Paradigm of coinfection with viral and bacterial agents causing bovine respiratory disease complex (BRDC) in Central Serbia

Viral and bacterial agents causing bovine respiratory disease complex (BRDC) were examined over a five-year period (2015– 2019). Thirty-eight farms of beef and dairy cattle from Central Serbia which showed symptoms of BRDC were observed. A total of 118 nasal swab specimens collected from diseased bullocks (n=55), cows (n=18) and calves (n=45) were chosen for the isolation of bacteria and identification by aerobic cultivation. The most commonly isolated bacterial pathogen was Pasteurella multocida (72.88% positive samples). Disk-diffusion method (antibiogram) showed that Amoxiclav and Enrofloxacin were the most efficient antibiotics against Pasteurella multocida isolates (66.67% and 54.17%, respectively). From all examined samples, using the Real Time RT-PCR and PCR methods, we determined the genome sequences of bovine respiratory syncytial virus (BRSV– 34.78% of tested samples (8/23), bovine viral diarrhea virus (BVDV, in 34.37% (11/32) and Mycoplasma spp. in 33.33% (8/24). The genome of BoHV-1 virus was not detected in any of the 19 samples tested by Real Time PCR method.


Introduction
BRDC is one of the most significant causes of cattle morbidity and mortality, both in beef and dairy cattle, causing enormous costs for the cattle industry, which are also reflected in reduced production, increased labor costs and reduced carcass value, regardless of the use of modern vaccines and antibiotics (Irsik et al., 2006;Gershwin et al., 2015). BRDC is caused by one or more of several viruses: bovine respiratory syncytial virus (BRSV), bovine herpes type 1 (BoHV-1) also known as infectious bovine rhinotracheitis (IBR)/infectious pustular vulvovaginitis (IPV), and bovine viral diarrhea virus (BVDV), which predispose animals to coinfection with one or more bacteria, including Pasteurella multocida (P. multocida), Mannheimia haemolytica (M. haemolytica), Mycoplasma bovis (M. bovis), and Histophilussomni (Gershwin et al., 2015). Viral agents such as BRSV and bovine parainfluenza virus 3 (BPIV-3) are designated as the most common challengers of BRDC. The pathogenesis of BRDC is still largely an enigma for researchers, particularly in the advanced stage of chronic disease, in addition to coinfection with other primary bacterial agents like M. bovis (Mehinagic et al., 2019).
BRDC is a multifactorial process due to infectious agents including viruses, bacteria and mycoplasma (Taylor et al., 2010). The development of BRDC is the result of complex and under-researched interactions between the environment, cattle, and pathogens (Setiyaningsih et al., 2018). There has been a more recent approach to addressing these gaps and authors identified signatures of tissue-specific transcriptional responses indicative of tropism in the coordination of host's immune tissue responses to infection by viral or bacterial infections, and showed that this tissue tropism in host transcriptional response to BRDC pathogens results in the activation of different networks of response genes. The results of the above mentioned research serve as a basis for the development of innovative therapeutic strategies and for the selection of cattle with enhanced resistance to BRDC (Behura et al., 2017).
The most commonly reported predisposing factors that facilitate the onset of BRDC are: stress, weaning, inadequate (mostly too high) temperature, inappropriate stocking density, dust, humidity, shipping and frequent dietary changes (Taylor et al., 2010). Other researchers have concluded that the major viral pathogens associated with BRDC that primarily cause respiratory lesions are BoHV-1, BRSV and BPIV-3 (Kirchhoff et al., 2014). Unique among the bovine respiratory viral agents is BVDV, because intrauterine infection can lead to cattle that are persistently infected (PI), chronically ill or dying in feedlots (Kurćubić et al., 2011). BVDV infections have an immunosuppressive effect and facilitate the onset of BRDC, primarily due to the creation of favorable conditions for the development of bacterial agents, the most common of which are: sixteen serotypes of M. haemolytica, P. multocida, Histophilus somni, Streptococcus pneumoniae, Staphylococcus aureus, Corynebacterium bovis, Streptococcus spp. and Micrococcus spp. (Taylor et al.,2010). Mycoplasmas caused serious disease in cattle, with significant negative economic and welfare impacts (Parker et al., 2018). M. bovisis currently recognized as one of the most important and frequently isolated Mycoplasma species associated with cattle disease worldwide (Bednarek et al., 2012;Fox, 2012). Other frequently isolated mycoplasmas are: Mycoplasma bovirhinis, Mycoplasma dispar, Ureaplasma diversum and even Mycoplasma canis (Szymańska et al., 2010).
The aim of our study was to discover the prevalence of bacterial and viral pathogens responsible for BRDC in fattening and dairy cattle of different age categories from a number of farms, as well as to determine the resistance of P. multocida to the most commonly used antibiotics/sulfonamides in Serbian veterinary practice.

