Ovis Infection Research Articles

Detection of Babesia spp., and Theileria spp., in sheep across diverse provinces of Iran

This study aimed to identify the diversity of Babesia and Theileria species in sheep across various regions of Iran using microscopic and molecular techniques, including species-specific PCR and enzymatic digestion. A total of 373 blood samples were collected from sheep during the tick vector activity period, from 2018 to 2021, in provinces such as Tehran, Alborz, Qazvin, Hamedan, West Azerbaijan, Kerman, and Fars.Results showed that 101 samples (27 %) exhibited piroplasms including 78 samples of Theileria spp. and 23 samples of Babesia spp. A molecular approach using general primers detected piroplasm parasites in 145 samples (38 %). Theileria ovis was notably present in 91 samples (24.39 %), followed by Theileria lestoquardi in 24 samples (6.43 %). Babesia ovis infection was detected in 30 samples (8.4 %). Despite extensive molecular evaluation, no other Babesia species, including Babesia motasi, were identified. Co-infections involving T. ovis and T. lestoquardi (4 samples; 1.07 %) and T. ovis and B. ovis (6 samples; 1.60 %) were observed. No Babesia spp. were detected in Kerman and Fars provinces, although T. ovis and T. lestoquardi were present.Blast analysis of the sequences indicated 100 % intra-species similarity, with inter-species similarities of 83.3 % (B. ovis and T. lestoquardi), 84.4 % (B. ovis and T. ovis), and 96.44 % (T. ovis and T. lestoquardi). In conclusion, B. ovis was the main cause of Babesiosis, while Theileriosis was predominantly attributed to T. ovis and T. lestoquardi. Molecular diagnostics play a key role in accurately distinguishing between these species, particularly in cases of co-infection involving Babesia spp. and Theileria spp.

Further Insights into The Pathogenic Mechanisms of Haemotropic Mycoplasma ovis.

In this study, we examined the effects of experimental intraperitoneal infection with haemotropic Mycoplasma ovis (0.5 mL of blood containing 80% parasitaemia) on selected serum biomarkers and cellular pathology in mice. After infection, M. ovis cells appeared in the blood films within one week. A dose-dependent peak of parasitemia was observed during the 3rd-week post-infection (pi), with a significant decrease in mean PCV between treatment versus control group at week 3 (t 14 = -3.693, P < 0.02), week 5 (t 14 = -2.096, P = 0.055), and week 7 (t 14 = -4.329, P = 0.001). There was a significantly (t 8 = -2.330, P = 0.048) lower serum oestrogen in treatment (10.38 ± 5.07) than control (17.43 ± 4.48), while serum progesterone was significantly (t 8 = 5.415, P = 0.001) increased in treatment (27.37 ± 2.17) than control (15.92 ± 4.20). Serum haptoglobin was significantly (t 8 = 8.525, P < 0.01) lower in treatment (8.72 ± 1.49) than control (18.16 ± 1.98) while the SAA was significantly (t 8 = 3.362, P = 0.01) higher in treatment (16.79 ± 2.71) than control (11.59 ± 2.15). Prominent lesions observed in the ovary include degeneration, necrosis, vacuolation, and hypertrophy of the lutein cells in corpora lutea. In the lymph nodes, diffused cellular hyperplasia of the lymphoid tissue in the cortex. In the liver, degeneration and necrosis accompanied by leucocytic cellular infiltration and Kupffer cell proliferation within the sinusoids. There were diffused leucocytic infiltrations and proliferative lesions in the glomerulus of the kidneys. The disturbance in progesterone and ovarian pathology highlights the potential role of haemotropic M. ovis in reproductive disorders. The observed changes in biomarkers and cellular reactions following M. ovis infection in the mouse may be further advanced in sheep and goats.

Tick control prevents carcass condemnations in lambs caused by Anaplasma ovis.

