Browsing by Author "Velathanthiri N"

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A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.
(Frontiers Media S.A., 2023-05-22) Ogbuigwe P; Roberts JM; Knox MA; Heiser A; Pita A; Haack NA; Garcia-Ramirez JC; Velathanthiri N; Biggs PJ; French NP; Hayman DTS; Xu R
Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.
Absence of Cryptosporidium hominis and dominance of zoonotic Cryptosporidium species in patients after Covid-19 restrictions in Auckland, New Zealand
(Cambridge University Press, 2021-09) Knox MA; Garcia-R JC; Ogbuigwe P; Pita A; Velathanthiri N; Hayman DTS
Coronavirus disease-2019 (Covid-19) nonpharmaceutical interventions have proven effective control measures for a range of respiratory illnesses throughout the world. These measures, which include isolation, stringent border controls, physical distancing and improved hygiene also have effects on other human pathogens, including parasitic enteric diseases such as cryptosporidiosis. Cryptosporidium infections in humans are almost entirely caused by two species: C. hominis, which is primarily transmitted from human to human, and Cryptosporidium parvum, which is mainly zoonotic. By monitoring Cryptosporidium species and subtype families in human cases of cryptosporidiosis before and after the introduction of Covid-19 control measures in New Zealand, we found C. hominis was completely absent after the first months of 2020 and has remained so until the beginning of 2021. Nevertheless, C. parvum has followed its typical transmission pattern and continues to be widely reported. We conclude that ~7 weeks of isolation during level 3 and 4 lockdown period interrupted the human to human transmission of C. hominis leaving only the primarily zoonotic transmission pathway used by C. parvum. Secondary anthroponotic transmission of C. parvum remains possible among close contacts of zoonotic cases. Ongoing 14-day quarantine measures for new arrivals to New Zealand have likely suppressed new incursions of C. hominis from overseas. Our findings suggest that C. hominis may be controlled or even eradicated through nonpharmaceutical interventions.
An observational study of farmer-reported clinical mastitis in New Zealand dairy ewes.
(Taylor and Francis Group, 2024-07-01) Chambers G; Laven R; Grinberg A; Ridler A; Velathanthiri N
AIMS: To describe the incidence, aetiology, treatment, and outcomes of farmer-reported clinical mastitis on New Zealand dairy sheep farms. METHODS: A prospective cohort study was conducted on 20 spring-lambing New Zealand sheep milking farms over the 2022-2023 season. Clinical mastitis was defined as a change in the appearance of milk and/or signs of inflammation in the gland. Farmers were required to report all cases of clinical mastitis and collect information on affected ewes' demographics, clinical features, treatments (where applicable), and outcomes. Milk samples from mastitic glands were submitted for microbiological culture and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF). RESULTS: Partial or complete clinical mastitis data were available for 236 cases from 221 ewes on 18/20 study farms. Clinical mastitis was diagnosed in 0-6% of ewes at the farm level, with an overall incidence of 1.8 (95% CI = 1.0-3.2)% using the study data, or 2.3 (95% CI = 1.6-3.3)% using the study data and farmer estimates that included unreported cases. Cases occurred mostly in early lactation, with 59% detected during the lambing period (August-October), at a median of 7 (IQR 3, 40) days in milk. The majority of cases featured clots in the milk (59%), swelling (55%), and unevenness (71%) of the glands. Pyrexia (rectal temperature ≥ 40.0°C) was diagnosed in 25% of cases and depression (lethargy, inappetence, or inability to stand) in 26% of cases. Treatment was given to 46% of cases, with tylosin being the most commonly used treatment (50% of treated cases). The most common outcome was immediate drying off to be culled without treatment (32%), followed by still milking and recovered but with lasting problems (25%). Nearly half of all the milk samples submitted were culture negative. Streptococcus uberis (14%), non-aureus staphylococci (12%), and Staphylococcus aureus (11%) were the most common isolates, found on 12, 8 and 8 of the 16 farms with microbiological data, respectively. CONCLUSIONS: Clinical mastitis affected up to 6% of ewes at the farm level. Systemic signs were observed in one quarter of affected ewes, suggesting a role for supportive treatment. Clinical mastitis can be severe and challenging to fully resolve in New Zealand dairy sheep. CLINICAL RELEVANCE: This is the first systematic study of clinical mastitis in New Zealand dairy ewes. It provides baseline information specific to New Zealand conditions for farmers, veterinarians, and other advisors to guide the management of mastitis for the relatively new dairy sheep industry in New Zealand.
