of fecal shedding and contamination from IAV infected waterfowl 

(Anseriformes) and shorebirds (Charadriiformes).The primary goal of the laboratory analysis was to isolate and 

investigate IAV in samples collected from water ponds in rural and urban Californian communities.  

There are two threats to the external validity of this study. The first threat to external validity is centered to the 

theoretical framework of this study. The theoretical framework for this study were based on the hypotheses 

proposed by Webster et al. (1992) and others that (a) migratory waterfowl are the natural reserve of influenza 

viruses, and (b) water-borne transmission of influenza virus occurs between migratory waterfowl and domestic 

waterfowl. The inclusion criteria of the study population were artificial recalculating water ponds in the 

geographic locations of rural and urban Californian communities. The geographical area was the state boundaries 

of California. Thus, the findings of this are limited to geographical locations along the migratory flyways of the 

waterfowl within the boundaries of California.  

To address this threat to the external validity of this study, the sampling and data analysis plan are presented in a 

fashion so the study may be reproduced accurately by others using the same or different geographical locations. 

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86 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

The second threat to external validity is a result of the inclusion criteria. Recirculation artificial water ponds 

require mechanical equipment for recirculation. This study does not include the type or design of the mechanical 

equipment used for recirculation as an independent variable. Thus, the mechanical equipment used for 

recirculation cannot be evaluated as a mediator or moderator of the outcome variables of this study.     

However, estimated surface area of the recirculating artificial water ponds as an independent variable may be of 

significant interest to future studies seeking to investigate factors that associate to the burden of IAV at the human-

animal interface in communities.  

The primary data collection of study is heavily weighted by laboratory instrumentation and molecular analysis. 

There are two threats to the internal validity of this study: instrumentation and laboratory assays. Firstly, 

inaccuracy of the instrument used for this study may systematically alter the data. Secondly, inaccuracy of the 

laboratory assays may fail to accurately detect IAV. To address the possible inaccuracy of the instrumentation, the 

same instruments were used on all samples. Thus, errors, if identified, may be corrected across all samples equally. 

To address the possible inaccuracy of the laboratory assays, positive and negative control samples were to be 

processed simultaneously as study samples. The positive control for IAV was not obtained. The negative control 

used for IAV was sterile normal saline.  

Threats to statistical conclusion validity have not been identified.  

Literature Review  

History of Influenza Surveillance  

In 1947, at the 4th International Congress for Microbiology held in Copenhagen, a group of virologist forwarded 

a recommendation to the Interim Commission of the World Health Organization an international program be 

initiated for influenza surveillance (Hampson, 1997). The World Health Organization Global Influenza 

Programme was established later that year (Fleming, van der Velden, & Paget, 2003).  

The 1947 influenza surveillance control programme is the oldest disease control program at the World Health 

Organization (World Health Organization, 2005).    

The World Health Organization stated the creation of the 1947 influenza surveillance control programme resulted 

from two concerns: first, the inevitable recurrence, at unpredictable intervals, of highly disruptive pandemics; and 

second, the significant health and economic impact of seasonal epidemics, which occur nearly every year (p. 

34).Hampson (1997) opinioned the objectives of the 1947 influenza surveillance control programme as: to gain 

an understanding of the epidemiology of influenza, and to promptly isolate influenza viruses from new outbreaks 

and distribute them for vaccine production (p. S8).Within four years of creation, the 1947 influenza surveillance 

control programme developed into a network of 60 laboratories across 40 countries.  

The influenza surveillance control programme became the WHO Global Influenza Surveillance Network 

consisting of 113 national influenza centers located in 84 countries (World Health Organization, 2005).Following 

the adoption by the World Health Organization of the Pandemic Influenza Preparedness Framework in May 2011, 

the Global Influenza Surveillance Network was changed to Global Influenza Surveillance and Response System.  

