**1. Lyme borreliosis**

### **1.1 Introduction**

Lyme borreliosis is the most prevalent tick-borne infection in the North hemisphere. It is a chronic and multisystem infectious disease caused by several species of *Borrelia burgdorferi* sensu lato complex. Three main species are known to cause the disease in Europe, namely *Borrelia afzelii*, *Borrelia garinii*, and *Borrelia burgdorferi* sensu stricto. The ability to cause persistent infection in humans and a wide variety of mammalian hosts is a common property of Lyme disease.

Early manifestations of Lyme disease include typical skin lesion erythema migrans as a sign of early localized infection and/or unspecific flu-like symptoms, acute meningitis or meningopolyneuritis, and acute arthritis, which are signs of early disseminated infection. Late manifestations may appear as chronic neurological abnormalities, chronic arthritis, or acrodermatitis chronica atrophicans, marking late disseminated infection.

Bulgaria is endemic for Lyme borreliosis and a country with mandatory notification of the disease. The reported incidence of Lyme borreliosis in Bulgaria is about 6/100,000 of the population, but the true incidence is most probably much higher, because the disease often is self-limited and mild cases go unrecognized.

#### **1.2 Clinical features of Lyme borreliosis in humans in Bulgaria**

Bulgarian study on clinical manifestations of 1257 patients with Lyme borreliosis, diagnosed by physicians and confirmed in laboratory (except for erythema migrans), showed that the most common clinical presentation is erythema migrans (EM), diagnosed in 868 (69.1%) of the patients, almost uniformly as a sole presentation and rarely as a part of multisystem involvement. Rashes had a median diameter of 11 cm (5–35 cm). The erythema rash was homogenous in 44% and with central clearing and peripheral border in 56%. In 14.7% of the patients, atypical rashes with a vesicular or ulcerated center were found. Flu-like symptoms, such as fever, headache, myalgia, arthralgia, fatigue, neck stiffness, were the most common signs associated with EM. Fever was found on physical examination in 133 (15.3%) of the EM patients. In parallel with EM, lymphadenopathy was detected in 284 (32.7%) of the EM cases. In addition, multiple EM was detected in 59 (6.8%) of the patients.

After EM, the second most frequent presentation of Lyme disease in Bulgaria is neuroborreliosis. It is usually presented as radiculoneuritis as a sole presentation (found in 72% of the patients with neuroborreliosis in Bulgaria) and much rarely manifested as cranial neuritis (mainly in children), myelitis, meningoradiculoneuritis, or encephalopathy.

The third most common presentation of Lyme disease in Bulgaria is Lyme arthritis. In the same study, it was diagnosed in 101 (8%) of the patients with Lyme borreliosis. Lyme arthritis was mainly presented by brief attacks of arthritis and rarely as chronic arthritis.

Rare manifestations of Lyme borreliosis in Bulgaria are those affecting heart and eyes (found in 1.1 and 0.9%, respectively). Very rare syndromes are borrelial lymphocytoma and acrodermatitis chronica atrophicans (0.3%).

Multiple organ involvement was found in 2.1% of the patients. Most commonly it was presented as neurological disorders and skin lesions or arthritis.

In addition, the highest proportion of the patients with Lyme borreliosis is in children aged 5–9. The disease shows two peaks in the seasonal distribution of the cases. Neuroborreliosis is a more frequent presentation than Lyme arthritis in Bulgaria.

#### **1.3 Borrelial C6 peptides as antigens for serological diagnosis of Lyme borreliosis**

Diagnosis of Lyme borreliosis depends on clinical signs supported by serological findings—enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Various *B. burgdorferi* sensu lato protein antigens are used for ELISA tests (OspA, OspC, FlaB, VlsE). VlsE is the most promising diagnostic antigen among them due to the conserved immunogenic epitopes.

