Severe Clinical Signs Research Articles

Experimental infection of non-immunosuppressed and immunosuppressed goats reveals differential pathogenesis of Babesia aktasi n. sp.

Babesiosis is an acute and persistent tick-borne disease caused by protozoan parasites of the genus Babesia. These hemoparasites affect vertebrates globally, resulting in symptoms such as high fever, anemia, jaundice, and even death. Advancements in molecular parasitology revealed new Babesia species/genotypes affecting sheep and goats, including Babesia aktasi n. sp., which is highly prevalent in goats from Turkiye's Mediterranean region. The objective of this study was to investigate the pathogenesis of B. aktasi infection in immunosuppressed (n=7) and non-immunosuppressed (n=6) goats. These animals were experimentally infected with fresh B. aktasi infected blood, and their clinical signs, hematological and serum biochemical parameters were monitored throughout the infection. The presence of parasites in the blood of immunosuppressed goats was detected by microscopic examination between 4 and 6 days after infection, accompanied by fever and increasing parasitemia. Goats that succumbed acute disease exhibited severe clinical signs, such as anemia, hemoglobinuria, and loss of appetite. However, the goats that survived showed milder clinical signs. In the non-immunosuppressed group, piroplasm forms of B. aktasi were observed in the blood within 2-5 days after inoculation, but with low (0.01-0.2%) parasitemia. Although these goats showed loss of appetite, typical signs of babesiosis were absent except for increased body temperature. Hematological analysis revealed significant decreases in the levels of red blood cells, leukocytes and platelet values post-infection in immunosuppressed goats, while no significant hematological changes were observed in non-immunosuppressed goats. In addition, serum biochemical analysis showed elevated transaminase liver enzymes levels, decreased glucose, and lower total protein values in the immunosuppressed group post-infection. Babesia aktasi, caused mild disease with minor clinical symptoms in non-immunosuppressed goats. However, in immunosuppressed goats, it exhibited remarkable pathogenicity, leading to severe clinical infections and death. In conclusion, this study provides valuable insights into the pathogenicity of the parasite and will serve as a foundation for future research aimed at developing effective prevention and control strategies against babesiosis in small ruminants. Further research is required to investigate the pathogenicity of B. aktasi in various goat breeds, other potential hosts, the vector ticks involved, and its presence in natural reservoirs.

Noticeable immune dysregulation-and-suppression in parvovirus affected dogs

Canine parvovirus type 2 (CPV-2) is one of the most common causes of infectious diarrhea in small animals, with high mortality and morbidity. Information on the specific treatment option(s) for CPV diseases (CPVD) is unachievably little. So, the treatment is mainly supportive one. Disruption of dog's innate immune system in viral diseases simply occurs; presumably, the CPV-2 may change the level of some TLRs, interleukins, CD4 and CD8 in the leukocytes of CPVD dogs, and disruptive activities of these immune molecules might be attributable to severe CPVD in dogs. Study on the role of the key immune molecules in CPVD is rare. Herein, by conducting and relating the clinical, para-clinical, immunological and molecular diagnostic tests, we tried to establish how some key immune molecules behave in blood of parvovirus affected dogs. As such, in the 1st study, the mRNA levels of TLR2, TLR4, TLR9, IL-1β, IL-6, CD4 and CD8 genes in the leukocytes of CPVD were assessed with quantitative (q)RT-PCR along with CPV-2 detection by rapid immunochromatography and PCR tests. In a 2nd study, the same measurements as in the 1st study were evaluated in two groups of mild versus severe clinical signs of CPVD. Both in the 1st and the 2nd studies leukopenia, much more pronounced in the severe CPVD, and immune dysregulation were observed. In the 1st study, a noticeable increase in the mRNA levels of TLR2 and TLR4 was detected with a slight decrease in TLR9 and a significant decrease in the expression of IL-1β, IL-6, CD4 and CD8 in leukocytes of CPV-infected dogs. Compared to the mild CPVD, the intense of downregulating effects on those immune molecules in the 2nd study was remarkably much more pronounced in the severe CPVD. Overall, it proves strong immune dysregulation and suppression/incompetence and potential T-cells exhaustion in severely CPV-2-affected dogs. Technically and clinically, this would be substantially applicable in canine medicine. By targeting those key immune molecules and their signaling pathways, new clinicodiagnostic approaches for CPVD can be evolved, and biotechnicoclinically this would be substantially applicable in all physiopathological conditions of dogs.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

