Evidence of Crimean–Congo hemorrhagic fever virus in livestock and wildlife in Northeastern Portugal

Abstract

Following the death of an 83-year-old man from the district of Bragança, in north-eastern Portugal due to Crimean–Congo hemorrhagic fever (CCHF), a serological survey was conducted to investigate domestic and wild ruminants. The survey included samples from cattle(n = 94), sheep(n = 30), goats(n = 4), and red deer(n = 10) collected within the affected region and neighboring areas where the human case was reported. CCHF antibodies were detected by ELISA in the serum of sheep, cattle and red deer, corresponding to seropositivity rates of 3.33%, 38.29%, and 60%, respectively, indicating significant exposure to the virus. Indirect immunofluorescence assays further validated the ELISA results. Most of the positive cattle originate from farms located in the Guarda district, which are located close to the Spanish border. None of the goats was positive for CCHFV-antibodies and viral-RNA was not detected in any of the samples. CCHFV-RNA was also not detected in 15 ticks from Dermacentor and Rhipicephalus genera collected from vegetation or cattle, on one of the positive farms. Our findings suggest that CCHFV is actively circulating in northeastern Portugal. Reports of human cases of CCHF in Spain, particularly near the border with Portugal, are consistent with the detection of CCHFV-RNA in ticks feeding on domestic and wild animals in western Spain, highlighting the potential for cross-border transmission and suggesting an established circulation of CCHFV in the Iberian Peninsula.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-10627-5.

Introduction

The Crimean–Congo Hemorrhagic Fever Virus (CCHFV) is the causative agent of Crimean–Congo Hemorrhagic Fever (CCHF), a tick-borne zoonotic disease. CCHFV infection usually presents as a subclinical disease, but in some cases can present as a hemorrhagic fever with a high mortality rate¹. As a result, CCHF is recognized by the World Health Organization as one of the top-priority diseases for research and development in the context of public health emergencies².

CCHFV belongs to the family Nairoviridae and genus Orthonairovirus, and it was first identified in the Crimean region in 1944. The Nairoviridae family is characterized by enveloped, spherical viral particles with diameters ranging from 80 to 120 nm. The virus has a tripartite, single-stranded, negative-sense RNA genome composed of Large (L), Medium (M) and Small (S) segments³. CCHF is primarily transmitted by the bite of infected ticks or through contact with the blood or tissues of infected animals, especially in agricultural or natural settings. Handling tick-infested animals and being close to vegetated areas with high tick densities are significant risk factors for CCHFV infection. While Hyalomma ticks are the main invertebrate hosts and vectors of the virus, CCHFV has been isolated from more than 30 different tick species. However, the mere detection of the virus does not confirm that all of these species are vectors for its transmission⁴.

The geographic range of the virus appears to be expanding since the beginning of the 21 st century, with its distribution closely associated with that of its primary tick vectors⁴ CCHFV has been identified in Africa, Asia, and Europe, namely in Turkey, Bulgaria, Kosovo, Hungary, Albania, Greece, France⁵, Italy⁶ and Spain³. In Portugal, the first serological evidence of CCHFV circulation in humans was found in 1985⁷. Later, in 2022, another serological study was carried out in sheep, which demonstrated a seropositivity rate of 0.4%⁸. Additionally, two more studies reported the presence of antibodies against CCHFV in red deer and wild boars from mainland Portugal, in 2021 and 2024, respectively^4,9.

In July 2024, the death of an 83-year-old man from the Bragança district of Portugal after a confirmed diagnosis of CCHF¹⁰ prompted further epidemiological investigations. Ruminants play a key role in CCHFV epidemiology by supporting tick populations and acting as virus carriers during transient viremia¹¹.

Therefore, to assess virus circulation in ruminants, a serological screening was conducted across several farms in the northeastern mainland of Portugal, as well as in red deer from hunting areas in the same regions. Sera from these animals, originally collected for Bluetongue virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) surveillance were reused for CCHFV surveillance.

The primary objective of our study was to enhance our understanding of the circulation and distribution of CCHFV in domestic and wild ruminants in northeastern mainland Portugal, updating existing data. This could raise awareness of disease risks and contribute valuable information to strengthen national animal health monitoring and control strategies.

Materials and methods

All methods were carried out in accordance with relevant guidelines and regulations, and experimental protocols were approved by the National Institute of Agricultural and Veterinary Research (INIAV I.P.).

Samples

A total of 138 serum samples from cattle, sheep, goats, and red deer (Cervus elaphus) were analyzed. In the case of domestic ruminants, blood was collected from the jugular or the tail vein, while in hunted wild animals it was obtained by cardiac puncture performed post-mortem. Blood was drawn into plain tubes and, after coagulation at room temperature, samples were centrifuged for 10 min at 1500 g at 4 °C. The serum was preserved at − 20 °C until analysis.

