Opening the Black Box of Host Range, Vectorial Diversity, and Genetic Variants of Genus Anaplasma: The Contributing Factors Toward Its Zoonosis.

Background: Genus Anaplasma of the family Anaplasmataceae possesses bacteria of hematopoietic origin, which are obligate intracellular Gram-negative bacteria transmitted mainly by tick vectors. The members of this group of infectious agents are not new as etiological agents of animal diseases worldwide. However, now, reports of their zoonotic potential have gained currency to study these pathogens. The emergence of new species of Anaplasma and the spread of existing species to new areas and hosts highlight the importance of monitoring and improving diagnostic and treatment options for zoonotic diseases caused by Anaplasma. Conclusion: This review focuses on the general and distinctive characteristics of Anaplasma spp., with particular emphasis on the novel species and their diverse spectrum of hosts as potential risk factors impacting its emerging zoonosis.

The first molecular identification and phylogenetic analysis of tick-borne pathogens in captive wild animals from Lohi Bher zoo, Pakistan.

Tick-borne pathogens are causing severe diseases in livestock, wild animals, and humans. Wild animals play a crucial role in tick-borne pathogens' transmission life cycle by serving as reservoir hosts or intermediate hosts, posing a continuous risk for domestic animals and humans. The presence of tick-borne pathogens is often ignored in wild animals kept in zoos, which is a public health concern. In the present study, we investigated these pathogens in tick-infested captive wild animals at the Lohi Bher zoo, Pakistan. Blood samples were collected from 22 animals, which include urials (4) (Ovis aries vignei), blackbucks (3) (Antilope cervicapra), fallow deer (1) (Dama dama), hog deer (6) (Axis porcinus), chinkaras (4) (Gazella bennettii), white tiger (2) (Panthera tigris tigris), a giraffe (Giraffa camelopardalis), and African lions (2) (Panthera leo). The samples were screened for Piroplasm and Anaplasma spp. by polymerase chain reaction targeting different gene loci. We detected three Theileria spp. and one Anaplasma sp. from the investigated captive wild animals. The Theileria sp. dama gazelle was detected from chinkara, Theileria sp. NG-2012b from chinkara and giraffe and T. parva from African lion, and Anaplasma bovis was identified in a giraffe. Moreover, Theileria sp. and Anaplasma sp. coinfection was detected in one giraffe. Overall, this study shows that Theileria spp. and Anaplasma spp. are circulating in captive wild animals, which can play an important role in their spread. Further studies are required to monitor tick-borne pathogens in zoo animals and their potential to spread from exotic wild captive animals to local wild and domestic.

The emergence of Dengue Fever in Sheikhupura, Pakistan: its seroprevalence and risk factors assessment during 2014-2017.

INTRODUCTION: District Sheikhupura encountered its first dengue outbreak in 2014 but lacked serological evidence and reports of risk factors associated with it. To assess this, a hospital-based study was conducted from January 2014 to December 2017. METHODOLOGY: Blood from 333 participants was collected, the serum obtained was tested for IgG and IgM antibodies against DENV using a commercially available ELISA kit. RESULTS: The results showed that out of all (n= 333) samples tested, 120 were turned up positive for DENV, making an overall prevalence of 36%. Of the 120 confirmed cases, 55% (n = 66) were recorded in 2014, 10% (n = 12) in 2015, 27.5% (n = 33) in 2016, and 7.5% (n = 9) in 2017. It was found that 68.3% (n = 82) were male and 31.7% (n = 38) were female, with 61% (n = 74) patients aged between 11-30 years. The highest prevalence of infection, 94.2% (n = 113), was noted after the rainy season. During the study, the highest number of cases appeared in Ferozewala Tehsil. The factors age, gender, and season were found statistically significant with the prevalence of infection (p < 0.05). CONCLUSIONS: The study is the first report on the detection of dengue in the Sheikhupura district. The survey anticipated its geographical expansion, determined associated risk factors, and suggests active disease surveillance in the area.

Molecular Survey of Tick-Borne Pathogens Reveals Diversity and Novel Organisms With Veterinary and Public Health Significance in Wildlife From a National Nature Reserve of China.

