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\begin{document}

\title{A case of early neonate tuberculosis in a three weeks old calf in
Ethiopia}



\author[1]{Getnet Mekonnen}%
\author[2]{Balako Gumi}%
\author[3]{Stefan Berg}%
\author[4]{Andrew Conlan}%
\author[3]{Martin Vordermeier}%
\author[5]{Gobena  Ameni}%
\author[6]{James Wood}%
\affil[1]{Affiliation not available}%
\affil[2]{Addis Ababa University}%
\affil[3]{Animal and Plant Health Agency}%
\affil[4]{University of Cambridge School of Clinical Medicine}%
\affil[5]{Akilu Lemma Institute of Pathology, Addis Ababa University, P.O.Box 1176, Addis Ababa, Ethiopia.}%
\affil[6]{University of Cambridge}%


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  \date{March 17, 2025}


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\begin{abstract}
Bovine tuberculosis is a chronic disease rarely observed in an early
age. Our observation of tubercle lesions in the lungs of a three week
old calf and confirmation of Mycobacterium bovis strains illustrate that
the progression of tuberculosis in neonates can be rapid under natural
conditions, contributing to transmission within-herds.%
\end{abstract}%



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\textbf{Key Clinical Message}

This report illustrates that calves may be infected with bovine
tuberculosis at early age under natural conditions and progression can
be rapid. Thus, testing of calves need to be considered in any control
program to reduce the risk of transmission.

\textbf{INTRODUCTION}

Bovine tuberculosis (bTB) is a chronic disease, caused primarily
by\emph{M. bovis} , affecting cattle, other domesticated animals,
certain wildlife species, non-human primates, and
humans.\textsuperscript{1-3}Cattle can be infected at any age, but
tubercle lesion development and subsequent clinical signs may appear at
a later stages. Calves can be born with tuberculosis congenitally when
infected with \emph{M. bovis}either via the umbilical vein from an
infected genital tract of the dam, from the infected placenta, or from
infected amniotic fluid.\textsuperscript{4} After birth, neonates can
also be infected even at an early stages following delivery, mainly by
inhaling infected droplets and/ or by ingesting raw milk or colostrum
from infected cows. Other routes of infection such as cutaneous, though
not common, are still important in situations where the prevalence of
bTB is high.\textsuperscript{5} As the course of the disease is slow
compared to many other infectious diseases, infected animals can spread
bTB to other herd mates and newborns before it begins to manifest
clinical signs. The transmission rate may be faster at late stages of
infection as the number of organisms being shed is larger in this
stage.\textsuperscript{1}

The observation of characteristic tubercle lesions in the lungs of a
three weeks old male calf led us to raise questions like `how the calf
could get the infection?' and `how fast is the disease progression under
natural infection?'. In order to get answers to these questions, further
investigation was made and this case report was compiled from the
pathological and molecular investigations conducted for the diagnosis of
the case.

\textbf{CASE HISTORY}

A dairy farm located in northern Ethiopia and with a herd size of 100
cattle (86 crossbreeds of Holstein-Frisian and Zebu breeds and 14 Zebu
breeds) was screened for bTB by the SICCT test. As it was learned from
its history, the farm was newly established just a year earlier through
the purchase of cows and heifers from dairy farms and markets in Mekelle
City and nearby towns, including Adigrat and Humera. The SICCT testing
was conducted on the herd in 2016 by Mekonnen \emph{et al.}
\textsuperscript{6} and a prevalence of 12\% (95\% CI: 7 to 19.8\%) was
recorded. One of the reactors was a three weeks old calf (ID 8A0597) and
its skin induration was 8 mm according to the calculation of the SICCT
test result. All positive reactors (including the calf) were removed
from the herd following disclosure of the test results. It was observed
from the husbandry of the farm that all calves in the farm were sharing
the same housing with the adults. Moreover, the calves were fed on
pooled milk obtained from milking cows in the herd including one SICCT
reactor cow, and looked after by the same individuals who were looking
after the adult cattle.

The dam of the calf (ID 8A0417) was a 10 years old cross of
Holstein-Frisian and Zebu breeds. The body condition of this cow was
rated as `Good' and it was negative for bTB by the SICCT test according
to the interpretation recommended by OIE.\textsuperscript{7} In
addition, nasal discharge was collected from the dam using a cotton swab
designed for such purpose, and the PBS wash of the swab was centrifuged
at 4000 rpm for 15 min. Genomic DNA was extracted from the pellet using
a Qiagen DNA extraction kit as per the manufacturer's
instruction,\textsuperscript{8} and the eluent tested by an RD4-based
PCR assay for specific identification of \emph{M. bovis} . The primers
defined by Brosch \emph{et al.} \textsuperscript{9} were used in the PCR
assay procedure described by Berg \emph{et al} .\textsuperscript{10}

\textbf{INVESTIGATION}

The body condition of the calf was moderate, but it was characterised
with some hair loss on various parts of its body and appeared depressed.
In post-mortem, multiple granulomas were observed in the apical lobe of
the lungs (FIGURE 1). A single \textasciitilde{}20 mm diameter of a
loosely encapsulated granuloma-like structure was also observed in the
diaphragmatic lobe. Lymph nodes and other vital organs appear normal.

