Study of molecular diagnosis and viremia of bluetongue virus in sheep and cattle

Abstract

Bluetongue virus (BTV) is an RNA virus that infects cattle and sheep. The objective of this study was to compare two real-time PCRs for the detection of BTV and to monitor Orbivirus viremia in sheep and cattle for 6 months. The PCR results showed the occurrence of infected animals throughout the experiment without records of clinical signs. The number of positive animals reduced during the experiment, but some animals were positive for BTV RNA during the entire experiment. The performance of the two RT-qPCRs for BTV detection techniques used in this work revealed a kappa index of 0.71 for cattle and 0.75 for sheep.

Introduction

Bluetongue virus (BTV) belongs to the genus Orbivirus, family Reoviridae [1]. This infectious agent is transmitted by vectors and can cause disease characterized by variable clinical signs, even with the possibility of asymptomatic animals. In mild cases, BTV causes swelling and cyanosis of the tongue, fever, and excoriations in the mouth that can lead to lesions caused by opportunistic bacteria. Severe cases might result in animal deaths due to loss of appetite, ruminal arrest with bloody diarrhea, and progressive weakness [2].

BT may occur in several ruminants, but sheep are normally the most susceptible animal to contract the disease. Transmission occurs mainly through bites from the insect of the genus Culicoides (Diptera: Ceratopogonidae) [3], which are remained mainly from tropical, subtropical, and temperate areas. The distribution of BT in the world is related to the presence of vectors in nature. It is influenced by climatic factors and their interference in insect behaviors [4]. Twenty-seven serotypes of this virus have already been characterized, and some of them might be transmitted via placental or vector dependent; nevertheless, these routes are not epidemiologically significant [3].

Although there are few records of clinical signs, BTV is considered endemic in Brazil, and the virus might be detected in domestic and wild ruminants [5]. The diagnosis of BT is of great importance to the World Organisation for Animal Health. This is a compulsory notification disease that leads to a wide range of sanitary problems. Serological and molecular tests are used in diagnosis, among them the real-time PCR technique [5]. Not only is this type of molecular test highly sensitive and specific but it offers the advantage of fewer sample manipulation steps reducing the risk of cross-contamination and false results.

The objective of this study was to validate and compare two RT-qPCR for the diagnosis of BTV viremia in sheep and cattle for 6 months.

All the experiment was done using samples sent to the Brazilian Federal Laboratory of Agriculture and Livestock Defense for diagnosing of bluetongue disease. Samples were collected for routine diagnose of a herd located on a farm in the city of Pedro Leopoldo, in the state of Minas Gerais, Brazil. Thirty-four cattle and 33 sheep classified as adult animals with non-defined breeds were analyzed. The herds were extensively monitored for 6 months, from January to June 2015. All animals were clinically evaluated by veterinarians before the start of the experiment and throughout the sample collection. Six collections (C1–C6) were performed on both cattle and sheep. The presence of antibodies against BTV was analyzed using agar gel immunodiffusion (AGID) [5].

Blood samples were properly collected, prepared, and submitted to serological and molecular tests for BTV detection. The RNA was extracted using Trizol, according to the manufacturer’s instructions (Invitrogen, USA). Extraction controls were added to this procedure to verify whether there was any contamination. The presence of inhibitors was evaluated by performing RT-qPCR for detection of the beta-actin mRNA [6, 7].

The RNA was extracted from all blood samples collected and molecular assays were conducted to detect BTV. Two RT-qPCRs were used for the detection of BTV. The first RT-qPCR targets the highly conserved genome segment 1 (RT-qPCR.S1) [8], while the second one targets the genome segment 10 (RT-qPCR.S10) [9] (Table 1, Fig. 1).

Table 1.

Results for RT-qPCR.S1 and RT-qPCR.S10 obtained for cattle and sheep during 6 months

	January	February	March	April	May	June	January	February	March	April	May	June
	RT-qPCR.S1 (bovine)						RT-qPCR.S1 (ovine)
Average Cq	31.87	33.58	33.79	35.14	35.22	34.67	33.838	36.22	34.4	34.91	33.99	35.43
Std. Dev	3.10408	3.062	2.959	3.852	2.209	1.654	3.75061	3.679	2.491	2.523	2.614	2.692
Max cq	37.9	40.26	38.95	38.98	37.26	37.68	39.35	40.87	38.32	37.78	37.58	39.09
Min Cq	25.62	28.45	27.06	24.61	30.86	31.89	29.86	28.99	30.65	30.6	31.43	30.46
	RT-qPCR.S10 (bovine)						RT-qPCR.S10 (ovine)
Average Cq	35.2913	35.07	35.63	37.48	40.29	38.84	33.4475	36.04	38.69	38.6	38.05	37.35
Std. Dev	2.76761	2.848	2.062	3.746	3.027	1.872	3.31498	3.146	3.437	2.752	3.624	3.301
Max cq	39.76	41	39.15	45.51	44.71	43.93	35.89	39.74	43.92	41.63	43.97	40.25
Min Cq	30.19	30.95	30.97	31.47	33.07	34.49	28.79	31.43	34.29	34.49	32.02	32.23

Fig. 1.

