Article

First Molecular Detection and Epidemiological Analysis of

Equine Influenza Virus in Two Regions of Colombia, 2020–2023
Juliana Gonzalez-Obando 1,2, Angélica Zuluaga-Cabrera 3, Isabel Moreno 4, Jaime Úsuga 4, Karl Ciuderis 4,
Jorge E. Forero 5, Andrés Diaz 6, Carlos Rojas-Arbeláez 2, Juan P. Hernández-Ortiz 4 and Julian Ruiz-Saenz 1,*

1 Grupo de Investigación en Ciencias Animales—GRICA, Facultad de Medicina Veterinaria y Zootecnia,

Universidad Cooperativa de Colombia, Bucaramanga 680002, Colombia;
juliana.gonzalezo@udea.edu.co
2 Grupo de Epidemiología, Universidad de Antioquia, Medellín 050010, Colombia; carlos.rojas@udea.edu.co

3 Grupo de Investigación GISCA, Facultad de Medicina Veterinaria y Zootecnia, Fundación Universitaria

Vision de las Américas, Medellín 050031, Colombia; angelica.zuluagac@uam.edu.co

4 GHI One Health Colombia, Universidad Nacional de Colombia, Medellín 050036, Colombia;

imorenol@unal.edu.co (I.M.); jausugar@unal.edu.co (J.Ú.); coord_cwohc@unal.edu.co (K.C.);

jphernandezo@unal.edu.co (J.P.H.-O.)
5 Grupo de Investigación en Microbiología Ambiental, Escuela de Microbiología, Universidad de Antioquia,

Medellín 050010, Colombia; jorge.forero@udea.edu.co

6 Pig Improvement Company Hendersonville, Hendersonville, TN 37075, USA; andres.diaz@genusplc.com

* Correspondence: julian.ruizs@campusucc.edu.co

Abstract: Equine influenza is a viral disease caused by the equine influenza virus (EIV), and accord-
ing to the WOAH, it is mandatory to report these infections. In Latin America and Colombia, EIV
risk factors have not been analyzed. The objective of this research is to perform an epidemiological
and molecular analysis of the EIV in horses with respiratory symptoms from 2020 to 2023 in Colom-
bia. Molecular EIV detection was performed using RT‒qPCR and nanopore sequencing. A risk anal-
ysis was also performed via the GEE method. A total of 188 equines with EIV respiratory symptoms
Citation: Gonzalez-Obando, J.;
were recruited. The positivity rate was 33.5%. The descriptive analysis showed that only 12.8% of
Zuluaga-Cabrera, A.; Moreno, I.;
Úsuga, J.; Ciuderis, K.; Forero, J.E.;
the horses were vaccinated, and measures such as the quarantine and isolation of symptomatic an-
Diaz, A.; Rojas-Arbeláez, C.; imals accounted for 91.5% and 88.8%, respectively. The variables associated with the EIV were the
Hernández-Ortiz, J.P.; Ruiz-Saenz, J. non-isolation of positive individuals (OR = 8.16, 95% CI (1.52–43.67), p = 0.014) and sharing space
First Molecular Detection and with poultry (OR = 2.16, 95% CI (1.09–4.26), p = 0.027). In conclusion, this is the first EIV investigation
Epidemiological Analysis of Equine in symptomatic horses in Colombia, highlighting the presence of the virus in the country and the
Influenza Virus in Two Regions of need to improve preventive and control measures.
Colombia, 2020–2023. Viruses 2024,
16, 839. https://doi.org/10.3390/ Keywords: epidemiology; molecular diagnostic; Colombia; risk factors; horses; equine influenza
v16060839

