Comparative evaluation of cell-mediated immune response in calves immunized with live-attenuated and killed Theileria annulata vaccines.

Tropical theileriosis is a tick-borne disease caused by the protozoan Theileria annulata and transmitted by numerous species of Ixodid ticks of the genus Hyalomma. The main clinical signs are fever, lymphadenopathy, and anemia responsible for heavy economic losses, including mortality, morbidity, vaccination failure, and treatment cost. Development of poor cell-mediated immunity (CMI) has been observed in the case of many bovine pathogens (bacteria, viruses, and parasites). Quantification of CMI is a prerequisite for evaluating vaccine efficacy against theileriosis caused by T. annulata. The current study evaluated the CMI in calves administered with two types of T. annulata vaccine (live attenuated and killed). We prepared a live attenuated T. annulata vaccine by attenuation in a rabbit model and also prepared killed vaccine from non-attenuated T. annulata. For the evaluation of immune response in experimental groups including control, 20 calves were divided into four different groups (A, B, C, and D). They were either inoculated subcutaneously with live rabbit-propagated-Theileria-infected RBCs (5 × 106) (group A) or with killed T. annulata vaccine (2 × 109 schizonts) with Freund's adjuvant (group B), along with an infected group (group C) and a healthy control group (group D). The protection of vaccinated calves was estimated with challenge infection. Our results showed that with a single shot of live-attenuated and killed vaccine with a booster dose elicited cell-mediated immune responses in immunized calves. We observed a significant elevation in CD4 + and CD8 + T cells in immunized calves. A significant difference in the CD8 + T cell response between the post-challenge stage of killed and live vaccine (p < 0.0001) was observed, whereas no other difference was found at both pre- and post-immunization stages. A similar finding was recorded for the CD4 + T cells at a post-challenge stage, where a significant difference was seen between killed and live vaccine (p < 0.0001). Another significant difference was observed between the CD8 + T cells and CD4 + T cells at the post-challenge stage in the live vaccine group, where there was a significantly higher induction of CD4 + T cell response (p < 0.0001).

Molecular detection and assessment of risk factors for Theileria lestoquardi in sheep from Balochistan, Pakistan.

This study aimed to determine the molecular prevalence and associated risk factors of theileriosis in sheep from Balochistan, Pakistan. For this purpose, a total of 408 blood samples were collected from tick-infested sheep in three different zones of Balochistan (i.e., Quetta, Zhob, and Loralai). All the collected samples were analyzed using conventional microscopy techniques, polymerase chain reaction (PCR), 18S small subunit rRNA gene sequencing, and phylogenetic analysis. The results of the microscopy and PCR confirmed the highest prevalence of Theileria species in district Zhob (14.22% and 15.68%) followed by district Loralai (11.52% and 13.97%) and district Quetta (10.29% and 12.00%), respectively. In addition, the prevalence of T. lestoquardi was higher in female sheep (84.12%), followed by adult sheep (74.71%) and the Hernai breed of sheep (28.23%) in the studied area. Similarly, the prevalence of theileriosis was higher in the summer season (40.59%), followed by the spring, autumn, and winter seasons. However, numerous risk factors such as age, sex, area, season, and breeds of the sheep were not significantly correlated (P > 0.05) with the presence of T. lestoquardi, except tick abundance and feeding pattern of animals (P < 0.05). Furthermore, sequencing and phylogenetic analyses of the isolated T. lestoquardi displayed 99% sequence similarity with isolates from Germany, Egypt, Iraq, India, Iran, and Pakistan. Altogether these results showed that T. lestoquardi is the main species causing ovine theileriosis in Balochistan. As a result, large-scale studies are required to design practical control approaches to reduce the risk of theileriosis infection in Balochistan, Pakistan.

Seroprevalence of toxoplasmosis in humans and animals in Pakistan: a systematic review and metadata analysis.

