Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 30/06/2025 Aceptado: 11/09/2025 Publicado: 17/10/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Revista Cienfica, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35730732 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Detecon of PRRS virus in housefly (Musca domesca) in a pig farm in southeastern Mexico Detección del virus de PRRS en la mosca domésca (Musca domesca) en una granja porcina en el sureste de México Gladys Noh-Cuxim 1 , Alejandro Alzina-López 1 , Guadalupe Ayora-Talavera 2 , Jesús Ek-Mex 3 , José Segura-Correa 1 ¹ Universidad Autónoma de Yucatán, Facultad de Medicina Veterinaria y Zootecnia, Campus de Ciencias Biológicas y Agropecuarias. Carretera Mérida-Xmatkuil, Km 15.5, Apartado postal 4-116, CP. 97100, Mérida Yucatán, México. ² Universidad Autónoma de Yucatán, Centro de Invesgaciones Regionales “Dr. Hideyo Noguchi”. C.43 s/n entre 96 Paseo de las Fuentes y 40 Col. Inalámbrica. Apartado postal 97225, Mérida Yucatán, México. ³ Universidad Autónoma de Baja California, Instuto de Invesgaciones en Ciencias Veterinarias.Carretera a San Felipe Km.3.5 Fraccionamiento Laguna Campestre, CP. 21386, Mexicali, Baja California, México. *Corresponding autor: jose.segura@correo.uady.mx ABSTRACT The goal of this study was to detect the presence of the porcine respiratory and reproducve syndrome virus in housefly (Musca domesca) in a pig farm. To know the porcine respiratory and reproducve syndrome virus circulaon in the farm, 1277 blood samples, and 26 samples of oral fluids from pigs housed in pens were taken to detect the nucleic acid of the porcine respiratory and reproducve syndrome virus using real me RT-PCR test. Three-hundred flies were collected, using an entomological net, and 50 samples of 6 flies each were formed. Data was analyzed using descripve stascs. From the sera tested, 95.74% (1224/1277) were posive in the ELISA test. The nucleic acid of the porcine respiratory and reproducve syndrome virus was detected in 34.6% (9/26) of the oral fluids and in 4% (2/50) of the fly samples analyzed. The real me- PCR test allowed detecng of the porcine respiratory and reproducve syndrome virus in houseflies. Therefore, further virus sequencing studies are needed to beer understand the role of flies in the porcine respiratory and reproducve syndrome transmission. Key words: Houseflies; PRRSV; real-me-PCR; swine RESUMEN El objevo de este estudio fue detectar la presencia del virus del síndrome respiratorio y reproducvo porcino en moscas doméscas (Musca domesca) en una granja comercial de cerdos. Para conocer la circulación del virus del síndrome respiratorio y reproducvo porcino en la granja, se tomaron muestras de sangre de 1277 y 26 muestras de fluidos orales de cerdos alojados en corrales para detectar el ácido nucleico del virus del síndrome respiratorio y reproducvo porcino ulizando la prueba de RT-PCR en empo real. Se recolectaron 300 moscas, ulizando una red entomológica, y se formaron 50 grupos de 6 moscas cada una. Los datos se analizaron mediante estadíscas descripvas. De los sueros analizados el 95,74% (1224/1277) fueron posivos en la prueba ELISA. El ácido nucleico del virus del síndrome respiratorio y reproducvo porcino se detectó en el 34,6% (9/26) de los fluidos orales y en el 4% (2/50) de las muestras de moscas analizadas. La prueba de PCR en empo real permió detectar el virus del síndrome respiratorio y reproducvo porcino en moscas doméscas. En conclusión, más estudios de secuenciación del virus son necesarios para comprender mejor la función de las moscas en la transmisión del síndrome respiratorio y reproducvo porcino. Palabras clave: Mosca domesca; PRRSV; PCR empo real, cerdos

