Risk factors and genetic characterization of bovine respiratory syncytial virus in the inner Aegean Region, Turkey

Abstract

Bovine respiratory syncytial virus (BRSV) is one of the causative viral agents of the bovine respiratory disease complex. This study was conducted to determine the seropositivity and risk factors associated with BRSV infection and to evaluate the phylogenetic relatedness of the BRSVs in the inner Aegean region of Turkey. In this cross-sectional study, serum samples (n = 557) and nasal swabs (n = 21) were collected from cattle herds (n = 43) between February 2018 and March 2019. A commercial indirect-ELISA kit was used for the detection of antibodies in the sera samples. Reverse-transcriptase PCR was used to detect viral RNA in nasal swabs. Nasal samples were also examined for the detection of bovine parainfluenza-3, bovine viral diarrhoea virus, and bovine herpesvirus 1 by molecular detection methods. Genetic characterization of the local BRSV field isolates was conducted by sequencing attachment glycoprotein (G) gene segment. Epidemiological data on potential risk factors were collected from each sampled herd during blood collection. All herds had at least one seropositive animal. After adjustment for assay sensitivity and specificity, the overall true seropositivity was 58.48% (95% CI: 53.32–63.47). BRSV RNA was detected in 2 of the 21 nasal swabs, whereas other infectious agents were not detected in the investigated samples. Phylogenetic analysis showed that the field isolates of BRSV obtained in this study belonged to subgroup III, but they were located on separate branch from previously characterised Turkish subgroup III isolates. BRSV field strains from this study displayed 3 new amino acid substitutions (P89S, D115G, and S165L) in the G protein chains compared to other main reference BRSV isolates, demonstrating that BRSV is still evolving. Generalised estimating equation model showed that there were positive associations between BRSV infection, age (OR = 2.36, p = 0.001), herd size (OR = 10.32, p < 0.001), herd type (OR = 8.97, p < 0.001), a past history of respiratory disease (OR = 4.06, p < 0.001). The results of this study revealed that BRSV infection is common among cattle herds in the inner Aegean region of Turkey. The obtained epidemiological and genetic data on BRSV infection from this study could be beneficial for designing effective biosecurity practices and vaccination strategies.

Introduction

Bovine respiratory syncytial virus (BRSV, renamed as bovine orthopneumovirus), a non-segmented negative-strand RNA virus, is a member of the family Pneumoviridae (ICTV, 2019). Its genome has nine structural proteins: the attachment glycoprotein (G), fusion protein (F), RNA polymerase (L), matrix protein (M), nucleoprotein (N), the polymerase cofactors M2-1 and M2-2, phosphoprotein (P), and small hydrophobic protein (SH) (Valarcher and Taylor 2007). The G and F genes are immunogenic antigens, and they have important roles in viral infectivity (Valarcher and Taylor 2007). Genetic analysis of the G and F proteins indicates that there are eight subgroups for the BRSV (Krešić et al. 2018).

BRSV infects sheep and goats, but infection usually occurs in cattle (Sarmiento-Silva et al. 2012). BRSV primarily affects young animals (especially 3 to 12-month-old calves) following the introduction of the virus into a herd. Although adult animals can be clinically or subclinically infected, severe clinical signs are seen mostly in young calves including upper and lower respiratory damage. The main symptoms are fever, coughing, loss of appetite, increased breathing rate, and nasal discharge (Valarcher and Taylor 2007). Furthermore, BRSV infection can increase the risk of secondary bacterial infections that can result in pneumonia (Brodersen 2010). The morbidity rate varies between 60 and 80%, and mortality rate varies between 2 and 3%, but it may reach up to 20% in calves (Elvander 1996; Valarcher and Taylor 2007).

Literature on the transmission of BRSV reported that the virus is mainly transmitted via aerosols, fomites, direct contact with infected animals, and humans as passive vectors (Norström et al. 2000; Valarcher and Taylor 2007). Infection is more prevalent in the autumn and winter seasons, and intensification practices, environmental stress factors, herd size, and age distribution can influence the occurrence of disease (Van der Poel et al. 1994; Norström et al. 2000; Sacco et al. 2014; Ferella et al. 2018). Although vaccination and biosecurity are effective methods of preventing BRSV infection, prevalence of infection is higher in develo** countries because of the cost of control programs and the absence of information on the risk factors associated with BRSV (Norström et al. 2000; Saa et al. 2012; Ferella et al. 2018).

