Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Molecular Detection of Respiratory Syncytial Virus (RSV) in Calves of Al-Anbar Provinces

Mohammed A. Hamad1*, Murtadha Abdule Mahdi Al-Mudhafar2, Alaa Fayeq Habeeb3, Firas R. Jameel1, Ismael Raheem Al-Muhana2, Najemaddin A. Hamad5

1Department of Biotechnology, College of Applied Science University of Fallujah, Al-Anbar 31002, Iraq; 2Department of Microbiology, Faculty of Veterinary Medicine, University of Al-Kufa, Al-Najaf 54001, Iraq; 3Department of Pathological Analyzes College of Applied Science, University of Fallujah, Anbar, Iraq; 4Department of Religious Education and Islamic Studies, Sunni Endowment Office, Fallujah, Iraq.

Received | August 25, 2024; Accepted | November 18, 2024; Published | December 09, 2024

*Correspondence | Mohammed A. Hamad, Department of Biotechnology, College of Applied Science University of Fallujah, Al-Anbar 31002, Iraq; Email: [email protected]

Citation | Hamad MA, Al-Mudhafar MAM, Habeeb AF, Jameel FR, Al-Muhana IR, Hamad NA (2024). Molecular detection of respiratory syncytial virus (RSV) in calves of Al-Anbar provinces. J. Anim. Health Prod. 12(s1): 306-311.

DOI | https://dx.doi.org/10.17582/journal.jahp/2024/12.s1.306.311

ISSN (Online) | 2308-2801

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Bovine pneumonia is a complex disease presenting with significant financial consequences. Among the major contributing causes of pneumonia in young animals are infections with the bovine respiratory syncytial virus (BRSV) and the bovine parainfluenza virus-3 (BPI-3). These viral agents either primarily cause pneumonia or predispose animals to the development of pneumonia (Kamdi et al., 2020).

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection in cattle worldwide and acute (ARI) in humans, particularly in children, the elderly, and the immunocompromised.

Several years following the initial identification and characterization of human respiratory syncytial virus (HRSV), a study was released suggesting that RSV may play a role in respiratory disease in cattle. In 1968, it was discovered that bovine serum carried neutralizing antibody against hRSV, indicating the possible existence of a comparable virus in cattle. In the early 1970s, studies were published on respiratory disease outbreaks in various European countries. These reports found that viruses recovered from cattle were closely related to HRSV (Doggett et al., 1968).

Bovine parainfluenza 3 virus (BPIV3) and Bovine Respiratory Syncytial virus (BRSV) are extremely similar viruses which play a significant role in bovine respiratory disease (BRD), a prevalent and economically impactful illness affecting cattle populations globally. Both viruses have similar shape and replication strategy characteristics, which are also shared with their human equivalents, HRSV and HPIV3. Thus, infections of BRSV and BPIV3 in cattle are regarded as valuable animal models for HRSV and HPIV3 infections in people (Makoschey and Berge, 2021).

Bovine Respiratory Disease (BRD) impacts juvenile males and females globally. The clinical presentation is marked by respiratory symptoms, which can vary in severity from moderate to severe and, in some cases, can result in fatality. It is more common for multiple germs to infect an individual simultaneously rather than just one (Makoschey and Berge, 2021).

Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus 3 (BPIV3) mostly spread by the transmission of airborne droplets and gain entry into the body through the respiratory system. Upon inhalation, the viruses enter the mucous and either penetrate or break it down. The mucous acts as the first defense of the innate immune system. Subsequently, the viruses attack the upper respiratory tract’s epithelial cells by attaching to sialic acid residues on the cell membranes. Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus type 3 (BPIV3) mostly reproduce in the respiratory system (Viuff et al., 1996; Ceribasi et al., 2014). Overall, BRSV exhibits lower virus replication levels than BPIV3 virus, both in vitro and in vivo (Salt et al., 2007).

Considered age-related variations in the development of BRSV infection in calves without antibodies, comparing those aged 1 day and six weeks. Neonatal calves exhibited more virus multiplication and lung consolidation, but displayed reduced pro-inflammatory responses, specific humoral immune responses, lung neutrophilic infiltration, and clinical symptoms compared to 6-weeks old calves (Ogunbiyi et al., 1988).

