Diagnostic Techniques for Infectious Bovine Rhinotracheitis: A Review

Review Article

Austin J Vet Sci & Anim Husb. 2022; 9(4): 1102.

Diagnostic Techniques for Infectious Bovine Rhinotracheitis: A Review

Dima C¹* and Abdisa K²

1Animal Health Institute, Sebeta, Ethiopia

2Ambo University, College of Agriculture and Veterinary Science, Ambo, Ethiopia

*Corresponding author: Chala Dima, Animal Health Institute, P.O. Box: - 04, Sebeta, Ethiopia

Received: August 24, 2022; Accepted: September 15, 2022; Published: September 22, 2022


Infectious Bovine Rhinotracheitis (IBR) is a group of the bovine respiratory disease multifaceted pathogens and a key disease of cattle, leading to significant economic losses to the dairy industry globally. The causative agent of IBR is Bovine herpesvirus type-1 (BoHV-1), which is a member of the genus Varicellovirus in the Alphaherpesvirinae subfamily, which belongs to the family Herpesviridae, order Herpesvirales. BoHV-1 can be categorized into three subtypes (BoHV-1.1, BoHV-1.2a, and BoHV-1.2b) that belong to one single viral species, which is a serologically indistinguishable strain. Therefore, a more optimal method for the rapid diagnosis of BoHV-1 infection is highly needed. Hence, the objective of this paper is to review the appropriate diagnostic techniques for the IBR virus in infected cattle. In this review, various rapid and confirmatory diagnostic methods used for the diagnosis of BoHV-1 infection were briefly described. BoHV-1 can be routinely detected by virus neutralization tests and enzyme-linked immunosorbent assays (indirect or blocking ELISA). IBRgEELISA is the most specific serological test for BoHV-1 and is recommended for marker vaccine to differentiate wild infection from vaccination schemes. Furthermore, virus isolation from tissue or swab samples by cell culture and DNA detection with LAMP, PCR, and real-time PCR techniques are all used to detect infected cattle. Direct sequencing of the entire genome using the Sanger sequencing method recently allowed for the differentiation of BoHV-1 subspecies and the distinction of the BoHV-1 field strain from vaccine strains based on single nucleotide polymorphisms. As the gold standard diagnosis for IBR is virus isolation in cell culture, commonly followed by BoHV-1 gene sequencing, it is also recommended.

Keywords: Bovine herpesvirus; Diagnostic techniques; ELISA; IBR; PCR; Virus neutralization


Infectious Bovine Rhinotracheitis (IBR) is one of the bovine respiratory disease multifaceted pathogens, which is the major cause of cattle death around the world [1]. IBR is a key disease of cattle, causing considerable economic losses to the dairy industry globally [2,3]. BoHV-1 is a member of the genus Varicellovirus in the Alphaherpesvirinae subfamily which belongs to the family Herpesviridae, order Herpesvirales [4,5] and it is an etiological agent responsible for the development of a severe respiratory form of infection known as IBR in all cattle [6,7], and Infectious Pustular Vulvo-Vaginitis (IPV) and Infectious Pustular Balanoposthitis (IPB) in cows and bulls respectively [8].

Bovine herpesvirus type-1 is also one of the most essential viral infections of buffaloes globally [9], except in the BoHV-1 free countries [10]. BoHV-1 can be sub-grouped into three-subtypes belonging to one single viral species [11], in which they are serologically indistinguishable strains (BoHV-1.1, BoHV-1.2a, and BoHV-1.2b) [12]. Those animals infected with BoHV-1 experience a variety of mild to severe clinical syndromes, including rhinotracheitis, vaginitis, balanoposthitis, abortion, conjunctivitis, and enteritis, as well as decreased milk production and weight gain [2], and cough, nasal discharge, and lachrymal discharge with mild diarrhea [13].

Clinical indicators, species affected, epidemiological pattern, post-mortem lesions, and laboratory confirmation by isolation of the etiological agent utilizing different serological and molecular approaches can all be used to make a preliminary diagnosis of IBR. Virus Isolation (VI), Fluorescent Antibody technique (FAT), antigen detection by ELISA, and immunoperoxidase assays are being employed in diagnostic virology laboratories to identify BoHV-1. The indirect fluorescent antibody test (IFA) detects immunoglobulin M (IgM) and G (IgG) antibodies in serum in a semi-quantitative, sensitive, and rapid manner [14]. BoHV-1 abortion is diagnosed by the existence of appropriate histological lesions, VI, and Immunohistochemistry (IHC) for viral antigen identification. With sensitivity similar to that of IHC, BoHV-1 has been detected in tissues [15].