Detecon of PRRS virus in housefly / Noh-Cuxim et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Porcine reproducve and respiratory syndrome (PRRS) virus is a pathogen of wide world distribuon, with negave effects for the swine industry [1]. The cost caused on pigs by the PRRS virus in the USA is around $664 million per year [2 , 3]. The PRRS virus, a single-stranded, posive-sense, enveloped RNA belongs to the family Arteriviridae, genus Arterivirus and order Nidovirales [4]. PRRS virus classified into two major genotypes, European or type 1 (PRRSV-1), Lelystad strain, and the North American or type 2 (PRRSV-2), isolated ATCC-VR-2332. Both genotypes share 63.4% identy at genomic level [5 , 6 , 7]. There are different control strategies for PRRS virus, such as vaccinaon, use of inoculates, natural infecon and the use of gilts grown in the own farm [8 , 9 , 10]. Transmission commonly occurs by direct contact between pigs because of high populaon density or by exposion to contaminated body fluids contaminated blood with PRRS virus, semen, secreons and saliva [4], contaminated needles, fomites (coveralls and boots) and vectors such as Diptera [11]. Houseflies (Musca domesca) are insects that belong to the Diptera order, which are considered potenal agents in the transmission of PRRS disease [12]. Flies are common insects in pig farms, where they reproduce and feed animal wastes, transming bacteria, viruses or parasites when they defecate or regurgitate the food, infecng other animals and humans with acquired pathogens [13]. Previous studies have demonstrated that houseflies could serve as vectors for PRRS virus; however, experimental studies indicate that this virus seems not to replicate in flies, which means that they could not act as a biological vector [11], 14 , 15 , 16]. However, experimental studies have limitaons, such as laboratory condions and the size of fly populaons. The goal of the present study was to detect, through RT-PCR test, the presence of PRRS virus in houseflies in a commercial pig farm. MATERIALS AND METHODS Study design, site and animals A transversal descripve study was carried out in a populaon of houseflies, in a replacement gilts barn, where gilts were distributed in 72 pens with approximately 11 animals per pen. The study was conducted in a pig (Sus scrofa domescus) farm located in Yucatán, Mexico. The climate of the region is tropical subhumid with average temperature, rainfall, and relave humidity of 26 °C, 1200 mm, and 78 %, respecvely [17]. The farm was a connuous flow and complete cycle farm with approximately 5500 sows, PRRS posive stable, according to the classificaon of Holtkamp et al. [18]. To control PRRS effects on animals, the farm produced its own gilt replacement, which are taken to an acclimazaon area. The farm did not applied vaccines against PRRS. Serum samples were taken from gilts before they were introduced to the breeding herd, at approximately 30 weeks of age. Only gilts posive to the ELISA test were kept. Biological samples from pigs and houseflies To detect PRRS virus anbodies in pigs in a commercial farm, 1227 gilt serum samples were taken. The samples were analyzed with a commercial kit (IDEXX HerdCheck PRRS X3, USA), and they were considered seroposive when the S/P rao was ≥ 0.4, according to the manufacturer instrucons. The ELISA test was carried out at the clinical laboratory of the Faculty of Veterinary Medicine and Animal Science of the Universidad Autónoma de Yucatán. Oral fluid samples from 26 pens, out of a total of 72 pens, were collected by allowing pigs to interact with 20 cm coon ropes for 20 min, using the protocol described by Pricke et al. [19], 20]. Thereaſter, the oral fluids were deposited in collecon tubes, stored in coolers, and then sent to the laboratory for analysis. Housefly samplings were carried out using an entomological net [21 , 22] throughout the length of the barn. Three-hundred houseflies were collected, placed in a mesh cage, and stored at −80 °C in an ultra-low temperature freezer (VWR model 5703, USA). Then pools of six flies were deposited in sterile Eppendorf tubes for a total of 50 samples [11 , 23]. Pools were pressed in the wall of the tube, added 500 μL of phosphate buffered saline (PBS) and centrifuge at 450 G per 5 min, to obtain the supernatant [11]. To detect PRRS virus nucleic acid, all oral fluid samples and housefly homogenates were tested by real-me RT-PCR (VetMAX™ PRRSVNA & EU Controls of Therma Fisher Scienfic, USA) following the manufacturer instrucons and using the sequence of the ORF7 gene. A PCR reacon was considered posive if the cycle threshold (Ct) level was obtained at <37 cycles. The