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Vol.6, Issue 1; January - Febuary 2021; 

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87 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

The Centers for Disease Control and Prevention and the European Influenza Surveillance Scheme (EISS) 

collaborate with the WHO Global Influenza Surveillance and Response System to monitor the evolution of 

influenza viruses and provides recommendations in various disciplines including laboratory diagnostics, vaccines, 

antiviral susceptibility and risk assessment (World Health Organization, 2013).   

Origin of Influenza (China) 

The literature suggests China, with its abundance of live poultry and swine markets, maybe the epicenter of novel 

influenza viruses. Wan et al. (2005) proclaimed that Southern China has been shown to be the avian influenza 

virus pool for flu outbreaks in history including H2N2 (1957), H3N2 (1968), H5N1 (1997 & 2003), and H9N2 

(1999).Webster et al. (1992) noted historical records and the appearance of the Asian, Hong Kong, and Russian 

pandemic strains of influenza virus in China suggest the majority of pandemics of human influenza since about 

1850 have originated in China.  

Perez-Ramirez et al. (2011) described how large parts of Asia support high densities of humans, backyard poultry 

(ducks, geese, and chickens), pigs, and wild birds. These high density areas provide opportunities for close 

interaction between influenza reservoir animals and create a unique environment for influenza evolution (Perez-

Ramirez et al., 2011).These unique environments may be considered an expansion of the mixing vessel theory; 

whereas these high density areas may be identified as “mixing vessel environments”.  

Migratory Waterfowl (Vector for Global Distribution)  

Austin and Hinshaw (1984) investigated feral duck species as a source of transmission of IAV and 

paramyxoviruses. The researchers collected swab samples from tracheal and cloacae from different species of 

feral ducks (Austin & Hinshaw, 1984). Austin and Hinshaw and were able to identify several viral strains of IAV.  

Additionally, Austin and Hinshaw asserted the alterations of influenza viral strains occur due to antigenic drift at 

point mutations and antigenic shift is caused by genetic reassortment.  

The authors contended it was possible these alterations occurred in the intestinal tract of ducks (Austin & 

Hinshaw, 1984).This article provides support to the hypothesis that migratory ducks are a source point for 

influenza viruses. Therefore, surveillance of healthy ducks and the aquatic environment they frequent may be of 

significant interest to monitoring and controlling IAV.  

As previously stated, the literature suggests novel influenza viruses originate in the Southern China region where 

migratory waterfowl become infected.  Infected asymptomatic migratory waterfowl may transport the novel 

influenza viruses in their intestinal tracts to the wetland breeding grounds of Alaska and Siberia.  The waterborne 

transmission of the influenza viruses to new hosts (migratory waterfowl from Northern and Southern American 

regions) may occur at these Alaskan and Siberian wetland breeding grounds.  The new host(s) may act as a mixing 

vessel for influenza viruses.  This may result in an antigenic shift or antigenic drift of influenza viruses resulting 

in novel strains.  Infected asymptomatic migratory waterfowl may then transport these influenza virus strains 

along the eastern Pacific flyway for migratory birds. This process provides a possible explanation for the global 

distribution of novel influenza strains and further supports the necessity of influenza virus surveillance at the 

human-animal interface.  

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88 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

Water Analysis  

Water analysis has been an important scientific approach to understanding the epidemiological triangle model of 

influenza viral diseases. In the early study by Cumming (1919), a case-control approach was used to better 

understand the transmission and infectivity of the causitive agent of influenza-pneumonia. The findings of 

Cumming study suggested the agent was the influenza-pneumonia organism, the route of transmission was 

contaminated eating utensils, and aquatic dishwashing was the environment.   

Webster et al. (1992) asserted waterborne transmission of influenza viruses occurs due to the viral shedding in 

the fecal material of waterfowl (Anseriforms) and shorebirds (Charadriiformes). Van Dalen et al. (2010) 

investigated waterborne transmission of influenza A H4N6 in mallards (Anas platyyrhychos) in a controlled 

laboratory environment. Lang et al. (2008) collected and analyzed sediment samples from three ponds in the 

Creamer’s Field Migratory Waterfowl Refuge, Alaska, a location used by a wide variety of migratory waterfowl. 