Ovine anaplasmosis is causing relevant economic losses in Spain due to icteric carcass condemnation in lambs. Anaplasma ovis infection occurs through grazing sheep that transfer ticks to their offspring. This study compared the efficacy of deltamethrin and cypermethrin pour-on treatments for tick control. A total of 250 PCR A. ovis-positive ewes and their offspring were divided into 5 groups. Group A (50 ewes/50 lambs) was kept as an untreated control group. In groups B (50/50) and C (45/93), the lambs were treated with deltamethrin pour-on and cypermethrin pour-on, respectively, one week after birth. In groups D (50/75) and E (51/68), the ewes were treated with cypermethrin pour-on and deltamethrin pour-on one week before the estimated parturition. External parasite assessment and A. ovis PCR were conducted before treatment and at 21 and 42 days post-treatment. Ewes were checked weekly for tick-detection until weaning. Lamb carcasses were examined at the slaughterhouse. Riphicephalus sanguineus sensu lato ticks were found in ewes throughout the study, with only one tick found in a control group lamb. Three lambs tested positive for A. ovis during the trial, with one condemnation at the abattoir due to jaundice. However, no significant differences were observed between treatment groups. Overall, a significant decrease in infected animals and condemned carcasses was observed compared to previous years, suggesting that deltamethrin and cypermethrin prevent A. ovis transmission from dams to lambs. Further studies are needed to confirm synthetic pyrethroids' effectiveness in controlling tick infestation and averting A. ovis transmission to lambs.

Evaluating serum electrolyte and trace element variations between Babesia ovis-infected and uninfected Lohi sheep

Babesiosis caused by Babesia ovis is a major threat to the livestock industry worldwide. In Pakistan, the Lohi sheep breed is an important economic resource, but limited information is available on the impact of B. ovis infection on this breed. This study aimed to investigate the serum electrolyte and trace element variations between B. ovis-infected and uninfected Lohi sheep from Pakistan. A total of 97 Lohi sheep was stratified based on the geographical distribution of Multan district, employing a multistage cluster sampling method. Blood and serum samples were collected from randomly selected sheep, and DNA extraction and PCR amplification were performed to detect B. ovis infection. Serum electrolyte and trace element levels were analyzed in infected (n=67) and uninfected (n=30) groups, stratified by age and gender. The study revealed a 69.07% overall infection rate of B. ovis in Lohi sheep. Infected sheep showed significantly elevated serum copper levels (p&lt;0.0001), with no substantial differences observed in serum iron, sodium, potassium, and chloride levels. However, age-wise variance analysis revealed statistically significant variations in sodium and potassium levels (p&lt;0.0001 and p&lt;0.05, respectively). Notably, serum chloride levels differed between infected and uninfected females in individuals younger than two years (p&lt;0.05). Serum iron levels remained consistent across different age groups. Comparative analysis indicated a higher prevalence of electrolyte imbalances, such as hyponatremia, hyperkalemia, hyperchloremia, and hypoferremia, in infected sheep compared to normal reference ranges, while instances of hypernatremia, hypokalemia, hypochloremia, and hyperferremia were less frequent. In conclusion, our study suggests that B. ovis infections could lead to alterations in serum electrolyte and trace element levels in Lohi sheep, emphasizing the importance of further research into the specific mechanisms driving these alterations to enhance disease management strategies tailored to this breed.

Seasonality of Oestrus ovis infection in sheep in the steppe zone of the Stavropol Territory

The purpose of the research is to study the seasonality of Oestrus ovis infection in sheep depending on the management conditions in the steppe zone of the Stavropol Territory.Materials and methods. The research to study the botfly flight seasonality was conducted on two farms in the Apanasenkovsky District and two farms in the Ipatovsky District of the Stavropol Territory. The number of sheep botflies was studied in the entire flight season by regular inspection and counting of the parasite adult stage on the outer surfaces of sheephouse walls and the side walls of sheds with a 15 day-interval.Results and discussion. The sheep botfly flight season was 5.5 months (from May 25–30 to October 31–November 11). Two increases in the number of botflies were observed during this period: in July and September–October. Infection of sheep with oestrosis in 1971 had two peaks (the first, May to June; the second, August to November). Due to the earlier spring 2022, the first peak started in the second half of March and lasted until the first half of July, and the second peak was observed from the second half of August to the end of November.