\textbf{(FIGURE 1 here)}

The culturing result (L\selectlanguage{ngerman}öwenstein-Jensen medium slant) from lung
tubercles of the calf showed two strains of typical \emph{M.
bovis}colony morphology. Heat-killed colonies were tested by RD4-based
PCR and confirmed that both strains were \emph{M. bovis} .
Spoligotyping\textsuperscript{11} then classified these two strains into
different spoligotype patterns, SB0134 and SB0133 (FIGURE 2). Genomic
DNA from the dam's nasal discharge was positive for \emph{M. bovis} by
the RD4-based PCR assay; however, spoligotyping was not sensitivity
enough to confirm the pattern.

\textbf{(FIGURE 2 here)}

\textbf{DISCUSSION}

\emph{M. bovis} strains identified in the present case, classified as
spoligotypes SB0134 and SB0133, suggesting mixed infection. Although
mixed infection has not been frequently described in Ethiopia, it is
more likely to happen in herds which have established their stocks from
different farms, as was the case for the present herd. Hence, the mixed
infection observed in this calf could be due to transmission from more
than one reactor, possibly recruited from different herds without prior
testing for bTB.

The time-frame for development of tubercle lesions in the investigated
calf is comparable with artificially infected calves via respiratory
route under experimental conditions.\textsuperscript{12} However, under
such conditions; the extent of pathology could vary depending on the
infective dose and factors specific to the animals and husbandry
systems. Interestingly, the gross pathological lesions in the present
calf were found only in specific lobes of the lungs.

Evidences have shown that the route of transmission of \emph{M.
bovis}could be inferred from lesions distribution.\textsuperscript{13}
In the present calf, tubercle lesions were localized in the lungs and no
tubercle lesions were observed in other tissues and organs, which could
suggest inhalation of infectious droplets as a possible route of
infection while ingestion of infected colostrum or milk might seem less
likely. Other possibilities of in-utero transmission through
hematogenous route and/or aspiration of \emph{M. bovis} contaminated
amniotic fluid \textsuperscript{14, 15} could not be justified as there
was no lesion suggestive of bTB in other vital organs, such as liver.
Tuberculosis transmitted hematogenously is typically characterised by
presence of hepatic tubercles or more disseminated form of the
disease,\textsuperscript{16} while through aspiration of infected
amniotic fluid, the primary tubercle could be in the lungs and/or the
gut.

The molecular test result imply that the dam was infected with \emph{M.
bovis} even though it was not a reactor to the SICCT test (the increase
at the PPD-B injection site was 3.2 mm while at the PPD-A site it was
2.9 mm). This could potentially be due to a development of anergic
status, in which the dam would either had been be in a late disease
stage or immunosuppressed attributable to the hormonal effects during
pre- and post-parturition. An animal with a chronic stage of the disease
is known to be highly infectious and would likely serve as a good source
of infection to a neonate by transmission in several possible
ways.\textsuperscript{17, 18} Therefore, it is highly likely that the
calf in this observed case got infected in its first week of life, while
suckling the colostrum by inhalation of aerosol primarily generated from
its dam, although the possibility of transmission from other infected
animals could not be ruled out as all reactors in this farm shared the
same housing and husbandry system as the calf. In fact, the latter could
explain why strains of the two spoligotype patterns were isolated from
its lungs.

In conclusion, although this is a single case, it illustrates that the
progression of bTB in calves can be rapid under natural conditions,
contributing to transmission within herds. This has important
implications in the appropriate structure of models used to estimate the
rate of transmission within herds. \textsuperscript{19} Calf can respond
to the tuberculin test at a very early age, and thus should be
considered in the routine bTB screening to reduce transmission within
herds.

\textbf{AUTHORS' CONTRIBUTIONS}

GAM performed the investigation, analyzed the data, and wrote the
report. BG, SB, AJKC, MV, GA, and JLNW contributed to the study
conception and design and reviewed the report.

\textbf{ACKNOWLEDGEMENTS}

This work was funded by the Biotechnology and Biological Sciences
Research Council, the Department for International Development, the
Economic \& Social Research Council, the Medical Research Council, the
Natural Environment Research Council and the Defence Science \&
Technology Laboratory, under the Zoonoses and Emerging Livestock Systems
(ZELS) program, ref: BB/L018977/1.

\textbf{COMPETING INTEREST}

The authors declare that they have no competing interests.

\textbf{ETHICAL CONSIDERATIONS}

This study was under the framework of a research protocol approved by
the Institutional Review Board (IRB) of Aklilu Lemma Institute of
Pathobiology, Addis Ababa University (Reference
number\emph{IRB/ALIPB/2018} ).

\textbf{AVAILABILITY OF DATA AND MATERIALS}

All data generated and /or analyzed during this study are not publicly
available but are available from the corresponding author on reasonable
request.

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Tuberculosis in Great Britain.\emph{PLoS Comput Biol} 8: e1002730.\selectlanguage{english}
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