16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method.

The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kakapo) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

Mammal-related Cryptosporidium infections in endemic reptiles of New Zealand.

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.

Validation of an Indirect Immunofluorescence Assay and Commercial Q Fever Enzyme-Linked Immunosorbent Assay for Use in Macropods.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

Gut microbiota of the threatened takahe: biogeographic patterns and conservation implications.

BACKGROUND: The Aotearoa New Zealand takahe (Porphyrio hochstetteri), once thought to be extinct, is a nationally threatened flightless rail under intensive conservation management. While there has been previous research into disease-related microbes in takahe, little is known about the microbes present in the gastrointestinal tract. Given the importance of gut-associated microbes to herbivore nutrition and immunity, knowledge of these communities is likely to be of considerable conservation value. Here we examined the gut microbiotas of 57 takahe at eight separate locations across Aotearoa New Zealand. RESULTS: Faecal samples, taken as a proxy for the hindgut bacterial community, were subjected to 16S rRNA gene amplicon sequencing using Illumina MiSeq. Phylogenetic analysis of > 2200 amplicon sequence variants (ASVs) revealed nine main bacterial phyla (Acidobacteriota, Actinobacteriota, Bacteroidota, Campilobacterota, Firmicutes, Fusobacteriota, Planctomycetota, Proteobacteria, and Verrucomicrobiota) that accounted for the majority of sequence reads. Location was a significant effect (p value < 0.001, 9999 permutations) that accounted for 32% of the observed microbiota variation. One ASV, classified as Lactobacillus aviarius, was present in all samples at an average relative abundance of 17% (SD = 23.20). There was strong evidence (p = 0.002) for a difference in the abundance of the genus Lactobacillus between locations. A common commensal bacterium previously described in takahe, Campylobacter spp., was also detected in most faecal samples. CONCLUSIONS: Location plays a pivotal role in the observed variation among takahe gut bacterial communities and is potentially due to factors such as supplemental feeding and medical treatment experienced by birds housed in captivity at one of the eight sampled sites. These data present a first glimpse of the previously unexplored takahe gut microbiota and provide a baseline for future microbiological studies and conservation efforts.

MYCOBACTERIAL DISEASE AND SUBSEQUENT DIAGNOSTIC INVESTIGATIONS IN A GROUP OF CAPTIVE PINNIPEDS IN NEW ZEALAND.

This case series includes a single case of disseminated tuberculous disease due to Mycobacterium pinnipedii in a New Zealand fur seal (Arctocephalus forsteri), which was being cared for by a zoo in New Zealand. The remaining five pinnipeds in the colony underwent extensive mycobacterial disease surveillance over the following 4 yr, involving a total of 26 anesthetic procedures and numerous diagnostic tests that included comparative intradermal tuberculin skin tests, mycobacterial antibody serology, respiratory and gastric lavages, and computed tomography (CT) scans. An additional case of chronic sinusitis due to Mycobacterium marinum and Pseudomonas aeruginosa was identified in a California sea lion (Zalophus californianus). Results from CT and the respiratory lavages were the most helpful antemortem diagnostic tests for active mycobacterial disease in this case series. Of the remaining four animals, two were euthanatized and two remain alive, and none of them had evidence of active mycobacterial disease. Further mycobacterial disease surveillance in staff and animals was performed, and no other case was identified. There are no validated mycobacterial surveillance tests available for pinnipeds and so it remains unknown whether the two surviving pinnipeds are truly negative or whether they have latent mycobacterial infection that could develop into active mycobacterial disease in the future. For this reason, increased levels of biosecurity and quarantine remain permanently in place for the pinniped colony.

Toxoplasmosis in Sand cats (Felis margarita) and other animals in the Breeding Centre for Endangered Arabian Wildlife in the United Arab Emirates and Al Wabra Wildlife Preservation, the State of Qatar.

