Mediterranean spotted fever in Salamanca, Spain. Epidemiological study in patients and serosurvey in animals and healthy human population

Mediterranean spotted fever is a spotted fever group rickettsiosis caused by Rickettsia conorii. The fever has a recognized incidence in large geographic areas, and its presence in Salamanca Province (Spain) has been well documented since 1981. This work presents the results of the centralized prospective survey carried out in this area and was designed to study the epidemiological behavior of the disease and the prevalence of antibodies against R. conorii among animals and healthy human population. In 110 patients with confirmed diagnosis by an immunofluorescent antibody test we have observed a bimodal temporal curve of incidence of Mediterranean spotted fever in our zone and their predominant origin in villages under 2000 inhabitants. The serosurvey in a statistically representative sample of the human population of Salamanca shows a rate of 73.5% of reactive sera and percentages as high as 82% in samples from small villages. The seroepidemiological study of 300 animals reveals a widespread response to rickettsial antigens throughout the province. Immunofluorescent antibody titers of greater than or equal to 1:40 are present in 93% of dogs and high rates and titers are detected in other domestic mammals, suggesting their feasible epidemiological role in Mediterranean spotted fever.     

Prevalence of antibodies to spotted fever group rickettsiae in humans and domestic animals in a Brazilian spotted fever-endemic area in the state of São Paulo, Brazil: serologic evidence for infection by Rickettsia rickettsii and another spotted fever group Rickettsia

In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibody titers against two rickettsial antigens were evaluated after cross-absorption. Sera from seven horses and two dogs contained antibodies specific for R. rickettsii, and one dog serum had antibodies against a Rickettsia species very closely related to R. africae. The latter may have been caused by infection with the recently identified COOPERI strain.     

Domestic dogs (Canis familiaris) as reservoir hosts for Rickettsia conorii

Rickettsia conorii is the causative agent of Mediterranean spotted fever (MSF) and Israeli spotted fever (ISF) transmitted by the brown dog tick Rhipicephalus sanguineus. In areas where MSF or ISF are prevalent, dogs have high prevalence of R. conorii -neutralizing antibodies. However, the true role of dogs in the persistence of the R. conorii transmission cycle is unknown, and their reservoir competence for this pathogen has remained untested. We assessed the ability of dogs infected with R. conorii to transmit the pathogen to previously uninfected Rh. sanguineus ticks. Dogs were infected either via needle-inoculation of cultured rickettsiae or naturally via infected tick bite. Dogs were monitored for clinical signs of infection, for rickettsemia by PCR, and for seroconversion and were subjected to infestation with uninfected ticks at different time points. Rh. sanguineus larvae and nymphs successfully acquired the agent from both needle-inoculated and tick-infected dogs and transmitted it transtadially. Tick-infected dogs remained infectious to ticks for at least a month postinfection. The molted ticks were, in turn, infectious to na?ve dogs. These results demonstrate that dogs are capable of acquiring R. conorii from infected Rh. sanguineus ticks and transmitting infection to cohorts of uninfected ticks, thus confirming for the first time that dogs are indeed competent reservoirs for R. conorii. In addition, dogs with different genetic backgrounds appear to differ in their susceptibility to R. conorii infection.     

Seroepidemiology of spotted fever group rickettsial infections in humans in Zimbabwe

In sera collected throughout Zimbabwe the prevalence of antibodies reactive with Rickettsia conorii (Kenya) and a Zimbabwean spotted fever group rickettsia (ZSFR) was investigated using an indirect immunofluorescence assay (IFA). A random sample of these sera was also tested using a commercial IFA test. There was close agreement between IFA titres using the African rickettsial antigens and the commercial antigen slides. When differences in titre were detected, these were rarely greater than a twofold serum dilution. In Western blot immunoassays, IFA positive human sera detected immunogens of ZSFR and R. conorii (Kenya) that could also be demonstrated with homologous mouse antisera. The overall seroprevalence was 52% to R. conorii (Kenya) and 55% to ZSFR. For both antigens the highest seroprevalence was recorded from the south of the country, where the highest incidence of clinical tick-bite fever was reported in a questionnaire survey to medical practitioners. No difference was found between the seroprevalence in males and females, but high titres of antibody were common in samples from young people.     