VlsE gene consists of expression site and 15 silent cassettes with a high degree of homology and high rate of reassortments. Each cassette consists of six variable and six invariable regions. The 26-amino-acid-long sixth invariable region (IR6) is immunodominant and much conserved among *B. burgdorferi* sensu lato species. It was shown that IgG antibodies to IR6 (C6) are often detected in early and late Lyme borreliosis [1, 2]. These two statements, i.e. that C6: (1) possess high immunogenicity and (2) is highly conserved among species of the complex B. burgdorferi sensu lato, we decided to test in practice with serum samples from Bulgarian patients with Lyme disease.

Four 26-amino-acid-long peptides were synthesized (ProteoGenix SAS, France), which corresponded to IR6 regions of VlsE proteins from *B. burgdorferi* sensu stricto, *B. garinii*, and *B. afzelii*. Because of the previously described difference in reactivity [1], two peptides were synthesized from two strains of *B. burgdorferi*

**89**

**Table 1.**

*Vector-Borne Infections in Bulgaria*

a patient with neuroborreliosis.

the developed peptide ELISA tests.

(**Table 1**).

peptides was high [3].

*DOI: http://dx.doi.org/10.5772/intechopen.81916*

sensu stricto—B31, isolated from a tick and 297, isolated from cerebrospinal fluid of

Test results showed that the two C6 peptides from *B. burgdorferi* sensu stricto had higher reactivity than the corresponding C6 peptides from *B. afzelii* and *B. garinii* with sera from patients with erythema migrans and those with Lyme arthritis. On the contrary, the C6 peptide from *B. garinii* was more reactive with sera from patients with neuroborreliosis [3]. The two peptides from *B. burgdorferi* sensu stricto showed different reactivity with sera from patients with erythema migrans

Concerning non-specific reactivity of the peptide antigens with sera from patients with syphilis, leptospirosis, rheumatoid arthritis, and sclerodermia, the lowest level of specificity (87.5%) was found for the C6 peptide from *B. afzelii*; specificity was higher (90% and 92.5%) with the C6 peptides from *B. burgdorferi* s.s. and highest (100%) with the C6 from *B. garinii*. Overall, specificity of the four

In order to test applicability of the C6 peptide for serological diagnosis, we used peptide ELISA tests for detection of antibodies in Lyme borreliosis. Four peptide antigens from the C6 regions of VlsE proteins from the three *Borrelia* species that mainly cause Lyme disease in Europe were tested. The findings were very promising since up to 80% of the patients with early Lyme borreliosis and neuroborreliosis and all patients with Lyme arthritis can be diagnosed by the peptide ELISA tests. In addition, overall specificity of the C6 tests was high (87.5–100%). Notably, the tests are easy to perform and cheap as the peptide synthesis is much more easy to imple-

The C6 peptides from *B. burgdorferi* sensu stricto showed the highest sensitivity in detection of specific anti-borrelia antibodies in patients with early Lyme disease

Erythema migrans (n = 50) 36 (72%) 39 (78%) 26 (52%) 29 (58%) Neuroborreliosis (n = 20) 11 (55%) 11 (55%) 16 (80%) 9 (45%) Lyme arthritis (n = 10) 10 (100%) 10 (100%) 8 (80%) 8 (80%)

**C6 297 (***B. burgdorferi* **s.s.)**

57 (71.3%) 60 (75%) 50 (62.5%) 46 (57.5%)

3/40 (92.5%) 4/40 (90%) 0 (100%) 5/40 (87.5%)

**C6 IP90 (***B. garinii***)**

**C6 PT7 (***B. afzelii***)**

*burgdorferi* **s.s.)**

*Reactivity of peptide C6 ELISA with serum panels of patients with Lyme disease in Bulgaria.*

ment than the production of recombinant protein antigens.