The sediment samples were collected using a time-series approach and analyzed for IAV RNA using reverse 

transcription-polymerase chain reaction (RT-PCR) methodologies (Lang et al., 2008).  

Zhang et al. (2006) collected and analyzed samples of ice or water from three northeastern Siberian lakes in the 

Koluma River region. The samples were analyzed for the presence of IAV using reverse transcription-polymerase 

chain reaction (RTPCR) methodologies (Zhang et al., 2006).Hinshaw et al. (1980) collected samples from 

waterfowl, unconcentrated lake water, and feces from lake shores near Vermillion, Alberta, Canada. These 

samples were used to isolate influenza viruses and to investigate whether influenza viruses continually circulate 

or whether the same or different strains are present from year to year (Hinshaw et al., 1980).   

Methodology 

Research Design   

The epidemiological triangle provided framework for the primary data collected. 

The epidemiological triangle has three vertices: host, agent, and environment. The host is the organism harboring 

the disease. For influenza disease, the reservoir hosts are migratory waterfowl and shorebirds (Franklin et al., 

2011).The agents are the IAV. The environments are the natural and artificial habitats where the hosts are found.  

The independent environmental variables of this study were the geographical locations of the artificial water 

ponds in either a rural or urban community. The dependent agent variables were IAV.IAV data included detection 

for M gene sequence by real time RT-PCR using World Health Organization recommended primer sequences.  

Sampling Methods and Procedures  

The study method was quantitative using a cross-sectional design. A convenience sampling approach was used. 

The geographical area was the state boundaries of California. Equal sample sizes from rural and urban 

communities were attempted. A representative sampling from each of the 21 counties considered rural areas, and 

37 counties considered metropolitan and not rural in California by California Business and Professions Code 

Section 19986(l) were attempted. The inclusion criteria of the study population were artificial recirculation water 

ponds in the geographic locations of rural and urban communities. 

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89 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

Water Sampling (Pre analytical Phase) 

Water samples were collected as previously published in the U.S.EPA Field Sampling Guidance Document 

#1225.A dip sampler for water collection was used. 

A dip sampler is useful for situations where a sample is to be recovered from an outfall pipe or along a pond bank 

where direct access is limited. The long handle on such a device allows access from a discrete location so as not 

to disturb wildlife (U.S. Environmental Protection Agency, 1999).The water volume per sample was 200 ml. 

Other researchers have collected 200 ml volume water samples for influenza studies (Zhang et al., 2011).   

Sample Preparation (Pre analytical Phase)  

Sample integrity was maintained until received in the laboratory for processing. Sample identification was 

maintained throughout the laboratory testing. Samples were filtered using Millipore Durapore 0.22 µm filters to 

remove particulate matter and other suspended contaminates. The filtered sample were poured into 250 ml 

centrifuge tubes and concentrated at 10,000 rpm for 20 minutes at 4°C using a Sorvall SL-1500 Super-Lite 

centrifuge rotor. 100 µL of RNA later RNA Stabilizer reagent (QIAGEN N.V.) was added to 100 µL of the 

concentrated pond water sample in a separate polypropylene micro vial for viral RNA extraction. Evers et al. 

(2007) studied the commercial preservative RNA later (QIAGEN N.V.) by evaluating against the current method 

of cryo-freezing, and ethanol preservatives for IAV samples. From the findings of the Evers et al. study, the 

authors asserted the commercial preservative RNA later held at ambient temperatures might be useful for the 

identification of IAV in samples collected from infected waterfowl.   

Real Time RT-PCR (Analytical Phase)   

Reverse transcriptase polymerase chain reaction (RT-PCR) is a powerful technique for the identification of 

influenza virus genomes (World Health Organization, 2007).   

