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Abstract Background Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic granulomatous enteritis that affects domestic and wild ruminants and camelids. The disease has rarely been reported in alpacas in Germany. This publication describes epidemiologically independent cases of paratuberculosis in two alpacas in Germany. Case presentation Two alpacas, a 26-year-old female zoo animal (case 1) and a 2.5-year-old breeding stallion from a private owner (case 2), presented with progressive emaciation, leading to death (case 2) or euthanasia (case 1) because of deteriorating general condition. In both cases typical granulomatous lesions in the intestinal mucosa and mesenteric lymph nodes were found. In case 2, other lymph nodes were severely enlarged and MAP was detected in the mandibular lymph node, lung, and liver by qPCR. The MAP isolates differed between the alpacas, with two distinct phylogenetic clades (Clade 1 and 8) within Subgroup A of the MAP-C type group and two distinct INMV profiles (INMV 2 and 1) found. These genotypes have been identified in cattle and goats in different regions in Germany. The genotype isolated from case 1 has been detected in goats from the zoo since 2011, indicating transmission between these species. Conclusions MAP can cause severe clinical disease in alpacas of variable age and under different husbandry conditions. Therefore, paratuberculosis should be considered for differential diagnosis in alpacas with emaciation and poor general condition. Although not definitely shown, cross-species infection between ruminant species and camelids is exceedingly likely.

This study investigated the intra- and inter-herd diversity of Mycobacterium avium subsp. paratuberculosis (MAP) isolates from four goat herds in Thuringia (Germany) that were affected by paratuberculosis for several years. The main focus was on the characterization and distribution of genotypes among animals and the environment of goat herd 1. This study included 196 isolates from the feces of 121 infected goats, various tissues from 13 clinically diseased goats, 29 environmental samples from herd 1, and additionally, 22 isolates of different origin from herds 2 to 4. The isolates, sampled between 2018 and 2022, were genotyped using short-sequence-repeat (SSR) analysis, mycobacterial-interspersed repetitive units–variable-number tandem repeat (MIRU–VNTR) analysis, and a single nucleotide polymorphism (SNP)-based assay for phylogenetic grouping. All the isolates belonged to the MAP-C group. In herd 1, one predominant genotype was determined, while two other genotypes were identified very rarely and only in fecal and environmental samples. One of three further genotypes was found in each of herds 2 to 4. The assignment of genotypes to different phylogenetic clades suggested six different infection strains. The results indicated no epidemiological links between the examined herds. Based on the current MAP genotyping data from Germany, possible sources of infection are MAP-contaminated barns previously used by infected cattle and the purchase of sub-clinically infected goats.

ABSTRACT A total of 50 birds diagnosed with mycobacteriosis were examined for pathomorphological lesions, coinfections, and causative agents. Mycobacterial species were identified and isolates differentiated using multilocus sequence typing (MLST) and mycobacterial interspersed repetitive-unit variable-number of tandem-repeat (MIRU-VNTR) analysis. Possible associations between mycobacterial species, pathomorphological findings, coinfections, bird orders, and husbandry conditions were evaluated statistically. Mycobacteria were isolated from 34 birds (13 of 22 Psittaciformes, 12 of 18 Passeriformes, five of six Columbiformes, and four other orders) belonging to 26 species in total. Mycobacterium genavense (Mg) was cultured from 15 birds, Mycobacterium avium subsp. avium (Maa) from 20 birds, and Mycobacterium avium subsp. hominissuis (Mah) from three birds; hence, four birds had mixed infections. About equal numbers of psittacines and passerines were infected with Ma and Mg. The genetic diversity differed; Mg isolates belonged to one MLST type, Maa to six, and Mah to three combined genotypes. Several coinfections were detected; viruses and/or endoparasites affected 44%, fungi 38%, and bacteria 29% of the birds. Pathological findings and mycobacteriosis-affected organs were independent of coinfections. Overall, gross pathological findings were more often seen in mycobacteriosis caused by Ma (95%) compared with Mg (66%). Organ distribution of mycobacteriosis was independent of the mycobacterial species. Pathomorphological changes were seen in the small intestine of 71% and the lung of 65% of the birds, suggesting oral or pulmonal ingestion of mycobacteria. There were no associations between mycobacterial species and bird orders or bird husbandry conditions. Not only Mg, but also Maa and Mah, were clearly identified as primary cause of mycobacteriosis in pet birds. IMPORTANCE In this study, the causative agents and confounding factors of mycobacteriosis in a set of pet and some wild birds from Germany were examined. Not only Mycobacterium genavense, but also M. avium subsp. avium and M. avium subsp. hominissuis, contributed to mycobacteriosis in these birds. Various coinfections did not affect the manifestation of mycobacteriosis. Due to different gross necropsy findings, however, a different pathogenicity of the two species was assumed. New strains of M. avium subsp. hominissuis originating from birds were identified and characterized, which is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between M. avium subsp. avium genotypes and virulence which will have to be confirmed by broader studies.