**Serum panel C6 B31 (***B.* 

Total number of reacted samples

Number of reactive sera from patients with other diseases (%

(% sensitivity)

specificity)

Four different peptide ELISA tests were developed based on IR6 regions of two *B. burgdorferi* sensu stricto strains (B31 and 297), of one *B. afzelii* (PT7) and one *B. garinii* (IP90) strain. Two different serum panels were tested. The first one consisted of serum samples from Bulgarian patients with Lyme borreliosis—50 sera from patients with erythema migrans (clinical hallmark of early Lyme borreliosis), 20 sera from patients with neuroborreliosis, and 10 sera from patients with Lyme arthritis. This serum panel was used to analyze sensitivity of the tests. It contained 40 serum samples from patients with known cross-reactive serological results patients with syphilis (n = 10), leptospirosis (n = 10), rheumatoid arthritis (n = 10), and sclerodermia (n = 10). The second serum panel was applied to test specificity of

#### *Vector-Borne Infections in Bulgaria DOI: http://dx.doi.org/10.5772/intechopen.81916*

*Vectors and Vector-Borne Zoonotic Diseases*

patients.

ritis, or encephalopathy.

rarely as chronic arthritis.

to the conserved immunogenic epitopes.

**1.2 Clinical features of Lyme borreliosis in humans in Bulgaria**

Bulgarian study on clinical manifestations of 1257 patients with Lyme borreliosis, diagnosed by physicians and confirmed in laboratory (except for erythema migrans), showed that the most common clinical presentation is erythema migrans (EM), diagnosed in 868 (69.1%) of the patients, almost uniformly as a sole presentation and rarely as a part of multisystem involvement. Rashes had a median diameter of 11 cm (5–35 cm). The erythema rash was homogenous in 44% and with central clearing and peripheral border in 56%. In 14.7% of the patients, atypical rashes with a vesicular or ulcerated center were found. Flu-like symptoms, such as fever, headache, myalgia, arthralgia, fatigue, neck stiffness, were the most common signs associated with EM. Fever was found on physical examination in 133 (15.3%) of the EM patients. In parallel with EM, lymphadenopathy was detected in 284 (32.7%) of the EM cases. In addition, multiple EM was detected in 59 (6.8%) of the

After EM, the second most frequent presentation of Lyme disease in Bulgaria is neuroborreliosis. It is usually presented as radiculoneuritis as a sole presentation (found in 72% of the patients with neuroborreliosis in Bulgaria) and much rarely manifested as cranial neuritis (mainly in children), myelitis, meningoradiculoneu-

The third most common presentation of Lyme disease in Bulgaria is Lyme arthritis. In the same study, it was diagnosed in 101 (8%) of the patients with Lyme borreliosis. Lyme arthritis was mainly presented by brief attacks of arthritis and

Rare manifestations of Lyme borreliosis in Bulgaria are those affecting heart and eyes (found in 1.1 and 0.9%, respectively). Very rare syndromes are borrelial

Multiple organ involvement was found in 2.1% of the patients. Most commonly

In addition, the highest proportion of the patients with Lyme borreliosis is in children aged 5–9. The disease shows two peaks in the seasonal distribution of the cases. Neuroborreliosis is a more frequent presentation than Lyme arthritis in Bulgaria.

**1.3 Borrelial C6 peptides as antigens for serological diagnosis of Lyme borreliosis**

Diagnosis of Lyme borreliosis depends on clinical signs supported by serological findings—enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Various *B. burgdorferi* sensu lato protein antigens are used for ELISA tests (OspA, OspC, FlaB, VlsE). VlsE is the most promising diagnostic antigen among them due

VlsE gene consists of expression site and 15 silent cassettes with a high degree of homology and high rate of reassortments. Each cassette consists of six variable and six invariable regions. The 26-amino-acid-long sixth invariable region (IR6) is immunodominant and much conserved among *B. burgdorferi* sensu lato species. It was shown that IgG antibodies to IR6 (C6) are often detected in early and late Lyme borreliosis [1, 2]. These two statements, i.e. that C6: (1) possess high immunogenicity and (2) is highly conserved among species of the complex B. burgdorferi sensu lato, we decided to test in practice with serum samples from Bulgarian patients with

Four 26-amino-acid-long peptides were synthesized (ProteoGenix SAS, France),

which corresponded to IR6 regions of VlsE proteins from *B. burgdorferi* sensu stricto, *B. garinii*, and *B. afzelii*. Because of the previously described difference in reactivity [1], two peptides were synthesized from two strains of *B. burgdorferi*

lymphocytoma and acrodermatitis chronica atrophicans (0.3%).

it was presented as neurological disorders and skin lesions or arthritis.

