Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencingle-number tandem-repeat analysis and whole-genome sequencing

Zoonotic tuberculosis is a risk for human health, especially when animals are in close contact with humans. Mycobacterium tuberculosis was cultured from several organs, including lung tissue and gastric mucosa, of three captive elephants euthanized in a Swiss zoo. The elephants presented weight loss, weakness and exercise intolerance. Molecular characterization of the M. tuberculosis isolates by spoligotyping revealed an identical profile, suggesting a single source of infection. Multilocus variable-number of tandem-repeat analysis (MLVA) elucidated two divergent populations of bacteria and mixed infection in one elephant, suggesting either different transmission chains or prolonged infection over time. A total of eight M. tuberculosis isolates were subjected to whole-genome sequence (WGS) analysis, confirming a single source of infection and indicating the route of transmission between the three animals. Our findings also show that the methods currently used for epidemiological investigations of M. tuberculosis infections should be carefully applied on isolates from elephants. Moreover the importance of multiple sampling and analysis of within-host mycobacterial clonal populations for investigations of transmission is demonstrated

Iron regulation in elderly Asian elephants (Elephas maximus) chronically infected with mycobacterium tuberculosis

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection

Clinical Chemistry and Haematology Values of a Captive Population of Humboldt Penguins (<i>Spheniscus humboldti</i>)

Reference intervals for clinical chemistry and haematology analytes are essential for the interpretation of blood results, but limited data are available for Humboldt penguins (Spheniscus humboldti) in zoological collections as well as in the wild. The aim of the present study was therefore to establish reference intervals for a series of clinical chemistry and haematology analytes in a single zoological population of Humboldt penguins. Furthermore, possible variations of the analytes influenced by the age and sex of the individuals were investigated. Lithium heparinized plasma and whole blood samples from 39 animals were analysed and statistically evaluated. The following clinical chemistry analytes were significantly higher in females: glutamate dehydrogenase (p = 0.0003), alanine transaminase (p = 0.0005), alkaline phosphatase (p = 0.0245), aspartate aminotransferase (p = 0.0043), creatine kinase (p = 0.0016), lactate dehydrogenase (p p = 0.0223), and sodium (p = 0.0415). No significant differences between males and females were found for any haematology analyte. Cholesterol (p = 0.0233; −0.39), white blood cell count (p = 0.0208; −0.40), and absolute heterophiles (p = 0.0148; −0.42) had significant negative correlations with the age of the penguins. The results indicate that the factors of sex and age should be considered when interpreting the results of haematological and clinical biochemistry assays, and further studies are needed to determine whether there are more differences in other age groups or seasons

Recurrent tongue tip constriction in a captive giant anteater (Myrmecophaga tridactyla)

A male giant anteater (Myrmecophage tridactyla) was treated twice for tongue tip constrictions. Clinical signs were partial anorexia, soft stool, bleeding from the mouth, and intermittent lingual discomfort. In the first presentation, wood fibers constricting the distal part of the tongue were detected by endoscopy and were removed. In the second presentation, bands of collagenous fibers were identified and resected. Dietary elements were responsible for both cases: elongated wood fibers were present in peat, which was included as a supplement to improve stool consistency, and collagenous fibers originated from fascias of lean meat, which served as a protein source in this diet. Preventive measures included sieving of the peat to eliminate long fibers and grinding of the meat, respectively, prior to diet presentation. A homogenous diet, utilizing cellulose rather than peat and dry cat food rather than meat, will avoid tongue tip constriction as described in these cases

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection

Prolonged luteal lifespan and pseudopregnancy in Asian elephants (Elephas maximus)

Pseudopregnancy is a physiological occurrence in mammals which have copulation induced ovulation, but is rarely described in spontaneous ovulating species. In this study, three cases of prolonged luteal lifespan are reported in non-pregnant Asian elephants (Elephas maximus). Case 1 was a 25-year-old female that had produced three calves previously; Case 2 was a nulliparous and 32-year-old at the start of the pseudopregnancy episode; and Case 3 occurred in a 49-year-old nulliparous elephant. Serum progesterone metabolite concentrations remained elevated for 10 months in Case 1. Urinary progestagens were high for >16 months in Case 2 and for five months in Case 3. In Case 1, multiple persistent corpora lutea were visualized monthly by ultrasonography. In all three cases, uterine leiomyoma were present and progestagen concentrations decreased spontaneously. In Case 1, the elephant became pregnant 3 years later, whilst with Case 2, the female resumed estrous cycling normally, and for the Case 3 female, there was continuation with another prolonged luteal phase before ovarian function was purposely suppressed. These examples indicate that persistently elevated progestagen concentrations may not always be indicative of pregnancy in elephants. The reasons for prolonged luteal lifespan are not understood, although serum prolactin concentrations quantified in the Case 1 female were elevated compared to values from previous reports and two other herd mates. Furthermore, all three elephants had varying degrees of uterine pathologies. It is believed that the resulting damage to the endometrium may have led to a reaction similar to implantation, which includes prolactin secretion. Prolactin may exert luteotropic properties and is thought to initiate luteal rescue during pregnancy in elephants.Japan Society for the Promotion of Science (JSPS) for their support of this research which allowed sample analysis in Tokyo, Japan for prolactin (grant # PE 11016).http://www.elsevier.com/locate/anireprosci2019-10-01hj2018Anatomy and Physiolog

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection

Polycystic kidney disease in the pygmy hippopotamus (Hexaprotodon liberiensis)

Polcystic kidney disease (PKD) was diagnosed at necropsy in a captive aged female pygmy hippopotamus (Hexaprotodon liberiensis), which presented with numerous cysts in both kidneys, liver, duodenum and one single in the pancreas. There were no premonitory clinical signs of a nephropathy observed prior to its death. Similar findings were made in the male partner animal half a year later. Both animals had been wild-caught. A literature review showed that another seven cases of PKD have been reported in pygmy hippopotamuses, and an additional screening of records available from the international studbook for the species revealed yet another six cases. In all cases, aged females were affected, and in several instances, affected animals were related to each other. These patterns suggested familiar transmission, similar to PKD in humans and other animals. The disease, and especially the presumptive bias in diagnosis towards females – the male animal of this report was to our knowledge the first case of PKD reported in a male pygmy hippopotamus -, warrant further investigation. The status of the kidneys with respect to PKD should be assessed (including histology) in every deceased pygmy hippopotamus

Occurrence of cataract and fatty liver in captive plains viscachas (Lagostomus maximus) in relation to diet

An increased incidence of cataract and fatty liver in plains viscachas (Lagostomus maximus) was noted for many years at the Zurich Zoo. Based on elevated serum fructosamine and glucose, diabetes mellitus was diagnosed, and these parameters normalized when the diet of the animals was changed from a low-fiber to a high-fiber diet. Here, we evaluate 177 necropsy reports from before and after the diet change for the incidence of cataracts and fatty liver. Sixteen of 56 animals (29%) that were born before the diet change developed cataract. In contrast, only two of 121 animals (1.65%) that were born after the diet change developed cataracts. The prevalence of cataract and fatty liver in animals born after the diet change (1% and 0%, respectively) was significantly lower than in animals born before (9% and 6%, respectively) the diet change. The results suggest that the plains viscachas at Zurich Zoo were affected by an alimentary induced diabetes mellitus (type 2) before the diet was changed