In Vitro Activity of Quaternary Ammonium in Prototheca Isolated from Clinical Bovine Mastitis Identified by Mass Spectrometry and PCR Sequencing of the cytb Gene Marker.

The in vitro algaecide activity of quaternary ammonium (QA) against Prototheca isolated from bovine clinical mastitis was investigated, in which the clinical severity was scored, milk samples were subjected to microbiological culture, and algal species were identified by molecular typing. A total of 4275 milk clinical samples of different cows from ten large dairy farms were used. Forty-four (1%) samples of cows from three dairy farms yielded growth of Prototheca, of which 88.6% (39/44) were identified as Prototheca bovis and 11.3% (5/44) as Prototheca sp. by MALDI-TOF MS, whereas 100% of the isolates were identified as P. bovis using PCR sequencing of the cytb gene. Among cows for which clinical severity scoring was available, 78.8% (26/33) and 21.2% (7/33) had mild and moderate infections, respectively, whereas no animal showed severe clinical signs. The algaecide activity of QA in Prototheca was observed in low concentrations among all isolates, in 20.4% (9/44) at 35 ppm, 36.4% (16/44) at 17 ppm, and 43.2% (19/44) at an 8 ppm, in addition to activity on three reference Prototheca strains. Overall, the study highlights the predominance of P. bovis as the causative agent of algal mastitis in bovines. Prototheca induced abnormalities preponderantly in the milk and mammary gland tissue of cows, and to our knowledge, our study is the first to apply clinical severity scoring in protothecal mastitis. In addition, the study underlines the activity of QA in low concentrations against Prototheca, indicating its potential use as an antiseptic/disinfectant in milking facilities and dairy environments.

Characterization of Rongchang piglets after infection with type 2 porcine reproductive and respiratory syndrome virus strains differing in pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the production and health of pigs and causes severe economic losses to the swine industry worldwide. Different pig breeds have been reported to have different levels of susceptibility to PRRSV, and different PRRSV strains may also influence the infectivity and pathogenicity of the virus. In this study, the susceptibility of Rongchang pigs (a prominent local pig breed in China) to PRRSV infection was thoroughly investigated. Rongchang piglets were exposed to two PRRSV strains: HuN4 (highly pathogenic PRRSV) and SD53-1603 (moderately virulent NADC30-like PRRSV). We observed that Rongchang pigs infected with HuN4 displayed significant clinical manifestations, including fever, reduced body weight, and interstitial pneumonia lesions. Routine blood tests revealed that HuN4-infected pigs exhibited slightly decreased levels of red blood cells, hemoglobin, reticulocytes, and a notable increase in monocytes than control pigs. Additionally, the Rongchang pigs exhibiting severe clinical signs presented a higher neutrophil-to-lymphocyte ratio and a lower lymphocyte-to-monocyte ratio. In contrast, SD53-1603 infection did not cause considerable harm to Rongchang pigs, only resulting in slightly elevated leukocytes and lymphocytes. Furthermore, these two PRRSV strains elicited divergent cytokine responses, such that SD53-1603 infection induced higher levels of TNF-α and IFN-γ, whereas HuN4 infection upregulated IL-1β. These dissimilarities in clinical symptoms, pathological changes, viremia, cytokine expression, and routine blood indices between HuN4 and SD53-1603 infections are critical in understanding the mechanisms of PRRSV infection and developing rational prevention and control strategies against PRRSV.

Construction of pseudorabies virus variant attenuated vaccine: codon deoptimization of US3 and UL56 genes based on PRV gE/TK deletion strain.