OBJECTIVE: To review the seroprevalence of toxoplasmosis in Pakistan. METHODS: The systematic review comprised search on Science Direct, Google Scholar, PubMed and Scopus databases for studies related to the seroprevalence of toxoplasmosis in Pakistan published between 2006 and 2020 which used serological diagnostic tests to detect Toxoplasma gondii. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used throughout the review and statistical analysis was done using forest plot and random effect model. RESULTS: Of the 7093 human studies initially found, 20(0.28%) were reviewed. Of the 16,432 animal studies, 16(0.09%) were selected for detailed review. The pooled seroprevalence of toxoplasmosis in humans, calculated in this review was found as (76%) (95% confidence interval: 69-83%). Seroprevalence of human toxoplasmosis was higher in Khyber Pakhtunkhwa (31.7%) than Punjab (20.4%). Pooled seroprevalence in animals calculated in this review was found as (69%) (95% confidence interval: 64-74%). Seroprevalence in animals was higher in Khyber Pakhtunkhwa (44.7%) than Punjab (29.4%). CONCLUSIONS: The seroprevalence of toxoplasmosis in both humans and animals should be studied it other parts of Pakistan as well.

DOCUMENTATION OF TRYPANOSOMA EVANSI IN CAPTIVE TIGERS AND LIONS IN PUNJAB (2016-2018), PAKISTAN.

Trypanosoma evansi is an important hemoparasite of a variety of animal species worldwide. This parasite is a threat to the health of domestic animals as well as wild animals, particularly those managed in captivity. The current study investigated the presence of T. evansi in captive tigers (Panthera tigris tigris) and lions (Panthera leo) in Pakistan. In total, 24 blood samples from 11 tigers and 3 lions (n = 14) were collected during the course of roughly 3 yr (2016-2018). Eighteen samples were subjected to both microscopic and molecular evaluation for the presence of T. evansi; the remaining 6 samples were processed for PCR only. Of the 18 samples tested by both methods, 3 (16%) and 8 (44%) were positive by microscopy and PCR, respectively. This highlights the higher sensitivity of PCR over microscopy for detection of trypanosomes. Of the 24 total samples evaluated by PCR, 12 (50%) were positive. The three sequences obtained showed 99% identity with variant surface glycoprotein genes of the different isolates of T. evansi. The sensitivity, specificity, positive predictive value, and negative predictive value of microscopy in identifying T. evansi was 37.5, 100, 100, and 66.7%, respectively, considering PCR as the gold standard. We recommend rigorous monitoring of captive tigers and lions for hemoparasites, particularly in winter and early spring in areas with high infection rate of this parasite, preferably via PCR.

Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

A systematic review on modelling approaches for economic losses studies caused by parasites and their associated diseases in cattle.

Parasites reside inside or outside their hosts and get host nutrition and blood. Here, we have emphasized economic losses in cattle caused by parasitic diseases due to ecto- and endo- parasites (flies, ticks, mites and helminths). We have outlined different methods/models including economic evaluation techniques and dynamic analysis as a major class, used for the calculation of economic losses caused by parasites in cattle. According to already conducted studies, a decrease in production is mentioned in quantity and percentage while financial losses are expressed in the form of account with respect to per head, herd or for the specific study area. The parasites cause the reduced production and financial losses due to control, treatment and mortality costs. We calculated the average decrease in milk production and organ condemnation as 1.16 L animal-1 day-1 and 12.95%, respectively, from overall cattle parasitic infections. Moreover, the average calculated financial and percentage losses were US$ 50.67 animal-1 year-1 and 17.94%, respectively. Economically important parasitic diseases mentioned here are caused by specific spp. of protozoans and helminths according to data collected from the literature. Protozoan diseases include tick-borne diseases, coccidiosis, neosporosis, trypanosomiasis and cryptosporidiosis. Losses due to tick-borne infections were encountered for decreased milk production, mortality, treatment and control. Losses from coccidiosis were due to decreased weight gain, treatment costs and mortality. While abortion losses were encountered in neosporosis. Trypanosomiasis caused losses due to a decrease in milk yield. Moreover, only diagnostic (conventional or molecular techniques) cost was taken into account for cryptosporidiosis. Economically important nematode parasites are Oesophagostomum spp., Cooperia spp., Trichostrongylus spp., Strongyloides spp., Ostertagia spp. and Haemonchus placei. Due to the zoonotic importance of echinococcosis, Echinococcus granulosus is the most economically important cestode parasite. Losses caused by echinococcosis were due to organ condemnation, carcass weight loss and decreases hide value, milk production and fecundity. While, fascioliasis is one of the most economically important trematodal disease, which causes cirrhosis of the liver due to parasite migration, and thus, the organ becomes inedible. So, it would be helpful for farmers and researchers to approach these methods/models for calculation of parasitic losses and should adopt suitable measures to avoid long-term economic losses.