Wildlife is involved in the maintenance and transmission of various tick-borne pathogens. The objective of the present study was to determine the occurrence and diversity of tick-borne pathogens in free-ranging wild animals collected from Tangjiahe National Nature Reserve of China. Blood or liver samples from 13 wild animals (5 takin, 3 Himalayan goral, 3 Reeves' muntjac, 1 forest musk deer, and 1 wild boar) were collected and screened for piroplasm, Anaplasma spp., Ehrlichia spp., and spotted fever group (SFG) rickettsiae by PCR-based on different gene loci. Three Theileria species, a potential novel Theileria parasite (Theileria sp. T4) and two Anaplasma species were identified in those wildlife. Theileria capreoli was found in Himalayan goral, Reeves' muntjac, and forest musk deer; Theileria luwenshuni, Theileria uilenbergi, and a potential novel, Theileria parasite (Theileria sp. T4), were identified in takin. Meanwhile, Anaplasma bovis was identified in Himalayan goral, takin, Reeves' muntjac, forest musk deer, and wild boar; Anaplasma phagocytophilum and related strains was found in takin, Reeves' muntjac, and forest musk deer. All wildlife included in this study was negative for Babesia, Anaplasma ovis, Anaplasma marginale, Ehrlichia, and SFG rickettsiae. Moreover, coinfection involving Theileria spp. and Anaplasma spp. was observed in eight wild animals. This study provided the first evidence of tick-borne pathogens in free-ranging wild animals from the nature reserve, where contact between domestic and wild animals rarely occurs.

Identification and evaluation of midgut protein RL12 of Dermacentor silvarum interacting with Anaplasma ovis VirD4.

Anaplasma ovis, a tick-borne intra-erythrocytic Gram-negative bacterium, is a causative agent of ovine anaplasmosis. It is known that Dermacentor ticks act as biological vectors for A. ovis. VirD4 is the machine component of Type IV Secretion System of A. ovis. To better understand the pathogen-vector interaction, VirD4 was used as a bait protein for screening midgut proteins of Dermacentor silvarum via yeast two-hybrid mating assay. As a result, a ribosomal protein RL12 was identified from the midgut cDNA library of D. silvarum. For further validation, using in vitro Glutathione S-transferase (GST) pull-down assay, interaction between the proteins, GST-RL12 and HIS-VirD4, was observed in Western blot analysis. The study is first of its kind reporting a D. silvarum midgut protein interaction with VirD4 from A. ovis. Functional annotations showed some important cellular processes are attributed to the protein, particularly in the stringent response and biogenesis. The results of the study suggest the involvement of the VirD4-RL12 interaction in the regulation of signaling pathways, which is a tool for understanding the pathogen-vector interaction.

Identification and evaluation of UL36 protein from Dermacentor silvarum salivary gland and its interaction with Anaplasma ovis VirB10.

BACKGROUND: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. METHODS: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. RESULTS: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. CONCLUSIONS: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.

In vitro influence of Theileria annulata on the functions of bovine dendritic cells for stimulation of T lymphocyte proliferation.

The present study was performed on antigen-presenting cells (APCs) of Theileria annulata transformed dendritic cells (TaDCs) and monocyte-derived dendritic cells (MoDCs) to compare differences in antigen presentation and stimulation of T lymphocyte proliferation. Antigen presentation for T lymphocyte proliferation was analysed by flow cytometry. Additionally, the level of mRNA transcription of small GTPases of the Rab family expressed in the TaDC cell line was analysed by quantitative real-time polymerase chain reaction (Q-RT-PCR). The endocytosis rate of TaDCs was significantly (P < 0.01) lower than in MoDCs. In contrast, when T lymphocytes were co-cultured with TaDC-APCs T cell proliferation was similar, while co-culture with MoDC-APC stimulated proliferation of CD4+ cells to a greater degree than CD8+ cells. However, the efficacy of TaDC-APCs to stimulate T lymphocytes dropped as the number of passages of TaDC-APC increased. Likewise, the transcription level of Rab family genes also significantly (P > 0.001) declined with progressive passages (>50) of the TaDC cell line. We conclude that initially the TaDC cell line efficiently presents antigen to stimulate T lymphocyte proliferation to produce a cellular immune response against the presented antigen.

First Molecular Evidence of Anaplasma bovis and Anaplasma phagocytophilum in Bovine from Central Punjab, Pakistan.

Obligate intracellular bacteria belonging to the genus Anaplasma spp. are responsible for causing a hemolytic disease called anaplasmosis in animals, as well as in humans. This study was aimed at the molecular identification and genetic analysis of responsible causative agents of anaplasmosis beyond those already reported. A survey was performed during July and August 2018 in the Jhang District, Punjab, Pakistan. Four hundred and fifty blood samples from asymptomatic, tick-infested cattle were collected on FTA cards and tested for the Anaplasma spp. presence using nested-polymerase chain reaction (PCR) methods. The 16S ribosomal RNA gene sequences generated from the positive samples were used for genetic analysis of Anaplasma spp. The nested-PCR results showed the presence of two Anaplasma spp. with an overall prevalence rate of 10.44%, where the prevalence of A. bovis and A. phagocytophilum was 7.78% and 2.66%, respectively. The study portrayed new molecular data on the prevalence of Anaplasma spp. in the studied cattle population, indicating a potential threat to the human population as well.