Academic Editors: Stephane Pronost,

Gang Lu and Romain Paillot
1. Introduction
Received: 4 April 2024
Revised: 22 May 2024 Equine influenza, which is caused by the equine influenza virus (EIV), is a highly
Accepted: 22 May 2024 contagious disease distributed worldwide [1–3], and cases must be reported to the World
Published: 24 May 2024 Organization for Animal Health (WOAH) [2,3]. The EIV belongs to Orthomyxoviridae; the
genus name is Alphainfluenzavirus, and the species is Alphainfluenzavirus influenzae. [4]; it
has an enveloped capsid and contains eight negative sense genomic segments of single-
Copyright: © 2024 by the authors. Li- stranded RNA. The surface proteins hemagglutinin (HA) and neuraminidase (NA) deter-
censee MDPI, Basel, Switzerland. mine the EIV subtype [5–7]. Currently, only EIV H3N8 causes significant economic losses
This article is an open access article in the equine industry [2].
distributed under the terms and con- Although a preference for α-2,3 sialic acid receptors is shown by equine influenza
ditions of the Creative Commons At- viruses [8,9], different evolutionary analyses have proposed an avian origin for the EIV
tribution (CC BY) license (https://cre- [10–12]. Furthermore, the EIV has been documented to jump species barriers and infect
ativecommons.org/licenses/by/4.0/). various animal species, such as cats, pigs, and dogs [13,14]. This phenomenon is facilitated

Viruses 2024, 16, 839. https://doi.org/10.3390/v16060839 www.mdpi.com/journal/viruses

Viruses 2024, 16, 839 2 of 14

by shared glycan receptors possessing sialic acid residues in α-2,3 bonds, enabling cross-
species infection. Cases of both spontaneous infections and experimental infections in an-
imal models have been reported, as evidenced by the 2004 case of influenza transmission
from equines to canines, which spread extensively throughout the United States [15]. Sim-
ilar occurrences in felines have also been documented through natural infections and ex-
perimental models [16,17].
The clinical signs of equine influenza include fever, enlarged lymph nodes, nasal dis-
charge, and cough [2,18]. Vaccination may reduce clinical symptoms [19,20]. The trans-
mission of the EIV between horses is associated with low vaccination rates, inadequate
isolation practices, attendance at competitions, and delayed diagnosis [18,20–22]. How-
ever, the epidemiology and risk factors for the EIV in the country and other Latin Ameri-
can countries are poorly understood.
In Colombia, the first equine influenza case was reported in 1982, followed by out-
breaks in the 1990s and 2000s [18,23]. Additionally, since 1999, EIV vaccination has been
mandatory for mobilizing horses for equestrian exhibitions or competitions [24]. Alt-
hough EIV outbreaks were reported in South America in 2018 and 2022 [18,23], the current
circulation of the EIV in Colombia is unknown. Therefore, the objective of this study is to
assess the presence of the EIV in Colombia using molecular diagnostics and to explore the
risk factors and symptoms associated with the disease in the country.

2. Materials and Methods

A descriptive cross-sectional study was carried out in equines from two regions in
Colombia (the Departments of Antioquia and Cundinamarca) from November 2020 to
April 2023. Antioquia and Cundinamarca are the regions (Supplementary Figure S1) with
the largest number of horses registered in the country and where most equestrian activi-
ties and horse-related events occur [25,26].
A convenience sampling approach was used in this study. Domestic equines that met
the inclusion criteria were selected and sampled. The inclusion criteria were Equidae spe-
cies (horses, mules, and/or donkeys) of all ages and of any sex who presented with symp-
toms of influenza-like illness (bilateral nasal discharge, cough, and positive cough reflex)
and who also lived at the selected sites (Supplementary Table S1).

2.1. Epidemiological Variables: Survey

A written questionnaire of 37 elements (survey form, Supplemental Table S1) was
used to record epidemiological variables and other characteristics, including individual
information on the animal, management and animal health practices, and location, includ-
ing animal characteristics related to age, sex, type of activity, and breed. The clinical char-
acteristics and treatment included fever (higher than 38.5 °C); decreased performance; in-
creased size of the retropharyngeal, cervical, and parotid lymph nodes; reduced food con-
sumption; loss of weight; respiratory distress; runny nose; cough; eye discharge; and treat-
ment. The herd characteristics included the number of stables, number of occupied sta-
bles, number of equines (horses, mules, and donkeys), and predominant breed in the herd.
The health management variables and possible risk factors include coexistence with other
animal species, such as canines, felines, pigs, and others, due to the possible risk of infec-
tion between species by EIV subtype H3N8, vaccination, type of vaccine, time of the first
vaccination, vaccination of pregnant mares, type of deworming, frequency of deworming,
product with which the disinfection of the herds is carried out, entry of new horses in the
last year, location from which they enter, clinical records, types of medical records (writ-
ten or virtual), participation of the herds in massive events, and number of horses sam-
pled. This survey was administered to the caregivers and veterinary doctors in charge.
Viruses 2024, 16, 839 3 of 14