Prevalence of Toxoplasma gondii oocysts through Copro-PCR in cats at Pet Center (UVAS), Lahore, Pakistan.

Toxoplasmosis is a major zoonotic disease of warm-blooded animals caused by Toxoplasma gondii. Cats are the only definitive host and they excrete environmentally resistant T. gondii oocysts in their faeces. Coproscopy was used to detect oocysts of enteric coccidians and then Copro-PCR was employed to test specifically for T. gondii in 470 cat samples. The prevalence of T. gondii oocysts was 2.3% (11/470) based on PCR. We observed 15 (3.2%) of 470 samples positive for coccidian oocysts by microscopy. The presence of Copro-DNA of T. gondii was found significantly higher (p<0.05) in males than females. We tested 11 samples of T. gondii oocysts in which 9 samples were from coccidian oocysts positive samples and 2 samples from negative faecal samples. Our results showed that PCR is the reliable method for the detection of faecal oocysts of T. gondii in cats as compared to microscopy. As per our knowledge, ours is first study for Copro-PCR prevalence of cats' T. gondii oocysts excretion in Pakistan.

Use of physiological biomarkers in diagnosis along with field trials of different trypanisidal drugs in camels of Cholistan desert.

The point prevalence of trypanosomiasis with different physiological biomarkers along with evaluation of the most responsive trypanosidal drug against trypanosomiasis under field conditions was studied. For this purpose a total of 300 free range camels were selected at different grazing and watering point in Cholistan desert. The study population of camels included 150 clinically suspected camels for trypanosomiasis and 150 healthy camels with normal temperature, pulse and respiration. For therapeutic trials 36 positively diagnosed animals were randomly divided into three experimental groups for therapeutic trials. Group A was treated with Imidocarb dipropionate (ID) @ 1.2 mg kg-1 body weight; Group B was treated with Diaminazine aceturate (DA) @ 3.5 mg kg-1 body weight and Group C was treated with Isometamidium chloride hypochloride (IC) @ 0.75 mg kg-1 body weight of camels. Data on risk factors of age,sex, ectoparasites, housing was also collected. Results revealed an overall 15% point prevalence of trypanosomiasis. There was significant (P < 0.05) decline in the values of physiological biomarkers of total erythrocyte counts, hemoglobin concentration, packed cell volume, serum total proteins and albumin while erythrocyte sedimentation rate was increased in infected camels as compared to healthy ones. Different hepatic enzymes including aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and alkaline phosphatase were also significantly increased in the infected animals. Therapeutic trials indicated that Isometamidium chloride hypochloride (IC) was more effective than Imidocarb dipropionate (ID) and Diaminazine aceturate (DA). It is concluded that haemato-biochemical parameters were important physiological biomarkers and IC was the most responsive therapeutic agent against trypanosomiasis in camels in field conditions. The risk factors analysis showed older camels (>5 years) showed highest infection while infection was found to be lowest in less than 1 year age group.

Induction of specific humoral immune response in mice immunized with ROP18 nanospheres from Toxoplasma gondii.

Toxoplasmosis is one of the most common zoonotic protozoal diseases. Recent advances in biotechnology have produced recombinant protein, which are immunogenic, and progress in nano-pharmaceutics has generated encapsulated protein in nanospheres, which are suitable for vaccine delivery. DNA was extracted from Toxoplasma gondii oocysts and was confirmed through nested PCR and sequencing. The 1665 bp of ROP18 was cloned into the easy vector system: pGEM-T by the T-A cloning method. DH5α bacteria were transfected with pGEM-ROP18. ROP18 was subcloned from pGEM-ROP18 into pET28-ROP18. BL21 bacteria were transfected with pET28-ROP18. Thus, rROP18 protein was expressed in BL21 bacteria by induction at different concentrations of isopropyl ß-D-1-thiogalactopyranoside. Protein expression was confirmed through SDS-PAGE and Western blotting. The immunoblot of rROP18 was recognized by anti-HIS antibodies and sera from infected mice at 67 kDa. Recombinant ROP18 protein was encapsulated in nanoparticles with PLGA and was characterized through scanning electron microscopy. Intraperitoneal immunizations with rROP18 protein and intranasal immunization of nanospheres were carried out in mice, and the immune response was detected by ELISA. Results showed that rROP18 in nanospheres administered intra-nasally elicited elevated responses of specific IgA and IgG2a as compared to groups inoculated intra-nasally with rROP18 alone, or injected subcutaneously with rROP18 in montanide adjuvant. It was concluded that nanospheres of ROP18 would be a non-invasive approach to develop vaccination against T. gondii. Further experiments are needed to determine the cellular response to these nanospheres in a mouse model for chronic toxoplasmosis.