Environmental samples are often used to classify the paratuberculosis status of cattle herds. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), predominantly through oral ingestion during infancy. In this explorative study, the presence of MAP was determined in the barn environment of a paratuberculosis-infected vaccinated dairy goat herd. A total of 256 bedding, dust, feed, and water samples were collected at eight time points and examined using culture and qPCR. Detection rates of both methods were compared, and factors determining MAP confirmation were identified. MAP was cultured from 28 bedding and one dust sample, while MAP DNA was detected in all materials (117/256). Samples from high animal traffic areas and those collected during the indoor season were more likely to yield positive culture and qPCR results. Cultivation of MAP from kidding pens indicated this area as a possible infection site. Dust proved to be the most suitable material for detecting MAP DNA, as bedding was for MAP culture. Environmental sampling was demonstrated to be an effective way to detect MAP in a dairy goat herd. qPCR results could confirm herd infection, while culture results provided insight into crucial areas for MAP transmission. These findings should be considered when designing farm-specific paratuberculosis control plans.

Bovine tuberculosis (bTB) not only poses a zoonotic threat to humans but also has a significant economic impact on livestock production in many areas of the world. Effective vaccines for humans, livestock, and wildlife are highly desirable to control tuberculosis. Suitable large animal models are indispensable for meaningful assessment of vaccine candidates. Here, we describe the refinement of an animal model for bTB in goats. Intrabronchial inoculation procedure via video-guided endoscopy in anesthetized animals, collection of lungs after intratracheal fixation in situ, and imaging of lungs by computed tomography (CT) were established in three goats using barium sulfate as surrogate inoculum. For subsequent infection experiments, four goats were infected with 4.7 × 102 colony-forming units of M. bovis by intrabronchial inoculation using video-guided endoscopy with spray catheters. Defined amounts of inoculum were deposited at five sites per lung. Four age-matched goats were mock-inoculated. None of the goats developed clinical signs until they were euthanized 5 months post infection, but simultaneous skin testing confirmed bTB infection in all goats inoculated with M. bovis. In tissues collected at necropsy, M. bovis was consistently re-isolated from granulomas in lymph nodes, draining the lungs of all the goats infected with M. bovis. Further dissemination was observed in one goat only. Pulmonary lesions were quantified by CT and digital 2D radiography (DR). CT revealed mineralized lesions in all the infected goats ranging from 10 mm in diameter. Small lesions

Oral intake of Mycobacterium avium subspecies paratuberculosis (MAP) in first days of life is considered to be the main route of infection for paratuberculosis. This can be related to a direct contact to contaminated feces or feeding of MAP containing colostrum. Colostrum is believed to become contaminated either by lactogenic shedding or introduction of MAP from environmental sources. In this pilot study, the presence of MAP in individual and bulk colostrum samples from a paratuberculosis-infected, vaccinated dairy goat herd in Germany and the effect of udder skin disinfection on the MAP load of colostrum were examined. In order to distinguish between lactogenic shedding and fecal contamination, 49 udder skin swabs were cultivated on solid medium whereas 29 swabs were additionally analyzed by qPCR. qPCR was applied on 110 individual colostrum samples collected from 55 goats, one before and one after disinfection with a mycobactericidal disinfectant, and 14 bulk colostrum samples. MAP DNA was detected in 10.3% (3/29) of the swab samples, but no viable MAP was cultivated from any sample. These results indicate a low-level MAP contamination of the udder skin and colostrum of milking goats suggesting a low risk of MAP transmission via these routes.

Regionally, the monitoring of paratuberculosis at the herd level is performed by the detection of specific antibodies in pooled milk samples by ELISA. The negative/positive cut-off S/P values applied for pooled milk samples are low and particularly vulnerable to variation in the test performance. In this study, a batch variation in the test performance of two ELISA tests was assessed to identify consequences for sample classification. A total of 72 pooled milk samples (50 from MAP-infected herds, 22 from one MAP-non-infected herd) were analyzed using three different batches, each of two different MAP antibody ELISA tests (A and B). Receiver operating characteristic (ROC) analysis was performed, with the results of each batch, S/P values of the samples and optical density (OD) readings of the negative and positive control samples included in the kits being compared between the batches of one test. ROC analysis revealed a considerable variation in the test performance of the batches of the two individual tests, caused by differences in the S/P values of the samples and resulting in different sensitivities at a specificity of 100%. Major sources of variation originate from the manufacturing processes of test batches. These sources have to be better controlled, and the test performance has to be revisited regularly.