The Sand cat (Felis margarita) is a small-sized felid found in sand and stone deserts ranging from the north of Africa to Asia, with the Arabian Peninsula as its centre of distribution. The Sand cat captive breeding program at the Breeding Centre for Endangered Arabian Wildlife (BCEAW), Sharjah, UAE, has experienced high newborn mortality rates, and congenital toxoplasmosis was recently recognized as one of the causes of this mortality. In the present study, one 18-month-old Sand cat (FM019) died of acute toxoplasmosis-associated hepatitis and pneumonitis acquired after birth; Toxoplasma gondii was demonstrated in histological sections which reacted with T. gondii polyclonal antibodies by immunohistochemistry (IHC). T. gondii DNA was found by PCR of extracted DNA from liver and lung tissues of this cat. Antibodies to T. gondii were found in serum examined in 1:1600 dilution in the modified agglutination test (MAT); its 2-year-old cage mate seroconverted (MAT titer 1:3200) at the same time. Another Sand cat (FM017) was euthanized because of ill health when 3 years old; its MAT titer was >1:3200, and T. gondii tissue cysts were found in brain, heart, ocular muscles and skeletal muscle, confirmed by IHC. Viable T. gondii was isolated by bioassays in mice inoculated with tissues of another chronically infected Sand cat (FM002); T. gondii was not found in histological sections of this cat. T. gondii antibodies were found in several species of animals tested, notably in 49 of 57 wild felids at BCEAW. A 7-year-old Sand cat (3657) from Al Wabra Wildlife Preservation (AWWP), Doha, State of Qatar died of acute visceral toxoplasmosis with demonstrable T. gondii tachyzoites by IHC, and T. gondii DNA by PCR, and a MAT titer of >3200. T. gondii antibodies were found in 21 of 27 of wild felids at AWWP. PCR-RFLP genotyping at 10 genetic loci revealed that these T. gondii isolates from Sand cat (FM002 and FM019) at BCEAW have an atypical genotype, which was previously reported in T. gondii isolates of dogs from Sri Lanka. The genotype from the cat from AWWP (3657) is a genetic Type II strain with a Type I allele at locus Apico. This is the first report of genetic characterization of T. gondii isolates from Middle East.

Fatal toxoplasmosis in sand cats (Felis margarita).

The sand cat (Felis margarita) is a small-sized felid occurring in the United Arab Emirates (UAE). The sand cat captive-breeding program at the Breeding Centre for Endangered Arabian Wildlife in Sharjah, UAE, has until recently been severely compromised by very high newborn mortality rates. Two different pairs of sand cats gave birth, respectively, to one and two litters (with a total of eight kittens) between 1999 and 2006. Seven out of eight kittens died between the third and 21st wk of life. Toxoplasmosis was confirmed as the cause of death in these two litters. Adult cats had high antibody titers to Toxoplasma gondii before pregnancy, suggesting that maternal immunity did not protect the kittens against infection with T. gondii and that maternal immunity might not have prevented transplacental transmission of the parasite. This observation contrasts with what is seen in domestic cats. To date, this is the first report on confirmed fatal toxoplasmosis and prevalence of T. gondii in sand cats.

Toxoplasmosis in sand fox (Vulpes rueppelli).

Fatal toxoplasmosis was diagnosed in a sand fox (Vulpes rueppelli) from United Arab Emirates. Toxoplasma gondii-like tachyzoites were found associated with necrosis in the intestine, spleen, liver, pancreas, lungs, mesenteric lymph nodes, and heart. Tachyzoites reacted positively with T. gondii-specific polyclonal antibodies. Antibodies to T. gondii were detected in 8 captive V. rueppelli assayed by the modified direct agglutination test in titers of 1:800 or higher.

Seroprevalence of antibodies to Toxoplasma gondii in Gordon's wildcat (Felis silvestris gordoni) in the Middle East.

Most species of felids tested have been found to be the definitive host for Toxoplasma gondii. Gordon's wildcat (Felis silvestris gordoni) is a threatened species found in the United Arab Emirates (UAE) and Oman. Antibodies to T. gondii were found in all 29 captive and 2 of 7 wild-caught F. s. gordoni in UAE examined by the modified agglutination test (MAT). Titers were 1:100 in 1, 1:200 in 5, 1:400 in 6, 1:800 in 10, 1:1,600 in 5, and 1:3,200 or higher in 4. None of these cats was ill, despite exhibiting high antibody titers. This is the first report of T. gondii infection in this host.

Toxoplasma gondii infection in Blanford's fox (Vulpes cana).

Fatal toxoplasmosis was diagnosed in a Blanford's fox (Vulpes cana) from the United Arab Emirates. Toxoplasma gondii-like tachyzoites were found associated with necrosis in intestine, spleen, liver, kidneys, lungs, skeletal muscle, brain and heart. Protozoal tachyzoites reacted positively with T. gondii-specific polyclonal antibodies. Antibodies to T. gondii were detected in 10 of 12 V. cana assayed by the latex agglutination or the modified direct agglutination test.