The ability to generate pro-inflammatory cytokines seems to rise with age, which could account for the variations in the development of BRSV dependent on age. Furthermore, in the case of the BPIV3 virus, it appears that the immune response plays a role in developing the disease. This is confirmed by the discovery that mast cells in the lungs of BPIV3-infected calves secrete higher quantities of histamine (Ogunbiyi et al., 1988). This study was setup to identify and to determine the prevalence of the respiratory syncytial virus, and the factors that contributed to the infection in calves using molecular techniques.

Materials and Methods

Collection of specimens

Fifty-two (n= 52) nasal swabs were collected from young cattle with age from 2 months and 1 years which suffer from respiratory tract infection collected by using viral transport media VTM The nasal swabs were dipped in test tubes containing 2 ml of cooled transport media.

The nasal swabs were collected from different areas of dairy cattle farms cow breeders in villages from the areas of Saqlawiyah, Al-Karma, and south of Fallujah in the Fallouja city in Al-Anbar province. For the PCR, samples were applied and run under previously described conditions (Maidana et al., 2012; Vilcek et al., 1994).

Viral RNA extraction and genome RSV amplification

RNA was isolated from the VTM a nasal-swab homogenate of 100μL with a real line extraction 100 kit (Germany) according to the manufacturer’s recommendations, extracted RNA served as the basis for the Real Star® RSV real time -PCR Kit 3.0. The quality of the extracted RNA has a significant impact on the overall performance of the test system. It has to be ensured that the nucleic acid extraction system is compatible with the Real-time PCR technique.

All reagents and samples were thoroughly thawed, mixed (through the use of pipetting or gentle vertexing), and briefly centrifuged. In this study we used the Real Star® RSV Real-Time-PCR Kit 3.0 (Germany) which includes a heterologous Internal Control (IC) that can be used as both a Real Time-PCR inhibition control and a control of the sample preparation procedure (nucleic acid extraction). Probes specific for RSV A RNA are labelled with a fluorophore Cy5 whereas the probes specific for RSV B RNA are labelled with the fluorophore FAM. The probe specific for Internal Control (IC) is labelled with the fluorophore JOE, to quantify RNA in the samples by stratagene MX3005P qPCR system (Germany).

Statistical analysis

For the statistical analysis, data was entered into a specialized statistical package for the social sciences (SPSS) version 26 for Windows (GraphPad Software, San Diego, California, USA). The mean ± SD was used to express the results. The Chi square test is used to compare two category variables, and the t-test was used to compare two means. A statistically significant result was defined as a p-value of less than 0.05, and a highly significant result as a p-value less than 0.001.

Results and Discussion

The results of the amplification curve for the detection of RSV via Real-Time RTPCR shown in (Figure 1), revealed the percentage of suspected RSV cases in male calves was 68%, which was higher than females (32%) shown in (Table 1).

 

Table 1: Presents the number of samples and the percentages of infection correlated with gender (samples included male and female).

Number of tested samples	Gender	% of infection
35	males	68%
17	females	32%
52		100%

 

Table 2 showed that there was significant difference between Holstein Friesian and Local-cross breed in the percentage of RSV viral infection (χ2 = 6.231, p = 0.013), which revealed that Holstein Friesian are more susceptible to RSV virus. Also, a significant difference was found between male and female in both breed in percentage of RSV viral infection (χ2 = 4.923, p = 0.027).

 

Table 2: Illustrates the difference between Holstein Friesian and Local-cross in rate of RSV viral infection.

	Holstein Friesian (n = 35)	Local-cross (n = 17)	P-value
Gender	Percentage %		χ2 = 4.923 P= 0. 027
Male (N%)	27 (77.1 %)	7 (41.1 %)
Female (N%)	8 (22.8)	10 (58.8 %)
Breeds	35 (67.3 %)	17 (32.7 %)	χ2 = 6.231 P= 0. 013

 

Table 3: Illustrates the relationship between the age and the rate of RSV viral infection.

	Local-cross Mean ± SD (n = 17)	Holstein friesian Mean±SD (n = 35)	T-test	P value
Age (month)	7.53 ± 3.826	5.37 ± 2.327	14.409	0.000**
PCR	1.24 ± 0.437	1.51 ± 0.507	20.570	0.000**

 

Table 3 indicate that there was highly significant relationship between ages of calves and percentage of RSV viral infection (p = 0.000).

Table 4 revealed that there was highly significant difference between the strains of RSV (p = 0.000). The infection of calves with genotype B was more than another 13 cases compared to 7 and 10 cases of genotype A and genotype AB, respectively.