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

Picada de Bothrops en Pinscher: Reporte de caso

The objective of this report was to describe the case of a Pinscher canine, seven years and four months old, residing in the municipality of Magé, Rio de Janeiro (Brazil), bitten by a snake of the Bothrops genus. Ophidian accidents involving pit viper bites on domestic animals are common in rural areas, with no preference for sex, and can lead to severe clinical signs that, if not treated immediately, may result in death. The guardians took the dog to the clinic along with the snake for gender identification by the veterinarian, which was essential for the start of treatment. The patient presented edema in the right frontal limb with perforation. For stabilization, diluted antivenom serum was administered in 500 mL of Sodium Chloride (NaCl 0.9%), as well as a corticosteroid, antibiotic, analgesic, antihistamine, and oxygen therapy. Subsequently, the dog was referred for hospitalization, where it remained for two days and showed bruises all over the body and hyporexia. After improvement of the clinical condition, the veterinarian advised continuing treatment at home. After 17 days, the canine returned to the clinic with no clinical abnormalities, completing its treatment.

SARS-CoV-2 Omicron variant infection affects blood platelets, a comparative analysis with Delta variant.

In November 2021, the SARS-CoV-2 Omicron variant of concern has emerged and is currently dominating the COVID-19 pandemic over the world. Omicron displays a number of mutations, particularly in the spike protein, leading to specific characteristics including a higher potential for transmission. Although Omicron has caused a significant number of deaths worldwide, it generally induces less severe clinical signs compared to earlier variants. As its impact on blood platelets remains unknown, we investigated platelet behavior in severe patients infected with Omicron in comparison to Delta. Clinical and biological characteristics of severe COVID-19 patients infected with the Omicron (n=9) or Delta (n=11) variants were analyzed. Using complementary methods such as flow cytometry, confocal imaging and electron microscopy, we examined platelet activation, responsiveness and phenotype, presence of virus in platelets and induction of selective autophagy. We also explored the direct effect of spike proteins from the Omicron or Delta variants on healthy platelet signaling. Severe Omicron variant infection resulted in platelet activation and partial desensitization, presence of the virus in platelets and selective autophagy response. The intraplatelet processing of Omicron viral cargo was different from Delta as evidenced by the distribution of spike protein-positive structures near the plasma membrane and the colocalization of spike and Rab7. Moreover, spike proteins from the Omicron or Delta variants alone activated signaling pathways in healthy platelets including phosphorylation of AKT, p38MAPK, LIMK and SPL76 with different kinetics. Although SARS-CoV-2 Omicron has different biological characteristics compared to prior variants, it leads to platelet activation and desensitization as previously observed with the Delta variant. Omicron is also found in platelets from severe patients where it induces selective autophagy, but the mechanisms of intraplatelet processing of Omicron cargo, as part of the innate response, differs from Delta, suggesting that mutations on spike protein modify virus to platelet interactions.

“Slow kill” treatment reduces DNA damage in leukocytes of dogs naturally infected with Dirofilaria immitis

Parasitic diseases are considered to be a cause of oxidative stress which leads to oxidative damage of various molecules including DNA. This can result in mutations, replication errors, and genome instability. Therefore, aim of this study was to measure DNA damage induced by Dirofilaria immitis in the single cells such as dogs’ leukocytes using the comet assay. Also, we monitored the effects of antiparasitic treatment on mitigation of sensitivity to DNA damage in leukocytes treated with H2O2 using the in vivo and ex vivo comet assay. The whole blood samples from 34 dogs from Serbia were used, both males and females, from one to 13 years old, both pure and mixed-breeds. A rapid immunochromatographic test (Antigen Rapid Heartworm Ag 2.0 Test Kit, Bionote, Minnesota, USA) was used for the detection of D. immitis antigens. The modified Knott’s test and PCR were used in the aim of detecting D. immitis microfilariae in dogs’ blood, and evaluating the number of circulating microfilariae during the treatment. The genotoxicity evaluation showed that D. immitis infection resulted in DNA damage in naturally infected dogs, with the highest DNA damage occurring in the group of dogs with severe clinical signs. Treatment with ivermectin and doxycycline decreased DNA damage in leukocytes of dogs in all groups, as the intensity of infection decreased due to applied therapy. Ex vivo comet assay results showed that leukocytes exhibited decreased sensitivity to H2O2-induced DNA damage during treatment. The results of the modified Knott’s test and PCR in our study showed that treatment with ivermectin and doxycycline was successful in decreasing the average number of microfilariae during the time and at the end eliminating them from the dogs’ blood.