**88**

Lyme disease.

sensu stricto—B31, isolated from a tick and 297, isolated from cerebrospinal fluid of a patient with neuroborreliosis.

Four different peptide ELISA tests were developed based on IR6 regions of two *B. burgdorferi* sensu stricto strains (B31 and 297), of one *B. afzelii* (PT7) and one *B. garinii* (IP90) strain. Two different serum panels were tested. The first one consisted of serum samples from Bulgarian patients with Lyme borreliosis—50 sera from patients with erythema migrans (clinical hallmark of early Lyme borreliosis), 20 sera from patients with neuroborreliosis, and 10 sera from patients with Lyme arthritis. This serum panel was used to analyze sensitivity of the tests. It contained 40 serum samples from patients with known cross-reactive serological results patients with syphilis (n = 10), leptospirosis (n = 10), rheumatoid arthritis (n = 10), and sclerodermia (n = 10). The second serum panel was applied to test specificity of the developed peptide ELISA tests.

Test results showed that the two C6 peptides from *B. burgdorferi* sensu stricto had higher reactivity than the corresponding C6 peptides from *B. afzelii* and *B. garinii* with sera from patients with erythema migrans and those with Lyme arthritis. On the contrary, the C6 peptide from *B. garinii* was more reactive with sera from patients with neuroborreliosis [3]. The two peptides from *B. burgdorferi* sensu stricto showed different reactivity with sera from patients with erythema migrans (**Table 1**).

Concerning non-specific reactivity of the peptide antigens with sera from patients with syphilis, leptospirosis, rheumatoid arthritis, and sclerodermia, the lowest level of specificity (87.5%) was found for the C6 peptide from *B. afzelii*; specificity was higher (90% and 92.5%) with the C6 peptides from *B. burgdorferi* s.s. and highest (100%) with the C6 from *B. garinii*. Overall, specificity of the four peptides was high [3].

In order to test applicability of the C6 peptide for serological diagnosis, we used peptide ELISA tests for detection of antibodies in Lyme borreliosis. Four peptide antigens from the C6 regions of VlsE proteins from the three *Borrelia* species that mainly cause Lyme disease in Europe were tested. The findings were very promising since up to 80% of the patients with early Lyme borreliosis and neuroborreliosis and all patients with Lyme arthritis can be diagnosed by the peptide ELISA tests. In addition, overall specificity of the C6 tests was high (87.5–100%). Notably, the tests are easy to perform and cheap as the peptide synthesis is much more easy to implement than the production of recombinant protein antigens.

The C6 peptides from *B. burgdorferi* sensu stricto showed the highest sensitivity in detection of specific anti-borrelia antibodies in patients with early Lyme disease


#### **Table 1.**

*Reactivity of peptide C6 ELISA with serum panels of patients with Lyme disease in Bulgaria.*

from Bulgaria. Our previous studies on borrelia infections of Bulgarian ticks have shown that the ticks are mostly infected with *B. afzelii*, followed *by B. burgdorferi* sensu stricto and *B. garinii* [4]. The discrepancy between the abundance of *B. afzelii* in our ticks and predominant *B. burgdorferi* sensu stricto reactivity of Lyme borreliosis patients could be explained by different pathogenic potential of the *Borrelia* species.