These samples originated from 35 farms and 10 hunting areas located in various regions of northern and eastern Portugal (Fig. 1). The sera, mainly from adult female cattle, were collected between July and October 2024 as part of the national surveillance programs for Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) and were submitted to the National Institute of Agrarian and Veterinary Research (INIAV, I.P.) for virological diagnosis. This opportunistic sampling focused on the Bragança region, where the human case was identified, as well as on nearby northeastern areas along the Spanish border, where locally acquired human cases have previously been reported.

Fig. 1.

Map of sampling areas in northern and eastern mainland Portugal comprising Bragança, Vila Real, Viseu, Guarda and Castelo Branco districts. Icons indicate the sampled animal species. Under each species the number of samples considered is indicated along with the number of farms from which they belong to.

Ticks (n = 9) were collected from the surrounding vegetation of a CCHFV seropositive cattle farm located in the municipality of Sabugal, within the Guarda district on September 3rd using the blanket dragging method, where white fabric strips were systematically dragged across vegetation in animal resting areas to attract and capture ticks¹². Additionally, ticks were also collected from cattle (n = 6) in the same farm in mid-October. All specimens were transferred to microtubes containing 70% ethanol (v/v).

Tick classification

Ticks were identified to the genus level based on morphological characteristics using a stereomicroscope and standard taxonomic keys^13,14. Identification focused on key distinguishing traits specific to Hyalomma species, the primary vectors of CCHF.

To enhance accuracy of the morphological classification, molecular confirmation was performed using 16 S ribosomal DNA sequence analysis, as previously described¹⁵.

Antibody ELISA

Antibody detection in sera was performed using the ID Screen CCHF Double Antigen Multispecies ELISA kit, following the manufacturer’s instructions (IDvet, Grabels, France). ​The Sample-to-Positive (S/P) ratio was calculated by dividing the optical density (OD) of each sample by the OD of the positive control, and then multiplying the result by 100 to express it as a percentage (%S/P) (Supplementary Table 1) to provide a quantitative measure of the sample’s antibody concentration relative to the control, with higher values indicating stronger antibody presence and potential exposure to the target pathogen. Samples with a %S/P > 30 were considered positive. Each ELISA plate validation was performed as recommended by the manufacturer.

Indirect Immunofluorescence assay

A subset of 9 ELISA-positive and 3 ELISA-negative bovine sera and 4 ELISA-positive red deer sera were tested by a commercial immunofluorescence biochip (Euroimmun, Lübeck, Germany).

The selected sera were tested under different dilution conditions to optimize the signal-to-noise ratio in the immunofluorescence assay. The serum samples were diluted to 1:50, 1:100, and 1:200, using both PBS-Tween and the sample buffer provided with the commercial kit. Concurrently, the FITC-conjugated secondary antibody was tested at dilutions of 1:100, 1:200, and 1:500 in PBS containing 1% BSA. All conditions were tested in quadruplicate to ensure the reproducibility and consistency of the results. The combination of conditions that yielded the strongest and most specific fluorescence signal with minimal background was subsequently adopted for final image acquisition.

Each serum was added to wells containing CCHFV-GPC (glycoprotein precursor), CCHFV-N (nucleocapsid protein) and EU 90 (control-transfected cells) antigens in three different locations. All the sera were diluted 1:50 in sample buffer from the kit and incubated for 45 min at 25 ºC in a humid chamber.

After two washes (1 min and 5 min) with shaking in PBS-Tween, the secondary antibody diluted 1:100 in PBS with 1% BSA (Thermo Fisher Scientific, MA, USA) was incubated for 30 min at 25ºC in a humid chamber followed by two washes (1 min and 5 min) with shaking using PBS-Tween. A Rabbit anti-Bovine IgG (H + L) Secondary Antibody, FITC (Invitrogen, MA, USA) and a Rabbit anti-Deer IgG (H + L) Secondary Antibody, FITC (Seracare, MA, USA) were used for bovine and deer serum samples, respectively.

Slides were mounted using antifade mounting medium with DAPI (Vectashield, CA, USA) and observed on a Nikon Eclipse TI-FL inverted microscope.

Nucleic-acid extraction and viral RNA detection

Individual ticks were homogenized at 20% (w/v) with phosphate-buffered saline (PBS) under mechanical homogenization with 0.5 mm zirconium beads (Sigma-Aldrich, St. Louis, Missouri, EUA) using four cycles of 15 s at 3000 rpm with an interval of 10 s (Precellys^® Evolution, Belgic) and then clarified at 3,000 g for 5 min.