Hefer valley virus: a novel ephemerovirus detected in the blood of a cow with severe clinical signs in Israel in 2022.

A novel ephemerovirus was identified in a Holstein-Friesian cow in the Hefer Valley, Israel, that showed severe and fatal clinical signs resembling an arboviral infection. A sample taken during the acute phase tested negative for important endemic arboviral infectious cattle diseases. However, sequencing from blood revealed the full genome sequence of Hefer Valley virus, which is likely to represent a new species within the genus Ephemerovirus, family Rhabdoviridae. Archived samples from cattle with comparable clinical signs collected in Israel in 2021 and 2022 tested negative for the novel virus, and therefore, the actual distribution of the virus is unknown. As this is a recently identified new viral infection, the viral vector and the prevalence of the virus in the cattle population are still unknown but will be the subject of future investigations.

ANALYSIS OF THE RELATIONSHIP BETWEEN INDICATORS OF OXIDATIVE STRESS AND THE AMINO ACID PROFILE OF BLOOD PLASMA IN PSORIASIS

Introduction. Psoriasis is an autoimmune disease that affects the skin, has a number of systemic manifestations and is associated with the devel-opment of a number of somatic pathologies. Significant systemic metabolic features of psoriasis are changes in the pool of free amino acids and oxi-dative stress. The purpose of the study: analysis of markers of lipid peroxidation and oxidative modification of plasma proteins and peripheral mononuclear cells, the amino acid spectrum of blood plasma, in relation to each other and clinical signs of severity and duration of psoriasis, body mass index and blood pressure indicators. Material and methods. The study group included 45 patients with psoriasis vulgaris. The control group included 46 donors. Determination of the lev-els of products of lipid peroxidation and oxidative modification of proteins was carried out by spectrophotometric methods. Blood plasma amino acid levels were determined by capillary electrophoresis. Results. In patients with psoriasis, an increase in the concentrations of primary and secondary products of lipid peroxidation and the level of metal-catalyzed oxidative modification of proteins in blood plasma, a decrease in the concentrations of end products of lipid peroxidation in peripheral mono-nuclear cells were revealed. In the amino acid profile of the blood plasma of patients with psoriasis, an increase in the relative concentration of sulfur-containing amino acids, concentrations of phenylalanine, leucine, cysteine, and a decrease in the relative concentration of oxyaminoacids were re-vealed. Relationships between the levels of amino acids and markers of oxidative stress with the duration of the course of the disease or exacerbation, as well as the severity of psoriatic lesions of the skin, were not revealed. Cysteine levels in patients with psoriasis are positively correlated with body mass index and diastolic blood pressure; the relative content of oxyaminoacids in blood plasma negatively correlates with body mass index. Conclusions. Changes in the amino acid profile of blood plasma in psoriasis occur in parallel with oxidative stress and skin inflammation, but are as-sociated with excess body weight for cysteine and oxyaminoacids, which allows us to consider the levels of cysteine and oxyaminoacids as promising prognostic and diagnostic markers of comorbid pathology associated with psoriasis.

A Newly Emerging Serotype A Strain in Foot-and-Mouth Disease Virus with Higher Severity and Mortality in Buffalo than in Cattle Calves in North Egypt.