2.2. Sample Collection

Nasopharyngeal swabs (NS) were taken from 188 equines with respiratory symp-
toms, mainly cough and nasal discharge, in 40 different equine herds (n = 32/40 in An-
tioquia and n = 8/40 in Cundinamarca) using a Medline® Swab with a 16” Rayon Tip (Med-
line Industries, LP. Northfield, IL, USA). After collection, the swab tips were stored in viral
transport media and frozen at −80 °C until RNA extraction.

2.3. Molecular Diagnosis and H3N8 Confirmation

RNA extraction was performed with a QIAmp RNA Viral Kit and an RNA Easy
Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s in-
structions. The quality and quantity of RNA were determined using a Nanodrop® ONE.
Then, the obtained RNA was stored at −80 °C until RT‒qPCR analysis.
Reverse transcription was performed using the RevertAid First Strand cDNA Syn-
thesis Kit (Thermo Scientific® Waltham, MA, USA) following the manufacturer’s instruc-
tions, and cDNA was stored at −20 °C. Then, qPCR was performed as previously described
by Heine et al. (2007) [27], but a TaqMan probe, directed against the matrix proteins of the
influenza virus type A, was used [28]. The TaqMan™ Gene Expression Master Mix Kit
(Applied Biosystems™ Life Technologies, Darmstadt, Germany) was used in the
QuantStudio™ Real-Time PCR System (Applied Biosystems ®) following the manufac-
turer’s instructions. CT values < 38 were considered positive. H3N8-positive cDNA, which
was generated by Stephanie Reedy from the University of Kentucky, was used as a posi-
tive control, and ddH2O was used as a negative control.
For the rt-qPCR-positive samples, multisegment RT‒PCR was performed by using a
SuperScript™ III One-Step RT‒PCR System with Platinum™ Taq DNA Polymerase and
using influenza-specific universal primers at the end of all 8 genomic segments, as previ-
ously reported [29]. Only three samples showed a clear electrophoresis band pattern. Pos-
itive samples were analyzed using sequencing performed on the Minion platform (Oxford
Nanopore sequencing) [30,31]. The amplicons were purified using Beckman Coulter™
Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) with an individual barcode for
each sample by using the SQK-LSk109 and EXP-NBD196 kits (Oxford Nanopore Technol-
ogies, Oxford, UK). The library was loaded on an R9.4 Oxford MinION flowcell and se-
quenced using a MinION Mk1B device. Base-calling and demultiplexing were performed
using Guppy (v6.0.1).
The reference genome used for homology-based assembly was A/eq-
uine/Ohio/OH21-6023/2021 (H3N8). HA sequences were registered in GenBank under ac-
cession numbers PP737049–PP737051. For the phylogenetic analysis, 93 representative
HA sequences were obtained from each EIV sublineage and the sequences in our study.
A maximum-likelihood phylogeny was constructed by using the stringent GTR + G algo-
rithm, which was identified by using the best model tool available in MEGA 7, by boot-
strapping with 1000 replications.