A constellation of studies have targeted the M gene sequence of the IAV genome by RT-PCR as an indicator of 

IAV positivity (Harmon et al. 2010; Lee et al., 2012; Magnard et al.,1999; Perez-Ramırez, et al., 2012).During 

the analytical phase of this study real time RT-PCR technique was utilized.  

Using real time RT-PCR allows for the detection of products as amplification is ongoing, allowing quantification 

(World Health Organization, 2007).The analytical phase was conducted following World Health Organization 

Real-time RT-PCR Protocol 2 for IAV (H5N1) detection.   

Data Collection and Analysis  

The primary data analysis was a cross-sectional approach comparing proportions recirculation artificial ponds in 

rural and urban geographical locations. The independent variable was: geographic community location (rural or 

urban). The dependent variable was: IAV detection by real time RT-PCR. The statistical analysis software was 

IBM SPSS Statistics 21 and Microsoft Office Excel 2007. Geographical graphing and mapping software was 

Google Earth. Data integrity was maintained by recording data and observations with ink into field notebook and 

laboratory notebook. Hand written entries were transferred or transcribed to Microsoft Office Excel 2007 

Spreadsheets. All electronic data had digital backup and was password protected. 

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90 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

Credibility and Validity 

Credibility is important to uphold society’s trust that scientific research results are an honest and accurate 

reflection of a researcher’s work (National Academy of Sciences, National Academy of Engineering, and Institute 

of Medicine of the National Academies, 2010). The materials and methods of this environmental baseline research 

study were built on the research of others.  

Environmental water sampling underscored the research design of this study. Therefore, the research adhered to 

the Code of Ethics and Standards of Practice for Environmental Professionals. The objectives of Environmental 

Professionals are to conduct their personal and professional lives and activities in an ethical manner (National 

Association of Environmental Professionals, n.d).  

Findings  

Evaluation of Research Data 

Artificial pond water samples were collected from 14 of the 21 counties considered rural areas, and from 25 of 

the 37 counties considered metropolitan and not rural in California by California Business and Professions Code 

Section19986(l). Urban counties’ pond water samples [NUrban = 100] were collected from 25 of the 37 counties 

considered metropolitan and not. Rural counties’ pond water samples [NRural=82] were collected from 14ofthe21 

counties considered rural and not metropolitan. 

Pond water samples were verified for their content of IAV genome by real time RT-PCR targeting the M gene 

sequence. The real time RT-PCR M gene assay was conducted using a Bio-Rad CFX96 Touch Real time PCR 

Detection System (BioRad, Hercules, California) instrument and utilized two fluorescent dyes: FAM and HEX. 

The analysis software used was Bio-Rad CFX Manager 3.1. The software setting was: End cycles to average [5] 

and Percent of Range [10.0]. The results for the  

FAM fluorophore were: [Lowest RFU value = -3.71], [Highest RFU value = 3.41], [Negative Control Average = 

-0.453], and [Cut Off Value = -0.0668]. The number of statewide samples [N = 182] called (+) positive using 

FAM [NFAM = 45, 45/182 =  

24.7%]. 

The results for the HEX fluorophore were: [Lowest RFU value = -1.93],  

[Highest RFU value = 1.46], [Negative Control Average = 1.42], and [Cut Off Value = 1.42].The number of 

statewide samples [N = 182] called (+) positive using HEX [NHEX = 1, 1/182 = 0.5%].The number of statewide 

samples [N = 182] verified for their content of IAV genome by real time RT-PCR targeting the M gene sequence 

is [n = 45] (see Figure 1.).The confidence interval formula for a proportion is commonly known as: CI = p ± Z α/2 

× √[ (p × q) / n] , (x, n – x ≥ 5), where p = x / n, q = 1 - p, α = 1 - (Confidence Level/100) x = Frequency, n = 

Sample Size, and Z α/2 = Z-table value.  