There is limited knowledge available about the prevalence of BRSV infection (Yavru et al. 2005; Yeşilbağ and Güngö, 2008; Kadiroğlu et al. 2020) and genetic features of BRSV strains circulating in Turkey (Hacıoğlu et al. 2019; Timurkan et al. 2019; Yazici et al. 2020). Furthermore, the possible risk factors for BRSV infection in cattle have not been studied in Turkey. To establish a successful control program, they are important to determine the infection prevalence and risk factors associated with disease. The aims of this study were therefore to determine the seropositivity and risk factors associated with BRSV infection and to identify genetic variations of the BRSVs circulating in the inner Aegean region of Turkey.

Material and methods

Study area

The study was carried out in cattle herds (n = 43) in the Afyonkarahisar and Uşak Provinces, located within 37° 45′ North and 39° 17′ North latitudes and 28° 97′ East and 31° 43′ East longitudes, in the inner Aegean region of Turkey (Fig. 1). The study area is one of the major livestock centres in the Aegean region with approximately 600,000 cattle (TUIK, 2019). According to the Köppen-Trewartha climate classification, the study area has a continental temperate climate (average low-temperature and average high-temperature is − 2.4 °C and 21.8 °C, respectively), and elevation of studied area varies between 907 and 1012 m (GDM, 2020).

Fig. 1

Location of sampled provinces in Turkey

Study design and sample collection

This cross-sectional study was carried out during the months of February 2018 and March 2019 on 43 cattle herds. The sample size was determined based on expected prevalence of 50% with 95% confidence level (CI) and a precision of 5%. In this study, a two-stage cluster sampling was used. The initial sample size (n = 384) was increased to 557 animals (6–54 months) with the design effect based on parameters used in sampling: number of herds = 43, number of sampling animals in each herd = 2, and design effect = 1.45 and intra-class correlation coefficient (ICC) = 0.28 which are values mentioned for BRSV (Segura et al. 2013). A random sampling programme (Win Episcope 2.0) was used to determine the number of animals by random sampling in each selected herd (Sergeant 2017). To avoid misinterpretation of results due to presence of maternal immunity among young cattle, cattle > 6 months of age were sampled.

For BRSV detection, nasal swabs were collected from 21 cattle (aged between 1 and 9 months) suspected to have BRSV infection with clinical signs including cough, ocular and nasal discharge. Samples originated from four beef herds in the Uşak Province and seven dairy herds in the Afyonkarahisar Province. On these herds, there were no vaccination records against BRSV. The medical history of sampled herds was obtained from questionnaire survey.

Serological assays

Blood samples were collected using a sterile vacuum tube without an anticoagulant from the jugular vein and transferred to the laboratory on frozen ice. Sera were separated by centrifugation (3000 rpm for 15 min at 20 °C) and stored in a freezer (at − 20 °C) until analysis. A commercial indirect-ELISA kit (BIO K 061/5, BioX, Rochefort, Belgium) was used for the detection of BRSV anti-F specific antibodies in the sera samples. The reported specificity and sensitivity of the kit were 87% and 93%, respectively. The optical density was measured using a spectrophotometer (BioTek Instruments Inc., Winooski, USA) at 450 nm. ELISA results were evaluated according to the manufacturer’s instructions.

Nucleic acid extraction and screening BRDC associated viruses

A commercial extraction kit (Cador Pathogen Mini Kit, Qiagen, Hilden, Germany) was used for the extraction of total nucleic acids from the nasal swabs. Total nucleic acids were tested by RT-PCR methods for detection of bovine parainfluenza-3 (BPIV-3), bovine viral diarrhoea virus (BVDV), and BRSV. The protocols described by Valarcher et al. (2000), Zhu et al. (

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.