A severe foot-and-mouth disease (FMD) epidemic struck several Egyptian provinces recently, causing significant losses among animals even in vaccinated farms. This study indicated the existence of the newly emerging foot-and-mouth disease virus (FMDV) and first investigated its effect on the Egyptian water buffalo (Bubalus bubalis) and cattle calves in the Beheira province, north Egypt. Twenty tongue epithelial samples from diseased calves in five infected farms were randomly collected, prepared, and propagated using baby hamster kidney-21 (BHK-21) cells. Whole genomic RNA was extracted from the cells of the third passage. A FMDV genome was detected and serotyped using one-step reverse transcription polymerase chain reactions (RT-PCRs). Nucleotide sequencing of the purified serotype-specific PCR bands was performed, and a maximum likelihood phylogenetic tree based on 600 base pairs of VP1 was constructed. The results identified FMDV, serotype A in all infected samples, whereas the serotypes O and SAT2 were negative. The obtained 20 sequences were identical to each other and similar to the newly reported strain in Egypt that belongs to the Europe-South America (Euro-SA) topotype. The epidemiological and clinical parameters associated with such a strain were fully recorded by veterinarians and analyzed in a single infected farm including 70 cattle and buffalo calves. It caused higher peracute mortalities in buffalo (25.7%; 95% CI: 13-43) than in cattle (8.6%; 95% CI: 2-24) calves. Severe clinical signs such as dullness, hypothermia, bradycardia, and cardiac arrhythmia were common to both except in fatal cases, whereas hyperthermia and respiratory signs were prevalent in cattle calves. In conclusion, we first characterized the newly emerging FMDV in the calves of Beheira as more fatal and severe in buffalo than in cattle calves.

Infectious keratoconjunctivitis in semi-domesticated reindeer (Rangifer tarandus tarandus): a questionnaire-based study among reindeer herders in Norway and Sweden

BackgroundThe effects of climate change, loss of pastureland to other land usage and presence of large carnivores are the main reasons for the increase in supplementary feeding of semi-domesticated reindeer (Rangifer tarandus tarandus) in Fennoscandia over the last decades. Feeding might expose reindeer to stress and increased animal-to-animal contact, leading to an increased risk of infectious disease transmission, such as infectious keratoconjunctivitis (IKC). As it can develop rapidly and be very painful, IKC is described as an important animal welfare concern and a potential source of economic loss. The aim of this study was to investigate the current presence of IKC and potential associations between IKC and supplementary feeding through an online questionnaire survey, distributed among reindeer herders in Norway and Sweden in 2021.ResultsSeventy-six reindeer herders (33 from Norway and 43 from Sweden) responded to the questionnaire, representing 6% and 4% of the registered reindeer herding groups in Norway and Sweden, respectively. Infectious keratoconjunctivitis was common, with 54 (71%) of the 76 herders that responded having observed clinical signs during the past 10 years. These signs were mainly observed as increased lacrimation, causing “wet cheeks”, but also as keratitis and conjunctivitis. Autumn and winter were the seasons in which IKC was observed most. The herders reported several measures, such as slaughter and isolation of affected reindeer, to counteract the spread of disease. The herding year 2019/2020 was associated with reports of outbreaks of IKC in herds as well as being the herding year where most herders (80%) had performed supplementary feeding. A significant association was found between IKC and feeding performed in an enclosure (odds ratio = 15.20), while feeding on free-range areas had a non-significant, negative, relationship with the appearance of IKC outbreaks (odds ratio = 0.29). Finally, there was a trend in the data suggesting that IKC affected calves especially.ConclusionsInfectious keratoconjunctivitis is a common disease, mainly observed in winter and autumn. It usually has mild to moderately severe clinical signs. Our results imply that IKC is associated with stress and feeding situations and that calves might be more susceptible than adults, however, this needs to be confirmed with further studies, preferably at an individual animal level.