Total nucleic acid was extracted from 200 µL of the clarified tick supernatant or blood samples, using the IndiMag^® Pathogen Kit (Indical, Leipzig, Germany) in a KingFisher Flex extractor (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Nucleic acids were preserved at −20 °C until use. The extraction process was validated using an 18 S rDNA qPCR¹⁶ and an RT-qPCR for the detection of spiked synthetic RNA (VLP-RNA EXTRACTION, Meridian Life Science, Memphis, TN, USA), which was added to the sample (4 µL/sample) prior to extraction.

Detection of the CCHFV genome in blood samples and in ticks was performed using RT-qPCR targeting the nucleoprotein gene (S segment), as described by Koehler et al.¹, with the One-step NZYSpeedy RT-qPCR Probe kit (NZYtech, Portugal).

Results

CCHFV seroprevalence

Antibody detection was carried out using the ID Screen CCHF Double Antigen Multispecies ELISA kit (IDVET), which identified 36 positive cattle samples out of 94 and 6 positive red deer samples out of 10 for CCHFV antibodies. The total sample set was collected from 19 cattle farms and 10 hunting areas. The positive cattle originated from two of the five districts investigated (Castelo Branco and Guarda), resulting in a mean seroprevalence of 38.3% (95% CI: 28.7–48.7). All 6 positive red deer originated from a single district (Castelo Branco), corresponding to a seroprevalence of 60.0% (95% CI: 26.2–87.8). In terms of farm-level positivity, 7 out of the 19 cattle farms (36.84%) had animals testing positive for CCHFV antibodies (Table 1).

Table 1.

CCHF ELISA results by species and sampling regions of the north-eastern Mainland Portugal.

District	No. of Farms/Hunting areas investigated	No. of Cattle tested Pos/Total (PP%)	No. of Sheep tested (Pos/Total) (PP%)	No. of Goats tested (Pos/Total) (PP%)	No. of Red deer tested (Pos/Total) (PP%)	CCHF-Ab Positive farms/hunting areas
Bragança	2	0/28 0%	0	0	0	0
Castelo Branco	23	2/8 25.0%	1/14 7.14%	0/4 0%	6/10 60%	8
Guarda	19	34/55 61.82%	0/16 0%	0	0	5
Viseu	1	0/2 0%	0	0	0	0
Vila Real	1	0/1 0%	0	0	0	0
Total	46	36/94 38.29%	1/30 3.33%	0/4 0%	6/10 60%	13

PP-percentage of positivity in the sample.

In cattle, seropositivity per district ranged from 0% (Brangança, Viseu and Vila Real) to 61.82% (Guarda district).

Twenty-four cattle that tested positive for CCHFV antibodies in the ELISA, originating from two farms (sera order number 1 to 3, 6, 8 to 13, 18,19, 22, 23 and 55 to 64, Supplementary Table 1) in the municipalities of Sabugal and Almeida within the Guarda district, were re-sampled and retested after two months, maintaining their seropositivity.

CCHF antibodies were detected in only 1 (serum order number 133, Supplementary Table 1) of the 30 sheep serum samples analyzed, while none of the 4 goats tested had a positive result in the ELISA.

The ELISA manufacturer reports 100% specificity and 96.9% sensitivity with no cross-reactivity to Hazara, Dugbe, or Nairobi viruses. Nonetheless, a small set of samples (including positive and negative samples) was used to be tested by Fluorescent Antibody Test (FAT) to confirm the CCHF ELISA serological results, given their novelty.

Nine ELISA-positive bovine samples (sera 1 to 3, with %S/P of 154.19, 176.73 and 156.77, respectively, serum 23 with a %S/P of 55.73, sera 59 to 62 with %S/P of 203.55, 113.29, 183.59 and 185.92, respectively, and serum 82 with a %S/P of 104.87, Supplementary Table 1) were tested by IFT, of which one (serum 23) was negative and one was weakly positive (serum 82), giving 88% concordance between the two tests. Three ELISA-negative samples were tested (sera 14 to 16, with %S/P of 5.76, 6.49 and 29.88, respectively, Supplementary Table 1), and all three were negative by IFT. Regarding red deer sera, only four ELISA-positive samples could be tested by IFT, namely sera 102 (%S/P of 163.99)), 109 (%S/P of 35.08), 110 (%S/P 151.63) and sera 138 (%S/P of 39.88), Supplementary Table 1), of which two were positive (sera 102 and 110) and two, classified as weak positive by ELISA, were negative by IFT (sera 109 and 138), representing a concordance between the two tests of 50%. This discrepancy may be explained by the lower sensitivity of IFT test¹⁷.

