Molecular identification of tick‐borne pathogens infecting cattle in Mymensingh district of Bangladesh reveals emerging species of Anaplasma and Babesia

Tick‐borne diseases are considered a major hindrance to the health and productive performance of cattle in Bangladesh. To elucidate the epidemiology of tick‐borne pathogens (TBP s) in local cattle, a cross‐sectional study was performed in the 12 subdistricts (Upazilas) of Mymensingh district in Bangladesh. Blood samples and ticks were collected from 384 clinically healthy cattle kept by 135 farmers from 96 randomly selected villages. DNA extracted from the blood samples was subsequently screened by polymerase chain reaction (PCR ) and a Reverse Line Blot (RLB ) hybridization assay using an in‐house prepared chemiluminescence solution for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria spp. A total of 2,287 ticks were collected from 232 infested cattle (60.4%, 232/384) and identified morphologically as Rhipicephalus (Boophilus) microplus (n  = 1,432, 62.6%) and Haemaphysalis bispinosa (n  = 855; 37.4%). The RLB results demonstrated that the majority of the cattle (62.2%) were infected with at least one TBP . Theileria orientalis infections were most common (212/384, 55.2%) followed by infections with Anaplasma bovis (137/384, 35.67%), Anaplasma marginale (16/384, 4.17%), Babesia bigemina (4/384, 1.04%) and Babesia bovis (2/384, 0.52%). A previously uncharacterized Anaplasma sp. (Anaplasma sp. Mymensingh) and Babesia sp. (Babesia sp. Mymensingh), which are genetically closely related to Anaplasma platys and B. bigemina , were detected in 50 of 384 (13.0%) and 1 of 384 (0.3%) of the blood samples, respectively. Key risk factors for the occurrence of T. orientalis , A. marginale and Anaplasma sp. Mymensingh were identified. In conclusion, this study revealed that cattle in Mymensingh district are mainly infested with R. microplus and H. bispinosa ticks and may carry multiple TBP s. In addition, two previously uncharacterized pathogens were detected in the bovine blood samples. The pathogenicity of these species remains to be determined.     

Molecular detection of tick‐borne pathogens in bovine blood and ticks from Khentii,Mongolia

Recent studies reported the detection of DNA from tick‐borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi‐locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district.     

Bovine anaplasmosis and tick‐borne pathogens in cattle of the Galapagos Islands

A cross‐sectional study was conducted to determine the species of Anaplasma spp. and estimate its prevalence in cattle of the three main cattle‐producing Galapagos Islands (Santa Cruz, San Cristóbal and Isabela) using indirect PCR assays, genetic sequencing and ELISA . Ticks were also collected from cattle and scanned for 47 tick‐borne pathogens in a 48 × 48 real‐time PCR chip. A mixed effects logistic regression was performed to identify potential risk factors explaining Anaplasma infection in cattle. A. phagocytophilum was not detected in any of the tested animals. Genetic sequencing allowed detection of A. platys ‐like strains in 11 (36.7%) of the 30 Anaplasma spp.‐positive samples analysed. A. marginale was widespread in the three islands with a global between‐herd prevalence of 100% [89; 100]_95%CI and a median within‐herd prevalence of 93%. A significant association was found between A. marginale infection and age with higher odds of being positive for adults (OR = 3.3 [1.2; 9.9]_{95% Bootstrap CI} ). All collected ticks were identified as Rhipicephalus microplus. A. marginale , Babesia bigemina , Borrelia theileri and Francisella ‐like endosymbiont were detected in tick pools. These results show that the Galapagos Islands are endemic for A. marginale .     