Applying the data values of this study into the confidence interval formula the proportion of the overall statewide 

samples [N = 182] called (+) positive for the IAV M gene sequence [n = 45] is P = 0.247, 95% CI [0.185, 

0.310].The effect size index Cohen’s g is the departure from P = 0.50 (Cohen, 1988).Thus, the non directional 

calculation for Cohen’s g is [g = |P – 0.50|].For the overall statewide samples, the calculation of Cohen’s g is [g 

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91 | A c a d e m i c  J o u r n a l  o f  S c i e n c e ,  E n g i n e e r i n g  a n d  T e c h n o l o g y  

|  https://topjournals.org/index.php/AJSET 

= |0.247 – 0.50| = 0.253]; a large effect size (Cohen, 1988). Using G*Power 3.1.5, the post hoc computed Power 

= 0.999 (two tailed). 

Urban samples assayed by real time RT-PCR  

The number of urban county samples [NUrban = 100] called (+) positive using FAM [NFAM = 36, 36/100 = 

36.0%].The number of samples [NUrban = 100] called (+) positive using HEX [NHEX = 1, 1/100 = 1.0%].The 

number of urban county samples [NUrban = 100] verified for their content of IAV genome by real time RT-PCR 

targeting the M gene sequence is [n = 36].Applying the confidence interval formula for a proportion: CI = p ± Z 

α/2 × √[ (p × q) / n] , (x, n – x ≥ 5), where p = x / n, q = 1 - p, α = 1 - (Confidence Level/100) x = Frequency, n = 

Sample Size, and Z α/2 = Z-table value, the proportion of the urban counties’ samples [NUrban = 100] called (+) 

positive for the IAV M gene sequence [n = 36] is P = 0.36, 95% CI [0.266, 0.454].  

The effect size index Cohen’s g is the departure from P = 0.50 (Cohen, 1988).Thus, the non directional calculation 

for Cohen’s g is [g = |P – 0.50|].For the urban counties’ samples, the calculation of Cohen’s g is [g = |0.36 – 0.50| 

= 0.14]; a medium effect size (Cohen, 1988).Using G*Power 3.1.5, the post hoc computed Power = 0.768 (two 

tailed)  

Rural samples assayed by real time RT-PCR  

The number of rural counties’ samples [NRural = 82] called (+) positive using FAM [NFAM = 7, 7/82 = 8.5%].The 

number of samples [NRural = 82] called (+) positive using HEX [NHEX = 2, 2/82 = 2.4%].The number of rural 

counties’ samples [NRural = 82] verified for their content of IAV genome by real time RT-PCR targeting the M 

gene sequence is [n = 9].Applying the confidence interval formula for a proportions: CI = p ± Z α/2 × √[ (p × q) / 

n] , (x, n – x ≥ 5), where p = x / n, q = 1 - p, α = 1 - (Confidence Level/100) x = Frequency, n = Sample Size, and 

Z α/2 = Ztable value, the proportion of the rural counties’ samples [NRural = 82] called (+) positive for the IAV M 

gene sequence [n = 9] is P = 0.11, 95% CI [0.042, 0.177].  

The effect size index Cohen’s g is the departure from P = 0.50 (Cohen, 1988).Thus, the non directional calculation 

for Cohen’s g is [g = |P – 0.50|].For the rural counties’ samples, the calculation of Cohen’s g is [g = |0.11 – 0.50| 

= 0.39]; a large effect size (Cohen, 1988).Using G*Power 3.1.5, the post hoc computed Power =  

1.000 (two tailed). 

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Figure 1: Statewide samples verified for their content of IAV genome. Google Earth map of California showing 

statewide pond water samples verified for their content of IAV genome by real time RT-PCR targeting the M gene 

sequence. [RED icon = (+) Positive for M gene sequence and GREEN icon = (-) Negative for M gene sequence].   

Analysis of Relevant Research Data 

In this study the proportion and probability of the presence of IAV was investigated in recirculation artificial 

ponds in rural and urban geographical locations. Rural ponds were viewed as one population and urban ponds as 

another population. The dependent agent variables are IAV.IAV data included: detection for M gene by real time 

RT-PCR using World Health Organization recommended primer sequences.   