Diagnosis of protozoa diarrhoea in Campylobacter patients increases markedly with molecular techniques.
(Public Library of Science (PLoS), 2023-05-30) Hayman DTS; Garcia-Ramirez JC; Pita A; Velathanthiri N; Knox MA; Ogbuigwe P; Baker MG; Rostami K; Deroles-Main J; Gilpin BJ; Standley C
Cryptosporidium and Giardia are major causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques. Here we investigate the level of protozoa detection by molecular methods in campylobacteriosis cases missed through antigen-based assays and investigate different molecular testing protocols. We report findings from two observational studies; the first among 111 people during a Campylobacter outbreak and the second during normal surveillance activities among 158 people presenting with diarrhoea and a positive Campylobacter test, but negative Cryptosporidium and Giardia antigen-based test results. The molecular methods used for comparison were in-house end-point PCR tests targeting the gp60 gene for Cryptosporidium and gdh gene for Giardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical Cryptosporidium positive sample dilutions down to 10-5. The Cryptosporidium prevalence was 9% (95% CI: 3-15; 10/111) and Giardia prevalence 21% (95% CI: 12-29; 23/111) in the 111 Campylobacter outbreak patients. The Cryptosporidium prevalence was 40% (95% CI: 32-48; 62/158) and Giardia prevalence 1.3% (95% CI: 0.2-4.5; 2/158) in the 158 routine surveillance samples. Sequencing identified Cryptosporidium hominis, C. parvum, and Giardia intestinalis assemblages A and B. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (95% CI: 35-37) for 1 oocyst, suggesting a high limit of detection. In conclusion in surveillance and outbreak situations we found diagnostic serology testing underdiagnoses Cryptosporidium and Giardia coinfections in Campylobacter patients, suggesting the impact of protozoa infections may be underestimated through underdiagnosis using antigen-based assays.
Isolation of Aerobic Bacterial Species Associated with Palpable Udder Defects in Non-Dairy Ewes.
(MDPI (Basel, Switzerland), 2024-08-09) Zeleke MM; Kenyon PR; Flay KJ; Aberdein D; Pain SJ; Velathanthiri N; Ridler AL; Zecconi A
The objectives of these studies were to identify associations between udder half defects (hard or lump) and bacteria isolated from milk or mammary tissue swabs, to compare with samples from normal udder halves at different physiological time points and to compare bacterial species isolated via milk and swabs of mammary tissue from within the same udder halves. A total of 1054 samples were aseptically collected from each udder half of 199 non-dairy breed (Romney) ewes from three different studies (Study A, n = 77; Study B, n = 74; and Study C, n = 48). Conventional bacterial culture and MALDI-ToF mass spectrometry were used for bacterial identification. Of the 225 samples from which bacteria were isolated, Mannheimia haemolytica and Streptococcus uberis were the dominantly identified species from defective udder halves, whereas coagulase-negative staphylococcus (CNS) species, mostly Staphylococcus simulans and Staphylococcus chromogenes, were more frequently isolated from normal udder halves. The ongoing presence of bacterial species over time was variable, although less frequently identified species showed less stability over time. A very high agreement (91.5%) of bacterial species identified was observed between the mammary tissue swab and udder half milk samples during post-weaning. In summary, palpable udder half defects were associated with bacterial positivity, and the ongoing presence of the bacteria over time was dependent on the species involved. Hence, culling ewes with palpable udder half defects that had more stable bacterial species could contribute to reducing the recurrence of palpable defects or mastitis.