In IFT antibody-positive samples, fine to coarse granular fluorescent structures were observed in the cytoplasm of cells transfected with the viral antigen, more evident with the CCHFV-GPC antigen. This distinct green fluorescence contrasts with the unstained surrounding non-transfected cells (Fig. 2A, B, D, E). In the control wells (EU 90 control-transfected cells), no cells showed the same type of fluorescence (Fig. 2C and F)). The same lack of fluorescence was observed in the ELISA antibody-negative bovine samples, where no cytoplasmic staining was detected against the three antigens (results not shown). Negative deer samples were not tested by IFT due to lack of material.

Fig. 2.

Immunofluorescence of ruminant sera for positive detection of CCHFV-antibodies. Antigens (GPC and N) or MOCK cells are indicated at the top of the figures. Green represents antibodies recognizing the viral antigens. Blue stains nuclei DNA. A, B, C- ELISA positive bovine sample (serum 2, Supplementary Table 1); D, E, F– ELISA positive deer sample (serum 102, Supplementary Table 1). 100x total magnification.

CCHFV RNA investigation of bovine blood

Eighty cattle blood samples, including all CCHF-antibody positive samples (representing about 85.11% of the samples tested by ELISA), along with 10 red deer blood samples, were also tested for CCHFV RNA by the method described by Koehler et al.¹⁸. None of the samples revealed to have CCHFV RNA.

Tick classification and virological evaluation

Twelve of the ticks collected from one of the bovine farms were classified into the Dermacentor genus and three into the Rhipicephalus genus by morphological observation. No ticks from the Hyalomma genus, which is the primary vector for CCHFV, were found.

Analysis of the partial 16 S rDNA region revealed sequences with > 98% homology with the species Rhipicephalus bursa and Dermacentor marginatus (results not shown), confirming the morphological observation.

Despite collecting ticks on two different days, the number of ticks obtained was very low (n = 15), with half being collected from the cattle and the rest from the surrounding vegetation. At the time of blood collection, the animals were in good health status and parasite-free. Notably, the tick samples were collected in September and October, which is outside the peak activity period for ticks, which occurs from May to September in Iberian Peninsula. As previously reported, 60% of Spanish patients were infected during the summer, while 40% were infected in the spring¹⁹. Tick activity begins to decline as temperatures drop, which could explain the low number of ticks collected and the absence of Hyalomma specimens.

Ticks collected during this study were tested for CCHFV RNA by the method described by Koehler et al.¹⁸, with no RNA detection.

Discussion

CCHF is recognized as the most geographically widespread tick-borne viral infection globally, with a concerning fatality rate of up to 62% in humans. This high lethality underscores the severity of the disease and highlights the urgent need for effective interventions. Unfortunately, there are currently no vaccines or treatments approved by regulatory agencies in the United States or Europe, leaving a significant gap in public health preparedness and response²⁰.

The disease caused by CCHFV is primarily human-specific, however livestock and wild animals can harbor the virus asymptomatically through transient viremia, which may persist up to 15 days. This asymptomatic viremia in animals complicates surveillance and control efforts, as it can remain undetected while still contributing to the virus’s transmission cycle. Notably, ruminants are recognized as key reservoirs of CCHFV and have been linked to human cases in endemic areas. Following the discovery of a human case in Portugal last year, and the findings of our present study demonstrating the circulation of CCHFV not only in wild ruminants but also in bovines, there is an urgent need to raise awareness about the risk of infection, particularly due to the widespread presence of Hyalomma ticks in the country²¹. Enhanced vector surveillance, particularly on tick samples from ungulates, such as red deer, and comprehensive serosurveys in wildlife, livestock and human populations, are urgently needed, as suggested by other researchers¹⁰.

In our study, we detected antibodies indicative of CCHF virus circulation in cattle, sheep and red deer in the Castelo Branco and Guarda districts of Portugal, using a commercial competitive ELISA. In positive sera %S/P values ranged from 55.73 to 241.52% in cattle and between 35.08% and 200.59% in red deer (Supplementary Table 1). To confirm the accuracy of our results, we validated the ELISA findings using the fluorescent antibody test (FAT), further ensuring the reliability and specificity of the serological tests. This provides solid evidence of CCHFV presence within local livestock populations.

The small number of sera from small ruminants analyzed does not allow for conclusions to be drawn regarding the circulation of the virus in goats and even in sheep (with only one positive result). However, previous studies conducted in our country have reported seroprevalence percentages in sheep around 0.74%⁸.