It is well known that different *Borrelia* species cause predominantly certain clinical manifestations: neuroborreliosis is associated with *B. garinii* and Lyme arthritis with *B. burgdorferi* sensu stricto [5]. This finding may explain the higher reactivity of sera from patients with neuroborreliosis with the C6 peptide from *B. garinii* as well as the predominant reactivity of the sera from Lyme arthritis with the C6 from *B. burgdorferi* sensu stricto.

### **1.4 PCR detection of** *Borrelia***,** *Ehrlichia***, and** *Rickettsia* **DNAs in** *I. ricinus* **ticks from Bulgaria**

A total of 298 *I. ricinus* ticks, collected by flag from the vegetation in 2000 and 2001, were examined by the reverse line blotting technique for *Borrelia, Ehrlichia,* and *Rickettsia* DNAs [6].

Prevalence of *Borrelia*, *Ehrlichia*, and *Rickettsia* in 202 ticks, collected in 2000, was as follows. Overall *Borrelia* prevalence in adult ticks was 41% (44% in males and 39% in females) and 10% in nymphs. *B. afzelii* was the predominant species. Its prevalence was 23% (26 of 112) in adult ticks and 6% (5 of 90) in nymphs, representing 56% (31/55) of all *Borrelia*-positive results. *B. burgdorferi* sensu stricto was detected in 15 (13%) of 112 adult ticks and in 1 (1%) of 90 nymphs. Prevalence of *B. garinii* was 3% in adult ticks and 7% in nymphs. *B. valaisiana* was detected in 3 and *B. lusitaniae* in other 3 of 202 examined ticks.

Overall *Borrelia* prevalence in adult *I. ricinus* ticks, collected in 2001, was 29% (21 of 72) in adult ticks (8% in males and 40% in females). No *Borrelia* infection was found in nymphs from 2001. *B. afzelii* was again the prevalent species—prevalence rate 15% (11 of 72) in adult ticks, representing 52% (11/21) of all *Borrelia*positive results. Prevalences of *B. garinii* and *B. valaisiana* were 7% and 8%, respectively.

*Anaplasma phagocytophilum* was detected in 36 (32%) of 112 adult ticks and in 5 (6%) of 90 nymphs, collected in 2000. Of the ticks, collected in 2001, *A. phagocytophilum* was detected in 28% of adult ticks (male 17% and female 33%) and 21% of nymphs.

Of 202 *I. ricinus* ticks, collected in 2000, 94 (47%) were found to carry *Rickettsia* DNA: 78% of males, 61% of females, and 19% of nymphs. Prevalence of *R. helvetica* was 28% (56/202) and prevalence of IRS4 rickettsia was 30% (60/202). *R. conorii* was found in only two of the ticks. Ticks, collected in 2001, showed also high *Rickettsia* infectivity rate, 40% (38 of 96): 83% in males, 29% in females, and 17% in nymphs. *R. helvetica* was again the prevalent species, detected in 18% and 23% of the ticks, respectively.

A high proportion of Bulgarian *I. ricinus* ticks contains *Borrelia* DNA. Analysis of *Borrelia* prevalence revealed that ticks, collected from the same location and even in the same month (May) but in 2 adjacent years, 2000 and 2001, had different *Borrelia* prevalence (41% and 29%, respectively). Apart differences in overall *Borrelia* prevalence, there were also differences in *Borrelia* prevalence in males, females, and nymphs, collected in the 2 years in the same place. In ticks from 2000, *Borrelia* was most prevalent in adult males, less so in adult females and least so in nymphs, while in ticks from 2001, the prevalence was higher in females and lower in males and nymphs.

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*DOI: http://dx.doi.org/10.5772/intechopen.81916*

**2. Crimean-Congo hemorrhagic fever**

A case of probable airborne transmission is reported [9].

has been recently reported in Iran [11].

**general population of Bulgaria**

human population of Bulgaria.