Mammal-related Cryptosporidium infections in endemic reptiles of New Zealand.
(Springer Nature, 2023-05-01) Garcia-R JC; Pita AB; Velathanthiri N; Pas A; Hayman DTS
New Zealand's endemic reptile fauna is highly threatened and pathogens causing infectious diseases may be a significant risk to already endangered species. Here, we investigate Cryptosporidium infection in captive endemic New Zealand reptiles. We found two mammal-related Cryptosporidium species (C. hominis and C. parvum) and six subtypes from three gp60 families (Ib, Ig and IIa) in 12 individuals of captive endemic Tuatara, Otago and Grand skinks, and Jewelled and Rough geckos. Cryptosporidium serpentis was identified in two Jewelled geckos using 18S. In New Zealand, C. hominis and C. parvum are associated with infections in humans and introduced domestic animals but have also been recently found in wildlife. Our finding of Cryptosporidium infection in endemic reptiles can help inform strategies to monitor the conservation of species and manage potential introductions of pathogens to in-situ and ex-situ populations.
Subclinical mastitis in New Zealand grazing dairy ewes 1: Prevalence and risk factors
(Elsevier Inc on behalf of the American Dairy Science Association, 2026-01-01) Chambers G; Lawrence K; Grinberg A; Velathanthiri N; Ridler A; Laven R
Our objectives were to describe subclinical mastitis and identify its risk factors among grazing dairy ewes in New Zealand. Gland-level milk samples were collected from ∼15 randomly selected ewes on each of 20 dairy sheep farms at early, mid, and late lactation in a repeated cross-sectional study. California Mastitis Tests (CMT; measured on a scale of 0, trace, 1, 2, or 3) and aerobic bacterial culture were performed at the gland level, and SCC at the ewe level using composite milk samples. Subclinical mastitis was defined at the ewe level as having 1 or 2 bacteriologically positive glands and SCC >500 × 103 cells/mL or a CMT score ≥1 (or both). Milk samples were collected from 893 ewes, and complete subclinical mastitis data were available for 856 ewes. Median (range) SCC was 128,000 (2,000–34,953,000) cells/mL. A CMT score ≥1 in one or both glands was found in 21.2% of ewes. Bacteria were isolated from 5.5% of glands, with the most common species being non-aureus staphylococci (4.0% of glands) and Staphylococcus aureus (0.6% of glands). The prevalence of subclinical mastitis was 6.4% (95% CI = 4.6%–8.7%) and was not strongly clustered within farms (intraclass correlation coefficient = 0.04). Ewes with moderate or severe teat end hyperkeratosis had 6.4 times higher odds of subclinical mastitis than ewes with no or mild hyperkeratosis, and ewes with asymmetric udders had 2.3 times higher odds. The odds declined across the 3 visits. The prevalence was low compared with studies of more intensively farmed ewes in the northern hemisphere, but the bacterial causes were consistent. Subclinical mastitis management should be focused at the ewe level before the farm level, given the weak clustering within farms. When addressing or preventing a subclinical mastitis challenge, producers should consider teat end hyperkeratosis and udder asymmetry as simple visual screening tools but not rely on them alone to identify ewes at risk of subclinical mastitis. We present new information for New Zealand grazing dairy ewes, examine udder asymmetry as a diagnostic tool for subclinical mastitis, and show that, although prevalence was lower in New Zealand, the dominant pathogens are consistent, supporting the broader relevance of these findings to international mastitis control, albeit with adaptations for pasture-based systems.
