Fatal interstitial pneumonia caused by bovine coronavirus in cows from Southern Italy

An outbreak of winter disease, complicated by severe respiratory syndrome, occurred in January 2020 in a high production dairy cow herd located in a hilly area of the Calabria region. Of the 52 animals belonging to the farm, 5 (9.6%) died with severe respiratory distress, death occurring 3-4 days after the appearance of the respiratory signs (caught and gasping breath). Microbiological analysis revealed absence of pathogenic bacteria whilst Real-time PCR identified the presence of RNA from Bovine Coronavirus (BCoV) in several organs: lungs, small intestine (jejunum), mediastinal lymph nodes, liver and placenta. Since being the only pathogen identified, BCoV was hypothesized to be the cause of the lethal pulmonary infection. Like the other CoVs, BCoV is able to cause different syndromes. Its role in calfhood diarrhoea and in mild respiratory disease is well known: we report instead the involvement of this virus in a severe and fatal respiratory disorder, with symptoms and disease evolution resembling that of Severe Acute Respiratory Syndromes (SARS). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 July 2020 doi:10.20944/preprints202007.0749.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Coronaviruses (CoVs) are enveloped viruses, with a RNA single-stranded large genome of 27.6-31 kb [1]. This big group of viruses, belonging to the order Nidovirales, suborder Coronavirinae, family Coronaviridae, subfamily Orthocornavirinae, is divided, (on the basis of the antigenic and genetic properties of these viruses, [1,2] into 4 genera: alpha, beta, gamma and deltacoronavirus.
Coronaviruses are responsible for enteric, respiratory and neurological diseases in both birds and mammals, including humans [3]. Bats and birds are major reservoir of CoVs and some CoVs are endemic in domestic animals in different countries [4]. Several coronaviruses are well known for their ability to change tissue tropism, to pass species barriers and to adapt to new ecological niches.
These abilities are related to two factors commonly occurring during RNA replication: accumulation of point mutations (one mutation for each round of replication) caused by the low fidelity of their RNA dependent RNA polymerase (RdRp), as well as homologous and heterologous recombination events [1,2]. These features promote the emergence of novel strains with novel biological properties in term of host range, tissue tropism and virulence [1,5]. The high mutation and recombination rates are indeed implicated in the emergence of new human coronaviruses, of animal origin, like occurred for appearance of the strains HCoV-229E and HCoV-OC43 (the only two human coronaviruses known before SARS emergence, as well as for the arising of SARS-CoV Middle East Respiratory Syndrome Coronavirus Infection, (MERS-CoV) and the recent global pandemic SARS-CoV-2 [1,3,[6][7][8]. CoVs infections in veterinary medicine are well known from a long time [1]. The most common animal CoVs are: infectious bronchitis virus (chickens), porcine transmissible gastroenteritis, porcine hemagglutinating encephalomyelitis and porcine epidemic diarrhoea CoVs (swine), bovine coronavirus (BCoV, cattle), canine enteric and canine respiratory CoVs (dogs), and feline coronavirus (cats) [3]. BCoV belongs, together with human coronavirus (OC43), human enteric coronavirus (4408) and canine respiratory coronavirus (CRCoV), to subgroup A of the betacoronavirus genera [9][10][11]. This virus is pneumoenteric and has a dual tissue tropism, infecting both intestine and respiratory apparatus (upper and lower tracts), a feature in common with human SARS and SARS-CoV-2 [5]. It is endemic in all the world and is considered one of the major causes of neonatal calf diarrhoea, with mortality related to virus ability to destroy the intestinal villi and to cause severe bloody diarrhoea [2]. In adult dairy cattle, BCoV is instead responsible for the well known winter dysentery (WD), which is associated with severe drop in milk production and consequent significant economic losses for the herds [2]. In cattle of various ages the virus is also responsible for respiratory infections (bovine respiratory disease complex, shipping fever of feedlot cattle [5]. In our study we report an outbreak of possible WD in a calf breeding of Southern Italy. The majority of animals showed gastrointestinal disease with bloody diarrhoea and mild respiratory symptomatology (cough, slight temperature increase, nasal discharge). Among them, 5 displayed a very severe respiratory illness, which evolved with animals death within very few days after the occurrence of the first respiratory difficulties. To ascertain the cause of death, animals underwent necroscopy and organs were analysed for the presence of possible bacterial and viral pathogens respectively with cultural and molecular methods.