2.4. Data Analysis

The analysis was carried out with the statistical program SPSS ver. 21 (IBM Corpora-
tion), which is licensed. A descriptive analysis of the collected data was carried out, in-
cluding the mean, median, standard deviation, and range. For qualitative variables, abso-
lute and relative frequencies were used. We used tabular methods to estimate the crude
association between exposure variables and the detection of the EIV using RT‒qPCR (pos-
itive or negative) in horses with respiratory symptoms and considered the association to
be significant when the p value for the chi-square test or Fisher’s exact test was lower than
0.05 with a 95% confidence level. Subsequently, only epidemiological variables that had a
value of 0.25 or less were ultimately chosen for inclusion in the GEE (Generalized Esti-
mated Equation) model, with the equine herd as a cluster (random effects) to estimate the
adjusted association between the EIV and RT‒qPCR detection of other exposures. For the
Viruses 2024, 16, 839 4 of 14

analysis of the model, all variables were dichotomized into zeros and ones for model fit-
ting. Additionally, all models demonstrated an improved model fit and parsimony using
QIC [32–35]. In addition, a bivariate analysis was performed in order to detect variables
that had an association with the confirmed molecular diagnosis, using the chi-square test
or Fisher test, and a p value below 0.05 with a 95% confidence level was considered an
association.

2.5. Ethical Statement

We followed all the international and Colombian ethical standards for biomedical
research with animal subjects established by bioethical and animal research. The study
was reviewed and approved by the Ethics Committee for Animal Experimentation of the
Universidad Cooperativa de Colombia in Bucaramanga (Minute number 044-2018). Writ-
ten informed consent was obtained from the animal owners.

3. Results
A total of 188 equines were tested from 40 equine herds between 2020 and 2023, and
these herds were analyzed only once. Eight herds were from Cundinamarca, and thirty-
two were from Antioquia, with 36% and 64% of the animals tested, respectively. Sixty-
three (33.5%) of the strains tested were positive according to RT‒qPCR, and all were de-
tected in 2022 (Table 1).

Table 1. Summary of the equine samples and herds included in the study and percentages according
to department and year.

Number of Number of
Positive Positive
Year Antioquia Herds of Cundinamarca Herds of
Herds Herds
Antioquia Cundinamarca
2020 0/21 (0%) 8 0 0 0 0
2021 0/38 (0%) 9 0 0 0 0
2022 43/59 (72%) 14 14 20/67 (29%) 8 7
2023 0/3 (0%) 1 0 0 0 0
121 32 14 67 8 7

3.1. Molecular Diagnosis and Phylogenetic Analysis

The overall percentage of positive samples obtained using RT‒qPCR was 33.5%
(63/188). The positivity rate in Antioquia was 35.5% (43/121), while that in Cundinamarca
was 29.8% (20/67). From the three sequenced samples, all of which belonged to the An-
tioquia Department, we confirmed the presence of the H3N8 subtype in Florida clade 1.
Our sequences formed a monophyletic clade with sequences from the United States with
a bootstrap value of 98. These results are also related to the sequences reported from the
southern cone outbreak in 2018 (Figure 1).
Viruses 2024, 16, 839 5 of 14

Figure 1. EIV Phylogenetic analysis. (A): Maximum likelihood for hemagglutinin (HA) gene nucle-
otide sequences from 93 equine influenza viruses (EIVs) encoded by the H3N8 subtype of the EIV
and Colombian sequences. Bootstrap values obtained after 1000 replicates showing the main clades.
Full tree. (B): Subtree showing Florida clade 1 clade highlighting Colombian (this study), Chilean,
Argentinean, and United states sequences, with bootstrap values obtained after 1000 replicates.

3.2. Zoographic Characteristics

Ninety-nine percent of the samples were from horses (186/188), and 1% were from
mules (2/188). A total of 82.4% (155/188) were Colombian Creole horses, and 71.8%
(135/188) were female (Supplementary Table S1). The median age was three years, and the
IQR was twenty-three. When the rt-qPCR results were distributed by age, it was possible
to find that the age group with the greatest number of positive animals was <2 years old
(8/27). According to sex, females were the group with the most positive cases (36.3%,
49/135), and activity competition was the group with the greatest number of positive ani-
mals (53.3%—16/30) However, for these differences by groups (age/sex/activity), no sta-
tistical difference was found (Table 2).
Viruses 2024, 16, 839 6 of 14

Table 2. Summary of positivity according to age, sex, and vaccination state categories.