Tick‐borne pathogens in ticks (Acari: Ixodidae) collected from various domestic and wild hosts in Corsica (France), a Mediterranean island environment

Corsica is a mountainous French island in the north‐west of the Mediterranean Sea presenting a large diversity of natural environments where many interactions between humans, domestic animals and wild fauna occur. Despite this favourable context, tick‐borne pathogens (TBPs) have not systematically been investigated. In this study, a large number of TBPs were screened in ticks collected over a period of one year from domestic and wild hosts in Corsica. More than 1,500 ticks belonging to nine species and five genera (Rhipicephalus, Hyalomma, Dermacentor, Ixodes and Haemaphysalis) were analysed individually or pooled (by species, gender, host and locality). A real‐time microfluidic PCR was used for high‐throughput screening of TBP DNA. This advanced methodology enabled the simultaneous detection of 29 bacterial and 12 parasitic species (including Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia and Theileria). The Crimean–Congo haemorrhagic fever (CCHF) virus was investigated individually in tick species known to be vectors or carriers of this virus. In almost half of the tick pools (48%), DNA from at least one pathogen was detected and eleven species of TBPs from six genera were reported. TBPs were found in ticks from all collected hosts and were present in more than 80% of the investigated area. The detection of DNA of certain species confirmed the previous identification of these pathogens in Corsica, such as Rickettsia aeschlimannii (23% of pools), Rickettsia slovaca (5%), Anaplasma marginale (4%) and Theileria equi (0.4%), but most TBP DNA identified had not previously been reported in Corsican ticks. This included Anaplasma phagocytophilum (16%), Rickettsia helvetica (1%), Borrelia afzelii (0.7%), Borrelia miyamotoi (1%), Bartonella henselae (2%), Babesia bigemina (2%) and Babesia ovis (0.5%). The high tick infection rate and the diversity of TBPs reported in this study highlight the probable role of animals as reservoir hosts of zoonotic pathogens and human exposure to TBPs in Corsica.     

Exposure to West Nile virus and tick‐borne encephalitis virus in dogs in Spain

In the past decade, the spread of emerging zoonotic flaviviruses (genus Flavivirus , family Flaviviridae ) has been reported in many regions worldwide, representing a threat to both human and animal health. A serosurvey was carried out to assess exposure and risk factors associated with antigenically related flaviviruses, particularly West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ), in dogs in Spain. Flavivirus antibodies were detected in 39 of 815 dogs (4.8%; 95% CI : 3.3–6.3) by bELISA . Significantly higher seropositivity was observed in hunting dogs compared to pet dogs. Virus neutralization tests confirmed WNV ‐specific and TBEV ‐specific antibodies in 11 and 14 bELISA ‐positive dogs, respectively. This is the first serosurvey of WNV and TBEV in dogs in Spain and the first report of TBEV circulation in this country. The seropositivity obtained indicates widespread, but not homogeneous, distribution of WNV and TBEV in dogs in Spain. In 2013 and 2015, WNV ‐seropositive dogs were detected in those areas of Andalusia where the highest number of WNV outbreaks were reported in both horses and humans. Antibodies against TBEV have been found in dogs sampled in two different periods and regions in Spain. Serosurveillance in dogs could be a complementary way of monitoring the activity of emerging flaviviruses in Spain.     

Specific detection and differentiation of tick‐borne encephalitis and West Nile virus induced IgG antibodies in humans and horses

Tick‐borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod‐borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV‐endemic regions co‐circulation of both viruses is increasing. Flaviviruses are structurally highly similar, which leads to cross‐reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false‐positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross‐reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV‐ and WNV‐assays with improved specificities.     

Ticks infesting dogs in rural communities of Yucatan,Mexico and molecular diagnosis of rickettsial infection

