Integrative serological and molecular survey of Crimean-Congo hemorrhagic fever in livestock animals (Cow, Sheep, and Goats) in Sulaymaniyah/Iraq

1. Introduction

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic virus transmitted by ticks that poses a substantial public health threat in Africa, Europe, and Asia Schulz et al. (2021). and Atwan et al. (2024) have highlighted its widespread distribution. Initially discovered in the Crimean region of Russia in 1944 and subsequently in the Congo Basin in 1967 (Belobo et al., 2021), CCHF exhibits sporadic occurrence with a mortality rate of approximately 30% (Fillâtre et al., 2019), which is directly related to the genus Hyalomma, as evidenced by several reported CCHF cases documented from the Middle East, Asia, Africa, Eastern, and Southern Europe. This is evident from the reported CCHF cases, documented in the Middle East, Asia, Africa, Eastern and Southern Europe, with a high mortality rate of 30% (Shahhosseini et al., 2021). Additionally, it is endemic to some areas of Asia, Africa, and Europe. Besides, there have been many reports of peak case numbers globally in various countries, including Afghanistan, Iran, Pakistan, Iraq, and Turkey (Tahir et al., 2024). CCHF virus is transmitted to humans through the genus Hyalomma and is transmitted by H. marginatum as the primary vector of the virus, and another method of transmitting the virus to humans is by direct contact with the blood and bodily fluids of infected animals or humans (Tahir et al., 2024).

Clinical manifestations in humans vary, ranging from non-specific febrile illness to severe contusion and hepatic dysfunction in the first report on CCHF in Turkey, with a mortality rate of 8-80% in humans (Kar et al., 2021). Notably, mortality rates differ across regions; Iran reports rates ranging from 2% to 50%. While asymptomatic in domestic and wild animals, CCHF can cause severe disease in humans (Chinikar et al., 2010; Papa et al., 2016).

The vital program implemented for CCHF virus prevention includes tick control, focused educational campaigns for livestock handlers, surveillance systems to monitor tick circulation, systematic assessments to identify vulnerable areas with timely intervention to control them, and regular treatment of animals with acaricides (Motashar et al., 2024).

CCHF virus belongs to the family Nairoviridae (order Bunyavirales) and is an enveloped, negative-sense RNA orthonairovirus. It exhibits antigenic diversity, classified into seven serotypes (Shahhosseini et al., 2021; Klucher et al., 2023). Ticks serve as natural reservoirs, transmitting the virus through vertical (tick-to-tick) and horizontal (via tick bites) routes, as well as through tissue or blood contact (Balinandi et al., 2021; Nasirian 2022). Although animals carry the CCHF virus without clinical signs, they contribute to viremia and humoral responses (Balinandi et al., 2021).

Diagnostic methods include serological tests and molecular detection by real-time PCR, which provide accurate detection of CCHF infection. IgM detection by enzyme-linked immunosorbent assay (ELISA) in the first 7 days of illness was followed by its disappearance after 4 months, after which IgG was detected for at least five years (Nyakarahuka et al., 2023). The serological tests give a specificity and sensitivity of IgM detection ranging from 87.8 to 100% (Fillâtre et al., 2019). Moreover, molecular detection provides rapid and accurate diagnosis by using primers that target known viral strains (Nurettin et al., 2022).

In Iraq, sporadic cases of CCHF have been reported since 1979, with the first eight cases isolated in 1981 (Alhilfi et al., 2023). Intermittent outbreaks occurred until 2010 (Majeed et al., 2012). Recent increases in cases, from 33 in 2021 to approximately six times the number in 2022, necessitate urgent control measures (Jafar et al., 2022; Alhilfi et al., 2023).

Our study focuses on Sulaymaniyah Governorate in Iraq, where several cases of CCHF were recorded in 2022, despite the lack of measures to control them. We aim to detect the CCHF virus in ticks (the vector) and identify the disease in domestic animals, raising awareness and preventing possible outbreaks.

2. Materials and Methods

2.2 Study area and sample collection

The study population consisted of cows, sheep, and goats, and the sampling frame was limited to local breeds; samples were taken from these herds. Ticks were transported to the Veterinary Teaching Hospital, College of Veterinary Medicine, University of Sulaimani, for morphological identification. They were identified at the species level under the light microscope, analyzing morphological characteristics using the taxonomic keys by Horak et al., 2018. Ticks were categorized into the genera Hyalomma, Haemaphysalis, and Ixodes based on morphology. All samples were taken randomly using a random number table. The sample size was calculated using WinEpi software. Blood samples were obtained directly from the jugular veins of cows, sheep, and goats (n=450). These samples were collected in a plain test tube for subsequent ELISA testing (Table 1).