Age (Years) n Positive % p-Value

<2 67 29 43.28
2–5 56 15 26.79
0.68
>5–10 38 11 28.95
>10 27 8 29.6
Total 188 63
Sex
Male 53 14 26.4
Female 135 49 36.3 0.20
Type of activity
Competition 30 16 53.3
0.12
No competition 158 47 29.7

3.3. Bivariate Analysis of Clinical Variables and Treatment

Clinical severity variables in animals positive for the EIV were bilateral nasal dis-
charge 95.2% (60/63), dry cough 93.7% (n = 59/63), enlarged retropharyngeal nodes 52.4%
(33/63), and fever 41.3% (26/63), These last three had a statistically significant difference.
Infrequent symptoms in positive animals included respiratory distress 7.9% (5/63), eye
discharge 3.2 (2/63), and productive cough 7.9% 5/63 (Table 3). A statistical association was
found in the bivariate analysis of the following variables: dry cough, fever, and enlarged
retropharyngeal lymph nodes. In the veterinary treatment, the use of mucolytics were the
most commonly used therapeutic strategy in positive animals 41.3% (26/63); however,
42.9% of the equines did not receive any treatment (27/63).

Table 3. Summary of the clinical variables and statistical associations according to bivariate analysis.

Variables n Positive % Negative % p-Value

Bilateral Nasal Discharge
Yes 181 60 95.2 121 96. 8
0.59
No 7 3 4.8 4 3.2
Dry Cough
Yes 155 59 93.7 96 76.8
0.04 *
No 33 4 6.3 29 23.2
Productive cough
Yes 13 5 7.9 8 6.4
0.69
No 175 58 92.1 117 93.6
Fever
Yes 48 26 41.3 22 17.6
0.01 *
No 140 37 58.7 103 82.4
Weight loss
Yes 33 14 22.2 19 15.2
0.23
No 155 49 77.8 106 84.8
Respiratory distress
Yes 16 5 7.9 11 8.8
0.84
No 172 58 92.1 114 91.2
Decrease in food consumption
Yes 56 24 38.1 32 25.6
0.09
No 132 39 61.9 93 74.4
Viruses 2024, 16, 839 7 of 14

Increase in the size of retropha-

ryngeal nodes
Yes 57 33 52.4 24 19.2
0.00 *
No 131 30 47.6 101 80.8
Eye discharge
Yes 10 2 3.2 8 6.4
0.35
No 178 61 96.8 117 93.6
Decreased performance
Yes 51 22 34.9 29 23.2
0.09
No 137 41 65.1 96 76.8
* Statistically significant p value.

3.4. Characteristics of the Horse Herds

The average number of horses per herd was 56.18 (rank 2–240). The average number
of stalls per herd was 53.77 (2–200), with 52.57 stalls being occupied (2–200). Stables
housed all of the horses (188/188).

3.5. Shared Space with Other Species

Considering the well-known interspecies transmission of the EIV to various animal
species, we examined the presence of different animals on the premises (Figure 2). Our
findings revealed that canines constituted the predominant animal species sharing space
with the horses under study, accounting for 96.8% (182 out of 188) of the horses. A similar
trend was observed among the EIV-positive animals. Additionally, cats (60.3%) and poul-
try (41.3%) were identified as other significant species in close proximity to the EIV-posi-
tive equines. However, none of these animals (dogs, cats, poultry) exhibited clinical signs
of influenza during the study period.
The category “sharing space with poultry” exhibited a statistically significant associ-
ation (odds ratio = 2.16, 95% confidence interval 1.09–4.26, p = 0.027), possibly related to
poor biosecurity practices in herds.

Figure 2. Coexistence of horses with other animal species susceptible to influenza A viruses. The
gray bars indicate the total number of horses that cohabit with dogs, pigs, cats, and poultry. The
blue bar indicates EIV-positive horses living with different animals.
Viruses 2024, 16, 839 8 of 14

3.6. Vaccination and Sanitary Measures

Thirteen percent (24/188) of the horses sampled in the study were vaccinated against
the EIV in the previous year, and 15.9% (n = 10) of the EIV-infected animals had been
vaccinated. When we analyzed the type of activity and vaccination status of the studied
animals, we found that those animals that required movement, such as shows, exhibitions,
competitions, and saddle types (136/188), did not have an updated vaccination schedule
for equine influenza at the time of sampling.