The in-vitro diagnosis of the disease based cell lines, on the other hand, is difficult to maintain, making the procedure inconvenient, time consuming, and expensive. IBR diagnostic procedures frequently employ VI in cell culture. The infectiousness of the viral particle is not required for enzyme immunoassays for antigen and antibody detection, but the results are harmed if the virus is destroyed. As a result, insufficient sample preservation and transit to the laboratory has a negative impact on the diagnosis [16]. The development of nucleic acid approaches for the identification of viruses in clinical material has received recent interest in diagnostic virology. Indirect Immunofluorescence tests (IFAT), Real-Time Polymerase Chain Reaction (RT-PCR), and nucleic acid hybridization are also often utilized for BoHV-1 diagnosis [17].

Although Polymerase Chain Reaction (PCR) in conjunction with southern blot hybridization has been shown to be better sensitive than VI and dot-blot hybridization screening, a large number of extended semen by these techniques is time-consuming. Because of its rapidity, sensitivity, and specificity, PCR has been developed as an ideal diagnostic tool for the detection of BoHV-1 in a variety of clinical samples [16]. As a result, efforts have been undertaken to standardize and verify a real-time PCR protocol [18] for detecting BoHV1 in naturally infected cattle and buffalo, as well as to use a sensitive, specific, and repeatable technique [19]. Also, a PCR technique was used to identify the BoHV-1 gB and gE genes in semen samples from naturally infected bulls, and the sensitivity was determined [8].

Generally in the context of animal health control and disease prevention, having access to accurate diagnostic tests having the ability of identifying early outbreaks and/or ensuring the nonexistence of the disease within vast territorial extensions is crucial [20]. As a result, early diagnosis of IBR virus-positive animals is critical for disease management and eradication efforts aimed at easing the burden on the dairy sector. Therefore, the goal of this paper is to review the appropriate diagnostic techniques recently in use for the diagnosis of infectious bovine rhinotracheitis virus in infected cattle.

Literature Review

Diagnostic Techniques for IBR

Currently, available diagnostic techniques are limited to the laboratory and require the use of sophisticated tools by specially trained staff members. To differentiate the BoHV-1 subtypes, numerous techniques have been utilized, including monoclonal antibody-specific antigen and DNA fingerprinting by recognizing the existence of the restriction site [21]. Bovine herpesvirus type-1 can be routinely diagnosed by cell culture, ELISA, virus neutralization tests, and molecular techniques by Polymerase Chain Reaction (PCR) [22].

Although in-vitro viral isolation in cell culture is still the gold standard for detecting BoHV-1, it has drawbacks in terms of sensitivity, sperm cytotoxicity, time-consuming, and expense. Several PCR approaches for detecting BoHV-1 have been shown to be effective [19]. The loop-mediated isothermal amplification (LAMP) assay has been proposed as a simple, quick, and alternative molecular pathogen diagnostic tool for field testing [23]. In order to differentiate BoHV-1 field strain from the vaccination strain based on Single Nucleotide Polymorphisms (SNPs), full genome sequencing was recently required [24,25].

Serological tests: The serological tests frequently used for the diagnosis of BoHV-1 antibodies in the serum samples of animals comprise the indirect ELISA, blocking ELISA, and the Virus Neutralization Test (VNT) [15]. Because of the short-time it takes to get a response, its convenience for screening large numbers of serum samples, and the best performance of any serological test used for IBR diagnosis, the indirect ELISA is utilized more often. Furthermore, because BoHV-1 viral latency is common, identifying serologically positive and otherwise healthy animals might be a good predictor of infection level in a herd. As a result, antibodiespositive animals must be categorized as BoHV-1 infected (with two exceptions: serological responses caused by inactivated vaccine immunization or colostral antibodies) [26]. Hence, IBRgE blocking ELISAs discriminate antibodies against the absent antigen, allowing infected and vaccinated animals to be distinguished (DIVA). Because the virus might reactivate during stress or sickness, blood should be drawn for antibody testing during the acute phase and again 2 - 4 weeks later [27].

A. Indirect ELISA: This diagnostic technique is used with coated antigens into the wells of a polystyrene plate. Antibodies bind to the coated antigen and are identified using enzyme-labeled antibovine immunoglobulins if present [28]. The number of antibodies in a sample can be represented in a variety of ways, but in general, the greater the Corrected Optical Density (COD), the more antibodies there are in the sample. Because of the backdrop of negative samples, cut-off numbers may vary, although they will be determined within each test [27] (as illustrated in Figure 1 below).