quality control of the real-me RT-PCR process included negave (nuclease-free water) and posive (PRRSV RNA) controls, provided by the kit (VetMAX TM PRRSV NA & EU Controls, Thermo Fisher Scienfic, USA) Stascal analysis Data were analyzed using descripve stascs through the Excel soſtware, and the kappa test to compare the diagnosc assays (oral fluid vs serology). RESULTS AND DISCUSSION A total of 1,277 serum samples from gilts distributed across six groups were analyzed. The ELISA test indicated that 1239 serum (97,02%) were seroposive. In the six groups, percentages of seroposive cases were 98,90% (179/181), 99,45% (181/182), 98,74% (236/239), 99,30% (283/285), 100% (194/194) and 84,69% (166/196), respecvely. This is the first study that detects the PRRS virus in housefly under field condions in Mexico. In addion, farm PRRS virus seroposive was confirmed by the presence of specific anbodies in replacement gilts. The high prevalence (>95%) of anbodies indicates the ters circulaon of the virus and the development of anbodies in an endemically infected farms [24], 25]. The highest percentage of posive animals could be because the gilts were sampled 19 weeks aſter staying in the same barn with pigs (lots) of different ages, ranging from 2 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 6 to 20 weeks of age. However, that does not mean that pigs were shedding the PRRS virus. The short duraon of passive immunity in piglets could make them suscepble to infecon or natural reinfecon. Geldhof et al. [26] found anbodies against PRRS virus in piglets, transmied through colostrum and sll detectable in serum up to 5 weeks of age. The gilts, in this study, were sampled at 200 days (d) of age, which agree with Flores- Mendoza and Hernández [27] results, who menon that the anbodies produced by the infecon with PRRS virus can remain for long periods but with low ters. The high percentage of seroposive gilts here found shows the presence of infecon or natural reinfecon. IgG anbodies could be found between 7- and 15-d post infecon, remaining constant for months and declining between 135- and 300-d post infecon [9 , 28 ,[29]. PRRS virus nucleic acid was detected by RT-PCR in 9 of the 26 oral fluid samples (34,6%). In fly homogenates, 2 of 50 samples were detected posive (4%). Diagnosc results from fly homogenates and oral fluid are shown in FIG. 1. The posive control amplified in cycle 29. FIGURE 1. Amplificaon of the ORF 7 gene by real-me PCR of oral fluids (a) from pigs and housefly homogenates (b). The cut point is represented by a doed red line. The posive and negave controls of the amplified ones are represented in each figure (yellow and red lines respecvely). Number 1 corresponds to a sample of oral fluid from pigs, and number 2 corresponds to a homogenate sample of houseflies. The “X” axis corresponds to the amplificaon cycles, and the “Y” axis is the fluorescence producon The 34,6% (9/26) posive samples detected in oral fluid by real-me PCR test, agree with De Regge and Cay [30] who found that oral fluid samples would be PCR posive when more than 30% of individual pigs are serum posive. On other hand, Olsen et al. [31] reported 90% posive animals to PRRS virus using the PCR test in oral fluid, when 36% of the pigs were vaccinated. Pricke et al. [19] tested samples of oral fluid and serum for the detecon of PRRS virus with pigs of 3, 5, 8, 12 and 16 weeks of age, that were infected with the PRRS virus and obtained a 77% concordance between oral fluid and serum samples. An explanaon of the low detecon of the virus in oral fluid, is that probably few animals were shedding the virus in the saliva, as the transmission of PRRS virus in the animals here studied occurs naturally, causing the moments of contact to be different. This generates a diluon with negave saliva, whereby oral fluid samples could be below the detecon limit of the test. Seddon et al. [32] reported that 80% of the pigs in a corral chew the rope within 30 min of its placement. The real-me PCR test in oral fluid has a high sensivity when 30% of the pigs are individually shedding the virus [30]. Several authors menon that when the viral load is very low, the samples deteriorate