Molecular detection and identification of tick-borne bacteria and protozoans in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China.

BACKGROUND: Small ruminants are important hosts for various tick species and tick-associated organisms, many of which are zoonotic. The aim of the present study was to determine the presence of tick-borne protozoans and bacteria of public health and veterinary significance in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. METHODS: The occurrence of piroplasms, Anaplasma phagocytophilum, A. bovis, A. marginale, A. capra, A. ovis, Ehrlichia spp. and spotted fever group rickettsiae was molecularly investigated and analyzed in 134 goats and 9 free ranging C. pygargus living in close proximity. RESULTS: Piroplasm DNA was detected in 16 (11.9%) goats and 5 C. pygargus. Sequence analysis of 18S rRNA sequences identified 3 Theileria species (T. luwenshuni, T. capreoli and T. cervi). Four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) were identified in goats and C. pygargus. Anaplasma ovis and A. bovis were detected in 11 (8.2%) and 6 (4.5%) goats, respectively; A. phagocytophilum, A. bovis and A. capra were found in 3, 7 and 3 C. pygargus, respectively. Sequence analysis of 16S rRNA sequences revealed the presence of 5 different genetic variants of A. bovis in goats and C. pygargus, while the analysis of 16S rRNA and gltA sequence data showed that A. capra isolates identified from C. pygargus were closely related to the genotype identified from sheep and Haemaphysalis qinghaiensis, but differed with the genotype from humans. Anaplasma/Theileria mixed infection was observed in 2 (1.5%) goats and 5 C. pygargus, and co-existence involving potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. All samples were negative for A. marginale, Ehrlichia spp. and SFG rickettsiae. CONCLUSIONS: These findings report the tick-borne pathogens in goats and C. pygargus, and a greater diversity of these pathogens were observed in wild animals. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The contact between wild and domestic animals may increase the potential risk of spread and transmission of tick-borne diseases.

Establishment and Expression of Cytokines in a Theileria annulata-Infected Bovine B Cell Line.

This study aimed to establish a pure single-cell Theileria annulata-infected B cell line for the assessment of cytokine production in transformed and lipopolysaccharide (LPS)-stimulated cells. Several studies have aimed to identify cell surface markers in T. annulata-transformed cells; however, no information on cytokine production in these cells is available. To investigate the potential of the transformed cells to produce cytokines and their potential responses to antigen-stimulation, we purified mature B cells (CD21) from the whole blood of cattle experimentally infected with the T. annulata Kashi strain by magnetic separation. The purity and specificity of the established cell line was assessed by the identification of specific cell surface markers (CD21, IgM, and WC4) by flow cytometry analysis. The transcript levels of the cytokines IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL16, LTA, TGFB1, TNFA, IFNA, and IFNB in transformed, buparvaquone (BW720c)-treated cells, and antigen-stimulated cells were analyzed by quantitative polymerase chain reaction (qPCR) using cDNA from these cells. A T. annulata-infected bovine B cell line was successfully established with a purity of ~98.8% (CD21). IL4 and IL12A were significantly (p < 0.01) upregulated in the transformed cells. In BW720c-treated transformed cells, IL12B, TGFB1, and IFNB were significantly (p < 0.01) upregulated. Notably, no significant (p > 0.05) upregulation of cytokines was observed in LPS-stimulated transformed cells. Moreover, IL1A, IL1B, IL8, and IL16 were significantly (p < 0.01) upregulated in LPS-stimulated B cells. Our data signify the potential use of this cell line for cytokine production, observance of immunoglobulins, and production of an attenuated vaccine against tropical theileriosis.

Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau.

Anaplasma species are tick-transmitted obligate intracellular bacteria that infect many wild and domestic animals and humans. The prevalence of Anaplasma spp. in ixodid ticks of Qinghai Province is poorly understood. In this study, a total of 1104 questing adult ticks were investigated for the infection of Anaplasma species. As a result, we demonstrated the total infection rates of 3.1, 11.1, 5.6, and 4.5% for A. phagocytophilum, A. bovis, A. ovis and A. capra, respectively. All of the tick samples were negative for A. marginale. The positive rates of A. phagocytophilum, A. ovis and A. capra in different tick species were significantly different. The positive rates of A. capra and A. bovis in the male ticks were significantly higher than that in the female ticks. Sequence analysis of A. ovis showed 99.5-100% identity to the previous reported isolates. The sequences of A. phagocytophilum had 100% identity to strains Ap-SHX21, JC3-3 and ZAM dog-181 from sheep, Mongolian gazelles, and dogs. Two genotypes of A. capra were found based on 16S rRNA, citrate synthase (gltA) gene and heat shock protein (groEL) gene analysis. In conclusion, A. bovis, A. ovis, A. phagocytophilum, and A. capra were present in the ticks in Qinghai Province. Anaplasma infection is associated with tick species, gender and distribution. These data will be helpful for understanding prevalence status of Anaplasma infections in ticks in Qinghai-Tibet Plateau.