Outbreak description
The herd consisted of a total population of 52 Italian Friesian cows. The cattle were housed under a single canopy and separated into production groups with feeders on the central corridor and drinking bowls inside each paddock. The production system was characterized by internal recovery.
No movements or purchases occurred in the last three years. At the beginning of January 2020 most of the cows, all vaccinated against bovine alphaherpesvirus 1 (BoHV-1) and bovine viral diarrhoea virus (BVD), began to experience symptoms related to winter disease. In the following days about 90% of the cows showed bloody diarrhoea and a decrease in milk production of up to two thirds.
Dry cows and juveniles showed no symptoms. After about 5 days, the majority of cows with diarrhoea began to have respiratory symptoms, with cough and catarrhal secretion, dyspnoea and moderate febrile rise. In some of them, the symptoms worsened with manifestation of pneumoderm and pneumothorax. In particular two lactating cows (of about 4 years old) in the fourth month of pregnancy started to show mild respiratory symptoms which aggravated becoming very serious in 1-2 days. The two bovines died in 3-4 days after the appearance of the first respiratory difficulties.
Later on, 3 more animals, (of about 7 years old), died after showing the same symptomatology. About ten days later, respiratory and gastrointestinal symptoms of the other animals started to regress. Died animals underwent necropsy to investigated the cause of death and organs together with faecal material were analysed for the presence of bacterial, viral and parasite pathogens.

Histopathological analysis
Tissues of the organs were fixed in 10% neutral phosphate-buffered formalin and processed by routinary methods into paraffin blocks which were cut into 3-4 μm thick sections and stained with hematoxylin and eosin.

Bacteriological and parasitological analysis
Lungs, liver, kidneys, spleen, meseraic and supramammary lymphnodes as well as cephalorachid fluid were tested by bacterial culture methods to determine the possible cause of death. Briefly, samples were inoculated on both MacConkey agar no. 3 (CM0115 Oxoid) plates and blood agar (7% v/v ovine blood in blood agar base, CM0271 Oxoid) plates. MacConkey agar plates were incubated at 37°C for 48 h, while blood agar plates were incubated at 37°C for up to 72 h in presence/absence of 5% CO2 Mycoplasma spp. was investigated in lungs and kidneys by inoculating samples in culture broth and plates following protocols usually employed in our laboratories. Faecal material of the animals during the disease was analysed for the presence of helminth eggs and coccidia oocysts with routinary methods.

Viral Nucleic acids extraction procedures
Each organ (25 mg of tissue in 1 ml Phosfate buffered saline solution) was homogenated by Tissue Lyser (Qiagen). Nucleic acid extraction was carried out from 200 µl of organ homogenate by using Qiasimphony automated extraction system (Qiagen) with the DSP Virus/Pathogen Mini kit (Qiagen) according to the manufacturer's instructions. Nucleic acids were eluted in 60 µl of elution buffer containing 40 unit/µl RNase inhibitor (Promega) and immediately analysed by Real-time PCR. respiratory syncytial virus (RSV) were analysed by Real-time reverse transcription polymerase chain reaction using AGPATH reaction kit (Thermo Fisher Scientific). All the reactions were carried with primers (TemaRicerca) and probes (Thermo Fisher Scientific) specific for the virus tested on a Quantstudio5 system (Thermofisher) with protocols routinely used in our laboratory. In particular BCoV was identified following literature [12] with BCoV positive control was kindly given by Friederich institute, Germany.

BCoV sequencing
In order to characterize the strains of BCoV identified, 5ul of nucleic acids underwent RT-PCR to amplify a 456 bp fragment of the RNA-dependent RNA polymerase (rdrp) gene [13]. The reaction was carried out with AGPATH reaction kit (Thermo Fisher Scientific), using 1.25 µl of each primer (10 µM) with the following thermal profile: 50°C for 30 minutes, 95°C for 15 min, 45 cycles of 94°C for 30 sec, 50°C for 30 sec, 68°C for 30 sec and a final elongation step of 72°C for 10 minutes. PCR products were analysed by Tape station (Agilent) using the D 1000 kit. Amplicons were sequenced by capillary electrophoresis as previously described [14]. The nucleotide sequence similarity searches were performed using the BLAST server (http://www.ncbi.nlm.nih.gov/genbank/index.html) and phylogenetic analysis was carried out using the Jalview and MEGA 7 softwares.