FIRST REPORT OF TRYPANOSOMA EVANSI INFECTION (SURRA) IN A PUMA (FELIS CONCOLOR) OF LAHORE ZOO, PAKISTAN.

The blood protozoan Trypanosoma evansi, which is transmitted by biting flies, is frequently neglected due to subclinical infections. This report describes a case of trypanosomiasis due to T. evansi in a 9-yr-old male puma (Felis concolor) housed at the Lahore Zoo in Pakistan. Early in January 2015, this male puma presented with chronic lethargy, weight loss, incoordination, hyperthermia, anorexia, sunken eyes, and unthriftiness. Microscopic examination of Giemsa-stained blood smears showed numerous Trypanosoma parasites. The puma was treated with diminazene aceturate subcutaneously twice. A few days later, a blood smear examination showed absence of trypanosomes. Five months later the cat presented with acute epistaxis and died. Postmortem examination showed emaciation, pale liver and kidneys, and hemorrhages on the spleen. Examination of a blood smear taken at the time of death showed numerous Trypanosoma parasites. PCR testing confirmed the presence of Trypanosoma DNA. DNA sequencing of two amplicons confirmed the presence of Trypanosoma in the blood smears with a 98-99% identity with the previously identified GenBank sequences. A phylogenetic tree was then constructed. Further studies are needed to improve our knowledge about the epidemiology and pathogenesis of T. evansi infection in wild animal species.

The antigen-specific response to Toxoplasma gondii profilin, a TLR11/12 ligand, depends on its intrinsic adjuvant properties.

Agonists that activate Toll-like receptors (TLR) are potential vaccine adjuvants. In particular, Toxoplasma gondii profilin (TgPRF) is recognized by TLR11/12 to generate an inflammatory response. Unlike most TLR ligands, TgPRF is also a protein and can therefore simultaneously induce innate and adaptive immune responses. We found that variations in the conformation of TgPRF can affect its ability to induce a TLR11/12-dependent inflammatory response. The secreted recombinant T. gondii (S2-profilin), produced by Schneider 2 cells, has lost its ability to generate an IL-12 response. Reduction of the intramolecular disulfide bonds in S2-profilin rescued the TLR11/12-dependent IL-12 response. Immunization of mice with reduced S2-profilin induced strong cellular and humoral responses compared to mice immunized with unreduced S2-profilin. A mixed Th1/Th2 response was induced with both S2-profilins. However, a more polarized Th1-type response, which was consistent with the IgG2a-polarized humoral response, was observed with reduced S2-profilin. In conclusion, the intrinsic adjuvant properties of TgPRF had significant consequences on the immune response against TgPRF.

Role of CD4+ Foxp3+ Regulatory T Cells in Protection Induced by a Live Attenuated, Replicating Type I Vaccine Strain of Toxoplasma gondii.

Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii type I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4(+) Foxp3(+) T regulatory cells followed by recruitment/expansion of CD4(+) Foxp3(-) CD25(+) effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4(+) Foxp3(+) cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition, in vivo Treg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.

Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico.

BACKGROUND: Histoplasma capsulatum and Pneumocystis organisms cause host infections primarily affecting the lung tissue. H. capsulatum is endemic in the United States of America and Latin American countries. In special environments, H. capsulatum is commonly associated with bat and bird droppings. Pneumocystis-host specificity has been primarily studied in laboratory animals, and its ability to be harboured by wild animals remains as an important issue for understanding the spread of this pathogen in nature. Bats infected with H. capsulatum or Pneumocystis spp. have been found, with this mammal serving as a probable reservoir and disperser; however, the co-infection of bats with both of these microorganisms has never been explored. To evaluate the impact of H. capsulatum and Pneumocystis spp. infections in this flying mammal, 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) were screened using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms. RESULTS: Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%). CONCLUSION: The data highlights the H. capsulatum and Pneumocystis spp.co-infection in bat population's suggesting interplay with this wild host.