 

Table 4: Illustrates the RT-PCR for RSV genotype A, B and AB.

Genotype of RSV	Case of infection	Mean± S.D.	P-value
Genotype A	7	29.1 ± 7.27	0.000
Genotype B	13	27.9 ± 7.71	0.000
Genotype AB	10	26.9 ± 6.0	0.000

 

The primary challenges in the respiratory system of cattle are viral infections, particularly Bovine Respiratory Syncytial Virus (BRSV). Samples were collected during the late winter period. This could be due to the animals recovering from infection or because most of the tissue samples presumed to be from animals with pneumonia did not have any viral particles. Nevertheless, additional in vivo trials would be necessary for other vaccines. Hence, conducting genotyping investigations on viruses is vital for molecular epidemiology and vaccine research.

Respiratory tract infections are predominantly characterized by a combination of several pathogens. These infections can be caused by viruses, bacteria, or other agents, or they might result from interactions between viruses or bacteria (Thonur et al., 2012; Hodgson et al., 2005). Infection with the respiratory syncytial virus occurs across the world, however, it is most common in the winter months. The majority of animals are infected from 6 weeks to 2 years of age. According to the findings of this study, the prevalence of detected virus infection in calves was 68%, which was higher than in male (32%). Our research shows that RSV infections are more common in animals under the age of one year old and during the months of October through January.

Reverse transcriptase polymerase chain reaction screening of 406 cattle and buffaloes revealed that of the 406 cases, twelve (2.95%) were positive for BRSV and fifteen (3.69%) cases were positive for BPI-3 (RT-PCR) (Kamdi et al., 2020). The majority of animals develop a primary respiratory syncytial virus infection by the time they are two years old, and reinfection with the virus persists throughout life. In our investigation, we found that RSV is a significant cause of Acute respiratory infections (ARIs) in small animals under a year old. RSV frequently results in a fever, cough, and shortness of breath; from there, the illness can progress to bronchitis and acute respiratory distress syndrome. This is caused by a weak immune system and low virus resistance, with the premature birth being one of the key contributing factors.

In the current study, we found that RSV is a significant cause of ARI in animals under a year old. RSV frequently causes fever, cough, and shortness of breath; from there, the disease may lead to bronchitis and acute respiratory distress syndrome. This is caused by a weak immune system and low virus resistance, with the premature birth being one of the key contributing factors. Premature infants have fewer alveoli and, as a result, fewer anatomical barriers to gas exchange, which increases the lethality of bronchiolitis and other pulmonary infections.

In the other research investigation, it was found that the virus is transmitted to humans through animals a number of samples were taken from small animals who had upper respiratory tract infections, primarily bronchiolitis, and a high rate of RSV infection was found by Real Time-PCR; of these 22.8% were identified as positive cases.

Therefore, the goal of the current investigation was to identify BRSV and BPI-3 and investigate the pathogenic mechanisms underlying pneumonia deaths. Pneumonia is mostly a concern in young calves under 6 months of age, peaking between 2 and 10 weeks, while it can be observed more frequently in calves up to 1 year of age. Therefore, young animals under 12 months of age were chosen (Sivula et al., 1996) found that RT-PCR identified BPI-3 and BRSV in pneumonic cases of cows, with an incidence of 3.69% and 2.95%, respectively (Kamdi et al., 2020).

The present study found that there was significant difference between Holstein Friesian and Local-cross breed in the percentage of RSV viral infection (χ2 = 6.231, p = 0.013), which revealed that Holstein Friesian are more susceptible to RSV virus. Also, a significant difference was found between male and female in both breed in percentage of RSV viral infection (χ2 = 4.923, p = 0.027). These results are in line with Hussain KJ who showed that the prevalence was significantly higher in imported cattle than in local breed, which may be due to the stress of transportation and different environmental conditions (Hussain et al., 2019). When calf BRSV is infected, they produce a mixture of pro-inflammatory cytokines, including as CXCL8 (IL-8) and IL-12p40. Research on the cells and lymph fluid of calves infected with BRSV reveals increased synthesis of IL-4 and IL-13 in the serum and tissues as early as the fourth day after infection, together with elevated levels of virus-specific IgE in the serum, suggesting the initiation of a Th2-type response. Additionally, calf IFNγ generating cells grow and serum cytokine levels rise, both of which are positively correlated with diseases prognosis (Miao et al., 2004).