Table 1. Three different places (Kalar, Sharazoor, and Ranya) and blood samples taken from three animal species (Cow, Sheep, and Goats) for ELISA testing.

Places	Cow	Sheep	Goats	Total
Kalar	50	49	51	150
Sharazoor	50	50	50	150
Ranya	50	50	50	150
Total	150	149	151	450

The animals were sourced from three locations within the Sulaymaniyah governorate: Kalar, Sharazoor, and Ranya (Fig. 1). Sampling occurred between September 2022 and March 2023, due to the early onset of summer in this region. Tick activity commenced earlier than usual, coinciding with increased human-animal interactions and a higher frequency of meat consumption among the population. Additionally, engorged ticks (n = 600) were randomly collected and taken individually from each animal using forceps or tick removal devices. We used a simple random sampling strategy. Sterile, disposable tools were used for each sample to avoid contamination. Work surfaces were decontaminated with bleach or ethanol between samples. Strict adherence to biosafety protocols was maintained, including working in a biosafety cabinet when handling potentially infectious material, and Individual ticks or pooled samples were washed with sterile phosphate-buffered saline (PBS) to remove external contaminants. The samples were promptly transported to the Sulaymaniyah Veterinary Laboratory in a cold package and stored at -20°C until analysis.

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Fig. 1.

Geographical location of (Kalar, Ranya, and Sharazoor) in Sulaymaniyah provinces on the map (yellow patch), where blood and tick samples were collected from (cows, sheep, and goats) for ELISA and PCR tests. http://www.maphill.com/iraq/sulaymaniyah/panoramic-maps/savanna-style-map/

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3. Results

3.1 Geographical variation in CCHF infection rates: Insight from ELISA results

Based on ELISA results, the prevalence of CCHF infection varied across geographical locations. Goats exhibited a slightly higher prevalence at 37.7% (OR: 1.61, RR: 1.38) compared to cows (27.3%) and sheep (31.5%). Among the regions, Kalar had the highest prevalence (34.7%, OR: 1.23, RR: 1.15), while Sharazoor and Ranya reported rates of 30% and 32%, respectively. The lowest infection rate occurred in Sharazoor (Table 2). The P value between infected species was (P=0.151) while the P value between regions was (P=0.686). No significant differences were found between infected species and the geographical locations.

Table 2. ELISA results of CCHF according to three different geographical distributions (Kalar, Sharazoor, and Ranya) and animal species (Cow, Sheep, and Goats).

		ELISA		RR [CI]	OR [CI]
		+ve	-ve
		Count [N%]	Count [N%]
Species	Cow	41 [27.3%]	109 [72.7%]	Reference	Reference
	Sheep	46 [31.5%]	103 [68.5%]	1.15 [0.8-1.64]	1.23 [0.74-2.01]
	Goat	58 [37.7%]	93 [62.3%]	1.38 [0.99-1.92]	1.61 [0.99-2.62]
Places	Kalar	52 [34.7%]	98 [65.3%]	1.15 [0.83-1.6]	1.23 [0.76-2]
	Sharazoor	45 [30.0%]	105 [70.0%]	Reference	Reference
	Ranya	48 [32.0%]	102 [68.0%]	1.06 [0.76-1.49]	1.09 [0.67-1.79]

RR – relative risk OR- Odds ratio CI- Confidence interval

Based on ELISA results, the functional rate of animal species with CCHF varied across geographical locations. Goats exhibited a concurrent increase in favorable cases, while sheep and cows showed lower rates. Analyzing ELISA data across three settings revealed that Kalar had the highest CCHF prevalence, although statistically significant differences were not observed. Consequently, authorities, veterinarians, and researchers should prioritize studying various goat species and focus on Kalar during their investigations (Fig. 2). These findings can guide veterinary and public health professionals in monitoring new cases based on animal species and geographic distribution, potentially leading to early intervention and improved outcomes (Fig. 3).

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Fig. 2.

ELISA test results for CCHF according to three geographical distributions (Kalar, Sharazoor, and Ranya) and animal species (cattle, sheep, and goats).

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Fig. 3.