Survey for Tick-Borne Zoonoses in the State of Espirito Santo,Southeastern Brazil

Blood samples collected from 201 humans, 92 dogs, and 27 horses in the state of Espirito Santo, Brazil, were tested by polymerase chain reaction, indirect immunofluorescence assays, and indirect enzyme-linked immunosorbent assay for tick-borne diseases (rickettsiosis, ehrlichiosis, anaplasmosis, borreliosis, babesiosis). Our results indicated that the surveyed counties are endemic for spotted fever group rickettsiosis because sera from 70 (34.8%) humans, 7 (7.6%) dogs, and 7 (25.9%) horses were reactive to at least one of the six Rickettsia species tested. Although there was evidence of ehrlichiosis (Ehrlichia canis) and babesiosis (Babesia canis vogeli, Theileria equi) in domestic animals, no human was positive for babesiosis and only four individuals were serologically positive for E. canis. Borrelia burgdorferi-serologic reactive sera were rare among humans and horses, but encompassed 51% of the canine samples, suggesting that dogs and their ticks can be part of the epidemiological cycle of the causative agent of the Brazilian zoonosis, named Baggio-Yoshinari Syndrome.     

Rickettsia parkeri: a Rickettsial pathogen transmitted by ticks in endemic areas for spotted fever rickettsiosis in southern Uruguay

At first Rickettsia conorii was implicated as the causative agent of spotted fever in Uruguay diagnosed by serological assays. Later Rickettsia parkeri was detected in human-biting Amblyomma triste ticks using molecular tests. The natural vector of R. conorii, Rhipicephalus sanguineus, has not been studied for the presence of rickettsial organisms in Uruguay. To address this question, 180 R. sanguineus from dogs and 245 A. triste from vegetation (flagging) collected in three endemic localities were screened for spotted fever group (SFG) rickettsiosis in southern Uruguay. Tick extracted DNA pools were subjected to PCR using primers which amplify a fragment of the rickettsial gltA gene. Positive tick DNA pools with these primers were subjected to a second PCR round with primers targeting a fragment of the ompA gene, which is only present in SFG rickettsiae. No rickettsial DNA was detected in R. sanguineus. However, DNA pools of A. triste were found to be positive for a rickettsial organism in two of the three localities, with prevalences of 11.8% to 37.5% positive pools. DNA sequences generated from these PCR-positive ticks corresponded to R. parkeri. These findings, joint with the aggressiveness shown by A. triste towards humans, support previous data on the involvement of A. triste as vector of human infections caused by R. parkeri in Uruguay.     

Short report: isolation and identification of two spotted fever group rickettsial strains from patients in Catalonia, Spain

Two rickettsial strains, 16B (previously isolated) and FB1, were isolated from blood from patients with Mediterranean spotted fever in Catalonia, Spain. These are the only 2 human rickettsial isolates of the spotted fever group obtained so far in Spain. These strains were identified by the polymerase chain reaction and sequence analysis of a fragment of the outer membrane protein A (ompA) gene. The partial ompA sequence was found to be 100% identical with that of Rickettsia conorii (Malish 7 strain) for both strains. These results confirm the presence of R. conorii in Catalonia, despite the fact that in a previous study, no R. conorii were isolated, but a new rickettsial strain of the spotted fever group (Bar29) was isolated from dog ticks (Rhipicephalus sanguineus) in Catalonia. Further studies are necessary to get a better knowledge of the epidemiology of rickettsiae in Catalonia.     

Prevalence of antibodies to spotted fever group rickettsiae in dogs from southeastern Australia

Recent epidemiologic data suggests that Rickettsia australis, the cause of Queensland tick typhus, is present in southeastern Australia. In order to further confirm this observation, a canine serosurvey was undertaken to determine if naturally occurring antibodies were present in pet and farm dogs from this newly-recognized endemic area. Thirty-five of 312 surveyed dogs (11.2%) had indirect immunofluorescent antibody titers of 1:64 or greater against R. australis antigen. Positive control sera were obtained from two dogs experimentally inoculated with R. australis. One of these dogs was serially sampled and a rickettsemia could not be documented. None of 26 control sera obtained from dogs from South Australia, New Zealand, western Victoria, or North Carolina had antibody titers greater than or equal to 1:64. These results suggest that spotted fever group rickettsiae are present in Southeastern Australia.     