Rickettsial infection in dog‐associated ticks in three rural communities of Yucatan, Mexico was investigated using qPCR and nested PCR assays. A total of 319 dogs were studied and ticks samples were collected. A total of 170 dogs were infested with ticks (frequency of 53.4%). Overall, 1,380 ticks representing seven species were collected: Amblyomma mixtum, A. ovale, A. parvum, A. cf. oblongoguttatum, Ixodes affinis, Rhipicephalus microplus, and R. sanguineus sensu lato. The most abundant species was R. sanguineus s.l. with a mean intensity of 7.4 ticks/host. Dogs in the communities of Chan San Antonio and Yaxcheku were 2.84 and 2.41 times more likely to be infected with R. sanguineus compared with Sucopo (p < 0.05). Adult pools of A. mixtum, A. parvum, I. affinis, R. microplus, and A. c.f. oblongoguttatum were negative to E. chaffeensis, E. ewingii, A. phagocytophilum, and R. rickettsii. However, pools of R. sanguineus s.l. adults and A. ovale adults, as well as nymphs of Amblyomma spp. were positive to E. canis. Sequencing analysis of the nested PCR products amplifying the 16S rRNA gene fragment of E. canis confirmed the results and revealed 100% identity with sequences of E. canis. This is the first report worldwide of E. canis infection in A. ovale by PCR. This finding does not necessarily indicate that A. ovale is a competent vector of E. canis because pathogen transmission of this specific tick to a naïve dog remains to be documented. This study documented that different tick species parasitize dogs in Yucatan, Mexico, where R. sanguineus s.l., A. ovale, and nymphs of Amblyomma spp. were shown to be infected with E. canis. These findings highlight the need for control strategies against tick infestations in dogs to prevent the risk of tick‐borne disease transmission among companion animal and probably human populations.     

Molecular prevalence and ecoregion distribution of select tick‐borne pathogens in Texas dogs

Tick‐borne diseases (TBD), caused by borrelial, rickettsial and babesial pathogens, are common across the United States and can cause severe clinical disease in susceptible hosts, such as domestic dogs. However, there are limited TBD molecular epidemiological reports for dogs in Texas, and none for the non‐Lyme borrelial pathogen responsible for causing tick‐borne relapsing fever (TBRF). Therefore, data to support the prevalence of TBRF in the canine population is inadequate. This study aimed to characterize the molecular prevalence of 11 causative agents responsible for three TBD groups within domestic dogs with an emphasis on pathogen distribution within Texas ecoregions. A total representative number of 1,171 whole‐blood samples were collected opportunistically from two Texas veterinary diagnostic laboratories. A layerplex real‐time PCR assay was utilized to screen the dog samples for all 11 pathogens simultaneously. The overall molecular infection prevalence of disease was 0.68% borrelial, 1.8% rickettsial and 0.43% babesial pathogens, for a TBD total of 2.73% across Texas. Higher molecular prevalence was observed when analysed by ecoregion distinction, including 5.78% rickettsial infections by Ehrlichia canis and Anaplasma platys in the Rolling Plains ecoregion, and an average of 1.1% Borrelia turicatae and 1.0% Babesia gibsoni across detected ecoregions. To our knowledge, our findings indicate the first molecular detection of A. platys in Texas, and the first report of coinfections with E. canis and A. platys in dogs of Texas. The zoonotic concerns associated with TBDs, in conjunction with dogs’ implication as an effective sentinel for human disease, highlight the importance of characterizing and monitoring regions associated with active infections in dogs. Surveillance data obtained from this study may aid public health agencies in updating maps depicting high‐risk areas of disease and developing preventative measures for the affected areas.     

Serological Evidence of Brucella abortus Prevalence in Punjab Province,Pakistan – A Cross‐Sectional Study

Twenty‐one villages in Punjab Province, seven from each region (North, Central & South) were randomly selected to determine the prevalence of B. abortus infection by two age categories (Group A: 0–2 years; Group B: >2 years) in cattle and buffalo populations. In each village, eight blood samples were collected from each age group of cattle and buffaloes. Sera from a total of 672 blood samples (336 each from cattle and buffaloes) were analysed for the presence of antibodies to B. abortus using rose bengal plate agglutination test (RBPT). Further confirmation of the RBPT‐positive samples was carried out using competitive ELISA. Overall, 43 of 672 (6.4%) sera were found positive for the presence of antibodies to B. abortus using RBPT. In cattle, the prevalence of B. abortus antibody was 5.06%, and in buffaloes, it was 7.74%. From the RBPT‐positive sera, 30 (70%) sera were confirmed for B. abortus antibodies using competitive ELISA. Results indicated that the prevalence of infection increased with the age of animals i.e. 3.27% and 9.52% in groups A and B, respectively. In cattle, incidence of these antibodies was 3.57% and 6.54% while in buffaloes, it was 2.98% and 12.50% in groups A and B, respectively. The region‐wise prevalence of B. abortus infection in buffaloes was 3.13%, 4.46% and 4.02% and in cattle, it was 3.13%, 1.79% and 2.68% in Northern, Central and Southern regions, respectively. This study provided baseline data on the occurrence of brucellosis infection in cattle and buffaloes at village level. This is the first study in Punjab province on the prevalence of brucellosis at village level.     