A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission

Classical swine fever virus (CSFV) infection leading to CSF outbreaks is among the most devastating swine diseases in the pig industry. Porcine circovirus type 2 (PCV2) infection, resulting in porcine circovirus-associated disease (PCVAD), is also a highly contagious disease affecting pig health worldwide. To prevent and control disease occurrence, multiple-vaccine immunization is necessary in contaminated areas or countries. In this study, a novel CSFV-PCV2 bivalent vaccine was constructed and demonstrated to be capable of eliciting humoral and cellular immune responses against CSFV and PCV2, respectively. Moreover, a CSFV-PCV2 dual-challenge trial was conducted on specific-pathogen-free (SPF) pigs to evaluate vaccine efficacy. All of the vaccinated pigs survived and showed no clinical signs of infection throughout the experimental period. In contrast, placebo-vaccinated pigs exhibited severe clinical signs of infection and steeply increased viremia levels of CSFV and PCV2 after virus challenge. Additionally, neither clinical signs nor viral detections were noted in the sentinel pigs when cohabitated with vaccinated-challenged pigs at three days post-inoculation of CSFV, indicating that the CSFV-PCV2 bivalent vaccine completely prevents horizontal transmission of CSFV. Furthermore, conventional pigs were utilized to evaluate the application of the CSFV-PCV2 bivalent vaccine in field farms. An adequate CSFV antibody response and a significant decrease in PCV2 viral load in the peripheral lymph nodes were observed in immunized conventional pigs, suggesting its potential for clinical application. Overall, this study demonstrated that the CSFV-PCV2 bivalent vaccine effectively elicited protective immune responses and the ability to prevent horizontal transmission, which could be a prospective strategy for controlling both CSF and PCVAD in commercial herds.

Comparative pathogenicity of single and mixed drug-resistant Trypanosoma brucei brucei and Trypanosoma congolense infections in rats

Drug-resistant trypanosomes are widespread in sub-Saharan Africa and in conjunction with the drug-sensitive phenotypes cause a serious endemic wasting disease in animals. We evaluated the pathogenicity of single and mixed drug-resistant Trypanosoma brucei brucei and T. congolense isolates in 35 female rats, randomly divided into seven groups (1–7) of five rats. Group 1 was the uninfected control. Groups 2 and 3 were infected with drug-sensitive T. brucei brucei and T. congolense, respectively, whereas groups 4 and 5 were infected with multidrug-resistant T. brucei brucei and T. congolense respectively. Group 6 were infected with drug-sensitive T. brucei brucei and T. congolense while group 7 were infected with multidrug-resistant T. brucei brucei and T. congolense. Parasitaemia kinetics, haematological parameters, body weight, clinical signs, survival time, gross and histopathological changes in the spleen were evaluated. Parasitaemia occurred between day 3–9 post-infection in all the infected groups. Rats in groups 4 and 7 had markedly prolonged (p < 0.05) pre-patent period, days to first peak parasitaemia, survival time, and lower (p < 0.05) parasitaemia level than groups 2 and 6 rats while these parameters were comparable for groups 3 and 5 rats. Anaemia was noted in the infected groups but the severity did not vary amongst the infected groups. Severe clinical signs and splenic lesions were noted in rats infected with drug-sensitive trypanosome species compared to the multidrug-resistant species. Therefore, we conclude that the trypanosome isolates were pathogenic. However, the drug-sensitive T. brucei brucei and mixed drug-sensitive trypanosome infections were more pathogenic than their multidrug-resistant counterparts.

Serum proteome profiling of naturally acquired Babesia rossi infection in dogs

Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, with Babesia rossi causing the most severe clinical signs of all the large Babesia parasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected with B. rossi (subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused by B. rossi have revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused by B. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.

Oleander Poisoning in Cattle in Devghat, Nepal- a Case Report

Oleander poisoning, though widely seen in the Mediterranean region, is rarely seen in Nepal. The poisonous effect of the fodder is due to the accidental ingestion of the different parts of ‘oleandrin’ which contains cardiac glycosides called cardenolides causing mortality in animals. In our study, 8 lactating cows were affected after oleander leaves were accidentally mixed and chopped with Napier. After the ingestion of the mixed fodder, out of 8 cows, 5 died instantly and the remaining 3 were showing severe clinical signs such as severe depression, anorexia, ruminal atony, diarrhea, serous nasal discharge, tachycardia, and irregular heartbeat. The diagnosis of the poisoning was made by finding the chopped leaves of oleander in the feed and ruminal content. The visual observation of the oleander leaves along with gross lesions specifically in the heart causing coagulative necrosis- as it acts as cardiac glycosides- we came to diagnose the oleandrin poisoning. As there was not the availability of digoxin-specific antibodies for the therapy, symptomatic and supportive treatment was done, and the good prognosis was seen on morbid animals within a week.