Prevalence and associated risk factors of ixodid tick species infesting cattle and sheep in Balochistan, Pakistan.

Tick infestation poses a serious threat to animal health, leading to significant losses in terms of vector-borne disease transmission, reduced live weight, lower quality hides, decreased milk production, and impaired reproduction in tropical and subtropical regions worldwide. This study aimed to determine the prevalence, seasonal variation, distribution pattern, and associated risk factors of Ixodid family tick species in the cattle and sheep population of three different districts in Balochistan, Pakistan. This study employed a convenient sampling method, collecting 4080 adult ticks from 816 cattle and sheep of various breeds, ages, and sexes. Specific morphological keys were used to identify the ticks up to the genus and species level. Among cattle, the highest prevalence was recorded for R. (B) annulatus (27.01%), followed by R. (B) microplus (24.02%), and H. anatolicum (20.54%). H. dromedarii (5.29%) was the least prevalent species observed in cattle. In the sheep population, H. anatolicum (30.34%) showed the highest prevalence, followed by H. marginatium (22.99%), and R. (B) annulatus (20.88%). H. dromedarii (6.96%) was the least prevalent species observed in sheep. The prevalence of R. (B) decoloratus, H. anatolicum and H. dromedarii was found to be significantly associated (P < 0.05) with the breed, age, and sex of both cattle and sheep. However, the presence of R. (B) annulatus, R. (B) microplus and H. marginatium tick species showed no significant association (P > 0.05) with these factors. In addition, the prevalence of ticks was higher in younger, female, and crossbred Friesian cattle compared to adults, males, and other breeds. Conversely, the prevalence of ticks was higher in adult, female and Hernai breed of sheep in the studied area. In conclusion, R. (B) annulatus and H. anatolicum are the dominant tick species infesting the cattle and sheep population in Balochistan. Consequently, this study provides valuable insights for developing practical and effective control measures against ticks and tick-borne diseases in the sheep and cattle population of Balochistan, Pakistan.

Newly optimized ELISA kit and LAT reveal significantly higher seroprevalence in sheep raised in agro-ecological zone as against range-ecological zone, with a significant association of meteorological parameters.

BACKGROUND: Toxoplasma gondii is a zoonotic and foodborne intracellular parasite capable of inducing congenital infections, stillbirths and abortions in humans and animals, globally. The consumption of undercooked or raw mutton is "one of the vital risks" for acquiring toxoplasmosis: an asymptomatic condition in healthy persons, while life-threatening in immunodeficient individuals like "HIV/AIDS" patients. OBJECTIVES: The current study has multiple objectives: to optimize a newly ELISA kit for Sheep, to find out the seroprevalence of ovine toxoplasmosis of two ecological zones of the Punjab, Pakistan through LAT and newly Optimized Sheep ELISA kit, to do the comparison of efficacies of various tests (LAT with newly Optimized ELISA kit and newly Optimized ELISA kit with commercial ELISA kit) and to determine the different meteorological parameters as the risk factors for T. gondii infection in sheep. METHODS: A cross-sectional study was conducted on 400 sheep sera, 200 were collected from sheep raised on open grazing system by local farmers in the adjoining areas of Civil Veterinary Dispensaries (CVDs) of range-ecological zone i.e. tehsil Kot Chutta (Dera Ghazi khan). Similarly, the remaining 200 were collected from agro-ecological zone i.e. tehsil Sharaqpur (Sheikhupura), to evaluate the comparative efficacy of LAT with optimized ELISA kit and newly optimized ELISA kit with commercial ELISA kit. FINDINGS: The newly ELISA kit optimized against a commercial ELISA kit was found to have 100% sensitivity, 97.6% specificity with 98% Positive Predictive Value, 100% Negative Predictive Value, Cut off value = 0.505, 28.28 LR+, 0.0104 LR-, and 2719.23 DOR. Seroprevalence of toxoplasmosis was detected significantly (P < 0.01; χ2) higher in Sharaqpur (44.5% by LAT; 35.5% by ELISA) as compared to that in Kot Chutta (39.5% by LAT; 31% by ELISA). The highest seroprevalence was seen in the sheep of the 1-2 years age group (P < 0.01; χ2), whereas the lowest in the oldest animals (≥ 4 years). Investigation of meteorological data of both the regions reveals that the zone with higher seroprevalence has relatively higher rainfall, higher humidity, lower environmental temperatures, and higher altitude as the critical factors, potentially behind the significant difference seen in seroprevalence level. The partial correlation of both tests (newly optimized ELISA kit and LAT) was 0.991 at maximum temperature in Sharaqpur while it was 0.981 in Kot Chutta. INTERPRETATION: A novel significant correlation was found between the meteorological parameters (relative humidity, minimum, maximum, and average temperatures) divided into yearly units of both the ecological zones, and year-wise seroprevalence (birth years of age-wise groups) of the corresponding regions. We hypothesize that such environmental conditions increase the risk of toxoplasmosis in grazing sheep, owing to a more favorable environment for coccidian oocyst survival. The ELISA kit optimized in this study will be helpful for the detection of seroprevalence of ovine toxoplasmosis in other ecological zones of Pakistan as well as of any other country in the world. More studies are recommended involving regions from other ecological zones of Pakistan to further explore the seroprevalence of ovine toxoplasmosis and to ratify the novel correlation of meteorological parameters with seroprevalence.