during the conservaon period, which could result in a false negave case, which was not very likely in this study, since the cold chain was taken care of [33], 34]. Houseflies are insects that belong to the order Diptera, considered potenal agents in the transmission of diseases [12]. They are common in pig farms, where they develop and feed on animal waste and this is where these insects can acquire bacteria, viruses or parasites, and disseminate by flies hairs, legs, body and proboscis (by ingesng liquids from animal ssues, including blood, serum, saliva, mucus and tear secreons). These insects, by defecang and regurgitang [16 , 35] can infect animals and humans with the acquired agents [13]. Previous studies have shown that flies act as vectors for pathogen transmission [36] as well as PRRS virus [11]. Previous studies reported that the houseflies are a mechanical vector of PRRS virus [37], which can transport the pathogen through its morphological structure [38]. PRRS virus do not require many viral parcles to cause an infecon, according to Yoon [39] who found that transmission only requires ≤20 virions. However, here, the nucleic acid of PRRS virus was detected in only 4% (2/50) of the samples, which could be due to the handling of the samples, because the frozen of the extracted RNA could have suffered some type of degradaon [40]. However, based on the sample size, the amount of viruses here found could be important in the disseminaon, as few viral parcles are required to establish an infecon [41]. A possible explanaon for the absence of PRRS virus in fly homogenates could be due to a low number of viremic pigs or low loads of the virus shedding by infected pigs [42]; and to the amount of virus in the flies that could be detected [37]. Schurrer et al. [16] propose that virus retenon in flies appears to be related to the inial viral load aſter ingeson and ambient temperature. Otake et al. [11] using real-me PCR observed that the PRRS virus could remain viable in the intesnal tract of the houseflies up to 12 h aſter feeding fluids from an infected pig, but only for a short period on outer surface of the flies. On the other hand, Schurrer et al. [16] found that the PRRS virus could remain 48 h in the fly aſter its exposure to the virus; however, the prevalence of posive flies and the viral load decreased with me. The ability of the PRRS virus to remain within the body 3 of 6

Detecon of PRRS virus in housefly / Noh-Cuxim et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico of a housefly suggests that it could protect the virus against certain environmental factors that affect the survival of this microorganism outside the host, such as ultraviolet light and drying factors [43]. However, Grübel et al. [44] menon that the midgut of the housefly has a pH of 3,1, which could be another explanaon for the low number of posive samples, since the PRRS virus remains viable at a pH between 6,0 and 7,65 [45], thus decreasing the possibility of finding the virus in the macerates. There are studies that menon that the flight capacity of housefly could increase the risk of the transmission of disease. Schurrer et al. [15] reported that the housefly flight exceeds 5 km, with a distance dispersion of up to 33 km. It is very relevant to menon that the farm here studied was in a high pig density area with a large populaon of pigs within 10 km. In the present study, the season effect was not evaluated; however, it could be a risk factor, since there are reports of increments in the number of outbreaks, with low incidence during spring and summer, and high during the fall and winter seasons [46]. CONCLUSION The porcine reproducve and respiratory syndrome virus was detected in domesc flies using the real-me PCR test; however, more epidemiological and molecular studies are needed before being able to reach a conclusion regarding the importance of domesc flies in the transmission of the porcine reproducve and respiratory syndrome virus among and between pig farms. Conflict of interest statement The authors proclaim that there is no conflict of interest with regard the publicaon of this manuscript. 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