However, prior research has also shown that there is no discernible difference in the incidence of BRSV in male and female animals (Hagglund et al., 2004). But compared to males, female animals were more susceptible to BRSV. This discrepancy may be explained by the fact that female animals are more productive than male ones, making them more resilient to stress. In comparison to animals from other age groups, this study found that animals between the ages of 6 and 11 months had the highest incidence of BRSV. These findings are comparable with those of other studies that recorded the seroprevalence of individual animals (Bugarski et al., 2011; Bidokhti et al., 2012; Chavez et al., 2012). Moreover, Valarcher and Taylor (2007 found that age is a significant risk factor for BRSV () which may be linked to the higher prevalence of BRSV antibodies in adult animals, which may be linked to a high sero-prevalence of BRSV due to the probability of re-infections and repeated exposure to the viral infection throughout their lives (). Age-dependent variations in respiratory syncytial virus pathogenesis may be explained by the ability to create pro-inflammatory TNF-a, which appears to rise with age and makes re-infection with BRSV less severe even though it can recur throughout life ().

Conclusion and Recommendations

We have identified that there is a relationship or close connection between the presence of BRSV in pneumonic cases of cattle in Iraq. These findings suggest conducting molecular epidemiology investigations in different regions to determine the country’s precise incidence of circulating viruses. Therefore, appropriate preventative and immunization efforts can be performed to avert the emergence of the disease. This study provides more data on the prevalence of Bovine Respiratory Syncytial Virus (BRSV) in the bovine population. Based on these finding, it is recommended that a wider use of the vaccine should be practiced in small animals less than 2 years with pneumonia and the periodic examination when symptoms first appeared. According to our results, the infected age groups ranged from 2 months to 1 year where vaccination is deemed critical.

Acknowledgements

They wish to convey their heartfelt appreciation to the Department of Biotechnology, University of Fallujah, and the Department of Microbiology, University of Al-Kufa, for their invaluable support and resources throughout this study. Special thanks to the veterinary staff and farm owners in Al-Anbar province for their cooperation in sample collection. This research would not have been possible without the dedication and hard work of all contributors.

NOVELTY STATEMENT

This study highlights significant differences in RSV infection rates between Holstein Friesian and Local-cross breeds, as well as between male and female calves, which have not been previously documented in this region. These findings could inform targeted vaccination strategies to mitigate the economic impact of bovine pneumonia.

AUTHOR’S CONTRIBUTION

MAH conceptualized and designed the study, supervised the research process, and contributed to data analysis and manuscript writing. MAA-M was involved in sample collection, molecular analysis, and data interpretation. AFH assisted in the statistical analysis and manuscript review. FRJ contributed to the experimental procedures and data collection. IRA-M and NAH participated in the data collection and provided critical revisions to the manuscript. All authors read and approved the final version of the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

REFRENCES

Antonis AFG, Jong MCD, Poel HMVD, Most RGVD, Zurwieden NS, Kimman T, Schrijver, RS (2010). Age-dependent differences in the pathogenesis of bovine respiratory syncytial virus infections related to the development of natural immune-competence. J. Gen. Virol., 91(Pt 10): 2497-2506. https://doi.org/10.1099/vir.0.020842-0

Bidokhti MR, Traven M, Ohlson A, Zarnegar B, Baule C, Belák S, Liu L (2012). Phylogenetic analysis of bovine respiratory syncytial viruses from recent outbreaks in feedlot and dairy cattle herds. Arch. Virol., 157(4): 601-607. https://doi.org/10.1007/s00705-011-1209-3

Bugarski D, Petrovii T, Milanov D, Lazic S (2011). Seroprevalence of bovine respiratory syncytial virus (BRSV) in Vojvodina. Arch. Vterinarske Med., 4(2): 23-29. https://doi.org/10.46784/e-avm.v4i2.183

Ceribasi AO, Ozkaraca M, Ceribasi S, Ozer H (2014). Histopathologic, immunoperoxidase and immunofluorescent examinations on natural cattle pneumonia originated from Parainfluenza type 3, respiratory syncytial virus, adenovirus type 3 and Herpesvirus type 1. Rev. Med. Vet., 165(7–8): 201–212

Chavez DF, Correa JCS, Marquez LJG, Rubio AP, Flores AGV (2012). Detection on antibodies and risk factors for infection with bovine respiratory syncytial virus andparainfluenza virus 3 in dual-purpose farms in Colima, Mexico. Tropical animal health and production, 44, 1417-1421.