Serological and entomological survey in a zoonotic visceral leishmaniasis focus of North Central Anatolia, Turkey: Corum province

In the present study, we aimed to carry out an epidemiological and entomological survey on a visceral leishmaniasis (VL) focus located on the northern central part of Anatolia, Turkey. Five villages of Corum province, where five confirmed cases of human visceral leishmaniasis (HVL) (one patient/village) were reported between June 1998 and August 2001 were included in the study. A total of 625 children and 131 dogs were sampled and the physical examination was carried out by authorized physicians and veterinarians. An indirect fluorescent antibody test (IFAT) was performed by standard procedures for human and dog sera, while the direct agglutination test (DAT) was only performed for dog sera. Sand fly collection was performed in three villages by CDC miniature light traps. Hepatosplenomegaly and hepatomegaly were detected in two and eight children, respectively. The seropositivity rate among children was found to be 0.16% (1/625) in the region. The seroprevalence of canine infection in these five villages ranged between 0.0% and 28.26%. In two villages, named Ahlatcik and Asagifindikli, no seropositive dogs were found. A total of 1218 sand flies were collected throughout the study. Six species of Phlebotomus were identified: P. transcaucasicus, P. neglectus, P. halepensis, P. tobbi, P. papatasi, and P. jacusieli. P. transcaucasicus was found to be the predominant species in Cevizli (47.44%; 343/723) and Ucoluk (79.95%; 351/439) villages, while P. tobbi was abundant in Kucukerikli (42.85%; 24/56).     

Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients

BACKGROUND: The pathophysiologic mechanisms that determine the severity of Mediterranean spotted fever (MSF) and the host-related and microbe-related risk factors for a fatal outcome are incompletely understood. METHODS: This prospective study used univariate and multivariate analyses to determine the risk factors for a fatal outcome for 140 patients with Rickettsia conorii infection admitted to 13 Portuguese hospitals during 1994-2006 with documented identification of the rickettsial strain causing their infection. RESULTS: A total of 71 patients (51%) were infected with the Malish strain of Rickettsia conorii, and 69 (49%) were infected with the Israeli spotted fever (ISF) strain. Patients were admitted to the intensive care unit (40 [29%]), hospitalized as routine inpatients (95[67%]), or managed as outpatients (5[4%]). Death occurred in 29 adults (21%). A fatal outcome was significantly more likely for patients infected with the ISF strain, and alcoholism was a risk factor. The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, obtundation and/or confusion, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated levels of hepatic enzymes and creatine kinase. Some, but not all, of these findings were observed more often in ISF strain-infected patients. CONCLUSIONS: Although fatalities and similar clinical manifestations occurred among both groups of patients, the ISF strain was more virulent than the Malish strain. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strongly associated with a fatal outcome.     

Serologic evidence for exposure to Rickettsia rickettsii in eastern Arizona and recent emergence of Rocky Mountain spotted fever in this region

During 2002 through 2004, 15 patients with Rocky Mountain spotted fever (RMSF) were identified in a rural community in Arizona where the disease had not been previously reported. The outbreak was associated with Rickettsia rickettsii in an unexpected tick vector, the brown dog tick (Rhipicephalus sanguineus), which had not been previously associated with RMSF transmission in the United States. We investigated the extent of exposure to R. rickettsii in the local area through serologic evaluations of children and dogs in 2003-2004, and in canine sera from 1996. Antibodies to R. rickettsii at titers > or = 32 were detected in 10% of children and 70% of dogs in the outbreak community and 16% of children and 57% of dogs in a neighboring community. In comparison, only 5% of canine samples from 1996 had anti-R. rickettsii antibodies at titers > or = 32. These results suggest that exposures to RMSF have increased over the past 9 years, and that RMSF may now be endemic in this region.     

Rickettsial spotted fever in capoeirão village, Itabira, Minas Gerais, Brazil

The present study investigated the infection by spotted fever rickettsia in an endemic area for Brazilian spotted fever (BSF; caused by Rickettsia rickettsii) in Minas Gerais State, Brazil. Human, canine and equine sera samples, and Amblyomma cajennense adult ticks collected in a rural area of Itabira City, Minas Gerais State were tested for rickettsial infection. Through Immunofluorescence Assay (IFA) we demonstrated the presence of antibodies anti-R. rickettsii in 8.2%, 81.3% and 100% of the human, canine and equine sera, respectively. None of the 356 tick specimens analyzed were positive for Rickettsia by the hemolymph test or Polymerase Chain Reaction technique (PCR) for the htrA and the gltA genes. Our serological results on horses and dogs (sentinels for BSF) appoint for the circulation of a SFG Rickettsia in the study area, however in a very low infection rate among the A. cajennense tick population.     