Subclinical mastitis in New Zealand grazing dairy ewes 2: Relationships among somatic cell count, California Mastitis Test, and milk culture, and risk factors for elevated aerobic plate count
(Elsevier Inc on behalf of the American Dairy Science Association, 2026-01-01) Chambers G; Lawrence K; Grinberg A; Velathanthiri N; Ridler A; Laven R
Our objectives were, in grazing dairy ewes, (1) to describe SCC, California Mastitis Test (CMT) score, and ewe-level milk aerobic plate count (APC), (2) to explore the relationship between CMT and SCC, (3) to identify risk factors for elevated APC, and (4) to find the optimal SCC threshold for diagnosis of IMI. Gland-level milk samples were collected from ∼15 randomly selected ewes on each of 20 New Zealand dairy sheep farms at early, mid, and late lactation in a repeated cross-sectional study. Aerobic bacterial culture and CMT (measured on a scale of 0, trace, 1, 2, or 3) were performed at the gland level, and SCC and APC at the ewe level using composite milk samples. Milk samples were collected from 893 ewes, 870 of which had complete SCC and culture data. Geometric mean SCC was 169,039 (95% CI: 153,921–185,641) cells/mL, varying between farms and decreasing across visits. A CMT score ≥1 in one or both glands occurred in 21.2% of ewes. Mean log10 SCC increased linearly with CMT score, but the correlation between the ewe's highest gland-level CMT score and SCC was moderate (Kendall's tau = 0.47, 95% CI 0.43–0.52). Bacteria were isolated from 86 (9.9%) ewes, with the most common bacteria being NAS (7.0% of glands) and Staphylococcus aureus (0.6% of glands). A SCC threshold of ∼400,000 cells/mL had the greatest Youden's index for diagnosing IMI using a single SCC measurement. The APC was below the limit of quantification (1 × 103 cfu/mL) in 78.0% of ewes, and <100 × 103 cfu/mL in 96.9% of ewes, and varied between visits and farms. Using a mixed Bayesian ordinal regression model, elevated CMT score and SCC, positive milk culture, and subclinical mastitis, but not udder asymmetry, were confirmed as risk factors for elevated APC. These findings provide baseline milk quality data for New Zealand grazing dairy ewes, confirm that udder health should be considered when investigating elevated bulk milk APC, and can be used to help producers manage SCC, subclinical mastitis, and APC, as well as informing further research. Findings specific to New Zealand's emerging sheep dairy industry offer a benchmark for pastoral systems internationally and highlight the importance of udder health to bulk milk quality.
Uncovering the genetic diversity of Giardia intestinalis in isolates from outbreaks in New Zealand
(BioMed Central Ltd, 2022-12) Ogbuigwe P; Biggs PJ; Garcia-Ramirez JC; Knox MA; Pita A; Velathanthiri N; French NP; Hayman DTS
BACKGROUND: Giardia intestinalis is one of the most common causes of diarrhoea worldwide. Molecular techniques have greatly improved our understanding of the taxonomy and epidemiology of this parasite. Co-infection with mixed (sub-) assemblages has been reported, however, Sanger sequencing is sometimes unable to identify shared subtypes between samples involved in the same epidemiologically linked event, due to samples showing multiple dominant subtypes within the same outbreak. Here, we aimed to use a metabarcoding approach to uncover the genetic diversity within samples from sporadic and outbreak cases of giardiasis to characterise the subtype diversity, and determine if there are common sequences shared by epidemiologically linked cases that are missed by Sanger sequencing. METHODS: We built a database with 1109 unique glutamate dehydrogenase (gdh) locus sequences covering most of the assemblages of G. intestinalis and used gdh metabarcoding to analyse 16 samples from sporadic and outbreak cases of giardiasis that occurred in New Zealand between 2010 and 2018. RESULTS: There is considerable diversity of subtypes of G. intestinalis present in each sample. The utilisation of metabarcoding enabled the identification of shared subtypes between samples from the same outbreak. Multiple variants were identified in 13 of 16 samples, with Assemblage B variants most common, and Assemblages E and A present in mixed infections. CONCLUSIONS: This study showed that G. intestinalis infections in humans are frequently mixed, with multiple subtypes present in each host. Shared sequences among epidemiologically linked cases not identified through Sanger sequencing were detected. Considering the variation in symptoms observed in cases of giardiasis, and the potential link between symptoms and (sub-) assemblages, the frequency of mixed infections could have implications for our understanding of host-pathogen interactions.