**2.1 Introduction**

*B. afzelii* is highly prevalent among Bulgarian *I. ricinus* ticks, giving more than half of the *Borrelia*-positive results, followed by *B. burgdorferi* sensu stricto, *B. garinii, B. valaisiana*, and *B. lusitaniae* in that order. Prevalence of coinfection is high (17–45%) in *Ixodes* ticks, representing double or even triple infection with *Borrelia*, *Ehrlichia*, and/or *Rickettsia*. Even coinfection with two different *Borrelia* or *Ehrlichia* species was often detected, showing that the tick hosts are infected with multiple tick-borne pathogens. Since these ticks were collected from vegetation, a risk for simultaneous transmission of these pathogens during the same tick bite exists.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne human viral disease with a fatality rate up to 30%. It is characterized by a sudden onset of fever and muscular pain, often progressing to hemorrhagic manifestations [7]. Various wild and domestic mammals are natural reservoir hosts of CCHF virus (CCHFV). Ticks of the genus *Hyalomma* are primary vector and serve also as reservoir hosts. *Rhipicephalus* and *Dermacentor* ticks may play an additional role in maintaining the circulation of CCHFV in an enzootic cycle between vertebrates and ticks. The vector-competent ticks stay infected through the molt ("transovarial transmission") [7]. Humans are infected by infected ticks and also by contact with tissues or body fluids of infected animals or patients. Nosocomial outbreaks are described [8].

CCHF is spread in over 50 countries in Africa, Southern Asia, the Middle East, and Southeastern Europe, including the Balkan Peninsula. Cases have been reported in Bulgaria, Turkey, Kosovo, Albania, and Greece. CCHFV (genus *Orthonairovirus*, family *Nairoviridae*) is a negative-stranded RNA virus with a three-segmented genome: large (L), medium (M), and small (S) segments. CCHFV strains belong to seven genetic lineages. Lineage V, also named Europe 1, contains pathogenic CCHFV strains. Lineage VI, called Europe 2, contains genetically different Greek AP92 strains and recently reported similar strains from Turkey, Greece, Kosovo, and Algeria. Besides the detection in ticks, CCHFV lineage Europe 2 has been detected in a mild CCHF case in Turkey [10]. A fatal case due to an AP92-like strain

With sporadic cases or small outbreaks, CCHF appeared in Bulgaria every year since 1950s. CCHFV was first detected in 1952 in Stara Zagora region [12]. Over 1500 cases have been reported in the country since then. For the last 10 years, 2009–2018, a total of 60 CCHF cases are officially recorded in the country. Case fatality rate of CCHF was an average 15.0%. There is a tendency for CCHF to spread in previously non-endemic areas. The strains causing CCHF in the country are closely related to others in the Balkan peninsula, belonging to lineage Europe 1 [13].

**2.2 Countrywide seroprevalence study on Crimean-Congo hemorrhagic fever in** 

To test current circulation of CCHFV in the country, we conducted a seroepidemiological study. The main objective of the study was to estimate the prevalence of IgG antibodies to CCHFV, as stable and long-persisting antibodies, in general

Serum samples were collected prospectively from 1500 residents of all 28 districts in Bulgaria. Participants were selected randomly among persons referred

#### *Vector-Borne Infections in Bulgaria DOI: http://dx.doi.org/10.5772/intechopen.81916*

*B. afzelii* is highly prevalent among Bulgarian *I. ricinus* ticks, giving more than half of the *Borrelia*-positive results, followed by *B. burgdorferi* sensu stricto, *B. garinii, B. valaisiana*, and *B. lusitaniae* in that order. Prevalence of coinfection is high (17–45%) in *Ixodes* ticks, representing double or even triple infection with *Borrelia*, *Ehrlichia*, and/or *Rickettsia*. Even coinfection with two different *Borrelia* or *Ehrlichia* species was often detected, showing that the tick hosts are infected with multiple tick-borne pathogens. Since these ticks were collected from vegetation, a risk for simultaneous transmission of these pathogens during the same tick bite exists.