云南家鼠鼠疫疫源地人群和指示动物血清鼠疫F1抗体调查

目的 了解云南省家鼠鼠疫疫源地静息期人和指示动物血清鼠疫F1抗体情况,分析鼠疫发生和流行的风险。方法 2019年7——8月,课题组选择云南省家鼠鼠疫疫源地弥勒市、芒市和梁河县作为研究区域,对8个曾经报告2次及以上鼠疫的自然村和8个从未报告鼠疫的自然村的人群及指示动物进行血清采集,每个自然村采集人血清不少于20份,指示动物血清不少于10份。用间接血凝法检测鼠疫F1抗体,并用上转发光法对阳性样本进行再次检测确认。结果 368份人血清中仅有1份鼠疫F1抗体阳性,307份犬血清和12份猫血清鼠疫F1抗体均为阴性。结论 云南家鼠鼠疫疫源地人群鼠疫F1抗体阳性率非常低,但鼠疫F1抗体阳性的自然村需要加强鼠疫监测。指示动物未检测出鼠疫F1抗体,当地发生鼠疫的风险不高。     

Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from northwestern Spain

The aim of the present work was to investigate the seroprevalence against Ehrlichia canis (Ec), Anaplasma phagocytophilum (Ap), Neorickettsia risticii (Nr), Rickettsia conorii (Rc), and Borrelia burgdorferi (Bb) in two different clusters of canine samples from Northwestern Spain. Cluster 1 included 479 dogs presented at veterinary clinics located in Ourense and Pontevedra. Cluster II included 170 dogs from the public kennel of Ourense. All 649 canine serum samples were analyzed by immunofluorescent antibody test. Prevalences against the above-mentioned agents in cluster I were: Rc (24.6%), Bb (6.26%), Ec (3.13%), Ap (5.01%), and Nr (1.04%), whereas for cluster II were: Rc (50%), Bb (8.8%), Ec (54.7%), Ap (45.3%), and Nr (4.7%). Rc was significantly associated with age and history of exposure to ticks, and Bb showed a statistical relationship with age and clinical status. Ec and Ap were related to the occupation of the dogs, with stray dogs being the most frequently seropositive. Furthermore, seroreactivity against Ec and Ap was significantly higher in Ourense than in Pontevedra. The univariate analysis demonstrated a significant concomitant seroreactivity between Ec and Ap and between Rc and Ec and Ap antigens. The seroreactivity to Nr must be interpreted very cautiously as this infectious agent has been seldom reported outside North America.     

Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii

Rhipicephalus sanguineus group ticks collected on dogs in Thailand were maintained in the laboratory over several generations to test methods to infect these ticks with Rickettsia conorii, the agent of Mediterranean spotted fever. Three methods were tested: (1) infection of adults and nymphs through artificially induced bacteremic rabbit, (2) capillary feeding of solution containing 5 x 10(3) and 5 x 10(5)pfu/mL of R. conorii to adult female ticks, and (3) immersion of engorged nymphs which were "one leg-cut," "two leg-cut," or "cuticle cut" in solution containing R. conorii. The most efficient method to infect adult ticks with R. conorii was infection of ticks through the bacteremic rabbit (71.4%). The best method to infect nymphs with R. conorii was immersion of "one leg-cut" engorged nymphs in solution containing R. conorii (30%). Interestingly, a high mortality of the ticks infected with R. conorii was observed regardless of the method used. The harmful effect of R. conorii on Rh. sanguineus group ticks from Thailand is discussed including the role of the geographic origin of the ticks and the difficulties to identify ticks within this group to the species level.     

Hepadnavirus DNA Is Detected in Canine Blood Samples in Hong Kong but Not in Liver Biopsies of Chronic Hepatitis or Hepatocellular Carcinoma

Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.     

Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST)

Leishmaniasis caused by Leishmania infantum is a prevalent disease in dogs and humans. A serological survey of Leishmania infection in dogs was carried out in the endemic region of Alto Douro (north Portugal). Two hundred and ninety-four dogs from the municipality of Peso da Régua were examined for clinical signs of canine leishmaniasis (CanL), and sera samples were evaluated by the direct agglutination test (DAT) and the fast agglutination screening test (FAST). The sero-prevalence of infection was 20.4%, after screening the study population by FAST and subsequent confirmation by DAT. The overall prevalence of disease was 3.1%. Only 15.0% of the sero-positive dogs had clinical signs of CanL. A high degree of agreement (88.4%; kappa value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test for canine Leishmania infection in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of CanL.     

Kinetics of canine antibody response to saliva of the sand fly Lutzomyia longipalpis

Zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum is transmitted from dogs to humans by sand flies and Lutzomyia longipalpis is a major vector of this disease. We studied the antibody response in dogs experimentally exposed to L. longipalpis females to characterize sand fly salivary antigens recognized by canine sera and to find out whether the level of specific anti-saliva antibodies reflects the intensity of exposure. Sera from repeatedly bitten dogs revealed up to six salivary protein bands with approximate molecular weight of 66, 55, 45, 37-39, 34, and 25 kDa in L. longipalpis salivary gland lysate. Anti-saliva immunoglobin (Ig) G and its subclasses were found to be useful markers of exposure to sand flies. Specific IgG, IgG1, and IgG2 were related to numbers of bloodfed L. longipalpis females, and increased antibody levels were detectable throughout the study, i.e. more than 6 months after the last exposure. In contrast, specific IgE response developed in some dogs only, and no correlation was observed between its level and the intensity of exposure. Screening of dog sera for specific IgG against salivary antigens of the vector is suggested as a useful epidemiological tool in VL foci. Monitoring canine antibody response to sand fly saliva also allows evaluation of the effectiveness of anti-vector campaigns.     

Rickettsia slovaca infection in humans in the northeast of Spain: seroprevalence study

Catalonia is an endemic area of Mediterranean spotted fever. In 1997, A. Lakos described a new tick-borne infectious disease called tick-borne lymphadenopathy. The causative agent is Rickettsia slovaca, which is transmitted by Dermacentor marginatus ticks. We have diagnosed human cases in Catalonia. The objective of this study was to determinate seroprevalence of R. slovaca infection in humans in the northeast of Spain. The population included 217 subjects from Catalonia, northeast of Spain and was stratified by age and living place (rural, suburban, and urban). Age, gender, residence area, contact with animals, occupation, and history of rickettsioses was surveyed. Immunoglobulin G was measured by indirect immunofluorescence assay. Titers >or= 1/40 were considered. Seroprevalence of R. slovaca was 5.5% at titers of 1/40-1/320. Eight (3.7%) sera had antibodies against R. slovaca exclusively. Four sera reacted also against Rickettsia conorii and/or Bar29. Seroprevalence of R. slovaca would range from 3.7% to 5.5%. The only statistically significant association was that between R. slovaca seropositivity and age. We present serologic evidence of R. slovaca infection among population of Catalonia, northeast of Spain.     

Seroprevalence of murine typhus and fièvre boutonneuse in certain human populations in Egypt

A study was conducted between 1984 and 1987 to determine the prevalence of Rickettsia typhi and Rickettsia conorii infections among humans residing in the Nile Delta, Suez Canal area and Nile Valley of Egypt. Serum specimens were obtained from garbage and rodent control workers, other unclassified occupational workers, and from patients with fever of undetermined aetiology. All sera were assayed for IgA + IgM + IgG (IgAMG) antibody mixture and if positive, reassayed for specific IgM antibody to rickettsia by the indirect fluorescent antibody technique. R. typhi antibody was found in 19% (33/178) of the garbage collectors, whereas only 1% (2/178) had demonstrable antibody to R. conorii. Among those with other occupations, R. typhi antibody was detected in 0.7% (2/295) and none had R. conorii antibody. The antibody prevalence rate for R. typhi among patients with febrile illness ranged from 25 to 41%, and from 2 to 15% for R. conorii, at three different locations in Egypt. In addition, IgM antibody to R. typhi was demonstrated in some patients showing symptoms compatible with rickettsial disease and in some patients who seroconverted, indicating that R. typhi was the cause of illness among some of these patients. These findings support previous observations that R. typhi and R. conorii are the causes of human rickettsial disease in Egypt, and that humans are commonly infected with R. typhi.