Examination of the Research Question   

The proportion of the urban counties’ samples [NUrban = 100] called (+) positive for the IAV M gene sequence [nu 

= 36] is Pu = 0.36, 95% CI [0.266, 0.454]. This value is numerically larger than the proportion of the rural counties’ 

samples  

[NRural = 82] called (+) positive for the IAV M gene sequence [nr = 9] is Pr = 0.11, 95% CI [0.042, 0.177].It is 

commonly accepted the two-sided 100 (1 - α) % CI for the difference between two proportions of unequal sample 

size is of the form: (^p1 – ^p2  

)± z1 – α/2 √ [ (^p1 (1 – ^p1) / n1 ) + (^p2 ( 1 – ^p2) / n2 ) ].  

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For this analysis; [^p1 = nu / NUrban], [^p2 = nr / NRural], and [z1 – α/2 = 1.96].The calculated difference in the 

proportion of urban counties’ samples called (+) positive for the IAV M gene sequence to rural counties’ samples 

called (+) positive for the  

IAV M gene sequence is directionally towards urban counties’ samples [(^p1 – ^p2= 0.2502, 95% CI (0.1278, 

0.3604), RD = 0.2502, 95% CI (0.1337, 0.3668), RR = 3.28, 95% CI (1.83, 7.79), OR = 4.563, 95% CI (2.042, 

10.193)].  

The effect size index of the difference between two proportions is Cohen’s h. Cohen’s h is the calculated 

difference between the arcsine transformations of the proportions (P) of the populations (Cohen, 1988).Thus, 

Cohen’s h = |φ1 – φ2| where φ = 2 arcsine √(P).The calculated effect size index for the difference in the proportion 

of urban counties’ samples to rural counties’ samples called (+) positive for the IAV  

M gene sequence is Cohen’s h = | (2 arcsine √(Pu)) – (2 arcsine √(Pr)) | = | (2 arcsine √(0.36)) – (2 arcsine √(0.11)) 

| = | 1.287 – 0.676 | = 0.611; a large effect size (Cohen, 1988).Using G*Power 3.1.5, the post hoc computed Power 

= 0.979 (two tailed) and actual α = 0.038.  

Summary and Implications   

Summary  

This research study has been an investigation into the proportion of IAV in artificial suburban neighborhood water 

ponds. No known research has analyzed the proportion and persistence of influenza viruses in these aquatic 

habitats.  

To investigate the proportion of IAV in recirculation artificial ponds, 182 pond water samples were collected from 

a representative sampling from 14 counties considered rural areas (NRural = 82), and 25 counties considered 

metropolitan and not rural (NUrban = 100) in California by California Business and Professions Code Section 

19986(l) was achieved. Field research data and laboratory data were transcribed to a Microsoft Office Excel 2007 

spreadsheet and statistically analyzed to answer the research question of this study. The analysis of the proportion 

of IAV in rural and urban water ponds favored the greater burden of IAV in urban community ponds over rural 

community ponds [(^p1 – ^p2= 0.2502, 95% CI (0.1278, 0.3604), Cohen’s h = 0.611, Power = 0.979, actual α = 

0.038].  

Implications  

The potential impact of this study can be recognized at the international level, the national and state level, the 

population level, and at the individual level. The results show that artificial water ponds in communities can be 

sources of IAV.It is commonly known that IAV can result in a zoonotic disease. Infectivity and sub typing of 

isolated IAV may identify strains previously known capable for animal-human transmission, or human-human 

transmission. 

As sources of IAV, the artificial water ponds in communities can be used to increase IAV surveillance and 

monitoring at the human-animal interface, expand and improve upon the IAV strain library for vaccine 

development, and may bring greater awareness to the individual, and thus, greater perceived susceptibility to IAV 

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infection leading to improved vaccination rates. International surveillance and monitoring of influenza viruses is 

a current global challenge.  

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