Serological and molecular epidemiology of foot‐and‐mouth disease viruses in agro‐pastoralist livestock herds in the kachia grazing reserve,Nigeria

The Kachia Grazing Reserve (KGR) is located in Kaduna state in north‐western Nigeria and consists of 6 contiguous blocks housing 744 defined households (HH), all engaged in livestock keeping. It is considered as a homogenous epidemiological unit and a defined study area. In 2012, all cattle and sheep of 40 selected HH were sampled to determine sero‐prevalence of antibodies to foot‐and‐mouth disease virus (FMDV) and of FMDV. The overall sero‐prevalence of antibodies to the non‐structural 3ABC protein (NSP‐3ABC ELISA) was 28.9% (380/1,315) (30.6% cattle; 16.3% sheep), and in 4.5% (62/1,380) (5% cattle; 0.6% sheep) of the examined sera FMD viral RNA could be detected by real‐time RT‐PCR (rRT‐PCR). Additionally, in 2012 and 2014 serum, epithelium and probang samples were collected from cattle in reported FMD outbreaks and the causative FMDVs were molecularly characterized. Approximately half (28/59) of the outbreak sera reacted positive in NSP‐3ABC ELISA, and 88% (52/59) of the outbreak sera contained detectable viral RNA. Overall, antibodies against five FMDV serotypes (O, A, SAT1, SAT2 and SAT3) were detected by solid phase competitive ELISA with combinations of two or more serotypes being common. Of the 21 FMDVs that could be isolated 19 were sequenced and 18 were confirmed as SAT2 (lineage VII) while one was characterized as serotype O (EA‐3 topotype). Phylogenetic analysis revealed a close relationship between Nigerian FMDV strains and strains in this region and even with strains in North‐Africa. Our findings indicate that FMD constitutes an endemic health problem to cattle rearing in the agro‐pastoralist community in the KGR and that the KGR is not a closed epidemiological unit. Insight into the local FMDV epidemiology and in the circulating FMDV serotypes/strains is of support to the relevant authorities in Nigeria when considering the need for an FMD control policy to improve animal production in grazing reserves.     

Clinical Characteristics of Breast Cancers in African‐American Women with Benign Breast Disease: A Comparison to the Surveillance,Epidemiology, and End Results Program

Benign breast disease (BBD) is a very common condition, diagnosed in approximately half of all American women throughout their lifecourse. White women with BBD are known to be at substantially increased risk of subsequent breast cancer; however, nothing is known about breast cancer characteristics that develop after a BBD diagnosis in African‐American women. Here, we compared 109 breast cancers that developed in a population of African‐American women with a history of BBD to 10,601 breast cancers that developed in a general population of African‐American women whose cancers were recorded by the Metropolitan Detroit Cancer Surveillance System (MDCSS population). Demographic and clinical characteristics of the BBD population were compared to the MDCSS population, using chi‐squared tests, Fisher's exact tests, t‐tests, and Wilcoxon tests where appropriate. Kaplan–Meier curves and Cox regression models were used to examine survival. Women in the BBD population were diagnosed with lower grade (p = 0.02), earlier stage cancers (p = 0.003) that were more likely to be hormone receptor‐positive (p = 0.03) compared to the general metropolitan Detroit African‐American population. In situ cancers were more common among women in the BBD cohort (36.7%) compared to the MDCSS population (22.1%, p < 0.001). Overall, women in the BBD population were less likely to die from breast cancer after 10 years of follow‐up (p = 0.05), but this association was not seen when analyses were limited to invasive breast cancers. These results suggest that breast cancers occurring after a BBD diagnosis may have more favorable clinical parameters, but the majority of cancers are still invasive, with survival rates similar to the general African‐American population.