Molecular and haematological investigations on Anaplasma ovis infection in goats in Ahvaz, Iran: Insights into infection rate, haemolytic effect and breed influences.

Anaplasma ovis (A. ovis) is the predominant causative agent of anaplasmosis in goats and sheep in most tropical and subtropical regions of the world. However, there is considerable variation in reported infection rates, breed susceptibility, and controversial findings regarding the haemolytic effects of A. ovis infection in goats. Thus, we investigated the molecular and haematological aspects of A. ovis infection in goats from Ahvaz city. One hundred and fifty apparently healthy goats (74 blacks and 76 Najdi goats) were randomly sampled from six flocks in the Ahvaz suburb during ticks' activity season. Haematological evaluation, smear microscopic (SM) examination and PCR assay were performed to assess A. ovis infection. Additionally, the percentage of parasitemia was determined from blood smears. SM examination revealed that 25.7% of the goats displayed erythrocyte Anaplasma-like inclusion bodies. PCR analysis indicated that 54% of the goats were positive for A. ovis infection (44.6% of blacks and 63.2% of Najdi goats). No significant difference in haematological values was observed between healthy and infected goats based on PCR testing. However, a significant difference in haematological indices was observed between the group with parasitemia level of 0.01-0.02% (SM and PCR positive) compared to the healthy goats (SM and PCR negative), particularly concerning Hb, PCV and RBC count (p < 0.01). When the parasitemia exceeds 0.01%, A. ovis infection may disrupt haematological parameters in infected goats. The high prevalence of A. ovis infection (54%) among the studied goats underscores the importance of giving special attention to implementing necessary measures for disease control in the Ahvaz suburb.

Characterization of three predicted zinc exporters in Brucella ovis identifies ZntR-ZntA as a powerful zinc and cadmium efflux system not required for virulence and unveils pathogenic Brucellae heterogeneity in zinc homeostasis.

Brucella ovis causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonotic Brucella species that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potential B. ovis zinc exporters, including the ZntA ortholog previously studied in B. abortus. Despite an in-frame deletion removing 100 amino acids from B. ovis ZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity for B. abortus ZntA, which, together with differences in ZntR-mediated regulation of zntA expression between B. ovis and B. abortus, could contribute to explaining why the ΔzntR mutant of B. abortus is attenuated while that of B. ovis is virulent. Additionally, B. ovis ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for the B. abortus ortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in B. abortus, behaved as the B. ovis parental strain in all tests performed. However, their overexpression in the ΔzntA mutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence of B. ovis ΔzntA, suggest that host-mediated zinc intoxication is not a relevant mechanism to control B. ovis infection.

Molecular detection of A. phagocytophilium and A. ovis infection in goat flocks

Molecular detection of A. phagocytophilium and A. ovis infection in goat flocks

Role of Rhipicephalus bursa larvae in transstadial transmission and endemicity of Babesia ovis in chronically infected sheep.

Babesia ovis, transmitted by Rhipicephalus bursa ticks, is the causative agent of ovine babesiosis, a disease characterized by fever, anemia, hemoglobinuria, and high mortality in sheep. This study investigates whether sheep that survived babesiosis without treatment can serve as a source of infection for B. ovis-free host-seeking R. bursa larvae in a later season. Three donor sheep were experimentally infected with B. ovis, and after six months, persistence of B. ovis was assessed through blood and tick transmission experiments. Blood from donor sheep was intravenously injected into three recipient sheep, while donor sheep were also infested with B. ovis-free R. bursa larvae. Engorged nymphs molted to adults, and new recipient sheep were infested with these ticks. All recipient sheep were monitored for B. ovis for 100 days using microscopic, serological, and molecular approaches. The presence of B. ovis was confirmed in the recipient sheep that received blood, leading to clinical infection in two. However, no B. ovis was detected in recipient sheep infested with ticks. These results suggest that sheep recovering from B. ovis infection do not serve as a source of infection for R. bursa larvae in subsequent seasons.

Molecular Detection of Tick-Borne Pathogens in Kumasi: With a First Report of Zoonotic Pathogens in Abattoir Workers.