3.7. Other Sanitary Measures Evaluated

According to our survey, important preventive interventions were not applied in
most of the herds. No separation of healthy and sick equines was performed in most of
the patients (88.8%, 167/188), 33.1% of whom were positive (62/167). Furthermore, 89.4%
(168/188) of the horses were housed on properties where quarantine was not required for
new horse arrival.

3.8. Herd Variables and Disinfection and Health Management

Overall, sodium hypochlorite (bleach) was the most commonly used disinfectant
(27.1%, 51/188). Glutaraldehyde and hydrated lime were the disinfectants with the lowest
percentages, at 9.0% (17/188) and 3.2% (6/188), respectively (Table 4).

Table 4. Herd variables and health management.

Disinfection %
Sodium hypochlorite 27.1% (51/188)
Creolin 22.3% (42/188)
Do not disinfect 13.8% (26/188)
Detergent 12.8% (24/188)
Ammonium 10% 11.7% (22/188)
Glutaraldehyde 9.0% (17/188)
Lime 3.2% (6/188)
Do not disinfect 13.8% (26/188)
Detergent 12.8% (24/188)
Veterinary management %
On request if a problem occur 54.8% (103/188)
Permanent 45.2% (85/188)

3.9. Veterinary Care and Health Management

We found that 54.8% (n = 103/188) of the animals sampled were housed on properties
where they only received veterinary assistance if a problem arose. The remaining herds
received veterinary care full time. In terms of positivity, 65.1% (n = 41/63) of the positive
animals lived in herds without a full-time veterinarian.

3.10. Sanitary Management Analysis at the Herd Level

Quarantine: in total, 87.5% (35/40) of the horse herds did not require quarantine for
new arrivals or had a separate area for new horses that came from another property or
after an event. Sick and healthy animals were separated in 85.5% (34/40) of the herds eval-
uated. Among the properties in which there were positive individuals, 90.5 (19/21) did not
quarantine the new individuals who entered (Figure 3).
Viruses 2024, 16, 839 9 of 14

Figure 3. Sanitary measures and health management at herd level. Percentages of the total herds.

Variables related to vaccination: fifty percent (20/40) of the herds vaccinated their
horses against equine influenza. However, 77.5% (31/40) of the herds did not vaccinate
pregnant females against the EIV, and 37.5% (15/40) started the vaccination program after
6 months of age. Twenty-five percent of the herds (10/40) only vaccinated their animals
before a competition. When analyzing the positive farms, 85.7% (18/21) of these farms did
not vaccinate against the EIV.
Participation in equestrian events: 65% of the herds (26/40) participated in equestrian
events (jumping competitions, exhibitions, horseback riding) and international events,
e.g., in the United States and Costa Rica. A total of 76% (16/21) of the participants with
positive properties reported that equines participated in equestrian events.
In total, 67.5% of the property-generated horses were horses from other geographical
areas (27/40), 72.5% (19/27) were from nearby towns, and 27.5% (8/27) were from other
countries, including horses from the United States, Costa Rica, and Venezuela. At the herd
level, 71.4% (15/21) of the positive herds reported the presence of new horses entering the
property.

3.11. Multivariate Analysis

Two variables were associated with EIV status (positive or negative) in horses with
respiratory disease in this study (Table 3). The odds of testing positive for the EIV were
greater in horses housed in herds that did not separate sick animals from healthy individ-
uals and those housed in herds that allowed contact with poultry. The odds of testing
positive for the EIV were eight times greater in horses housed in herds where sick animals
were not separated from healthy individuals after adjusting for the effects of fever, en-
larged lymph nodes, and shared space with poultry (Table 5 and Supplementary Table
S2).

Table 5. Bivariate and multivariate analysis of equine influenza virus symptoms and risk factors.