Dengue fever is an emerging public health concern in the city of Multan, Pakistan: its seroprevalence and associated risk factors.

The prevalence of dengue IgG and IgM antibodies was investigated in 689 patients with suspected dengue. Of the 689 suspected cases, 373 (54.1%) were found to be positive for dengue antibodies, IgM being dominant. There was a significant relationship between incidence of dengue fever and season: all cases were reported during the rainy season, especially the post-monsoon season (89.5%), with none during the dry season. More male (79.3%) than female individuals were positive cases and the incidence was highest in the 21-49 year age group (63%). This is the first seroprevalence study reported from Multan, Pakistan.

Seasonal Distribution and Container Preference Ratio of the Dengue Fever Vector (Aedes aegypti, Diptera: Culicidae) in Rawalpindi, Pakistan.

Aedes aegypti (L.) and Aedes albopictus (Skuse) are known vectors of dengue, chikungunya, and other pathogens; however, their ecology and role in virus transmission has not been well studied in Pakistan. Here, we report on an intensive survey of potential breeding sites of Ae. aegypti in Rawalpindi, Punjab Province, Pakistan. The study continued for 11 mo and was divided into three seasons: January to June (pre-monsoon), July to September (monsoon), and October-November (post-monsoon). Larval mosquitoes were collected from all wet containers present in and around the houses. Altogether 5,570,418, 2,930,508, and 1,507,111 water-filled containers were examined during each season, of which 2,703, 8,843, and 3,439 were found positive for Ae. aegypti larvae or pupae, yielding Breteau indices of 0.46, 2.92, and 1.99%, respectively. Among 14 container types examined, the breeding preference ratio during all seasons was highest for roof-top water tanks and room evaporative coolers, followed by discarded tires and urban trash. The study concluded that increased urbanization, insufficient water supply and inefficient removal of urban trash resulted in increased numbers of nonbiodegradable containers around human dwellings, thereby creating ideal breeding habitats for Ae. aegypti. Measures such as integrated vector management, minimization of the breeding potential of Ae. aegypti by water management, proper disposal of discarded tires and urban trash, and health education were recommended for control of Ae. aegypti.

Spatial distribution and insecticide susceptibility status of Aedes aegypti and Aedes albopictus in dengue affected urban areas of Rawalpindi, Pakistan.

BACKGROUND & OBJECTIVES: Dengue is one of the most common arthropod-borne viral diseases which is transmitted mainly by two vector species, Aedes aegypti (Linnaeus 1762) and Ae. albopictus (Skuse, 1894) worldwide. As there is no effective medicine and vaccine available, vector control remains the most effective measure to prevent its transmission and outbreak. The aim of the study was to confirm the co-occurrence of Aedes aegypti and Ae. albopictus populations in the different localities of Rawalpindi, Pakistan and examine their susceptibility status against different groups of insecticides. METHODS: Ovitraps were randomly placed in the study localities. The number of eggs from all the ovitraps were counted and incubated for hatching in Medical Entomology and Disease Vector Control (MEDVC) insectarium for rearing up to adult stage. The adults were then identified by using the pictorial keys. Spatial distribution and aggregation of both Ae. aegypti and Ae. albopictus populations was determined by using Index of dispersion or variance to mean ratio and k values of the negative binomial distribution. The susceptibility status of both the species against different insecticides was assessed by using the World Health Organization (WHO) standard bioassay tests. RESULTS: The results showed that there was coexistence among Ae. aegypti and Ae. albopictus populations and the aggregation of their eggs was also observed in all the localities studied in Rawalpindi. Larval bioassays of both the populations exhibited incipient resistance against temephos while adult susceptibility testing results showed that both the species were resistant to DDT, malathion, bendiocarb and permethrin. INTERPRETATION & CONCLUSION: The results suggested that all the field populations of Ae. aegypti and Ae. albopictus existed together and showed qualitative changes in their susceptibility status. Resistance against deltamethrin and lambdacyhalothrin was not confirmed and further investigation was recommended to confirm the change in their susceptibility status. This study could help public health authorities to apply simultaneous control activities on both species due to their coexistence and also resistance management strategies should be formulated to slow down the process of development of resistance.