Tick-borne pathogens continue to infect humans and animals worldwide. By adapting to the movement of livestock, ticks facilitate the spread of these infectious pathogens. Humans in close contact with animals that could be amplifying hosts are especially at risk of being infected with tick-borne pathogens. This study involved the collection of dry blood spots (DBSs) to determine tick-borne pathogens occurring in slaughtered livestock and abattoir workers in Kumasi. This study employed the use of conventional PCR, RT-PCR, and Sanger sequencing to detect and identify the tick-borne pathogens. The resulting data was analysed using Stata version 13. A total of 175 DBSs were collected from goats (76), cattle (54), and sheep (45) in the Kumasi abattoir (130, 74.29%) and Akwatia Line slaughter slab (45, 25.71%). The pathogens identified were mostly bacterial including Anaplasma capra (9.71%), Anaplasma phagocytophilum (1.14%), and Rickettsia aeschlimannii (0.57.%). The only parasite identified was Theileria ovis (9.14%). A significant association was seen between A. capra (p < 0.001) infection and female sheep sampled from the Akwatia Line slaughter slab. Again, there was a significant association between T. ovis (p < 0.001) infections and female sheep from the Kumasi abattoir. From the human DBS (63) screened, the pathogens identified were all bacterial including Coxiella burnetii (1.89%), Rickettsia africae (1.89%), and R. aeschlimannii (1.89%). This study reports the first detection of R. aeschlimannii in livestock as well as the occurrence of the above-mentioned pathogens in humans in Ghana. Animals can serve as amplifying hosts for infectious pathogens; hence, there is an increased risk of infections among the abattoir workers. Continuous surveillance effort is essential, and abattoir workers need to protect themselves from tick bites and infectious tick-borne pathogens.

The Molecular Detection of the Anaplasma ovis Pathogens of the Serological Samples in Small Ruminants and Ixodid Ticks in Azerbaijan

A total of 704 blood samples (561 sheep, 143 goats) were examined for the Anaplasma ovis infection during a 1-year period. PCR and ELISA were used for the detection of the A. ovis antibodies. PCR assay identified A. ovis in 20 (8.1%) sheep and 15 (6.09%) goats. Using ELISA assay, 8.53% (31) were positive (21 sheep, 10 goats). A total of 246 blood smears were examined for the presence of intra-erythrocytic inclusions using Giemsa stain. Among the collected specimens, 60 were found positive with an overall prevalence of 24.3%. Among the 60 positive animals, 26 (43.3%) were sheep and 34 (56.7%) were goat. In the peripheral blood samples, the other piroplasmids — Babesia ovis, Theileria ovis, Th. recondita — were followed in an associative form. The ticks were collected, and the species composition was appointed in order to determine the ticks parasitized and have a pathogenic lifestyle in small ruminants. The intensive infection with the ticks of 2 genera — Rhipicephalus and Hyalomma was followed in small ruminants. It was determined that 45.8% of sheep and 35.1% of goats were infected intensively with the ticks of the Hyalomma genus. 110 samples prepared from the ticks of the Hyalomma genus were tested from the PCR test according to the Anaplasma ovis pathogen. 45 samples (40.9%) were assessed positively that 21 samples of them belonged to sheep and 24 of them to goats. 80 samples prepared from the internal organs of the ticks were examined according to the A. ovis parasite and the obtained results were analyzed. In 5 out of 35 samples which detected the parasites, A. ovis was followed, and in 30 samples, the associative parasites: Th. ovis, B. ovis piroplasmids. The Rickettsia and Coxiella pathogens were also detected in the samples.

Molecular Detection of Hemoplasma in animals in Tamil Nadu, India and Hemoplasma genome analysis.