CRUDE
Variables 95% CI p-Value OR adjusted using GEE 95% CI p-Value
OR
Do not isolate sick
from healthy 13.66 1.52 122.2 0.19 8.16 1.52 43.67 0.014 *
animals
Share space with
2.43 1.26 4.68 0.007 2.16 1.09 4.26 0.027 *
poultry
Live on property
2.57 1.25 5.28 0.01 1.32 0.612 2.86 0.47
with equines that
Viruses 2024, 16, 839 10 of 14

participate in eques-
trian events
Competition
2.7 1.22 5.99 0.12 1.92 0.75 4.9 0.17
equine
Do not quarantine 1.90 1.01 3.54 0.044 1.88 0.41 8.52 0.16
* Statistically significant p value.

4. Discussion
In the Latin American context, this is the first study looking for variables associated
with EIV infection in sick horses. In addition, in Colombia, this is the first study to confirm
the H3N8 subtype in horses. The percentage of positive samples was 33.5% (63/188) in
symptomatic animals, which might be high for a country in which vaccination has been
mandatory for more than 25 years. Our study is the first to confirm the circulation of the
H3N8 subtype in the Colombian equine population through next-generation sequencing.
One of the variables associated with EIV detection was a lack of ability to separate
sick and healthy horses, with OR = 8.16, in horses belonging to herds where horses with
respiratory symptoms were not separated from healthy horses. This finding is consistent
with the results of Amjad Khan et al. [36], who reported that a greater number of horses,
which made it impossible to separate sick from healthy horses, were infected with equine
influenza virus [36], suggesting the need to separate symptomatic equines from suscepti-
ble and asymptomatic equines. In addition, isolation should be accompanied by twice-
daily body temperature monitoring [37]. Clinical variables with greater severity, such as
fever, dry cough, and increased lymph node size, were strongly associated with the pres-
ence of the virus. This is strongly expected because fever is an immune system response
to the presence of the virus [2], increased lymph node size has been strongly associated
with the cellular immune response in EIV-infected horses [20], and dry cough has been
reported as one of the most frequent symptoms that persists over time [5]. These three
clinical variables could have great predictive value for suspected cases of the EIV.
The high positivity rate in our sample could be explained by the fact that the studied
population consisted of symptomatic individuals [20] and that the sampling period coin-
cided with the 2021–2022 outbreak, which had a global distribution, largely in America,
Europe, and Asia [7]. Furthermore, in 2022, there was an increase in rainfall in Colombia,
mainly between April and May [38], the months in which the outbreak was reported,
which may explain the high degree of virus transmission because sick and healthy horses
have closer contact, mostly with young susceptible individuals [39,40]. Furthermore, hu-
mid and rainy environments are widely recognized to promote outbreaks of influenza A
in regions with low latitudes, such as tropical and subtropical zones such as Colombia.
This is attributed to the effective transmission of the virus through large droplets and/or
aerosols, coupled with the potential for virus survival aided by salts and proteins present
in respiratory droplets during humid and rainy conditions. This assertion is supported by
extensive research into the survival capabilities of influenza viruses in aerosols, revealing
that the maximum survival duration in droplets varies between 1 and 24 h, depending on
the relative humidity and the specific influenza strain [41].
Approximately 66.5% of horses exhibiting respiratory symptoms and testing nega-
tive for the EIV might have been affected by alternate viral agents such as adenovirus or
herpesvirus, both of which are considered potential alternative diagnoses for the EIV
[40,42–44]. Otherwise, it is possible that the infectious and shedding period of the EIV had
elapsed.
Equestrian shows and exhibitions were reopened globally and locally after the SARS-
CoV-2 pandemic. Fifteen out of sixty-three (23.8%) horses positive for the EIV were in
herds that participated in equestrian shows in the United States. The positive samples
found in Colombia could be related to a multifactorial outbreak of respiratory disease oc-
curring in the Americas [45]; however, further sequencing and phylo-evolutionary analy-
sis are needed to better understand the origins of the EIV in Colombia. Previous outbreaks
Viruses 2024, 16, 839 11 of 14