In contrast, the high seroprevalence of CCHFV in cattle, underscores their role as maintenance hosts for the virus. Additionally, the detection of CCHFV seropositive cattle aged three years, well beyond the period when maternal antibodies would typically have waned, suggests that the virus has been circulating in the region in the recent years. Also, none of the seropositive cattle analyzed for the presence of viral RNA tested positive, suggesting that the viremia phase had already passed and that the infections had occurred some time ago. Testing of blood samples collected two months later from 24 CCHF seropositive cattle confirmed the persistence of CCHFV-specific antibodies.

Six of the ten red deer tested were found to be positive for CCHF antibodies, a finding that is consistent with results obtained in Spain where wild Cervidae are known to play a key role in the transmission cycle of the virus²².

Wild ungulates, in particular red deer, serve as key hosts for Hyalomma ticks in Iberia, which are the primary competent vector of CCHFV. As noted by previous studies²², red deer may be an ideal model for understanding the ecological factors influencing CCHFV transmission and for predicting human infection risks, given that they are the main host for Hyalomma lusitanicum.

In our study, no ticks from the Hyalomma genus were collected likely due to the timing of tick sampling in relation to their seasonal activity, despite their presence in the country²³. Furthermore, it cannot be ruled out that ethanol preservation may have compromised nucleic acid integrity, contributing to the negative PCR results obtained in the specimens collected.

While the detection of antibodies in cattle and red deer confirms the virus’s presence, our study could not determine the exact duration of its circulation. Additionally, the small number of samples analyzed, originating from only a few farms and districts within the country, does not allow for a comprehensive overview of the virus’s circulation across the entire mainland territory.

Animals infected with CCHFV experience short periods of viraemia (approximately 5 days) without showing signs of disease, and although vertebrates develop these brief viremias, they play a vital role in maintaining tick vector populations by providing blood meals and enabling the transmission of CCHFV to ticks²⁴.

Although the sample size was reduced due to the opportunistic nature of sampling, the high percentage of positive animals detected in few farms and hunting areas of Guarda and Castelo Branco districts provided valuable insights into the spread of the virus among livestock. These findings combined with evidence from wild Cervidae, suggests that both livestock and wildlife may serve as reservoirs for CCHFV in these regions. Moreover, the findings of this study highlight that cattle and red deer are excellent sentinels for CCHFV serological surveillance. Cattle samples can be easily collected through existing disease monitoring programs in the country, therefore incurring no additional costs or animal handling for sampling. Additionally, cattle movements are either geographically restricted or can be traced with ease, which is critical for drawing reliable epidemiological conclusions. As a result, healthcare professionals should remain vigilant for potential new cases of CCHF in these high-risk areas.

As in Portugal, human cases of CCHFV infections were also reported in Spain in 2024, specifically in the Salamanca region¹⁹, which is located approximately 100 km from the Portuguese border.

In light of these findings, the implementation of active surveillance strategies by national authorities appears to be a crucial measure to better identify high-risk areas in mainland Portugal. Furthermore, public awareness campaigns should emphasize preventive measures to reduce tick exposure, with particular focus on livestock owners, animal health professionals, and individuals engaged in outdoor activities.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

M.D.D. and S.S.B. wrote the main manuscript; M.D.D., S.S.B., and F.A.A.S. delineated the study. F.A.A.S. performed the Indirect Fluorescent Antibody Test (IFT) and prepared Figure 2; D.M. extracted DNA from ticks; S.S.B., F.A.A.S., A.D., and M.H. carried out the Enzyme-Linked Immunosorbent Assays (ELISAs); A.C. and A.L. collected blood from bovines; F.A.A.S., M.D., and A.L. collected the ticks in the field; L.B. and M.P. conducted the morphological characterization of the ticks; A.R.V. performed molecular analyses of the ticks; S.S.B, F.A.A.S., M.D.D. analysed the data. All authors reviewed the manuscript.​.

Funding

This research received no external funding.

Data availability

The datasets generated and/or analysed during the current study are not publicly available due to the fact that molecular data used solely for tick species confirmation were not retained or deposited, as they were limited to partial 16 S rDNA sequencing and are not relevant to the study’s conclusion but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study was conducted in full compliance with all relevant institutional and national guidelines and regulations, as well as the ARRIVE guidelines. Serum samples from domestic animals (cattle and sheep) were obtained as part of routine veterinary surveillance activities conducted under official animal health programs, and no animals were specifically handled or sampled for the purpose of this research. Serum samples from wild Cervidae (red deer) were obtained from animals legally hunted during the official hunting season, in compliance with national wildlife and hunting regulations. No animals were euthanized specifically for this study.