Hemoplasma are small pleomorphic wall-less Gram-positive bacteria that infect erythrocytes of various mammalian hosts. They generally cause asymptomatic or chronic anaemia but occasionally causes overt life-threatening hemolytic anaemia. In the present study, 316 cattle, 115 sheep, 61 goats and 6 buffalo blood samples were collected from various villages or organized farms located in nine districts of Tamil Nadu to detect the hemoplasma by PCR. Overall prevalence of 43.04%, 65.22%, and 44.26% hemoplasma DNA was observed in cattle, sheep and goats, respectively. In total, 21 hemoplasma positive samples were sequenced for 16S rRNA gene which revealed 8 Mycoplasma wenyonii, 11 'Candidatus Mycoplasma haemobos' and one Mycoplasma ovis infection. Sheep blood samples from Chennai district were infected with 'Ca. M. haemobos' whereas sheep sample from Thiruvannamalai district was infected with M. wenyonii. At least 50% genes in the hemoplasma genomes were paralogous genes whose functions were not known. Only 'Ca. M. haemolamae' genome contained one primitive CRISPR system without any cas genes. Antimicrobial resistance genes (ARG) could not be identified in any of the hemoplasma genomes but homologous ARG were identified in all the genomes. Adhesion related gene EF-Tu was detected in all 14 hemoplasma genomes but enolase gene was detected only in 'Ca. M. haemohominis' SWG34-3 genome. This is the first report on the prevalence of hemoplasma infection in cattle, sheep and goat in India.

Seroprevalence and risk factors of Streptococcus ovis infection in Tibet, China.

Sheep septicemic streptococcosis is an acute, febrile, and septic zoonotic disease caused by Streptococcus ovis. There are few reports of this disease especaillyat high-altitude areas of Tibet, China. A cross-sectional study was conducted at five different counties of Shigatse area (a cityinTibet territory)from 2021 to 2022. Sera from 1458 randomly selected sheep and goats from 50 flocks were tested for antigens of Streptococcus ovis using a commercial ELISA kit. The individual animal level prevalence was 6.93% (95% CI: 5.7-8.4), and the flock prevalence level was 84% (95% CI: 70.9-92.8). At the individual animal level, year and management type were significantly associated with seropositivity by binary logistic regression analysis. Prevalence of Streptococcus in 2021 significantly differed from 2022 (OR = 2.16, 95% CI: 1.39-3.39). The prevalence of Streptococcus varied between management types with herds having higher odds of disease than intensive farms (OR = 2.64, 95% CI: 1.71-4.10). Hence, our study reports for the first time on seroepidemiology and risk factors of Streptococcus ovis infection, laying a theoretical foundation for preventing and controlling streptococcosis in Tibet area.

Molecular prevalence and phylogeny of Anaplasma marginale, Anaplasma ovis and Theileria ovis in goats and sheep enrolled from a hill station in Punjab, Pakistan.

Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

Molecular Epidemiology, Seasonality and Phylogenetic Investigations of Anaplasma ovis in Small Ruminants from Diverse Agro-Climatic Regions of Punjab, Pakistan.

Anaplasma (A.) ovis is the most important cause of anaplasmosis in small ruminants. The current study was planned to estimate the molecular prevalence, risk factors, and phylogenetic analysis of A. ovis infection in sheep and goats from different agro-climatic regions of Central and Southern Punjab, Pakistan. A total of 400 jugular blood samples were collected from asymptomatic goats (n = 200) and sheep (n = 200) from the Jhang and Dera Ghazi Khan districts from January 2021 to February, 2023. Two hundred blood samples were collected from each district. Ten union councils (UC) were randomly chosen from each district, and 20 samples were collected from each UC based on the multistage cluster sampling technique. The samples were analyzed with PCR targeting the major surface protein (msp4) gene of A. ovis. The overall molecular prevalence of anaplasmosis was 57.5%. The disease occurrence was higher in Dera Ghazi Khan (61.5%) than in the Jhang district (53.5%). Infection positivity was greater in goats (65.5%) than in sheep (49.5%). Multivariate logistic regression analysis indicated that host species [sheep; Odds Ratio (OR) = 3.212; p = 0.000, Confidence Interval (CI) = 1.968-5.242], age (adult; OR = 2.606; p = 0.003, CI = 1.398-4.858), and acaricide use (never; OR = 13.671; p = 0.000, CI = 6.414-26.283) were significantly higher risk for A. ovis in small ruminants (p< 0.05; OR > 1). The sequencing and phylogenetic analysis of four representative isolates in the current study (Genbank numbers; Goats: OQ302202, OQ302203; Sheep: OQ319592, OQ319593) revealed novel strains of A. ovis with 97-100% similarity from different countries. The msp4-based goat isolates showed greater genetic diversity, while sheep genotypes showed homology with isolates from Italy, Spain, Hungary, Cyprus, Spain, Iran, and China. The current surveillance study will help in devising prevention and control strategies regarding anaplasmosis in small ruminants. However, there is a need for further study on the clinicopathological and vector competence aspects of these genotypes.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72-3.46) and 1.46% (CI 0.87-2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43-14.90) and 0.33% (CI 0.02-1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01-0.51) and 2.08% (CI 1.25-3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey.