This graphic illustrates the association between geographical distributions (Kalar, Sharazoor, and Ranya), and animal species (cow, sheep, and goats) with the ELISA results.

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3.2 PCR screening reveals CCHF infection patterns in three districts

During the PCR screening, 600 samples were collected from three distinct districts: Kalar, Sharazoor, and Ranya. Each location contributed 200 samples. Among these, 10 tested positive, with 3, 3, and 4 positive cases in Kalar, Sharazoor, and Ranya, respectively. The PCR result was an amplified 536-bp fragment of the S-segment of the CCHF viral genome (Fig. 4). Additionally, the partial sequence of the S-segment fragment from CCHF ticks was submitted to GenBank under accession numbers and strain names: OR713752- CCHF/Kalar, OR713753-Ranya, and OR713754-Sharazoor.

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Fig. 4.

Amplification of the partial S segment of the CCHF virus genome from tick samples using RT-PCR was visualized on an agarose gel. Lane M: 100 bp DNA ladder, Lane NC negative control, Lane PC positive control, Lanes 1 to 3 represent positive samples from infected ticks (536 bp each), and Lane 4-7 negative samples.

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3.3 Genetic similarities and variations in CCHF strains: Insight from DNA amino acid analysis

Based on our analysis of the three field strains (CCHF/Kalar/Iq, CCHF/Ranya/Iq, and CCHF/Sharazoor/Iq), there is a similarity range of 99.22% to 99.61% for DNA and 98.84% to 99.42% for amino acids. Additionally, the results indicate that the S segment sequence of these three strains closely resembles the Iran (KU242341.1), India (MN866211.1), Oman (DQ211645.1), and Emirate (JN108025) CCHF strains, with amino acid identities ranging from 97.67% to 98.84%. However, the DNA identities are 94.38%, 93.20% to 93.60%, 91.80% to 92.60%, and 91.00% to 91.47%, respectively (Table 3).

Table 3. Accession numbers in different countries, with the percentages of DNA and amino acid identities according to the genotypes, and information about strains.

Accession no.	Year/strain	Countries	DNA identity %	Amino acid identity %	Group	Genotype
MN866211	2019/MCL-19-T	India	93.20-93.60	97.67-98.84	Asia1	IV
KU242341	2015/IR-T3-RSP	Iran	94.38	97.67-98.84	Asia2	IV
KU242339	2015/IR-T1-HMA	Iran	91-91.70%	97-98.20	Asia1	IV
DQ211645	1997/Oman	Oman	91.80-92.60	97.67-98.84	Asia1	IV
AJ538196	1978//Baghdad	IRAQ	91-91.80	97-98.26	Asia1	IV
HM452305	2009/Afg09	Afghanistan	92.20-93	97.67-98.84	Asia1	IV
MG659724	2017/WJQ16206	China	91.91.80	97-98.26	Asia1	IV
AY223475	2004/HOZHA	Russia	91.28	96.51-97.67	Asia2	IV
KT384399	2013/Amreli	India	91.09	97-98.26	Asia2	IV
JN108025	1979/Dubai 616	Emirate	91-91.47	97.67-98.84	Asia2	IV
DQ076416	2005/SPU4/81	South Africa	87.20-87.40	94.77-95.94	Africa 1	III
DQ211640	1972/ArD15786	Senegal	83.30-83.40	94.19-94.77	Africa 3	I
DQ076413	2005/Semunya	Uganda	86.20-86.60	97-98.26	Africa2	II
DQ144418	Congo 3010	Congo	87.20-87.60	97-98.26	Africa2	II
GU477489	1981/V42/81	Bulgaria	87.40-87-80	93-93.60	Europe1	V
DQ206447	2002/ROS/HUVLV	Russia	87.20-88	94.77-95.30	Europe1	V
DQ133507	2005/Kosovo Hoti	Kosovo	87.80-88.20	94.77-95.35	Europe1	V
OQ454824	2015/5_OE	Turkey	87.37-87.75	94.77-95.35	Europe1	V
OQ954468	2019/YOZGAT-C49	Turkey	88.20-88-60	94.77-95.35	Europe1	V
DQ211638	1975/AP92	Greek	79.80-80.20	89-89.53	Europe 2	VI
KF793333	2012/Daral	Mali	86.80-87.20	94.77-95.93	Africa 1	III

Furthermore, in the multiple sequence alignments, a distinct nucleotide alteration was detected at position 441 (G→A) in only two field strains (CCHF/Kalar and CCHF/Sharazoor). This alteration leads to an amino acid change from Arginine (Arg) to Lysine (Lys) at position 147, as illustrated in Fig. 5. The previous Iraqi strain (“AJ581986.1 IRAQ/Baghdad” is grouped within the Asia-1 (Genotype IV) clade, located on a separate branch from the current three Iraqi isolates (field strain), indicating notable genetic differences. This divergence could suggest geographic, temporal, or ecological factors that have influenced the evolution of these strains within Iraq.

