Highly pathogenic Bovine Respiratory Syncytial virus variant in a dairy herd in Italy

Abstract Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production worldwide. Usually, it is detected in outbreaks of respiratory disease, most often during the winter period. During the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm comprising about 300 heads located in Central Italy. The herd was affected by a severe flu‐like syndrome unresponsive to any antibiotic treatment. Within 3 weeks, 39 adult animals died, and 12 abortions occurred. Direct and indirect laboratory tests were performed to detect the main pathogens causing the respiratory disease of the affected cattle. The results of laboratory investigations provided evidence of an acute and severe BRSV syndrome characterized by unusual mortality. In order to investigate the molecular underpinnings of this syndrome, phylogenetic analysis of the BRSV strain detected from the outbreak was carried out. The sequence analysis showed that the strain was genetically divergent from BRSV strains previously identified in Italy, as it showed high sequence similarity of more than 97% with strains isolated during a major BRSV epizootic that occurred in Sweden, Norway and Denmark during 2010–2011. The infection of the herd in Italy with this BRSV strain was likely due to the introduction of animals imported into Italy from abroad.


Bovine respiratory syncytial virus (BRSV) belongs to the genus
Orthopneumovirus, within the Pneumoviridae family; it is responsible for an acute respiratory disease syndrome in beef and dairy calves, particularly during winter periods. Globally circulating BRSV has a significant negative impact on the cattle industry due to economic losses because of the associated morbidity, mortality, costs of treatment and prevention, loss of production and reduced carcass value (Schneider, Tait, Busby, & Reecy, 2009;Valarcher, Schelcher, & Bourhy, 2000). Bovine respiratory syncytial virus is an enveloped virus, and its genome contains 10 mRNAs, which are transcribed and translated into 11 viral proteins (Valarcher & Taylor, 2007). Viral glycoprotein (G protein) has been suggested to be the major viral antigen that induces host immune response and confers a good level of protection. Variation of some G protein regions is believed to be antigenically important (Elvander et al., 1998;Prozzi et al., 1997). Biological DOI: 10.1002/vms3.312

Highly pathogenic Bovine Respiratory Syncytial virus variant in a dairy herd in Italy
significance of antigenic variation might be relevant to the efficacy of vaccines (Prozzi et al., 1997). The gene encoding G protein, as well as those encoding nucleoprotein (N) and fusion (F) protein have been used as targets of phylogenetic and molecular epidemiological studies (Valarcher & Taylor, 2007). Based on this analysis, eight subgroups (I-VIII) were assigned to represent both the temporal and geographical distribution of known isolates (Bertolotti, Giammarioli, & Rosati, 2018;Krešić et al., 2018;Valarcher & Taylor, 2007).
Moreover, genetic characterization showed a strict geographic correlation between virus variants and the emergence of new variants in Europe (Bidokti Medhi et al., 2012). There is limited information about the similarity and evolutionary relationship among BRSV isolates, particularly in those isolated from an Italian cattle population.
Two different clades have been identified so far in Italy, namely subgroup III and subgroup VII (Bertolotti et al., 2018). In this study, we describe identification and genetic characterization of a BRSV strain that was responsible for unusual mortality and relevant morbidity during an outbreak in a dairy farm in Central Italy.

| Outbreak and sample collection
In the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm of about 300 heads of Friesian breed, located in Central Italy. This breed is reared predominantly for milk production, and to a lesser extent, fattening of beef calves.
The non-vaccinated herd was affected by severe and unusual flu-like manifestations, including dyspnoea, anorexia, hyperthermia, loss of appetite, poor mobility and suffering, drastic reduction in milk production and severe respiratory symptoms, which were not relieved by any antibiotic therapy. Within 3 weeks, 39 adult animals died, and 12 abortions occurred at various gestational ages, especially in the animals with more severe symptoms. Unfortunately, the aborted fetuses were not submitted to laboratory for abortion diagnosis, and consequently the potential causes were not identified. Necropsies were performed by an on-site veterinary practitioner during this 3-week period as mortality occurred. Samples of the upper respiratory tract, lungs and nasopharyngeal swabs of the 35 animals that died with clinical respiratory disease were sent to the laboratory for pathological and laboratory investigations for the presence of the main pathogens causing the respiratory disease of cattle. In addition, serum samples were randomly collected from 20 convalescent animals to detect antibodies against the main viral agents of bovine respiratory disease complex.

| Sequencing and phylogenetic analysis
Total RNA was extracted using a QIAamp Viral RNA mini kit (Qiagen) and used as a template for the amplification of a 541-bp region encoding the G protein. Amplification was performed by using a onestep RT-PCR kit (Qiagen) following the manufacturer's instructions, applying a previously published nested protocol (Vilcek, Elvander, Ballagi-Pordány, & Belák, 1994). In addition, a fragment of a 731bp region encoding the N protein was amplified and sequenced to confirm the subgroup association (Bertolotti et al., 2018;Valarcher et al., 2000). Sense and antisense strands of two PCR-positive BRSV samples were sequenced in three independent reactions for each isolate. Sequence data were analysed using SeqManPro program from DNAStar package. Clustal X.2 was used to align the sequences with respect to the amino acid-coding frame with a set of reference sequences from GenBank, including representatives of the eight subgroups proposed previously (Bertolotti et al., 2018;Krešić et al., 2018;Valarcher et al., 2000). BioEdit sequence alignment editor version 7.0.5.2, was used for editing (Hall, 1999). The phylogenetic tree was drawn by applying the Kimura 2-parameter model with gamma distribution (K2 + G), using MEGA v.7. The robustness of the clusters was assessed by performing 10,000 bootstrap replicates, and branches with bootstrap values exceeding 70% were grouped together (Kumar, Stecher, & Tamura, 2016).

| RE SULTS AND D ISCUSS I ON
Overall, the pathological examination showed tracheal congestion, fibro-purulent bronchopneumonia, marked oedema and interstitial emphysema characterized by consolidation of the apical and middle lobes of the lungs. The results of laboratory investigation are presented in Table 1 We reported an outbreak with high mortality that occurred in a dairy farm in Central Italy and was attributed to a BRSV strain never described in the country. This strain had extremely negative effects on herd health and animal performance, as it had induced a rather high mortality and severe signs of respiratory disease. In few samples from lungs BRSV was detected together with either Mycoplasma spp. or Mannheimia haemolytica.
After the diagnosis, an emergency vaccination plan was set up with a live attenuated BRSV vaccine, which was administered intra-nasally. Nevertheless, the animals continued to die. Live and inactivated vaccines against BRSV are generally used, but their efficiency has been questioned (Valarcher et al., 2000). In mid-November, the health status of survivors stabilized, and milk production partially recovered. During the epidemiological investigation, it was established that in the end of September 2018, 22 Friesian cattle purchased from Germany had been introduced into the farm. They were already in lactation and enjoyed good health. About a couple of weeks after their introduction into the farm, one of these subjects presented a mild self-resolving respiratory syndrome 10 days before the beginning of the disease outbreak.
The BRSV strain characterized in this study did not cluster together with the viruses previously identified in Italy, but it of the vaccines is also recommended.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C S S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethics review committee approval has been received.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in NCBI at www.ncbi.nlm.nih.gov, reference numbers LR699790.1, LR699781.1.