Material and methods
The cattle included in the experiment were chosen from 38 farms of fattening bulls and dairy cattle from Central Serbia. Nasal swabs were sampled at the request of the owners, who reported in anamnestic data that the diseased animals showed symptoms of respiratory disease, which seemed to justify the suspicion of BRDC. In addition to routine clinical diagnostics, tests were performed to isolate bacterial and viral pathogens and determine the most effective antibacterial therapy. The anamnestic data were generally very similar for cattle suspected of BRDC: rectal temperature above 40.1 ˚C, loss of appetite accompanying weight loss, rapid, saccadic breathing, strong dry cough, nasal discharge of serous to mucopurulent appearance, eye discharge in some animals, less mobility and depression. Clinical examination involving auscultation of the lung revealed wheezing.
After anamnesis taking and clinical examination, sampling was performed immediately, from the nasal mucosa, using sterile swabs and test tubes.After sampling, all samples were properly transported in hand-held refrigerators to the receiving department of the Kraljevo Veterinary Specialized Institute, and examined at the Department of Laboratory Diagnostics.
A total of 118 nasal swab specimens taken from diseased bullocks (n=55), cows (n=18) and calves (n=45) were chosen for the isolation of bacteria and identification by aerobic cultivation,with subsequent biochemical identification and determination of the isolated strains (Laboratory Diagnostics Manual; standardization of diagnostic methods for bacterial, viral and parasitic diseases whose control is prescribed by law (1984) and Quinn et al. (2002).
Susceptibility testing of isolated bacterial strains to antibiotics/sulfonamides (antibiogram) was performed using the disk diffusion method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2015).
Detection and quantitation of BRSV were performed by Real Time RT-PCR (Boxus et al., 2005). Detection and quantitation of BVD virus were also conducted by Real Time RT-PCR OIE Chapter 2.4.8, section 1., d). Virus genomes were extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germany). Real Time RT-PCR reaction was performed using the Superscript III Platinum ® The genome of the virus IBR/IPV was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany). Real Time PCR reaction was performed using the Maxima™ Probe qPCR Master Mix (Fermentas, Lithuania), the Real Time PCR machine AriaMx (Agilent), according to the protocol described in the OIE Manual, Chapter 2.4.13 section 1., c).

One-Step Quantitative Real
Time RT-PCR System (Invitrogen, USA), the Real Time PCR machine AriaMx (Agilent), according to the protocol described by Baxi et al. (2006).
Rapid detection of Mycoplasma spp. was performed using a Bio Rad PCR machine according to the method described by Hotzel et al. (1996). DNA fragments were separated by horizontal electrophoresis in a 2% agarose-gel (E-Gel iBase Power System, Israel), at 48 V, 0.8 A, 50/60 Hz for around 30 min. Gels were illuminated and photographed in a chamber (Gel Doc XR System with Image Lab, Bio-Rad) using UV light.

Results and discussion
Results of the microbiological tests and susceptibility testing of P. multocidaisolates to antibiotics and sulfonamides (antibiogram) are presented in the Tables below (1-2).
The most common isolated bacterial pathogen was P. multocida (72.88%-86/118), and we tested its susceptibility to antibiotics and sulfonamides (Table 2). Other tested bacterial agents were less present, as shown in descending order: Streptococcus spp.   The presence of the IBR/IPV virus genome in our study was not determined in any of the 19 examined samples, during 2017. and 2018. The BRSV genome was determined in 34.78% of examined samples (8/23). The findings of Brodersen (2010) showed that BRSV is a major cause of respiratory disease and a major contributor to BRDC. In our examination, the BVDV genome was detected in 34.37% of the tested samples (11/32). Mycoplasma spp. was found in 33.33% of the examined samples (8/24), in our study. More recently, BPIV-3 has been found to be the predominant causative agent of single infections in 39 cases, and of coinfection with M. bovisin 39 cases as well. The comparison of the detection methods for M. bovis showed that Real Time PCR was more specific and sensitive than immunohistochemistry (IHC). BPIV-3 and M. bovis persisted in chronic BRDC, unlike BRSV, suggesting permanent damage to the defense mechanisms in the lungs (Parker et al., 2018;Mehinagic et al., 2019).
The data presented in Table 2 indicate that the most efficient antibiotics against P. multocida isolates were Amoxiclav and Enrofloxacin (66.67% and 54.17% sensitive isolates, respectively), in contrast to previous studies by Kurćubić et al. (2013), in which P. multocida was most sensitive to Enrofloxacin and Floron (100% of examined isolates sensitive to both antibiotics).

Conclusions
The most common bacterial findings were P. multocida, Streptococcus spp., Corynebacterium spp., M. haemolytica, Neisseria spp., Staphylococcus epidermidis and Klebsiella spp., suggesting their greater importance for BRDC in Serbian cattle compared with M. haemolytica, which is the predominant pathogen worldwide. In the future, research will be focused on determining how these pathogens interact with each other.According to Real Time RT-PCR findings, BRSV and BVDV are common viruses in Serbian cattle herds suffering from BRDC (the BoHV-1 genome was not identified in our study). Mycoplasma spp. was found in 33.33% of the examined samples (8/24), in our study. The most efficient antibiotics against P. multocida isolates were Amoxiclav and Enrofloxacin (66.67% and 54.17% sensitive isolates, respectively).