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Fig. 5.

Amino acid alignment was performed, comparing the partial sequence of the S segment from CCHF field virus strains with various reference strains from different countries and genotypes.

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The new virus strain’s close relationship to other Asia-2 (Genotype IV) isolates from Iran and Dubai points to shared cross-border transmission routes, potentially influenced by livestock movement or migratory patterns of tick hosts. The ELISA positive rate was 32.2%, which was higher than the PCR positive rate of 1.66%. Notably, the sole record of CCHF in Iraq was identified in 1978 with GenBank accession number AJ538196. The sample originated from a human source. Our current study analyzed this sequence and found that it corresponds to Asia 1 and Genotype IV (Table 3 and Fig. 6).

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Fig. 6.

A phylogenetic study of field-isolated CCHF virus strains, focusing on the partial S segment (highlighted in a bold red rectangle), was conducted using the neighbor-joining method. The analysis included representative strains from various genotypes, and MEGA X software was utilized to construct the phylogenetic tree through a 1000-bootstrap experiment with the ClustalW alignments algorithm.

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4. Discussion

According to the WHO report, CCHF is classified as a top-priority public health disease. Further research and cross-sectional surveys are needed to evaluate the risk of infection spreading to humans upon contact with sheep, goats, and cattle, mainly through serological testing (ELISA) and RT-PCR assay. While most research on the role of animals in transmitting and maintaining dates back to the late 1960s and 1970s (Jessica et al., 2016), sero-epidemiology studies in sheep, goats, and cattle provide valuable insights into the virus’s prevalence in endemic areas and help identify high-risk regions.

Interestingly, seroprevalence studies reveal that animals without active infection can still carry antibodies, indicating their potential as reservoirs for the CCHF virus. Positive serum IgG detection can persist longer than asymptomatic viremia (7-15 days) (Nurettin et al., 2022). However, detecting antibodies in the virus that causes CCHF remains challenging due to cross-reactivity with other Orthoviruses and the substantial genetic variation associated with the virus’s geographic spread (Desmedt et al., 2023).

According to a report by Alhilfi et al., 2023, the CCHF outbreak began in April 2022 in Iraq. Most confirmed cases were male individuals residing in southern Iraq, with an average age of 34.5 years and a case fatality rate of 16.4%. The outbreak remains ongoing. The increased incidence of CCHF in Iraq is associated with tick infestations among sheep, goats, and cattle. Additionally, CCHF has been endemic in Iraq since 1979, with repeated outbreaks in 2019 and 2022 (Al-Abri et al., 2017; Dalal Al-Rubaye 2022).

In 1990, an outbreak of CCHF occurred in Saudi Arabia. Researchers hypothesized that the disease was introduced via tick-infested animals in Jeddah. A recent study investigated CCHF-IgG antibodies in cattle, sheep, and goats at three locations (Kalar, Sharazoor, and Ranya). The results showed varying prevalence rates: 37.7% for goats, and 27.3% and 31.5% for cows and sheep, respectively. Several risk factors influence variation in the prevalence of CCHF among animal species. These include seasonal age, geographical location, severe tick infestations, method of diagnosis, type of husbandry management, and poor sanitary conditions of livestock sites. Notably, studies in neighboring countries, such as Iran, have reported varying prevalence rates: 58.7% in sheep, 25% in cows and goats, with the lowest prevalence at 24.8% (Mostafavi et al., 2013; Phonera et al., 2021). Turkey’s seroprevalence rates were 36.21% in cattle, 6.27% in sheep, and 6.67% in goats (Ozan and Ozkul 2020). Recent data suggest an increase in CCHF prevalence among sheep and goats imported from Turkey and Syria (Altaliby. Interestingly, the Sharazoor had the lowest infection rate. Researchers have found that temperature and moisture play a role in the distribution of CCHF among animals, tick survival, and virus circulation (Messina et al., 2015) (Zylinski 2015; Jessica R. Spengler 2016; Khwarahm 2023). The genetic divergence between the Baghdad strain and the northern Iraqi isolates highlights the need for localized surveillance to monitor the diversity of circulating CCHF virus strains within Iraq. This analysis supports the hypothesis of independent introductions or divergent evolution within different regions of Iraq. Understanding the genetic differences can guide region-specific diagnostic tools, treatments, and prevention strategies.