Molecular prevalence and associated infection risk factors of tick-borne protozoan and rickettsial blood pathogens in small ruminants

BackgroundTick-borne blood pathogens cause highly pathogenic diseases, which are associated with substantial economic losses in ruminants. Despite this, epidemiological research on these pathogens remains neglected in many countries. This study initiated a regional epidemiological survey that included the detection of molecular prevalence, associated risk factors, and gene sequencing, combined with phylogenetic analysis, targeting the two main tick-borne blood protozoan and rickettsial pathogens of Babesia, Theileria, and Anaplasma that infect small ruminants. One hundred blood samples were collected from 76 sheep and 24 goats.ResultsMicroscopic examination of Giemsa-stained blood films revealed that 73% of the samples were infected with at least one species of the three blood pathogenic organisms. Molecular diagnosis based on the 18 S rRNA for Babesia and Theileria species and the major surface protein 4 (msp4) for Anaplasma species, revealed that 43% of the small ruminants were infected with at least one of these pathogens. The animal’s sex was the most significant associated risk factor, with 49.4% of female animals infected compared with only 4% of male animals (P < 0.05). The open breeding system recorded the highest infection rate for tick-borne blood pathogens. Homology-based and phylogenetic analyses indicated that the specific isolate species were Babesia ovis (B. ovis), Theileria ovis (T. ovis), and Anaplasma ovis (A. ovis), with sequences showing significant identities with isolates from sheep, goats, and other animal species, and geographically diverse countries in Africa, Asia, and Europe, in addition to Egypt.ConclusionThis was the first molecular evidence of B. ovis, T. ovis, and A. ovis infections in sheep and goat populations in the North Coast region of Egypt. More extensive studies are required to develop an epidemiological map of blood pathogenic organisms, while more effective control strategies are required to reduce the burden of tick-borne pathogens on small ruminants.

Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence

BackgroundHelcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes.ResultsThe resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase).ConclusionsIn this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium’s pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

Molecular prevalence and associated risk factors of Anaplasma ovis in Pakistani sheep.

Majority of Pakistani population lives in rural areas and raising animals, especially the small ruminants, is their primary source of income. Anaplasma ovis is known to infect small ruminants globally and causing significant economic losses to livestock owners, however prevalence of Anaplasma ovis has been least investigated from Pakistan despite having a huge sheep population. The present study was conducted from June 2021 till December 2021 to report the PCR based prevalence of Anaplasma ovis in the blood samples of sheep (n = 239) that were collected from District Dera Ghazi Khan in Pakistan. Out of 239 samples, 30 (12.5%) amplified a 347 bp fragment specific for the msp4 gene of Anaplasma ovis. Represented partial msp4 gene sequences were confirmed by Sanger sequencing and deposited to GenBank (OP620757-59). None of the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, and composition of herd) showed an association (P > 0.05) with the Anaplasma ovis infection in enrolled sheep. Analysis of the amplified partial mSP4 sequence of Anaplasma ovis revealed that this gene is highly conserved as all three sequences were identical and phylogenetically resembled with the msp4 sequences amplified from small ruminants in China, Kenya, and Germany, Turkey, Portugal, Tunisia and India. In conclusion, for the first time, we are reporting a moderate prevalence of Anaplasma ovis prevalence in Pakistani sheep and this data will help in developing the integrated control policies against this newly reported tick-borne disease that is infecting our sheep breeds.