Cq variation for each month for both RT-qPCR and species tested. The last graphic shows weather data for the period covered in this study

Periodic clinical evaluations of cattle and sheep did not detect any sign of infectious disease. During 6 months, there were no records of clinical signs similar to BT. All animals, except for one bovine and two sheep, were serologically positive for BTV, even though they did not present any clinical signs during the whole period of the study. The discrepancy between the serological and the molecular tests can be explained by the sensitivity of the tests. Low viremia can make BTV detection difficult. Some of the animals may have developed positive serology before collecting material for molecular testing. Also, AGID may have cross serology other orbiviruses, especially those against the epizootic hemorrhagic disease (EHD) [5]. During the execution of the work, EHDV was detected by RT-PCR (data not shown).

Analysis by RT-qPCR did not show the same pattern of positives samples throughout the study, despite the high incidence of viremia on the herd. The number of positive cattle in at least one of the RT-qPCR reduced from 82.8 to 60% from the first to the last collection. The number of positive samples was significantly lower in sheep. The results showed 34% from the first to the fifth collection and 31% in the last one. The performance of the two RT-qPCR techniques used in this work was evaluated by the kappa index. The analysis revealed an index of 0.71 for cattle and 0.75 for sheep.

Animals from both herds with a Cq value below 32 showed positive results from the start to the end of the experiment. A reduction of these values was observed in the course of the months (Fig. 1). The Cqs values decreased slowly, sometimes being quite similar between the months, and then being reduced in 1 or 2 Cq cycles. Animals with initial Cqs equal to or over 32 revealed an increase of these values. It was observed a variation between 37 and 39 in the third collection, but, subsequently, this value changed to no Cq, which means that the same animals were diagnosed as negative in the fourth collection. Only two animals from the cattle herd were negative in the RT-qPCR in the first collection. However, they became positive in the second collection and this result was kept for the remainder of the experiment.

Although BTV is endemic in Brazil, the presence of clinical signs in both sheep and cattle is considered unusual [10]. BTV presents persistent viremia, especially in cattle with a high infection rate. The period of viremia in cattle seems to be long according to our study and other publication. The viral RNA was detected during this 6-month study. Although different experiments state that it could last up to nine weeks [11] or up to 22 weeks, the virus infections can be also isolated for up to 6 weeks [12].

Data from this work indicate that the viral RNA persisted in the host for more than 9 weeks, as was confirmed by two RT-qPCRs. The amount of BTV nucleic acid decreases with time. Probably, animals with Cq above 32 had previously been infected and the virus was eliminated from the bloodstream after the first four collections (from January to April) (Fig. 1). This result could be partially explained by the increase in the number of mosquitos during the rainy period in Brazil, which begins in October and extends until March of the following year. There is a high proliferation of Culicoides during these months, which facilitates the chances of the animal being infected by BTV [13, 14].

BTV is widely distributed in Brazil. Serological data indicate a prevalence of 1% in the South region and up to 40% in the Southeast region, and this factor is associated with the presence of the vector in the environment [15]. However, in endemic areas, animals develop few or no clinical signs [16]. The circulation of BTV may occur even after the rainy season, given the long viremia and the incidence of the vectors even in small populations. In this, only one sheep has shown evidence of a new infection. The RT-qPCR results change from negative (C1–C4) to positive (C5 and C6). The Cq values were 37 and 38 (C5) and 33 and 36 (C6). The high values of Cq and its reduction in the subsequent tests corroborate the possibility of a new infection [17].

No clinical signs were detected in this study, neither at the beginning of the experiment nor in prolonged infections. This molecular tool has been utterly important to diagnose BTV. However, the selection of the best genomic region to be used in diagnosis methods needs to be considered carefully. At some point in this study, both techniques failed to detect positive animals, especially after the third month of viremia. It is still necessary to determine if the higher Cq values have any clinical relevance since previous studies indicate that the virus is no longer infectious [12].

The epidemiology of BTV in Brazil is still not completely understood. Although this study used as reference literature based on conventional methods in diagnosis, such as serological tests or conventional PCR, it is important to outline that the RT-qPCR technique is extremely useful to verify the occurrence of the virus on animals and its circulation in nature. Through RT-qPCR, not only are researchers able to investigate deeply asymptomatic viremia from blood samples but they may also determine the viral load, a factor that cannot be measured with end-point PCR.

Author contributions

Acquisition, analysis, or interpretation of data: Marcela Gasparini, Mateus Laguardia-Nascimento, Érica Bravo Sales, Anna Gabriella Guimarães Oliveira, Zélia IP Lobato, Antônio Augusto Fonseca Júnior. Drafted the work or revised it critically for important intellectual content: Antônio Augusto Fonseca Júnior.

Funding

This work was funded by Ministério da Agricultura, Pecuária e Abastecimento, INCT Pecuária, and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico) SAGRES Project: 457417/2012–9.

Data availability

Not applicable.

Code availability

Not applicable.

Declarations

Ethical approval

No animals were harmed during this study. Samples were collected as a routine for diagnostic of viral diseases in the official laboratory of the Brazilian Ministry of Agriculture, Livestock, and Supply.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.