were also reported in the US between 2020 and 2021 [46]. It can be speculated that the EIV
was introduced to Colombia by individuals who took part in those international shows,
but further sequencing data are needed to support or rule out this hypothesis. The associ-
ation between EIV infection and horse movement between countries has been previously
documented [21–23].
Vaccination reduces the likelihood of infection for horses that require mobilization in
the country. However, in this study, low vaccination coverage (12.8%) was found. Low
vaccination rates or a lack of immunity can be associated with the rapid spread of the EIV,
similar to the situation in Chile in 2018, which was related to low herd immunity [47], and
Canada [48] and the United States [39]. Low vaccination rates not only favor virus trans-
mission but also may allow for the introduction of new strains [49].
Nevertheless, we found that 15.9% of vaccinated individuals were positive for the
EIV. The vaccine used in those animals (Equilis® Prequenza Te—MSD Animal Health) was
inactivated, and it included viruses from both clades 1 and 2 of the Florida sublineage.
Positive cases in vaccinated equines could be associated with virus evolution (antigenic
drift) or noncompliance with the entire vaccination protocol, as has been reported in Latin
America, Italy, and Croatia [50,51]. As reported by Oladunni et al. in 2021 [20], the con-
stant evolution of viruses, vaccine breakdown, and vaccination-induced short-lived im-
munity have become constant challenges for achieving full protection against EIV-in-
duced disease, highlighting the need for the constant study of EIV evolution and the host
immune response [1,52].
We also found a statistically significant association between being positive for the EIV
and living with poultry (OR = 2.16). This could be because EIV-positive horses live in
herds with low levels of biosecurity, favoring the coexistence of various animal species in
herds [2,53]. It has been suggested that practices involving the cohabitation of mixed spe-
cies sharing specific receptors, coupled with the absence of segregation between infected
and healthy individuals, could facilitate the local dissemination of the equine influenza
virus and its transmission among susceptible populations [54,55].

5. Conclusions
This study represents the first investigation of equine influenza in Colombia, a coun-
try renowned for its equine industry, confirming the presence of the EIV in symptomatic
individuals across two regions of the nation. This is the first instance of confirmation
through next-generation sequencing of the EIV H3N8 subtype. Furthermore, our findings
highlight the absence of adequate sanitary measures that facilitate the presence and dis-
semination of the virus. These measures include the failure to segregate infected and
healthy individuals, low rates of vaccination, and the coexistence of various susceptible
species within herds, such as poultry and dogs.

Supplementary Materials: The following supporting information can be downloaded at

https://www.mdpi.com/article/10.3390/v16060839/s1; Figure S1: Geographical location of the study
area in Colombia; Table S1: Descriptive analysis; Table S2: Bivariate analysis.
Author Contributions: Conceptualization: J.E.F. and J.R.-S.; methodology, J.G.-O., A.Z.-C., C.R.-A.,
A.D., I.M., J.Ú., K.C., J.P.H.-O. and J.R.-S.; software: J.G.-O.; validation, J.G.-O., J.P.H.-O. and C.R.-
A.; formal analysis, J.G.-O.; investigation, J.G.-O. and J.R.-S.; resources, J.G.-O. and J.R.-S.; data cu-
ration, J.G.-O.; writing—original J.G.-O.; writing—review and editing, J.G.-O. and J.R.-S.; visualiza-
tion, J.G.-O. and J.R.-S.; supervision, J.E.F., C.R.-A., A.D., J.P.H.-O. and J.R.-S.; project administra-
tion, J.R.-S.; funding acquisition, J.R.-S. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by CONADI-UCC, grant number INV2623 to J.R.-S. The APC
was funded by CONADI-UCC.
Institutional Review Board Statement: The study was approved by the Institutional Ethics Com-
mittee of the Universidad Cooperativa de Colombia, protocol code 044 from 3 September 2018.
Viruses 2024, 16, 839 12 of 14

Written informed consent was obtained from the owners for the participation of their animals in
this study.
Data Availability Statement: All the data are presented in the paper.
Acknowledgments: We thank the Animal Virology Group of the National University of Colombia,
especially Maria Fernanda Naranjo, Stephany Reeds, Marie Garvey, and Ann Cullinane for their
support.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu-
script; or in the decision to publish the results.

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