Genetic characteristics of waterfowl-origin H5N6 highly pathogenic avian influenza viruses and their pathogenesis in ducks and chickens.

Waterfowl, such as ducks, are natural hosts for avian influenza viruses (AIVs) and act as a bridge for transmitting the virus to humans or susceptible chickens. Since 2013, chickens and ducks have been threatened by waterfowl-origin H5N6 subtype AIVs in China. Therefore, it is necessary to investigate the genetic evolution, transmission, and pathogenicity of these viruses. In this study, we determined the genetic characteristics, transmission, and pathogenicity of waterfowl-origin H5N6 viruses in southern China. The hemagglutinin (HA) genes of H5N6 viruses were classified into the MIX-like branch of clade 2.3.4.4h. The neuraminidase (NA) genes belonged to the Eurasian lineage. The PB1 genes were classified into MIX-like and VN 2014-like branches. The remaining five genes were clustered into the MIX-like branch. Therefore, these viruses belonged to different genotypes. The cleavage site of the HA proteins of these viruses was RERRRKR/G, a molecular characteristic of the H5 highly pathogenic AIV. The NA stalk of all H5N6 viruses contained 11 amino acid deletions at residues 58-68. All viruses contained 627E and 701D in the PB2 proteins, which were molecular characteristics of typical bird AIVs. Furthermore, this study showed that Q135 and S23 viruses could replicate systematically in chickens and ducks. They did not cause death in ducks but induced mild clinical signs in them. All the infected chickens showed severe clinical signs and died. These viruses were shed from the digestive and respiratory tracts and transmitted horizontally in chickens and ducks. Our results provide valuable information for preventing H5N6 avian influenza outbreaks.

Survival and Hematologic Benefits of Romiplostim After Acute Radiation Exposure Supported FDA Approval Under the Animal Rule

Patients exposed to acute high doses of ionizing radiation are susceptible to dose-dependent bone marrow depression with resultant pancytopenia. Romiplostim (RP; Nplate) is a recombinant thrombopoietin receptor agonist protein that promotes progenitor megakaryocyte proliferation and platelet production and is an approved treatment for patients with chronic immune thrombocytopenia. The goal of our study was to evaluate the postirradiation survival and hematologic benefits of a single dose of RP with or without pegfilgrastim (PF; Neulasta, granulocyte colony stimulating factor) by conducting a well-controlled, treatment-concealed, good laboratory practice-compliant study in rhesus macaques that was compliant with the United States Food and Drug Administration Animal Rule regulatory approval pathway. Irradiated male and female rhesus macaques (20/sex in each of 3 groups: control, RP, and RP+PF) were subcutaneously administered vehicle or RP (5 mg/kg, 10 mL/kg) on day 1 in the presence or absence of 2 doses of PF (0.3 mg/kg, 0.03 mL/kg, days 1 and 8). Total body radiation (680 cGy, 50 cGy/min from cobalt-60 gamma ray source) occurred 24 ± 2 hours previously at a dose targeting 70% lethality for the control cohort over 60 days. The study examined 60-day survival postirradiation as the primary endpoint. Secondary endpoints included incidence, severity, and duration of thrombocytopenia and neutropenia, other hematology parameters, coagulation parameters, and body weight change to provide insights into potential mechanisms of action. Compared with sham-treated controls, treated animals demonstrated a 40% to 55% survival benefit compared with controls, less severe clinical signs, reduced incidence of thrombocytopenia and/or neutropenia, earlier hematologic recovery, and reduced morbidity from bacterial infection. These results were pivotal in obtaining Food and Drug Administration approval in January 2021 for RP's new indication as a single administration therapy to increase survival in adults and pediatric patients acutely exposed to myelosuppressive doses of radiation.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.