Doggett JE, Taylor-Robinson D, Gallop RG (1968). A study of an inhibitor in bovine serum active against respiratory syncytial virus. Arch Gesamte Virusforsch. 23: 126–137. https://doi.org/10.1007/BF01242120

Hagglund S, Hu KF, Larsen LE, Hakhverdyan M, Valarcher JF (2004). Bovine respiratory syncytial virus ISCOMs-protection in the presence of maternal antibodies. Vaccine, 23(5): 646-655. https://doi.org/10.1016/j.vaccine.2004.07.006

Hodgson PD, Aich A, Manuja A, Hokamp H, Roche FM, Brinkman FSI, Potter A, Babiuk LA, Griebel PJ (2005). Effect of stress on viral-bacterial synergy in bovine respiratory disease, novel mechanisms to regulate inflammation. Comp. Funct. Genom., 6: 244–250. https://doi.org/10.1002/cfg.474

Hussain KJ, Al-Farwachi MI and Hassan SD, 2019. Seroprevalence and risk factors of bovine respiratory syncytial virus in cattle in the Nineveh Governorate, Iraq. Veterinary World 12: 1862-1865. https://doi.org/10.14202/vetworld.2019.1862-1865

Kamdi B, Singh R, Singh V, Singh S, Kumar P, Singh KP, Dhama K (2020). Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India. Microb. Pathogen., 145: 104165. https://doi.org/10.1016/j.micpath.2020.104165

Kamdi B, Singh R, Singh V, Singh S, Kumar P, Singh KP, Dhama K (2020). Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India. Microbial Pathogen., 145: 104165. https://doi.org/10.1016/j.micpath.2020.104165

Maidana SS, Lomonaco PM, Combessies G, Craig MI, Diodati J, Rodriguez D, Romera SA (2012). Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis) in Argentina. BMC Vet. Res., 8: 1-9. https://doi.org/10.1186/1746-6148-8-83

Makoschey B, Berge AC (2021). Review on bovine respiratory syncytial virus and bovine parainfluenza–usual suspects in bovine respiratory disease. A narrative review. BMC Vet. Res., 17(1): 1-18. https://doi.org/10.1186/s12917-021-02935-5

Miao C, Woolums AR, Zarlenga DS, Brown CC, Brown JC Jr, Williams SM, Scott MA (2004). Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on cytokine messenger RNA expression following viral challenge in calves. Am. J. Vet. Res., 65: 725–733. https://doi.org/10.2460/ajvr.2004.65.725

Ogunbiyi PO, Black WD, Eyre P (1988). Parainfluenza-3 virus-induced enhancement of histamine release from calf lung mast cells effect of levamisole. J. Vet. Pharmacol. Ther., 11(4): 338–344. https://doi.org/10.1111/j.1365-2885.1988.tb00193.x

Salt JS, Thevasagayam SJ, Wiseman A, Peters AR (2007). Efficacy of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV and BRSV in experimentally infected calves. Vet. J., 174(3): 616–626. https://doi.org/10.1016/j.tvjl.2006.10.007

Sebeta Hawas Agricultural Office (2011). Annual socio economic profile R.T.N debele, 1999. And Statistical Abstract Report.

Sivula NJ, Ames TR, Marsh WE, Werdin RE (1996). Descriptive epidemiology of morbidity and mortality in Minnesota dairy heifer calves. Prevent. Vet. Med., 27(3-4): 155-171. https://doi.org/10.1016/0167-5877(95)01000-9

Thonur L, Maley M, Gilray J, Crook T, Laming E, Turnbull D, Nath M, Willoughby K (2012). One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine parainfluenza virus 3. BMC Vet. Res., 28: 37. https://doi.org/10.1186/1746-6148-8-37

Valarcher JF, Taylor G (2007). Bovine respiratory syncytial virus infection. Vet. Res., 38(2): 153-180. https://doi.org/10.1051/vetres:2006053

Vilcek S, Elvander M, Ballagi-Pordany A, Belak S (1994). Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples. J. Clin. Microbiol., 32(9): 2225-2231. https://doi.org/10.1128/jcm.32.9.2225-2231.1994

Viuff B, Uttenthal Å, Tegtmeier C, Alexandersen S (1996). Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization. Vet. Pathol., 33(4): 383-390. https://doi.org/10.1177/030098589603300403