IEg67 kDa Bovine Hydatid Cyst Antigen: A Candidate for Developing Sero-Diagnostic Assays for Cystic Echinococcosis, a Disease of One Health Importance.

Cystic echinococcosis (hydatidosis) is a world-wide zoonotic disease of mainly humans, livestock and dogs, caused by Echinococcus granulosus. The disease can negatively impact food production and animal welfare and causes socio-economic hardship. Here, we aimed to identify the local bovine hydatid cyst fluid (BHCF) antigen for developing a sero-diagnostic assay to be used for the pre-slaughter screening of food animals. In total, 264 bovines approved for slaughter in Pakistan were subjected to serum collection and post-mortem screening for hydatid cysts. These cysts were assessed microscopically to assess fertility and viability, and by PCR for molecular confirmation of species. A BHCF antigen was identified from positive sera via SDS-PAGE, confirmed by Western blot, and quantified via a bicinchoninic acid (BCA) assay. The quantified crude BHCF antigen (iEg67 kDa) was then used in ELISA screening to test all sera collected from known positive and negative animals based on hydatid cyst presence/absence. Of the 264 bovines examined, 38 (14.4%) showed hydatid cysts during post-mortem examination. All of these individuals, plus an additional 14 (total: 52; 19.6%) tested positive based on less time-consuming ELISA examination. Based on ELISA, occurrence in females (18.8%) was significantly higher than in males (9.2%) and was higher in cattle (19.5%) compared to buffalo (9.5%). The infection rate increased with age in both host species: cumulatively, 3.6% in animals aged 2-3 years, 14.6% in 4-5-year-olds and 25.6% in 6-7-year-olds. The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to their livers (5.5%), whereas the opposite was true in buffalo (6.6% livers, 2.9% lungs). For both host species, most cysts in the lungs were fertile (65%), while the majority in the liver were sterile (71.4%). We conclude that the identified iEg67 kDa antigen is a strong candidate for the development of a sero-diagnostic screening assay for the pre-slaughter diagnosis of hydatidosis.

First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records.

Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.

Characterizing Pneumocystis in the lungs of bats: understanding Pneumocystis evolution and the spread of Pneumocystis organisms in mammal populations.

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.

Depletion of CD25⁺ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice.

The interleukin (IL)-2R alpha chain (CD25) is expressed on regulatory T cells (Treg), which constitute more than 85% of the CD25+ T cell population in a naïve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.

Lumpy skin disease is expanding its geographic range: A challenge for Asian livestock management and food security.

In recent years, lumpy skin disease virus has extended its geographical range outside of endemic sub-Saharan countries to the Middle East and Asia indicating transboundary spread. Recently, lumpy skin disease (LSD) outbreaks have been reported in Asian countries such as Bangladesh, India, China, Nepal, Bhutan, Vietnam, Myanmar, Sri Lanka, Thailand, Malaysia, Laos and for the first time and represent a cause of serious concern for their livestock and dairy industries. This report summarizes information on the recent outbreaks of LSD in southern Asia and emphasizes the threat it poses to neighbouring countries. Various strategies and actions needed to control outbreaks of this emerging disease in Asia are also suggested.