Determining tick populations and pathogens is crucial for controlling tick-borne diseases. The natural cycle of CCHF involves transstadial, transovarial, and reproductive stages. Ticks, which infest both domestic and wild animals, play a significant role in spreading the virus (Tekin et al., 2012; Orkun et al., 2017). Studies suggest that ticks serve as long-term reservoirs for CCHF, while animals contribute to maintaining the virus cycle (Ozan and Ozkul 2020).

CCHF is susceptible to zoonotic endemism in domestic animals (sheep, goats, and cows). A survey was conducted on 2,205 animals from three similar animal regions. In Iraq, the prevalence rates were 57% in sheep, 49% in goats, and 29% in cattle, while in Iran, the rates were 38%, 36%, and 18% in sheep, goats, and cattle, respectively (Mendoza, et al. 2018).

CCHF is typically asymptomatic in humans, and together, they serve as a reservoir for the virus, affecting both domestic and wild animals. Introduced diseases have been shown to manifest themselves in three main ways: infected tick vectors, livestock, and wildlife (Fanelli and Buonavoglia 2021). It is essential to recognize that the CCHF virus is not transmitted via respiratory droplets or contaminated food or water. However, identifying the route of transmission is crucial for implementing preventive measures to reduce the spread of the CCHF virus, which primarily occurs through tick bite transmission. Direct contact with infected animals, as well as nosocomial and laboratory-acquired infections (Al Salihi, Mahmoud et al., 2023).

Genetic analysis of the field virus sequences revealed high identity (99.22% to 99.61%) and minimal divergence (1.78% to 1.39%), indicating that the three viruses are of the same type and originate from a common source. The phylogenetic tree (Fig. 6) supports this finding. Notably, the results closely resemble the Iranian strain (97.67 to 98.84%), suggesting Iran as the primary source. This is further supported by the extensive border between Sulaymaniyah and Iranian cities, as well as the presence of illegal animal trade between the two countries. Such high identity throughout a complete or partial genome strongly suggests recent or ongoing transmission between populations, even though mutation and evolution occur over time. Numerous studies indicate that the virus frequently circulates across borders and moves geographically through livestock and tick vectors (Shahhosseini et al., 2021; Papa et al., 2016).

Comparing and genetically analyzing field virus strains with the previous Iraqi strain revealed an identity of 91% to 91.80%. Divergent field virus strains showed an identity of 91% to 91.80% with a divergence of 9% to 8.20%. This suggests that the genetic material of the CCHF virus is not stable, leading to the emergence of different groups and genotypes over time. Further studies are needed to detect and identify the virus in other animals, allowing us to pinpoint the exact source of the disease. Doing so can prevent potential outbreaks from any infection source in the area surrounding Sulaymaniyah Governorate. The phylogenetic tree has been illustrated in Fig. 6. The 43 CCHF virus isolates from the viral genome were classified into seven groups and six genotypes (I-VI). Analysis of the partial S segment of CCHF and the strains in this study revealed that three field virus sequences belonged to Group Asia2 (Genotype IV) and clustered with the Dubai 616/1979 and IR-T3-RSP/2015 isolates (Fig. 6). It is crucial to use a reliable test for serology and avoid mixing substances used during sample collection, as this can lead to increased false positives and cross-contamination. We suggest using IDScreen CCHF Double Antigen Multi-species ELISA kits, as they have the highest specificity (100%) and excellent sensitivity (98.9%) across multiple species.

5. Conclusions

CCHF is a zoonotic disease with an expanding distribution, attributed to climate change and the transportation of livestock. Tick prevalence plays a crucial role in increasing the risk of CCHF virus infection. Our study confirms that goats have the highest infection rate among animal species, and Kalar exhibits the highest prevalence. Positive cases across species and locations highlight the potential outbreak risk and underscore the potential for an outbreak. The findings of this study can enhance our understanding of the CCHF virus in Iraq by presenting new data and information to authorities and researchers. Tick control and limitation of animal contact, together with observing borders, will be a suitable protocol for prevention and control. This study presents details on CCHF prevalence in Sulaymaniyah, Iraq.