Necroscopical findings
Of the 52 cows present in the breeding, 5 (9.6%) died with severe respiratory distress, death occurring 3-4 days after the appearance of the respiratory signs (caught and gasping breath).
Anatomo-pathological inspection of the dead cows showed similar frames herein described. We first of all observed congested explorable mucous membranes and haemorrhagic lymphadenitis of the supra-mammary glands. Jejunum was affected by hemorrhagic enteritis with the rectal ampoule containing little hemorrhagic diarrhoea. There was catarrhal lymphadenitis of the meseraics. Liver showed increased volume and was characterised by sub-glissonian haemorrhages and diffuse centro-lobular necrosis. Moderate splenomegaly with sub capsular extravasations at the visceral surface was observed. Kidneys showed red infarcts involving cortical and medullar tissues ( Figure   1A). Foetus of around 4 months old was characterized by diffuse subcutaneous haemorrhages. With respect to thoracic cavity we observed diffuse fibrinous-hemorrhagic pleurisy; thymus was studded with hemorrhagic petechiae. Respiratory apparatus showed severe imposing tracheo-bronchitis and muco-hemorrhagic bronchiolitis with the deposition of fibrin molds. Lungs ( Figure 1B

Histopathology
Microscopic examination of the lungs (

Sequencing analysis of BCoV
As shown in Figure   analyses were conducted in MEGA7 [16]. Sequence obtained in the present study (Bovine ITALY 2020) is indicated with an asterisk.

Discussion
In our study we described a strong pulmonary infection, likely provoked by bovine coronavirus, which lead to the death of 5 animals in a high genealogy Friesian dairy cows breeding. The infection involved 90% of the animals with gastrointestinal and mild respiratory symptoms but for few animals the pathogen showed to be more aggressive leading to a dead prevalence of 5.2%.
Since BCoV was the only pathogen identified, its role in the fatal pneumonia appeared unequivocal.
As a matter of fact while the involvement of BCoV in enteric infections is well known, its role in bovine respiratory disease is controversial and principally related to mild respiratory symptomatology. The virus has been indeed found in respiratory samples from both healthy and sick cattle [11,[17][18][19][20][21] and more frequently identified in association with other respiratory pathogens [11,18,[22][23][24]. Only few papers reported instead BCoV as the only recognized cause of the disease [9,22,25]. Furthermore, there are various studies in literature describing attempts to provoke clinical respiratory symptoms by infecting healthy cattle with BCoV inocula. Results showed in most experiments no symptomatology in some cases the developing of gastrointestinal disease [26][27][28][29], with only few reports describing slight respiratory problems [30,31]. Our results seem to corroborate the role of this virus in respiratory outbreaks and to point out the ability of the virus to provoke, in the same animal or within the same outbreak, both enteric and respiratory symptoms, confirming what already reported by Choulienko et al. [32].  [1,2,32]. In less than twenty years we assisted to the emergence of three highly pathogenic CoVs all of zoonotic origin. These viruses with reservoirs in bats and rodents, have passed the species barriers jumping to humans through intermediate animals [1]. BCoV shares with the human highly pathogen CoVs (SARS, MERS and SARS-CoV-2) ,the feature to have a broad host range and to be competent of infecting multiple species thus showing a likely zoonotic potential [3,5,34]. As a matter of fact, many bovine-like CoVs has been indeed identified as enteric and/or respiratory pathogens in both livestock and wildlife species (wild ruminants, captive ruminants as well as water buffalo, camelids, [5,10,[35][36][37][38][39]. With respect to the possibility of BCoV-like viruses to infect humans, in literature it has been described a case of child acute enteritis caused by a human CoV found genetically and antigenetically more closely related to BCoV than to HCoV-OC43 [40]. What's more HCoV-OC43 has been supposed to derive from an ancestral BCoV strain (considering their close genetic and antigenic similarity) which jumped the species barrier and had passed from rodents to humans through cattle [41][42][43]. Interestingly, phylogenetic analysis showed that our strain, with respect to the part of rdrp gene analysed, was very closely related to an enteric HCoV-4408 described in a patient in Germany (see Figure 4).
Interspecies transmission via wildlife and livestock host animals are key factors for the emerging of new, highly pathogenic, human coronaviruses. For this reason it is of utmost important to focus on and carefully investigate the outbreaks in which animal coronaviruses are involved, with particular regard to clinical symptomatology and genetic classification. What's more, in the case of BCoVs, it is necessary to recommend the farmers to strictly follow all the protection and prevention biosafety measures necessary to limit the diffusion of such viruses among animals and to avoid any possible